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Abstracts: Turkish Society of Molecular Medicine
Third International Congress of Molecular Medicine
May 5–8, 2009, Istanbul, Turkey
Third International Congress of Molecular Medicine, Abstracts: 273–389
Abstracts: Turkish Society of Molecular Medicine, Third
International Congress of Molecular Medicine, May 5–8, 2009,
Istanbul, Turkey
Welcome to the Third International Congress of Molecular Medicine
Prof. Dr. Turgay Isbir
Department of Molecular Medicine, Institue of Experimental Medical Research, Istanbul University, Istanbul, Turkey
Prof. Dr. Turgay Isbir
Chairperson of Turkish Society of Molecular Medicine
ISSN 1521-6543 print/ISSN 1521-6551 online
DOI: 10.1002/iub.183
Dear Colleagues,
On behalf of the Turkish Society of Molecular Medicine, it is
my great pleasure to welcome you to our III. International Congress of Molecular Medicine. The comprehensive scope of our
meeting, addressing the interests of physicians, scientists and
other healthcare professionals and the industry, may be challenging to all at the same time. We aim to merge our interest in molecular medicine and to establish a link between basic and clinical sciences. For the clinician, there is the opportunity to focus
on advances in the basic sciences forming the basis of clinical
practice. For the scientist, there is a perspective to appreciate the
wide-reaching clinical relevance of scientific discovery.
A large part of the programme is devoted to invited speakers
and poster presentations. The Molecular Medicine Congress participants will receive CME credits from the Turkish Medical
Society. We are confident that your stay in Istanbul will be
both academically and socially rewarding, and we look forward
to meeting with you personally at the III International Congress
of Molecular Medicine.
Contents
President’s Message . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Committees and Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Department of Molecular Medicine, Institute of Experimental Medicine, University of Istanbul . . . . . . . . . . . . . . . . . . . . . .
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Opening Lecture: Kurt Wüthrich (Nobel Prize Winner) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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PLENARY LECTURES
Plenary Lectures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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SYMPOSIA
S1. History of Molecular Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
285
S2. Genomic Analysis and Molecular Basis of Cancer (sponsored by IUBMB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
285
S3. G-Protein-Coupled Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
288
S4. Thrombosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
290
S5. Targeted Therapies in Ovarian Cancer (Organized by North Eastern German Society
of Gynaecological Oncology (NOGGO)) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
291
S6. Molecular Mechanisms of Heart Preconditioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
293
S7. Molecular Markers in Tumor Diagnosis and Therapy
(Organized by International Institute of Anticancer Research (IIAR)) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
295
S8. Phenotype, Response to Therapy and Molecular Genetics in Various Fields of Pediatric Endocrinology . . . . . . . . . . . . .
300
S9. Public Health Genomics and Nutrigenetics (Organized by The Turkish Society of Public Health Genomics
and Nutrigenetics, Public Health Genomics European Network, GENAR Institute) . . . . . . . . . . . . . . . . . . . . . . . . . . .
301
S10. Round Table Discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
303
S11. Drug Development, From Idea to Clinic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
304
S12. Biochemical Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
305
S13. Metabolic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
313
S14. Biochemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
314
S15. Redox Regulation of Cell Signaling in Ageing and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
317
S16. Emerging Vector-Borne Diseases in Istanbul and Surrounding Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
317
S17. Young Investigators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
319
S18. School of Molecular Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
322
S18-1. Molecular Medicine and Diagnostic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
322
S18-2. Genetics and Statistics, Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
323
S18-3. Reproductive Medicine (Organized by: VKV American Hospital Department of Genetics) . . . . . . . . . . . . . . . .
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POSTER SESSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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INDEX
THIRD INTERNATIONAL CONGRESS OF MOLECULAR MEDICINE
COMMITTEES
HONORARY PRESIDENT
Yunus Soylet
CONGRESS PRESIDENT
Turgay Isbir
VICE PRESIDENTS
Necip Ilhan, Nezih Hekim
INTERNATIONAL SCIENTIFIC STEERING COMMITTEE
Franz Theuring
Aldo Tomasi
John G. Delinassios
Kurt Wüthrich
Robert Huber
Angelo Azzi
Efstathios S. Gonos
Shant Kumar
Bedia Agachan
Nilüfer Bozkurt Yigit
Ilhan Eraltan
Atike Tekeli
Bülent Yigit
Mehmet Isbir
Olcay Isbir
Didem Kafadar
Tomris Ozben
ORGANIZATION COMMITTEE
Tülin Ozturk
Nevin Ilhan
Selim Isbir
Umit Zeybek
Alper Tunga Akarsubasi
H. Arzu Ergen
Uzay Gormus
Hulya Yilmaz
Guzide Yucebilgic
Kemal Altas
Hüseyin Bagci
Ahmet Belce
Turgay Budak
Mahmut Carin
Esen O. Dural
Kemal Erbil
Stefano Frosina
Kishorchandra Gohil
Emin Kansu
Güldal Kirkali
Zehra Bugra
Nesrin Emekli
Figen Gurdol
Ilker Durak
Gultekin Yucel
Nedret Kilic
SCIENTIFIC COMMITTEE
Mehmet Kurtoglu
Adile Muz
Fatma Oguz
Moshe Phillip
Ahmed El Sohemy
Yaman Tekant
Seyhan Tukel
Engin Ulukaya
Gary Williamson
Selma Yilmazer
Kadirhan Sunguroglu
Aysen Yarat
Turay Yardimci
Cihat Kucukhuseyin
Levent Karaca
Pakize Pasaoglu
Emine Kokoglu
Ertugrul Kahraman
Fehmi Narter
Oguz Ozturk
Lulufer Tamer
Tamer Inal
Makbule Aydin
Sertac Kip
Yesim Negis
Figen Aksoy
Nejat Akar
Luca Barella
Hakan Berkkan
Peter Butterworth
Ayhan Dagdeviren
David Dunger
Tevfik Dorak
Guido Frosina
Müjdat Uysal
Mustafa Kecer
Nazmi Ozer
Tevfik Akoglu
Kubilay Karsidag
Kilic Aydinli
Aysel Aricioglu
Nilgun Altan
Mine Erdem Inal
Alexander Levitzki
Jose M. Ordovan
Nesrin Kartal Özer
Henrik Enghusen Poulsen
Asuman Sunguroglu
Barbara Tudek
Bahattin Adam
Turkey
Turgut Ulutin
Mark Wheatley
Akin Yesilkaya
Odysseas Zoras
Ayse Ozer
Emel Akoglu
Canan Efendigil Karatay
Nurten Turkozkan
Serdar Ozturk
Mustafa Keçer
Filiz Aydin
Ayça Vitrinel
Aysun Pabuçcuoglu
Meral Tolun
CONSULTATIVE COMMITTEE
Werner Lichtenegge
George Deliconstantinos
Berlin, Germany
Athens, Greece
Abdülaziz A. Al-Khedhairy
Riyadh, S. Arabia
Joseph Molnar
Szeged, Hungary
Ferit Gursu
Turkey
Mhamad Battikhi
Zarga, Jordan
Masato Nakamura
Kanakawa, Japan
Ahmet Dirican
Turkey
George Birkmayer
Vienna, Austria
Edith Olah
Budapest, Hungary
Ferdane Kutlar
Augusta, GA, USA
Fatima Cardoso
Brussels, Belgium
Duran Üstek
Turkey
Siriwat Klinbunga
Bangkok, Thailand
Özcan Erel
Turkey
Filiz Aydin
Turkey
Yunus Luqmani
Safat, Kuwait
Mehmet Gürtekin
Turkey
Rolf F. Barth
Columbus, OH, USA
Kyriaki Mystakidou
Athens, Greece
Hyung Hoan Lee
Seoul, South Korea
Asli Baykal
Turkey
Fatma Oguz
Turkey
Ömer Küçük
Detroit, MI, USA
Bela Bodey
Reseda, CA, USA
Kia Rashidan
Montreal, Canada
ACKNOWLEDGMENTS
The Organizing Committee would like to express their gratitude to the following companies for their generous support.
• ISTANBUL UNIVERSITY
• TUBITAK
• INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (IUBMB)
• INTERNATIONAL INSTITUTE OF ANTICANCER RESEARCH (I.I.A.R.)
• INTERNATIONAL FEDERATION OF CLINICAL CHEMISTRY AND LABORATORY MEDICINE (IFCC)
Congress Secretariat
TIJOEVENTS
Emekli Subayevleri Tevfik Erdonmez Sk.
18. Blok D3 Esentepe-34394-Istanbul
T. 190 212 275 46 63 PBX
F. 190 212 275 46 68
[email protected]
277
Department of Molecular Medicine, The Institute of Experimental
Medicine, University of Istanbul
Turgay Isbir
Department of Molecular Medicine, Institue of Experimental Medical Research, Istanbul University, Istanbul, Turkey
Turgay Isbir is the Director of the Department of Molecular
Medicine at the Institute For Experimental Medical Research at
_
Istanbul
University. He received his B.Sc. and Ph.D degrees from
the Ankara University in the years of 1965 and 1969 respectively.
Between the years of 1977–1978 he worked at the Department of
Pharmacology in the Brain Research Council of Edinbrough University as a Research Fellow. He was nominated to Assoc. Prof. in
Biochemistry in 1979 in the Medical Schoool of the Cukurova University in Adana. Between the years of 1985–1986 he worked as a
Research Fellow at the Department of Heamatology of the Children’s Hospital associated with the School Of Medicine of the University of Pennsylvenia. Dr. Isbir became Professor in Biochemistry
in 1989 at Çukurova University in Adana. In 1997 he worked as a
visiting Professor at the Department of Molecular Medicine in the
Mayo Clinic located in Rochester. Currently his research interests
are focused mainly on the relation between genetic polymorphisms
and various types of cancer, lipid metabolism and genetic tendencies to cardiovascular diseases and diabetes in the Turkish population. He has authored over 100 articles and abstracts as well as one
book chapter. Dr. Isbir is member to several International Scifientific Societies and is the Chairperson of the ‘The Turkish Society of
Molecular Medicine in Turkey. Ó 2009 IUBMB
Keywords
Molecular medicine; institution.
Address correspondence to: Turgay Isbir, PhD, Department of Molecular Medicine, The Institute of Experimental Medicine, Capa 34390,
Istanbul, Turkey. Fax: 190 212 635 19 59.
E-mail: [email protected]
INTRODUCTION
The Department of Molecular Medicine at the Istanbul University Institute of Experimental Medical Research is an interdiciplinary scientific branch that analyzes the molecular biology
of cells and organisms, particularly from a medical point of
view. Recent advances in molecular and cellular biology have
required enormous amounts of research and practice. In all
areas of medicine, molecular biology has played a key role and
its importance to medical research is increasing every day. It is
therefore impossible to disregard this fast-growing area. Biotechnology and molecular biology will certainly play major
roles inclarifying the causes of some of the unsolved mysteries
of modern medicine, such as heart disease, hypertension, major
pyschiatric illness, rheumatic disease, and many others. Work in
these fields may also help us to gain insight into broader aspects
of human biology including development, aging, and evolution,
adding to the importance and necessity of molecular medical
research.
POSTDOCTORAL AND GRADUATE
STUDENT PROGRAM
Graduate students
Masters and Ph.D. programs are offered in the Department
of Molecular Medicine under the aegis of the Institute of Health
Sciences. Graduate students at the department carry out thesis
research under the supervision of staff members. Generally,
these students have completed their major course work requirements for the M.S. and doctoral degrees and are engaged in
full-time research. During the past few years, 13 students have
worked here, supported by training grants to the University of
Istanbul or by investigation research funds from the state. During the past 4 years, six students have completed their degrees
in the Department of Molecular Medicine.
The program allows young investigators to develop APOE,
ACE, LPL, AGN, CETP, PON1, END1 and MTHFR skills in
research methodology and interpretation. It is generally agreed
that establishment of high technology in both medicine and
biology will help Turkey in becoming a developed state. In
278
other words, the status of a country that exports more than it
imports isachieved only by maintaining enough technical
knowledge of molecular biology, physiology, biophysics, biochemistry, genetics, and immunology, etc. (1–74).
CURRENT RESEARCH OVERVIEW
The University’s Department of Molecular Medicine is
involved in many aspects of metabolic diseases that affect several million people worldwide, including atherosclerosis, hypertension, diabetes, and Alzheimer’s disease. Another branch of
the department is dedicated to cancer research. In all of these
areas, research on free oxygen radicals is being conducted, as
well as molecular biological analyses of several substances such
as apolipoprotein E, angiotensin, angiotensinogen, lipoprotein
lipase, and cholesterol ester transfer protein poly-morphism. In
the laboratory the application of molecular biological methods
is used to assessthe prognosis and treatment of cancer. The
main areas in our cancer research include myc protein, casein
kinase, p53 protein, and glutation S-transferase polymorphism.
REFERENCES
1. Unür M, Demirez E, Ağaçhan B, Görmüş U, Ergen A, Dalan B, Isbir
T. (2008) The relationship of oral disturbances of diabetes mellitus
patients with paraoxonase gene polymorphisms. Cell Biochem Funct.
11. [Epub ahead of print].
2. Dalan AB, Ergen A, Yilmaz H, Karateke A, Isbir T. (2008) Manganese
superoxide dismutase gene polymorphism, MnSOD plasma levels and
risk ofepithelial ovarian cancer. J Obstet Gynaecol Res. 34, 878–884.
3. Tekeli A, Isbir S, Ergen A, Görmüş U, Bozkurt N, Timirci O, Arsan S,
Isbir T. (2008) APE1 and XRCC3 polymorphisms and myocardial infarction. In Vivo. 22, 477–479.
4. Gözü A, Ergen A, Dayicioglu D, Yaylim I, Ozsoy Z, Isbir T. (2008) Lmyc polymorphism in head and neck nonmelanoma skin and lower lip
cancers. Arch Otolaryngol Head Neck Surg. 134, 725–728.
5. Yaylim-Eraltan I, Bozkurt N, Ergen A, Zeybek U, Ozturk O, Arikan S,
Erbil Y, Uslu I, Camlica H, Isbir T. (2008) L-myc gene polymorphism
and risk of thyroid cancer. Exp Oncol. 30, 117–120.
6. Ozger H, Kilicoglu O, Yilmaz H, Ergen HA, Yaylim I, Zeybek U, Isbir
T. (2008) Methylenetetrahydrofolate reductase C677T gene polymorphism in osteosarcoma and chondrosarcoma patients. Folia Biol
(Praha). 54, 53–57.
7. Ergen A, Isbir S, Tekeli A Isbir T. (2008) Investigation of ABCA1
C69T and G-191C polymorphisms in coronary artery disease. In Vivo.
22, 187–190.
8. Gormus U, Ergen A, Yaylim-Eraltan I, Yilmaz H, Turna A, Bozkurt N,
Isbir T. (2007) Fas-1377 A/G polymorphism in lung cancer. In Vivo.
21, 663–666.
9. Ergen HA, Narter F, Timirci O, Isbir T. (2007) Effects of manganase
superoxide dismutase Ala-9Val polymorphism on prostate cancer: a
case-control study. Anticancer Res. 27, 1227–1230.
10. Gormus U, Ergen A, Yilmaz H, Dalan B, Berkman S, Isbir T. (2007)
Fas-1377A/G and FasL-844 T/C gene polymorphisms and epithelial
ovarian cancer. Anticancer Res. 27, 991–994.
11. Isbir SC, Tekeli A, Ergen A, Yilmaz H, Ak K, Civelek A, Zeybek U,
Arsan S. (2007) Genetic polymorphisms contribute to acute kidney injury
after coronary artery bypass grafting. Heart Surg Forum. 10, E439–E444.
12. Ak K, Isbir S, Tekeli A, Ergen A, Atalan N, Dogan S, Civelek A, Arsan
S. (2007) Presence of lipoprotein lipase S447X stop codon affects the
magnitude of interleukin 8 release after cardiac surgery with cardiopulmonary bypass. J Thorac Cardiovasc Surg. 134, 477–483.
13. Aydin M, Ozkok E, Ozturk O, Agachan B, Yilmaz H, Yaylim I, Kebabcioglu S, Ispir T. (2007) Relationship between interleukin-8 and the oxidant-antioxidant system in end-stage renal failure patients. Exp Clin
Transplant. 5, 610–613.
14. Yigit B, Bozkurt N, Narter F, Yilmaz H, Yucebas E, Isbir T. (2007)
Effects of ACE I/D polymorphism on prostate cancer risk, tumor grade
and metastatis. Anticancer Res. 27, 933–936.
15. Yaylim-Eraltan I, Arzu Ergen H, Arikan S, Okay E, Oztürk O, Bayrak
S, Isbir T. (2007) Investigation of the VDR gene polymorphisms association with susceptibility to colorectal cancer. Cell Biochem Funct. 25,
731–737.
16. Zeybek U, Yaylim I, Yilmaz H, Ağaçhan B, Ergen A, Arikan S, Bayrak
S, Isbir T. (2007) Methylenetetrahydrofolate reductase C677T polymorphism in patients with gastric and colorectal cancer. Cell Biochem
Funct. 25, 419–422.
17. Kafadar AM, Yilmaz H, Kafadar D, Ergen A, Zeybek U, Bozkurt N,
Kuday C, Isbir T. (2006) C677T gene polymorphism of methylenetetrahydrofolate reductase (MTHFR) in meningiomas and high-grade gliomas. Anticancer Res. 26, 2445–2449.
18. Zeybek U, Isbir T, Ergen HA, Yilmaz H, Hekim N, Akoglu E. (2006)
Effects of cholesteryl ester transfer protein TAQ1B polymorphism in renal transplant patients. Transplant Proc. 38, 1382–1384.
19. Yigit B, Bozkurt N, Yaylim I, Titiz I, Isbir T. (2006) Analysis of Lmyc gene polymorphism in patients with renal failure outcome to renal
transplant. Transplant Proc. 38, 1267–1269.
20. Yigit B, Bozkurt N, Berber I, Titiz I, Isbir T. (2007) Analysis of CC
chemokine receptor 5 and 2 polymorphisms and renal transplant survival. Cell Biochem Funct. 25, 423–426.
21. Bilge I, Sirin A, Agachan B, Emre S, Sadikoglu B, Yilmaz H, Sucu A,
Isbir T. (2007) Is paraoxonase 192 gene polymorphism a risk factor for
membranoproliferative glomerulonephritis in children? Cell Biochem
Funct. 25, 159–165.
22. Kafadar AM, Ergen A, Zeybek U, Agachan B, Kuday C, Isbir T. (2006)
Paraoxonase 192 gene polymorphism and serum paraoxonase activity in
high grade gliomas and meningiomas. Cell Biochem Funct. 24, 455–
460.
23. Hekim N, Ergen A, Yaylim I, Yilmaz H, Zeybek U, Oztürk O, Isbir T.
(2007) No association between methylenetetrahydrofolate reductase
C677T polymorphism and breast cancer. Cell Biochem Funct. 25, 115–
117.
24. Okay E, Karadenizli A, Müezzinoglu B, Zeybek U, Arzu Ergen H, Isbir
T. (2005) N-acetylcysteine attenuates bacterial translocation after partial
hepatectomy in rats. J Surg Res. 127, 164–170.
25. Agachan B, Yilmaz H, Ergen HA, Karaali ZE, Isbir T. (2005) Paraoxonase (PON1) 55 and 192 polymorphism and its effects to oxidant-anti-
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oxidant system in turkish patients with type 2 diabetes mellitus. Physiol
54, 287–293.
Yilmaz H, Isbir S, Agachan B, Ergen A, Farsak B, Isbir T. (2006)
C677T mutation methylenetetrahydrofolate reductase gene and serum
homocysteine levels in Turkish patients with coronary artery disease.
Cell Biochem Funct. 24, 87–90.
Biyikli NK, Alpay H, Yildiz N, Agachan B, Ergen A, Zeybek U, Bozkurt N, Ispir T. (2006) Paraoxonase 1 192 and 55 polymorphisms in nephrotic children. Pediatr Nephrol. 21, 649–654.
Karaali ZE, Agachan B, Yilmaz H, Isbir T. (2004) Angiotensin-converting enzyme I/D gene polymorphisms and effects of left ventricular
hypertrophy in Turkish myocardial infarction patients. Acta Cardiol. 59,
493–497.
Yilmaz H, Agachan B, Ergen A, Karaalib ZE, Isbir T. (2004) Methylene tetrahydrofolate reductase C677T mutation and left ventricular hypertrophy in Turkish patients with type II diabetes mellitus. J Biochem
Mol Biol. 37, 234–238.
Yilmaz H, Unlüçerçi Y, Gürdöl F, Isbilen E, Isbir T. (2004) Association
of pre-eclampsia with hyperhomocysteinaemia and methylenetetrahydrofolate reductase gene C677T polymorphism in a Turkish population.
Aust N Z J Obstet Gynaecol. 44, 423–427.
Yilmaz H, Isbir T, Agachan B, Karaali ZE. (2005) Effects of cholesterol
ester transfer protein Taq1B gene polymorphism on serum lipoprotein
levels in Turkish coronary artery disease patients. Cell Biochem Funct.
23, 23–28.
Arzu Ergen H, Hatemi H, Agachan B, Camlica H, Isbir T. (2004) Angiotensin-I converting enzyme gene polymorphism in Turkish type 2 diabetic patients. Exp Mol Med. 36, 345–350.
Agachan B, Yilmaz H, Isbir T, Akoglu E. (2004) Paraoxonase 192
polymorphism and its relationship to serum lipids in Turkish renal transplant recipients. Transplant Proc. 36, 1385–1386.
Oktem F, Sirin A, Bilge I, Emre S, Ağaçhan B, Ispir T. (2004) ACE I/
D gene polymorphism in primary FSGS and steroid-sensitive nephrotic
syndrome. Pediatr Nephrol. 19, 384–389.
Yilmaz H, Agachan B, Karaali ZE, Isbir T. (2004) Taq1B polymorphism of CETP gene on lipid abnormalities in patients with type II diabetes mellitus. Int J Mol Med. 13, 889–893.
Agachan B, Yilmaz H, Karaali Z, Isbir T. (2004) Paraoxonase 55 and
192 polymorphism and its relationship to serum paraoxonase activity
and serum lipids in Turkish patients with non-insulin dependent diabetes
mellitus. Cell Biochem Funct. 22, 163–168.
Gürdöl F, Işbilen E, Yilmaz H, Isbir T, Dirican A. (2004) The association between preeclampsia and angiotensin-converting enzyme insertion/
deletion polymorphism. Clin Chim Acta. 341, 127–131.
Oztürk O, Isbir T, Yaylim I, Kocatürk CI, Gürses A. (2003) GST M1 and
CYP1A1 gene polymorphism and daily fruit consumption in Turkish
patients with non-small cell lung carcinomas. In Vivo. 17, 625–632.
Agachan B, Isbir T, Yilmaz H, Akoglu E. (2003) Angiotensin converting enzyme I/D, angiotensinogen T174M-M235T and angiotensin II
type 1 receptor A1166C gene polymorphisms in Turkish hypertensive
patients. Exp Mol Med. 35, 545–549.
Yaylim I, Bozkurt N, Yilmaz H, Isbir T, Isik N, Arikan S. (2003) The
apolipoprotein E epsilon 4 allele is not a risk factor for Turkish breast
cancer patients. Cancer Genet Cytogenet. 146, 86–87.
Isbir T, Yilmaz H, Agachan B, Karaali ZE. (2003) Cholesterol ester
transfer protein, apolipoprotein E and lipoprotein lipase genotypes in
patients with coronary artery disease in the Turkish population. Clin
Genet. 64, 228–234.
Yilmaz H, Agachan B, Isbir T, Akoglu E. (2003) Is there additional
effect of MTHFR C677T mutation on lipid abnormalities in renal allograft recipients? Transplant Proc. 35, 1390–1392.
Isbir T, Yaman A, Yaylim I, Yilmaz H, Oztürk O. (2003) Association
between Apo B signal peptide gene polymorphism and NIDDM. Cell
Biochem Funct. 21, 189–190.
279
44. Isbir T, Yaylim I, Arikan S, Kaytan E, Karşidağ T, Bayrak S, Camlica
H. (2002) Close correlation between restriction fragment length polymorphism of L-myc gene and susceptibility to gastric cancer. Cancer
Detect. 26, 454–457.
45. Isbir T, Yaylim I, Arı̂kan S, Küçücük S, Camlı́ca H. (2002) Increased
frequency of the S-allele of the L-myc oncogene in breast cancer. Mol
Med. 8, 521–524.
46. Yaylim I, Isbir T, Oztürk O, Turna A, Işitmangil T, Zonüzi F, Camlica
H. (2002) Is there any correlation between restriction fragment length
polymorphism of the L-MYC gene and metastasis of human nonsmall
cell lung cancer? Cancer Genet Cytogenet. 134, 118–122.
47. Yaylim I, Isbir T. (2002) Enhanced casein kinase II (CK II) activity in
human lung tumours. Anticancer Res. 22, 215–218.
48. Oztürk O, Yaylim I, Aydin M, Yilmaz H, Agaçhan B, Demiralp E, Isbir
T. (2001) Increased plasma levels of interleukin-6 and interleukin-8 in
beta-thalassaemia major. Haematologia. 31, 237–244.
49. Bozkurt N, Oztürk O, Isbir T. (2001) New MCAD gene mutation, not
previously reported in other nations, found at A1161G in Turkish population. Am J Med Genet. 103, 255–256.
50. Isbir T, Agaçhan B, Yilmaz H, Aydin M, Kara I, Eker D, Eker E.
(2001) Interaction between apolipoprotein-E and angiotensin-converting
enzyme genotype in Alzheimer’s disease. Am J Alzheimers Dis Other
Demen. 16, 205–210.
51. Yilmaz H, Isbir T, Ağaçhan B, Aydin M. (2001) Is epsilon4 allele of
apolipoprotein E associated with more severe end-organ damage in
essential hypertension? Cell Biochem Funct. 19, 191–195.
52. Isbir T, Agaçhan B, Yilmaz H, Aydin M, Kara I, Eker E, Eker D.
(2001) Apolipoprotein-E gene polymorphism and lipid profiles in Alzheimer’s disease. Am J Alzheimers Dis Other Demen. 16, 77–81.
53. Ildan F, Göçer AI, Tuna M, Polat S, Kaya M, Isbir T, Cetinalp E.
(2001) The effects of the pre-treatment of intravenous nimodipine on
Na(1)-K1/Mg12 ATPase, Ca12/Mg12 ATPase, lipid peroxidation
and early ultrastructural findings following middle cerebral artery occlusion in the rat. Neurol Res. 23, 96–104.
54. Oztaş B, Kiliç S, Dural E, Ispir T. (2001) Influence of antioxidants on
the blood-brain barrier permeability during epileptic seizures. J Neurosci Res. Nov 66, 674–678.
55. Oztaş B, Erkin E, Dural E, Isbir T. (2000) Influence of antioxidants on
blood-brain barrier permeability during adrenaline-induced hypertension.
Int J Neurosci. 105, 27–35.
56. Isbir T, Agaçhan B, Yilmaz H, Aydin M. (2000) Angiotensin converting
enzyme gene polymorphism in Alzheimer’s disease. Cell Biochem
Funct. 18, 141–142.
57. Koç M, Ozener IC, Tezcan H, Bihorac A, Isbir T, Akoğlu E. (2000) Left
ventricular hypertrophy and angiotensin-converting enzyme gene polymorphism in renal allograft recipients. Transplant Proc. 32, 542–544.
58. Isbir T, Yilmaz H, Ağaçhan B, Aydin M, Isbir CS. (1999) Association
between angiotensin-converting enzyme gene polymorphism and coronary artery disease. IUBMB Life. 48, 205–207.
59. Isbir T, Yaylim I, Aydin M, Oztürk O, Koyuncu H, Zeybek U, Ağaçhan
B, Yilmaz H. (2000) The effects of Brassica oleraceae var capitata on
epidermal glutathione and lipid peroxides in DMBA-initiated-TPA-promoted mice. Anticancer Res. 20, 219–224.
60. Aydin M, Cengiz S, Agaçhan B, Yilmaz H, Isbir T. (2000) Age-related
changes in GM1, GD1a, GT1b components of gangliosides in Wistar
albino rats. Cell Biochem Funct. 18, 41–45.
61. Isbir T. (1999) The Department of Molecular Medicine at the University
of Istanbul, Institute of Experimental Medical Research. Mol Med. 5,
69–70.
62. Isbir T, Yilmaz H, Bihorac A, Akoglu E. (1997) Mild-to-moderate
hypertension and apolipoprotein E gene polymorphism. Am J Hypertens.
10, 827–828.
63. Koçak R, Başlamişli F, Güvenç B, Tamer L, Aikimbaev KS, Isbir T. (1996)
Bencyclane as an anti-sickling agent. Br J Haematol. 92, 329–331.
280
64. Ildan F, Polat S, Göcer AI, Oner A, Isbir T, Mete UO, Kaya M, Karadayi A. (1996) The effects of the pretreatment of intravenous high dose
methylprednisolone on Na(1)-K(1)/Mg(12) ATPase and lipid peroxidation and early ultrastructural findings following middle cerebral artery
occlusion in the rat. Acta Neurochir. 138, 338–345.
65. Ildan F, Polat S, Oner A, Isbir T, Cetinalp E, Kaya M, Karadayi A. (1995)
The effect of the treatment of high-dose methylprednisolone on Na(1)K(1)/Mg(12) ATPase activity and lipid peroxidation and ultrastructural
findings following cerebral contusion in rat. Surg Neurol. 44, 573–580.
66. Ildan F, Polat S, Oner A, Isbir T, Göçer AI, Tap O, Kaya M, Karadayi
A. (1995) Effects of naloxone on sodium- and potassium-activated and
magnesium-dependent adenosine-5’-triphosphatase activity and lipid
peroxidation and early ultrastructural findings after experimental spinal
cord injury. Neurosurgery. 36, 797–805.
67. Iteğin M, Günay I, Loğoğlu G, Isbir T. (1995) Effects of static magnetic
field on specific adenosine-5’- triphosphatase activities and bioelectrical
and biomechanical properties in the rat diaphragm muscle. Bioelectromagnetics. 16, 147–151.
68. Ildan F, Oner A, Polat S, Isbir T, Göcer AI, Kaya M, Karadayi A.
(1995) Correlation of alterations on Na(1)-K1/Mg12 ATPase activity,
69.
70.
71.
72.
73.
74.
lipid peroxidation and ultrastructural findings following experimental
spinal cord injury with and without intravenous methylprednisolone
treatment. Neurosurg Rev. 18, 35–44.
Isbir T, Tamer L, Taylor A, Isbir M. (1994) Zinc, copper and magnesium status in insulin-dependent diabetes. Diabetes. 26, 41–45.
Yuregir GT, Donma O, Dikmen N, Isbir T, Cinar M. (1987) Population studies of hemoglobin S and other variants in Cukurova, the
southern part of Turkey. Nippon Ketsueki Gakkai Zasshi. 50, 757–
765.
Isbir T, Unal M, Yesilsoy C, Tukel S. (1987) Action of maprotiline
on the erythrocyte membrane Na1-K1/Mg11 ATPase and Ca11/
Mg11 ATPase in depressed patients. Int J. 34, 15–17.
Franklin SG, Wolf SI, Ozdemir Y, Yuregir GT, Isbir T, Blumberg BS.
(1980) Albumin Naskapi variant in North American Indians and Eti
Turks. Proc Natl Acad Sci U S A. 77, 5480–5482.
Reading HW, Isbir T. (1980) The role of cation-activated ATPases in
transmitter release from the rat iris. Q J Exp Physiol Cogn Med Sci. 65,
105–116.
Reading HW, Isbir T. (1979) Action of lithium on ATPases in the rat
iris and visual cortex. Biochem Pharmacol. 28, 3471–3474.
281
Opening Lecture
Kurt Wüthrich (Nobel Prize Winner)—Structural
Genomics-A New Platform for Drug Discovery
Kurt Wüthrich
The Scripps Research Institute, La Jolla, CA, USA, and ETH Zurich, Zurich, Switzerland
The determination of the human genome and the genomes of
a large number of other species carries great promise with
regard to improving the quality of life worldwide. New advances are expected in many different fields, including agriculture,
nutrition and healthcare. Realization of these advances will
have to be based on detailed knowledge of the proteome and
other gene products, in addition to genomic DNA sequences.
This lecture will reflect on strategic and practical aspects of this
post-genomic research, with a special focus on establishing new
platforms for drug discovery and drug design.
282
PLENARY LECTURES
L-1
EMBRYONIC STEM CELL-DERIVED DOPAMINERGIC NEURONS
FOR THE THERAPY OF PARKINSON’S DISEASE
Ahmed Mansouri1, Tanja Kuhlmann2, Ralf Dressel3, Claudia Trenkwalder4,
and Walter Paulus4
1
Department of Molecular Cell Biology, MPI for Biophysical Chemistry,
Germany, 2Department of Neuropathology, University of Göttingen,
Germany, 3Department of Molecular Immunology, University of Göttingen,
Germany, 4Department of Clinical Neurophysiolgy, University of
Göttingen, Germany
Cell replacement therapy remains one of the most promising future
approaches for treatment of Parkinson’s disease (PD). Several sources have been
evaluated for the generation of dopaminergic neurons. Currently, embryonic stem
cells and possibly the recently induced pluripotent (iPS) cells represent an unlimited source for the derivation of all embryonic cell types. We have used mouse
embryonic stem cell-derived dopaminergic neurons for transplantation into the 6hydroxydopamine (6-OHDA)-rat model for Parkinson’s disease. Our findings
corroborate with results from other studies showing remarkable functional benefit
in rat and primate model of Parkinson’s disease. However, serious concerns are
related to tumors due to the presence of few pluripotent cells. To eliminate such
cells by specific labeling of DA neurons or their progenitors generally requires
genetic manipulations. Therefore, one strategy to overcome these difficulties may
consist in the derivation of neuronal precursor cells from embryonic stem cells,
and to transplant these directly or after differentiation into DA neurons. We have
isolated such cells from mouse and human embryonic stem cells. These neuronal
stem cells (NSC) can be propagated in vitro and frozen for at least one year.
They express the neural stem cell markers nestin, CD133 and musashi. Interestingly, the pluripotency marker Oct4 is not detected in these cells. Moreover,
they have the potential to differentiate into dopaminergic neurons. Preliminary
experiments indicate that mouse ES cell-derived neural stem cells do not form
teratomas 3 months after injection into immunodeficient mice and rat. We are
currently using mouse and human ES-cell derived neural stem cells for the transplantation into the 6-Hydroxydopmaine lesioned rat model for Parkinson’s disease. Our studies will be presented. Acknowledgements: This project is supported by the Max-Planck society, the German Ministry of Research and Education (BMBF 01GN0510) and the Dr. Helmut Storz and Alte Leipziger Stiftung.
L-2
PROTEOMICS AS DIAGNOSTIC MARKERS
Aldo Tomasi, Elisa Bellei, Stefania Bergamini, Aurora Cuoghi,
and Emanuela Monari
Integrated Activity, Department of Laboratory Diagnostics and Forensic
Medicine Services, University of Modena and Reggio Emilia, Italy
The analysis of expressed proteins is an important developing area of
research. Advances in technologies have led to a rapid increase in applications to
a wide range of biological samples, such as plasma, urine, tissues, etc. The most
widely used techniques for analysis of clinical samples are two-dimensional gel
electrophoresis (2DE), to separate and visualize proteins, and mass spectrometry
(MS) for protein identification. Furthermore, recently a complementary highthroughput proteomics platform, the Surface- Enhanced Laser Desorption Ionization (SELDI), has been developed. A primary aim of clinical proteomics is the
identifications of biomarkers for early diagnosis, prognosis and therapeutic intervention of disease, by comparing the proteomic profiles of control and disease,
and differing physiological states. Protein profiling is a promising tool for tumor
characterization and the detection of biomarkers for prostate, bladder, lung,
breast and ovarian cancer. For example, SELDI technique has been used to identify cancer-specific proteins in prostate cancer and to identify potential new
markers in pancreatic adenocarcinoma, ovarian cancer and several potential urinary biomarkers in bladder cancer. The concept presented in these findings is
that the diagnostic endpoint for cancer detection is not a single analyte, but a
proteomic pattern that is composed of many individual proteins, each of which
independently cannot differentiate diseased from healthy individuals. Moreover,
proteomic analysis has been extensively applied to the discovery of potential
diagnostic markers for other diseases, such as neurological disorders, Alzheimer’s disease and profiling of urinary proteins to asses renal function. Several
recent and ongoing studies have applied urinary proteomics to biomarker discovery for kidney diseases as well as other disorders that may have systemic alterations in metabolic and biochemical profiles that can affect urinary protein excretion. In diagnostic field, proteomic analysis could represent the right approach
for discover and to identify early and predictive biomarkers of several and important diseases.
L-3
MOLECULAR MECHANISM OF ALPHA-TOCOPHEROL ACTION
Yesim Negis, Jean-Marc Zingg, Mohsen Meydani, and Angelo Azzi
USDA Human Nutrition Center, Tufts University, Boston, MA, USA
Vitamin E is a typical antioxidant when studied in a test tube. However, like
other antioxidants, in vivo this molecule has different properties, which are used
to trigger cell information transfer and modulate gene transcription. We have
shown that alpha-tocopherol (a-T) but not the very similar beta-tocopherol, is
able to inhibit a master switch in cells, protein kinase C and consequently cell
proliferation, inhibition of the NADPH oxidase assembly and inhibition of O2production. Inhibition by a-T of the transcription of the gene coding for the scavenger receptor CD36 was the first observation, which was followed by a number
of studies that have shown that several genes are regulated by vitamin E and
none of them codes for antioxidant enzymes. Such an expression would be
expected as a compensatory mechanism as a consequence of vitamin E diminution, in case that its action where that of an antioxidant. All the above findings
support the hypothesis that a-T does not act as an antioxidant. Recently, we have
searched for an a-T derivative, more active than a-T. Alpha-tocopheryl phosphate
(a-TP) has been found in normal tissues. a-TP is synthesized and hydrolyzed in
animal cells and tissues; it modulates also several cell functions. While it is similar to a-T, a-TP appears to be more potent than a-T in inhibiting cell proliferation, down regulating CD36 transcription, inhibiting atherosclerotic plaque formation etc. a-TP does not act by liberating a-T; rather, the intact molecule
appears to be more potent than a-T. Administration of a-TP to cells or to animals
requires its transfer through membranes, an event that cannot occur by simple
diffusion due to the size and the charge of the molecule but requires a transporter. Specific transport inhibitors, glybenclamide and probenecid showed no inhibition of cell proliferation or cytotoxicity. However, both compounds prevented, dose-dependently a-TP inhibition of cell proliferation. The data indicate
that a-TP enters cells via a glybenclamide and probenecid-sensitive transport system. Both, members of the ABC transporter family and of the organic anion
transporters (OAT), appear to be sensitive to these two inhibitors. However,
since ABC transporters function to export cell solutes and a-TP is imported,
a-TP transport may occur via an OAT family member. Finally, by GeneChip
U133 Plus 2.0 Gene Array (Affymetrix) 202 genes in THP-1 cells were found to
be significantly regulated by a-TP.
L-4
JAUNDICE AND HYPERBILIRUBINEMIA
Ayca Vitrinel
Yeditepe University Medical School, Istanbul, Turkey
Hyperbilirubinemia is one of the most common manifestations to occur in
neonatal period. Although hyperbilirubinemia is usually benign and self-limited,
elevated levels of indirect, unconjugated bilirubin are potentially neurotoxic.
Neurological sequelae, kernicterus are preventable conditions if the cause and
levels of hyperbilirubinemia are detected earlier. In the neonatal period, bilirubin production is increased because of larger red blood cell volumes, shortened
erythrocyte life span, increased ineffective erythropoiesis and decreased uridine
diphosphate glucuronyl transferase activity. An imbalance between the production and elimination of bilirubin results in varying levels of hyperbilirubinemia
in neonates. There is a long differential diagnosis for jaundice according to the
time if it is detected after birth. Jaundice is designated as physiological if it
appears on the second and third day and indirect bilirubin levels are not above
12 mg/dl. The diagnosis of physiological jaundice can be established only by
excluding known causes of jaundice based on history, clinical findings and laboratory data. Jaundice and the underlying hyperbilirubinemia are considered pathological if the time of appearance, duration or pattern differ from the physiologic
jaundice. Search for the cause should be made if jaundice appears in the first 2436 hrs of life, if serum bilirubin rises at a rate 5mg/dl/24 hr, if serum bilirubin is
more than 12mg/dl in full term infants and if jaundice persists after 10-14 days
of life. Sometimes it may not be possible to determine the precise cause of an
abnormal elevation of unconjugated bilirubin and the term ‘‘exaggerated physiologic jaundice’’ or ‘‘none physiologic jaundice with unexplained cause’’ are used.
Primary problem is probably a deficiency or inactivity of bilirubin glucuronyl
transferase and mutations in the gene for this enzyme. For preventing the development of bilirubin-induced neurological dysfunction in newborns with an unexplained cause of hyperbilirubinemia and even in healthy newborns additional
genetic studies should be performed in order to follow up the development and
pattern of hyperbilirubinemia.
283
oxidative burst are observed during the attack-free period. During fever attacks,
there are usually neutrophilia and production of a brisk acute-phase protein such
as serum amyloid A (SAA), and substantial influx of polymorphonuclear leukocytes into the affected tissues. It is well known that the persistent respiratory
burst caused by activated neutrophils may generate oxidative damage to cellular
macromolecules including proteins, lipids and DNA. Free radicals can cause oxidative macromolecular damage, which is implicated in mutagenesis, carcinogenesis and aging process. We hypothesized that the well-known persistent inflammation in FMF patients may cause oxidative damage to cellular macromolecules.
To test this hypothesis, we investigated, in the present study, oxidative stress in
polymorphonuclear leukocytes of patients with FMF during the attack-free period
with the goal of identification and quantification of typical products of free radical-induced lipid protein and DNA damages.
L-7
THE IMPORTANCE OF GENETIC VARIATIONS FOR ANESTHESIA
L-5
GENOMIC BIMARKES IN NEURODEGENERATIVE DISORDERS
Borut Peterlin, and Andrej Kastrin
UCML, Institute of Medical Genetics, Slovenia
Neurodegenerative disorders like Parkinson, Alzheimer, and other neurodegenerative diseases are becoming an increasing burden in the aging populations
worldwide. They generally begin late in life; slowly but inexorably they cause
progressive neuronal degeneration, and result in disability or death. There is an
urgent need for biomarkers to diagnose neurodegenerative disorders early in their
course, when therapy is likely to be most effective and to monitor responses of
patients to new therapies. In addition to clinical, imaging and biochemical tools,
recent studies have shown different patterns of gene expression in brain of several neurodegenerative disorders analyzed so far. It is often not clear whether
these differences represent a specific change in gene expression related to the
pathogenesis of a disorder, or a non-specific reaction to brain damage or an error
in the experiment. Moreover, due to obvious problems of obtaining brain tissue,
non-invasive genomic approaches would be advantageous. Indeed, several studies
indicated that there might be specific profiles in gene expression in the blood of
patients with Parkinson’s disease, Huntington’s disease, Down’s syndrome, multiple sclerosis, and others. However, most of the studies have included a small
number of patients and have not been validated. Here, we will describe the outcome of the meta-analysis of the available brain and blood genomic studies and
the potential impact on genomic biomarker development for neurodegenerative
disorders.
L-6
OXIDATIVE STRESS IN FAMILIAL MEDITERRANEAN FEVER
Guldal Kirkali
Department of Biochemistry, School of Medicine, Dokuz Eylul University,
Izmir, Turkey
Familial Mediterranean fever (FMF) is an autosomal recessively inherited
disorder characterized by recurrent, inflammatory self-limited episodes of fever
and localized inflammation, affecting serosal membranes, joints and skin. FMF is
caused by more than 25 mutations in the gene MEFV, which is located on
human chromosome 16p and encodes a protein named pyrin or marenostrin. This
protein has 781 amino acids and at least four different domains. It is expressed
in granulocytes, cytokine-activated monocytes, and serosal and synovial fibroblasts. Pyrin seems to have an important role in regulation of interleukin (IL)-1b
activation, exerting both positive and negative regulatory effects depending on
the experimental conditions. Genotype-phenotype correlations in FMF have not
been resolved definitely. The percentage of patients with a clinical phenotype fitting the diagnosis of FMF, but with no causative genetic mutation and no definitive diagnosis is still very high. The influence of unknown environmental factors
and/or the presence of other genetic changes are necessary to explain the phenotypic variation of the disease and the development of amyloidosis. In FMF
patients, the up-regulation of neutrophil and monocyte phagocytic activity and
Olcay Isbir
Department of Anesthesia, School of Medicine, University of Acibadem,
Istanbul, Turkey
The differences in responses to therapeutic agents between individuals are
now known to have strong hereditary basis; pharmacogenetics is a discipline
aimed at understanding such variations. A similar variability can be observed in
the risk of adverse effects of a drug or a chemical agent, such as the anesthetics.
For example, many factors are known to contribute to the differences in opioid
responsiveness including environmental, psychological and genetic. The responsible genes are thought to be the ones expressing drug-metabolising enzymes such
as cytochrome p450, drug-transporting proteins as p-glycoproteins and/or the
drug targets such as receptor proteins. In daily anesthetic practice, because of
genetic polymorphisms, many drugs display important variability in their effects
as unpredictable duration of action, side effects, toxicity, etc. There are some
known clinically relevant polymorphisms: Beta1-adrenergic receptor (ADRB1)
mediates inotrophic and chronotrophic responses to cathecolamines whose
genetic polymorphisms may affect the tolerability by patients of beta-blocker
therapy. Beta2-adrenergic receptor (ADRB2) is another important parameter for
hemodynamic regulation having several genetic polymorphisms identified.
CYP2D6 is the liver enzyme of cytochrome p450 family that catalyses more
than 100 kinds of drugs and the gene coding for this gene is known to be highly
polymorphic. Cathecol-O-methyltransferase gene encodes an enzyme in dopamine and norepinephrine metabolism influencing pain perception by patients;
some polymorphisms of this enzyme are known to affect the morphine doses
given to chronic cancer patients. l-opioid receptor encoded by genetic locus
OPRM1 is the primary site of action for endogenous opioid peptides such as
beta-endorphin and enkephalin all known to have roles in pain perception. As in
those instances, it is obvious that in future, pharmacogenetics will elucidate the
interpatient variability and it will be easier to predict anesthetic responsiveness
of each patient.
L-8
ADENYLYL CYCLASE EXPRESSION IN WILD TYPE AND PKD21/VASCULAR SMOOTH MUSCLE CELLS (VSMCS) AND ITS
RELATIONSHIP TO FORMATION OF ANEURYSMS OBSERVED
IN PATIENTS WITH AUTOSOMAL DOMINANT POLYCYSTIC
KIDNEY DISEASE (ADPKD)
S. Kip, Q. Ren, V. E. Torres, G. C. Sieck, and Q. Qian
Nephrology, Mayo Clinic College of Medicine, Rochester, MN,
United States
Many inherited disorders result in renal cyst development and the most common form, is autosomal dominant polycystic kidney disease (ADPKD), which is
a disorder most often diagnosed in adults and caused by mutations both in PKD1
or PKD2. The PKD1 protein, polycystin-1, is a large receptor-like protein,
whereas polycystin-2 is a transient receptor potential channel. The polycystin
complex localizes to primary cilia and acts as a mechanosensor essential for
maintaining the differentiated state of epithelia lining tubules in the kidney and
biliary tract. ARPKD is the neonatal form of PKD, which is associated with
284
enlarged kidneys and biliary dysgenesis that is characterized by highly variable
phenotypes, ranging from neonatal death to late presentation with minimal kidney disease. ARPKD is caused by mutation in PKHD1, and the ARPKD protein,
which is fibrocystin, is localized to cilia/basal body and complexes with polycystin-2. Rarely, PKD also include defects of the eye, central nervous system, digits,
and/or neural tube. ADPKD, one of the most common monogenetic disorders,
with an incidence of 1:400–1000, is diagnosed in adults and, with an incidence
of 1:400–1000. The disease is characterized by the progressive, bilateral development and enlargement of focal cysts ultimately resulting in end-stage renal disease (ESRD), approximately 5% of patients require dialysis or transplant.
ADPKD is a systemic disease, with cyst development also occurring in the liver,
pancreas, seminal vesicles, and arachnoid. Other notable phenotypes in ADPKD
involve the vasculature. Intracranial aneurysms are five times more common in
these patients than the general population with associated significant morbidity/
mortality secondary to rupture of aneurysms. ADPKD is genetically heterogeneous with two genes identified, PKD1 (16p13.3) and PKD2 (4q21). Mutations in
PKD1 cause a more severe disease than PKD2 and account for about 85% of the
cases. In addition, the average age at onset of ESRD is 20 years younger in
PKD1 mutations as opposed to PKD2 [54.3 years versus 74.0 years], and the
cysts develop at an early age. Both PKD1 and PKD2 can be associated with
severe PLD and vascular abnormalities. Intracranial aneurysm (ICA) formation,
thus a major vascular complication in ADPKD patients, is associated with vascular smooth muscle cells (VSMCs) apoptosis. Pkd21/- VSMCs develop a reduced
store operated Ca21 channel (SOCC) activity, a higher cAMP, and an elevated
apoptosis. We show cAMP triggers VSMC apoptosis, suggesting a pathogenic
significance. cAMP is generated from ATP by adenylyl cyclase (AC); 9 mammalian isoforms are identified; their expression is cell type specific. We characterized AC isoform expressions in w/t and Pkd21/- mouse aortic VSMCs and tested
the hypothesis that a reduction in SOCC mediated Ca21 entry, as in Pkd21/VSMCs, weakens AC5/6 inhibition and raises cAMP. Using isoform specific primers, AC1-9 mRNAs, isolated from primary cultured w/t and Pkd21/- VSMCs
generated from congenic littermates, were quantified by real time Rt-PCR (LightCycler). The major isoforms in w/t and Pkd21/- VSMCs were AC3, 5, 6, 7, and
9. The Ca21-stimulatable isoforms: AC1 and AC8 were nearly undetectable,
while the Ca21-inhibitable isoforms: AC5 and AC6 were highly expressed, inferring Ca21 is inhibitory to ACs in VSMCs. This pattern was confirmed at protein
level by quantitative Western analyses using isoform specific antibodies. The
level of each isoform in w/t VSMCs is (mRNA) AC6[AC5[AC7[AC9[AC3,
and (protein) AC6/5[AC7[AC9[AC3, in Pkd21/- VSMCs (mRNA)
AC5[AC6[AC7[AC9[AC3 and (protein) AC5/6[AC7[AC9[AC3. Compared to w/t, Pkd21/- VSMCs have higher levels of AC5 ([10-fold mRNA; [2fold protein expression) and AC6 ([ 2-fold mRNA; [1.5-fold protein expression). In vitro studies show SOCC-Ca21 entry reduces cAMP by inhibiting AC5
and AC6. To determine if a SOCC reduction, as in Pkd21/- condition, reduces
this inhibition, we treated w/t VSMCs with SOCC blocker (SKF96365, 25 mM,
x30 min). SKF96365 markedly rose ([10-fold) cAMP concentration, consistent
with SOCC inhibiting AC in this cellular system. AC3, 5, 6, 7, and 9 are the
major isoforms expressed in VSMCs. Pkd21/- VSMCs express higher levels of
Ca21 inhibitable AC5 and 6. Functionally, SOCC inhibition raises VSMC
cAMP. These findings suggest cAMP accumulation detected in Pkd21/- VSMCs
can be attributed, at least in part, to the AC activation due to a defect in SOCC
mediated Ca21 entry.
L-9
DIVERSE EFFECTS OF ANTIOXIDANTS ON THE CYTOTOXICITY
OF CHEMOTHERAPEUTIC DRUGS
Tomris Ozben
Department of Biochemistry, Akdeniz University, Medical Faculty, Antalya,
Turkey
There are conflicting views on the use of antioxidants in cancer patients, and
their potential interactions with radiation and chemotherapy. Some chemotherapeutic agents and all radiation therapies induce oxidative stress by generation of
oxygen free radicals (ROS) which might be an alternative mechanism for their
cytotoxic effect via inducing apoptosis. A question has logically developed as to
whether antioxidants taken concurrently during chemotherapy could reduce the
beneficial effect of chemotherapy on malignant cells by inhibiting ROS and preventing apoptosis of cancer cells. In order to clarify the roles of antioxidants in
chemotherapy, we investigated Quercetin (3,3’,4’,5,7-pentahydroxyflavone) and
N-acetylcysteine (NAC) in different cell types treated with anticancer drugs. We
studied cytotoxic activity of Topotecan alone and/or in combination with Quercetin in two human breast cancer cell lines, MCF-7 and MDA-MB-231. We also
investigated the effect of NAC on MRP1-mediated doxorubicin and vincristine
cytotoxicity in Human Embryonic Kidney (HEK293) and its MRPI transfected
(293MRP) cells. Our data indicated increased oxidative status in MCF-7 and
MDA-MB-231 cells exposed to Topotecan. Treatment with Quercetin did not inhibit ROS generation, and enhanced cytotoxicity of Topotecan in both cells. In
contrast, NAC enhanced resistance against doxorubicine and vincristine in MRPl
overexpressing cells. Our study demonstrates that Quercetin and NAC have
diverse effects in the cytotoxicity of chemotheurapeutic drugs. We conclude that
whether an antioxidant supplement would be helpful, harmful or neutral depends
in part on the specific antioxidant (and its dose), the chemotherapy drugs being
used, the type and stage of cancer being treated. Well designed randomized controlled trials are needed to fully elucidate the impact of single antioxidant and
antioxidant combinations in conventional cancer therapy.
285
SYMPOSIA
S1. History of Molecular Medicine
S1-1
ASCLEPIADES OF BITHYNIA: THE FATHER OF MOLECULAR
MEDICINE
Christos Yapijakis
Oral and Maxillofacial Surgery, University of Athens Medical School,
Greece
Hippocrates of Kos (460-377 BCE) is universally recognized as the father of
Clinical Medicine, which is based on observation of clinical signs and rational
conclusions. Before him, therapeutic attempts were based on religious or magical
beliefs. But about 350 years later, Asclepiades of Bithynia (124-40 BCE) became
the first known physician who spoke about what is known today as Molecular
Medicine. He was born in Prousias, Bithynia in the northwest region of Minor
Asia (modern Turkey). He was educated in philosophy at the Epicurean School in
Athens and in medicine at the Mouseion in Alexandria. At the age of 33 years he
moved to Rome, where he first taught philosophy and later on practiced medicine
being the first famous physician who established Hellenic Medicine in the capital
of Roman Empire. Asclepiades did not accept the Pythagorean dogma of four elements and humors and the theory of the ‘‘benevolent nature’’ that Hippocrates’ followers believed in, and, as an epicurean, he adhered to atomic theory, change and
evolution. He suggested that the human body is composed of void spaces
(‘‘poroi’’), as well as molecules (‘‘meri’’ or ‘‘corpuscula’’) made of atoms
(‘‘anarmoi ongoi’’). According to Asclepiades, diseases are caused by alteration of
form or position of a patient’s molecules. In order to restore health status, he
favored mild therapeutic methods such as healthy diet, exposure to light, hydrotherapy, massage, physical exercise, and above all the friendly support of patients.
Asclepiades was the first physician who coined the highly important division of
diseases in ‘‘acute’’ and ‘‘chronic’’ ones and the first to perform an elective nonemergency tracheotomy. He was a pioneer in treating women (previously thought
to be inferior beings) and in the humane treatment of patients with mental disorders, using labor and music therapy. He was probably the first physician who
assumed that in stagnant waters ‘‘invisible tiny animals’’ live (microbes) which, if
inhaled, may cause disease. His humane and naturalistic approach, as well as his
medical skills gave him a great reputation. His influence lasted for six centuries
through the Methodic medical school, which was established by his students. Famous among them are Themison, Titus Aufidius, Antonius Musa, Cornelius Celsus
and Soranus of Ephesos. Some ideas of Asclepiades have been rediscovered in the
last century and represent the molecular basis of Medicine.
S1-2
ADVANCES IN MOLECULAR MEDICINE
Turgay Isbir
Istanbul University, Institute for Experimental Medical Research,
Department of Molecular Medicine, Istanbul, Turkey
The employment of molecular biology and gene technology has enhanced the
understanding of human diseases creating a new branch of research - that of
"molecular medicine". Molecular Medicine is an essential area connecting diagnostic aspects using classical genetics procedures to clinical biochemistry with the
aim of a better disease diagnosis and treatment. Genes and gene products do not
function independently but participate in complex, interconnected pathways, networks and molecular systems that, taken together, give rise to the functioning of
cells, tissues, organs and organisms. Genetics seeks to correlate variation in DNA
sequence with phenotypic differences. The greatest advances in human genetics
have been made for the traits associated with variation in a single gene. However,
most of the disorders have more complex origins, involving the interplay between
multiple genetic factors and non-genetic factors (environmental influences). Defining these systems and determining their properties and interactions is crucial to
the understanding of how biological systems function. Understanding biological
pathways, networks and molecular systems requires information from several
areas and branches. Beside the patient-oriented classical medicine and modern
biology, the area of molecular medicine clarifies the molecular basis of diseases
by connecting the genetic, biochemical and clinical aspects. The application of
research involving gene technology, gene therapy, molecular structural analysis,
genetic epidemiology and molecular and clinical pharmacology has made unprecedented progress and precision of great potential in the understanding, prevention,
diagnosis and treatment of human diseases. In the future, we will be able to determine individual genetic variations and develop nutrigenetics and pharmacogenetics for preventive and therapeutic aims. For all those advancements, genetic
risk information will be useful to individualized therapies and prevention; treatment plans leading to improved health, will be associated with healthcare cost
reduction and diminution of therapy side effects. Such research should be interdisciplinary and use the tools and expertise of many fields, including genomics,
health education, healthcare, economics, bioinformatics, etc.
S2. Genomic Analysis and Molecular Basis of Cancer
S2-1
SIGNAL TRANSDUCTION THERAPY OF CANCER
Figure. Asclepiades of Bithynia (Capitoline Museum, Rome).
Alexander Levitzki
Unit of Cellular Signaling, Department of Biological Chemistry,
The Alexander Silverman Institute of Life Sciences, The Hebrew
University of Jerusalem, Jerusalem 91904, Israel
286
In view of the success of targeted cancer therapy since the early studies on tyrphostins (1988) we have enhanced our efforts to improve the performance of targeted therapies by following new paradigms. In this talk I will present three
ongoing studies in our laboratory. (1) The development of novel substrate competitive PKB/Akt inhibitors and how they perform in the treatment of prostate cancer
and brain cancer in nude mice; (2) the development of novel allosteric IGF1R
inhibitors that lead to the degradation of the IRS proteins and their highly potent
in vivo efficacy against disseminated ovary cancer, breast cancer, brain cancer and
prostate cancer in nude mice; and (3) the eradication of EGFR over-expressing
disseminated tumors by EGFR targeted long chain dsRNA (PolyIC). This
approach is highly effective due to the targeted ‘‘bystander’’ effect induced by the
PolyIC that is inserted selectively into the EGFR over-expressing tumor cells.
S2-2
BASE EXCISION REPAIR MODULATION AS A RISK FACTOR FOR
HUMAN CANCERS
Barbara Tudek
Department of Molecular Biology, Institute of Biochemistry and
Biophysics, Polish Academy of Sciences, Poland
Oxidative stress is involved in the pathology of human cancers. The major
pathway for oxidative DNA damage repair is Base Excision Repair (BER). Functional assays performed in blood leukocytes of cancer patients and matched controls show that specific BER pathways are decreased in cancer patients, and may
be risk factors. These include 8-oxoguanine (B-oxoG) repair in lung and head
and neck cancer patients, as well as repair of lipid peroxidation (LPO) derived
exocyclic adducts in patients lung and colon cancers. Regulation of BER proteins
activity is related to changed transcription rate, gene polymorphism, interactions
between BER system partners and signaling proteins, as well as post-translational
modifications. Transcription activation of several DNA glycosylases and AP-endonuclease may be due to oxidative stress. In rodent model this is accompanied
by the appearance of preneoplastic colon lesions, aberrant crypt foci. Polymorphisms of DNA glycosylases may change their enzymatic activities. Some polymorphisms increase the risk of inflammation-related cancers, colorectal, lung and
other types. Polymorphisms of BER platform protein, XRCCl may change the
rate of B-oxoG repair. BER activity is enhanced in colon cancers in comparison
to unaffected surrounding tissues. In lung tumors, repair rate of etheno adducts is
similar to that in unaffected lung tissue, but B-oxoG repair is decreased. These
changes may be due to dysregulation of BER proteins activity by mutated critical
genes products, e.g. APC or TP53, stimulation of transcription or post-translational modifications of BER enzymes. Modulation of BER enzymes activities
maybe, then, an important factor determining the risk of cancer and also may
participate in cancer development.
S2-3
LONGEVITY ASSURANCE MOLECULAR PATHWAYS IN
HUMAN CELLS
Efstathios Gonos
Department Molecular and Cellular Ageing, National Hellenic Research
Foundation, Greece
Ageing and longevity are two multifactorial biological phenomena whose
knowledge at molecular level is still limited. We have developed a senescence
induced cellular system and we have cloned several genes including a novel survival
factor, namely Clusterin/Apollpoproteln J (CLU). CLU is found over-expressed in
vitro under a variety of stress conditions and in vivo in samples from patients suffering from various age-related diseases as well as in primary tumours, which have
acquired chemotherapeutic drug resistance (Int J Cancer 120, 611-622, 2007). In
addition, we have demonstrated that inhibition of endogenous CLU expression by
RNA interference induces growth retardation, higher rates of endogenous cellular
death and sensitizes human cells to stress (Cancer Res 64, 1834-1842, 2004). Our
recent findings indicate that CLU binds to the cytoplasmic Ku70/Bax nexus and suppresses Bax activation and relocation to mitochondria (Clin Cancer Res, in press,
2008). We have also studied proteasome function in replicative senescence and cell
survival. We have observed reduced levels of proteasome content and activities in
senescent cells due to the down-regulation of the catalytic subunits of the 20S com-
plex (J Biol Chem 278, 28026-28037, 2003). In support, partial inhibition of proteasomes in young cells by specific inhibitors induces premature senescence, which is
p53 dependent (Aging cell 7, 717-732, 2008). Stable over-expression of catalytic
subunits or POMP resulted in enhanced proteasome assembly and activities and
increased cell survival following treatments with various oxidants. Importantly, the
developed ‘‘proteasome activated’’ human fibroblasts cell lines exhibit a delay of
senescence by approximately 15% (J Biol Chem 280, 11840-11850, 200S).
S2-4
DIETARY FLAVONOIDS AND THE RISK OF CANCER
Gary Williamson
Chair of Functional Food, Procter Department of Food Science,
University of Leeds, Leeds, LS2 9JT, UK
Flavonoids are found at high levels in many fruits and vegetables, and in
foods and beverages derived from plant foods. There is a strong interest in their
action to reduce cancer risk, and several recent epidemiological studies support
this, especially in situations where there is a clear carcinogenic stress such as
smoking. Typical flavonoids which have been studied for their anticarcinogenic
activity are green tea catechins ((-)-epicatechin, (1)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate), quercetin (found at
high levels in onions, apples and tea), flavones (e.g. apigenin found in some
herbs), anthocyanins (the coloured polyphenols in berry fruits) and proanthocyanidins (found in many fruits and also cocoa). Mechanistically, depending on the
structure, they inhibit COX-2, induce phase II detoxification enzymes and modify
cell signalling processes. In animal studies, many flavonoids are protective
against a carcinogenic insult. However, there are very few human intervention
studies on anti-carcinogenesis. Most human studies to date are on populations
who have already manifested some early signs of high risk, such as colon polyp
formation, where a preventive mechanism is difficult to demonstrate. In general,
the evidence support a protective effect against carcinogenesis, where selected
flavonoids act on certain types of cancers, but more evidence is required especially in human intervention studies using early biomarkers of cancer risk.
S2-5
PROPHYLAXIS OF OXIDATIVELY DAMAGED DNA BY
HETEROLOGOUS REPAIR PROTEINS
Guido Frosina, Paolo Degan, Mara Foresta, Ilaria Pettinati,
and Monica Ropolo
Molecular Mutagenesis & DNA Repair, Istituto Nazionale Ricerca Cancro, Italy
Objective: A significant contribution to human mutagenesis and carcinogenesis may come from oxidatively damaged DNA. For instance, defective repair of
this damage has been frequently associated to lung cancer. Oxidatively damaged
DNA may also be important in neurodegenerative processes such as those occurring in the transcription/repair defective disorder Cockayne syndrome. DNA
repair of oxidatively damaged DNA is rather efficient in human cells but a certain amount of damage inevitably escapes mending. Bacterial proteins that repair
oxidatively damaged DNA [e.g., formamidopyrimidine DNA glycosylase (FPG)
or endonuclease III (NTH)] are catalytically more efficient than their human
counterparts. The main objective of this work has been to enhance DNA repair
of oxidatively damaged DNA in human cells and reduce oxidative mutagenesis
via expression of heterologous DNA repair proteins.
Methods: cDNAs for bacterial (FPG, NTH) or Drosophila (dS3) DNA repair
proteins have been cloned in mammalian expression vectors containing the
enhanced green fluorescent protein (EGFP) tag gene. The EGFP-fused repair proteins have been expressed in human cells from normal individuals or patients
affected by Cockayne syndrome.
Results: Expression of EGFP-FPG enhanced DNA repair of oxidized purines (8oxoguanine) in normal human cells and caused a 10-fold reduction in mutations
induced by the oxidizing agent potassium bromate. In Cockayne syndrome cells,
expression of EGFP-FPG completely corrected the repair defect for 8-oxoguanine
and alleviated the repair defect for oxidized pyrimidines (5-hydroxycytosine). On the
contrary, expression of EGFP-dS3 repair protein was toxic to the cells. Studies with
the E.coli NTH protein that primarily repairs oxidized pyrimidines are ongoing.
Conclusions: Expression of bacterial DNA repair proteins enhances repair of
oxidatively damaged DNA in human cells and abates mutagenicity of oxidizing
agents. Their use in vivo as a protective mechanism from certain forms of lung
cancer and neurodegenerative processes will be explored.
S2-6
287
different mechanism involving inactivation of p53 in a manner similar to that of
the SV40 large T antigen is involved, and elevation of p73 is mechanism to
compensate for the lost of 53 activity. DNA micro array analysis has shown that
the E6 proteins of HPV 11 and HPV 18 have different effects on cellular genes.
OXIDATIVELY DAMAGED DNA, THE SPECTRUM OF
MODIFICATIONS AND THEIR IMPORTANCE
S2-8
H. E. Poulsen, M. Pedersen, and A. Weimann
Department of Clinical Pharmacology, Q7642, Rigshospitalet,
20 Tagensvej, DK-2200 Copenhagen N
OXIDATIVELY GENERATED SINGLE AND CLUSTERED DAMAGE
TO CELLULAR DNA
There are several methods for analysing oxidatively modified DNA. Direct
chemical methods include HPLC with electrochemical detection or mass spectrometry. Indirect methods are alkaline elution and the Comet Assay. Estimations of oxidatively modified DNA can be done in nuclear or mitochondrial DNA, and it can
be dome in body fluids. When measuring in DNA the concentrations are about one
modified base in one million unmodified bases. This calls for special precautions to
avoid artificial oxidation. For example: an 0.1% oxidation does not represent a
problem for biological methods such as PCR, however for chemical determination
a 0.1% oxidation would give oxidation of 1 bases among 1000, and the true value is
about 1 per million. For measuring in body fluids, such as plasma and urine, the analytical challenges are different, because the concentrations appear in the low nanomolar range and because of a large number of interfering substances. This requires
chromatography to be particularly elaborate. For measuring oxidatively modified
DNA bases in DNA, interlaboratory quality control schemes using synthetic oligonucletides have been developed. For urine measurements there are not defined
interlaboratory quality control schemes still need to be developed. There is some
discrepancy between the values obtained with direct and indirect methods; however, during the last years the values reported by the two methods are getting closer.
The urinary excretion of oxidatively modified nucleosides is a validated method to
estimate oxidative stress to DNA. Some diseased are of particular interest in relation to oxidative stress to DNA, among these are haemachromatosis and diabetes.
Recently, it has been suggested that the mechanism of action of certain antibiotics
include a free radical medicated oxidation of DNA, and this also seems to be the
case in humans. Oxidative stress to DNA seems to be important in the pathogenesis
for certain diseases, and its measurement may be relevant for estimating prognosis
and in some cases also the effect of drug treatment.
S2-7
THE ROLE OF HPV IN SQUAMOUS CELL CARCINOMA
OF THE OESOPHAGUS
M. Iqbal Parker
International Centre for Genetic Engineering and Biotechnology (ICGEB),
Wernher and Beit Building (South), UCT Campus, Anzio Road,
Observatory 7925, Cape Town, South Africa
Several studies have implicated p53 mutations in the aetiology of squamous
cell carcinoma of the oesophagus, but such mutations have been shown to be distinctly absent in South African oesophageal cancer patients. Since several studies
have shown the presence of HPV in South African patients with squamous cell
carcinoma oesophagus, we investigated whether an alternate mechanism of HPV
E6 mediated inactivation of p53 could play a role in carcinogenesis. 114 patients
with squamous cell carcinoma of the oesophagus were evaluated and HPV DNA
was shown to be present in 39% of cases using PCR consensus primers to the
L1 gene and in 30% of patients using primers to the E6 gene. Immunohistochemical analysis with an anti -p53 antibody showed intense staining in 59% of
the cases, while in histologically normal neighbouring epithelium, less than 10%
of the cells stained positive for p53. The levels of nuclear p73, a structural
homologue of the p53 that has been shown to possess anti-apoptotic activity was
significantly elevated in tumour cells (61% of patients) while in normal neighbouring epithelia elevated p73 expression was observed in only 8% of patients.
Co-expression of both p53 and p73 was statistically significant (r 5 0.48, P \
0.05) in 54% of tumours. No statistically significant relationship was observed
between p53 protein accumulation and HPV status (P50.16), while p73 was
higher in HPV DNA positive patients than in HPV DNA negative samples
(P50.01). Over-expression of p53 is usually correlated with the presence of p53
gene mutations, therefore, these results suggest that it is unlikely that HPV contributes to tumourigenesis via p53 degradation in oesophageal cancer. Perhaps a
Jean Cadet, Thierry Douki, and Jean-Luc Ravanat
Laboratoire des Lésions des Acides Nucléiques, INAC/SCIB,
CEA Grenoble, France
Emphasis has been placed in the recent years on the elucidation of oxidation
reactions of DNA and related model compounds in order to provide the basis for a
better understanding of the molecular mode of action of diverse endogenous and
exogenous processes such as oxidative metabolism, inflammation, solar light, ionizing radiation and xenobiotics. A large body of information is now available on
several major degradation pathways of purine and pyrimidine bases in both isolated and cellular DNA upon exposure to OH radical, singlet oxygen and one-electron oxidants such as high intensity UVC laser pulses. This was achieved in cells
by applying the highly accurate HPLC-electrospray ionization tandem mass spectrometry method and the modified comet assay that involves the use of DNA
repair glycosylases to reveal classes of modified purine and pyrimidine bases.
Thus several oxidized bases that can be used as biomarkers of dedicated oxidation
events have been detected in cells and human skin. There is now an increasing interest devoted to the search of more complex DNA lesions that may arise from
one radical hit. Evidence has been provided at least in isolated DNA for the formation of tandem base lesions that involves intramolecular addition of pyrimidine
hydroperoxyl radical on a vicinal guanine moiety. Another relevant example of
clustered lesions that involve both a base modification and an altered 2-deoxyribose residue deals with purine 50 , 8-cyclonucleosides whose radiation-induced levels of formation in cellular DNA remain to be established. It has been also shown
that radiation-induced OH radical hydrogen abstraction from C40 of the sugar moiety of cellular DNA gives rise to addition products to cytosine and purine bases.
S2-9
NUTRITION, ANGIOGENESIS AND CANCER
Mohsen Meydani
Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts
University, Boston, MA
Objective: Cancer is the second highest cause of mortality around the world.
The majority of cancers results from exogenous damages to genetic materials
over time including tobacco use, radiation, infectious agents, environmental pollutants and carcinogenic agents in food and drinks.
Methods: We know that nutrition plays an important role in modulating several processes associated with cancer development including DNA repair, cell
proliferation, hormonal regulation, inflammation, immunity and carcinogen metabolism. A large body of data at cell and molecular levels as well as at clinical
and population levels attest the importance of nutrition in reducing cancer risk.
Fruit and vegetable consumption is associated with reduced cancer risk. Nutrient
components of fruits and vegetables such as vitamins A, D, C, folate, selenium,
as well as bioactive components such as genistein, EGCG, curcumin, quercetin,
and carotenoids have been identified to interfere with cell cycle and malignant
cell division stages. Mounting evidence shows that bioactive food components
may modulate cancer development by affecting carcinogenesis and modulating
angiogenesis, a necessary process for tumor growth and metastasis.
Results: We have found that the antiangiogenic capacity of flavonols such myricetin, quercetin, kaempferol and galangin is associated with the number of
–hydroxyl group (-OH) on these flavonols’ chemical structure. Interestingly, certain
polyphenols such as curcumin and EGCG may also suppress adipose tissue growth
and prevent nutritional obesity, also associated with increased cancer risk. We have
advanced our understanding of molecular mechanisms by which EGCG modulates
progression of angiogenesis in endothelial cells. Our in vitro results indicated that
oat avenanthramides anti-inflammatory properties might contribute to reduction of
colon cancer risk associated with the high fiber content of oats.
288
Conclusion: While nutrients and bioactive foods play important roles in carcinogenesis, high dose and supplemental intake of bioactive food components
may up-regulate enzymatic systems involved in carcinogenesis. Supported by
USDA contract #58-1950-7-707.
S2-10
TUMOR SUPPRESSORS-GOOD AND BAD GUYS
Maciej Olszewski, Dawid Walerych, Alicja Zylicz, and Maciej Zylicz
International Institute of Molecular and Cell Biology in Warsaw
Most of the tumor suppressor genes are expressed at low levels in various tumor types. The p53 protein, however, is quite unique in that regard and its activity is regulated at additional levels except for its abundance. In non-cancer cells,
MMD2 downregulates p53 by binding to its transactivation domain, thus inhibiting its transcriptional activity and targeting it for proteasome-mediated degradation. It has recently been shown that MDM2 not only acts as a negative regulator
of p53, but is also required for the synthesis of p53 protein and its folding. In a
large proportion of tumors, missense mutant forms of p53 are expressed at high
levels and may contribute to oncogenesis. In such cases, radiation and chemotherapy trigger tumor promotion and metastasis. What is the molecular and functional basis for mutant p53 gain of function? Several options will be discussed
including inhibition of wt p53 by interaction with mutant forms of p53 and/or
involvement of mutant p53 in regulation of other oncogenes or in repression of
growth-inhibiting genes. During this talk, we will challenge our original hypothesis that mutant p53 proteins in cancer cells form a different multichaperone complex than in normal cells. In cancer cells, formation of this multichaperone complex prevents ubiquitylation and, in consequence, degradation of mutant p53.
Dramatic increase in the amount of partially unfolded p53 mutant proteins could
trigger Unfolded Protein Response and subsequently the induction of expression
of Hsp70. We show that Hsp70s not only are potent antiapoptotic factors but
could also be involved in mutant p53 aggregation. Such protein aggregates could
potentially sequester other transcription factors possibly involved in repression of
proliferation of cancer cells. In conclusion, recent reports challenge conventional
chemo- and radiotherapy as activating wild-type p53 and, in consequence, suppressing tumor growth. In case of mutated p53 protein, such treatment could
lead to its activation and, paradoxically, promote tumor progression.
the magnitude of the transcriptional activation of IFN-responsive genes could form
the basis for cell-specific transcriptional signatures.
S2-12
INTERSECTING PATHWAYS REGULATING ANGIOGENESIS AND
LYMPHANGIOGENESIS IN CANCER
Tuomas Tammela and Kari Alitalo
Molecular/Cancer Biology Laboratory, Biomedicum Helsinki,
University of Helsinki, Finland
Angiogenesis, the growth of new blood vessels from pre-existing vasculature,
is insufficient in pathological conditions such as ischemic heart disease and critical
lower limb ischemia. Vascular endothelial growth factors (VEGFs) stimulate
angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells. VEGFR-3 is present on all endothelia during development, and in the adult, it becomes restricted to the lymphatic endothelium.
VEGFR-3 is activated by VEGF-C and VEGF-D. VEGF-C expression in human
tumors has been correlated with poor prognosis, and increased incidence of lymph
node metastases. We have showed that blocking VEGF-C/VEGFR-3 results in
suppression of lymph node metastasis in preclinical tumor models. Furthermore,
we show that VEGF-C induces growth of lymphatic capillaries and collecting vessels in adult tissues and that this therapy can increase lymphatic drainage and
reduce tissue edema. VEGF-C therapy also improved the outcome of lymph node
transplantation in a mouse model of axillary lymph node dissection. We also show
that VEGFR-3 expression is induced during blood vascular angiogenesis also in
adult mice, and elucidate the role of VEGFR-3 signaling in settings of physiological and pathological angiogenesis. We demonstrate that VEGFR-3 is highly
expressed in angiogenic vessel sprouts, and that blocking VEGFR-3 signaling
results in decreased sprouting, vascular density, vessel branching and endothelial
cell proliferation. Furthermore, disruption of the Notch signaling pathway leads to
widespread endothelial VEGFR-3 expression and excessive sprouting, which is
inhibited by blocking VEGFR-3 signals. These results highlight the utility of the
VEGF-C/VEGFR-3 pathway in the treatment of cancer and lymphedema.
S3. G-Protein-Coupled Receptors
S3-1
S2-11
IDENTIFICATION OF GENES SELECTIVELY REGULATED BY IFNs
IN ENDOTHELIAL CELLS
EVALUATION OF V1A RECEPTOR LIGAND SELECTIVE
SIGNALLING AS A POTENTIAL THERAPY FOR SMALL CELL
LUNG CANCER
Stefano Indraccolo
Istituto Oncologico Veneto-IRCCS, Padova, Italy
Alison MacKinnon1, Mark Wheatley2, and Tariq Sethi1
1
Centre for Inflammation, University of Edinburgh, UK, 2School of
Biosciences, University of Birmingham, UK
IFN-a is the prototype of anti-angiogenic cytokines, and this activity contributes to its therapeutic effects in many tumor models. The effects of IFN-a on the
vasculature have been mainly attributed to inhibition of production of angiogenic
factors by the tumor cells, including bFGF, VEGF and IL-8. On the other hand,
there is evidence that IFN-a also has direct effects on endothelial cells (EC), including impairment of their proliferation and migration. We recently investigated the
gene expression profile induced by IFN-a in EC, and observed that several genes
encoding negative regulators of angiogenesis are up-modulated, thus providing an
amplification mechanism for this biological activity. We found that in HUVEC as
well as in other EC types 175 genes were upregulated ([2-fold increase) by IFNs,
including genes involved in the host response to RNA viruses, inflammation, and
apoptosis. Interestingly, several genes showed a much higher induction by IFN-a in
EC compared to human fibroblasts; among them, the genes encoding the angiostatic
chemokines CXCL10-11, along with other genes associated with angiogenesis regulation, including TRAIL, and guanylate binding protein 1 (GBP-1). These results
were extended to the analysis of the transcriptional effects of IFN-a in subpopulations of cancer cells with stem cell features, and some relevant quantitative differences compared to the gene expression profiles obtained in EC emerged. Treatment
of transplanted or spontaneous tumors with IFN-a was followed by up-regulation of
CXCL10-11 and GBP-1 expression in the tumor microenvironment and this was
associated with striking anti-vascular effects. Overall, these findings show that
IFN-a triggers an anti-angiogenic transcriptional program in EC which may explain
its anti-angiogenic activity. Moreover, we suggest that quantitative differences in
Arginine vasopressin (AVP) and the V1A vasopressin receptor are frequently
upregulated in small cell lung cancer (SCLC) and are hallmarks of the transformed phenotype. In addition, the inevitable development of chemoresistance to
conventional chemotherapy in SCLC is accompanied by an increased sensitivity
to AVP. Our work has identified several analogues based on substance P such as
[D-Arg6,D-Trp7,9,NmePhe8]-substance P (6-11) (SP-G) which block mitogenic
signalling by AVP but activate V1A receptor mediated cell death. These analogues are antiproliferative in SCLC in vitro and in vivo. Moreover, SP-G is
more potent in chemoresistant SCLC cell lines and can sensitise SCLC to chemotherapy. Therefore SP-G may have additional benefit as an adjunct to chemotherapy or as a second line treatment of resistant disease. SP-G, has been taken
into Phase 1 clinical studies for SCLC where therapeutic concentrations were
achieved with minimal toxicity. However, its precise mechanism of action
remains to be elucidated. Epithelial cells expressing human V1A receptors
undergo cellular transformation and exhibit serum and adhesion independent
growth characteristic of the transformed phenotype. Moreover, these cells show
increased sensitivity to the antiproliferative effects of SP-G. We show that SP-G
directs receptor signalling via the V1A vasopressin receptor, causing an inhibition
of V1A receptor coupling to Gq resulting in an inhibition of AVP stimulated PLC
activation and intracellular Ca21 elevation, whilst activating ERK via a stimulation of V1A receptor coupling to Gi. Studies with chimeric V1A/V2 receptors
show that the second intracellular loop of V1AR is essential for AVP-stimulated
PLC and ERK activation but not for SP-G-induced ERK activation. This study
provides experimental evidence for agonist selective V1A receptor conformations
and gives mechanistic insight into the therapeutic utility for V1A receptor signal
selective agonists as anticancer agents for SCLC. Such pharmacological agents
may offer another level of selectivity for drug design with reduced toxicity.
289
binding, there are probably concerted movements involving transmembrane
(TM) helices 3 and 6. In TM 6, a proline halfway down the helix appears crucial and may act as a pivot, allowing the helix to change conformation. At the
base of TM3, a glutamate appears to be important in activation; it may interact
with nearby basic residues in TM2. The C-terminal tail is important for receptor internalisation.
S3-2
ASSOCIATION BETWEEN SUPPRESSOR OF CYTOKINE SIGNALING
AND DISEASE
S3-4
Bilkay Basturk
Department of Immunology, Faculty of Medicine, Gazi University, Turkey
SIGNALLING THROUGH FAMILY A GPCRs
Cytokines are secreted glycoproteins, which regulate diverse biological process through their interaction with multi-subunit receptor complexes. The suppressor of cytokine signaling (SOCS) family comprises proteins induced on cytokine stimulation, which bloc further signaling in a classic feedback loop. The
SOCS protein family consists of eight members; SOCS1-7. Common familial
features include a central SH2 domain and a conserved c-terminal SOCS box.
SOCS2 might have a role in regulating the expression of other SOCS proteins.
SOCS1 has been the archetypal member of this family and besides being the
most potent inhibitor of cytokine signaling. Multiple studies have demonstrated
that over-expression of SOCS1 can inhibit signaling downstream of multiple
cytokines that use JAK kinases for their intracellular signaling. SOCS1 deficiency is associated with a wide range of acute and chronic inflammatory disorders suggesting an important non-redundant role for SOCS1 in autoimmune disease. SOCS3 is induced by various inflammatory and anti-inflammatory cytokines, such as IFN-gamma, IL-3, IL-10 and granulocyte colony-stimulating
factor. In rheumatoid arthritis patients Stat3 and SOCS3 levels are elevated. Elevated SOCS3 expression again suggests that SOCS3 may also have a regulatory
role in ulcerative colitis and Chron’s disease. SOCS4 is the least studied SOCS
family member. SOCS4 levels are up regulated on EGF stimulation. SOCS4 and
SOCS5 are the only SOCS family members that markedly reduce EGFR level.
SOCS6 and SOCS7 are most closely related SOCS proteins. SOCS1 and SOCS3
are associated with allergy. Increasing evidence supports a role for SOCS1,
SOCS3 and SOCS5 in coordinating T helper Th1/Th2 cellular profiles. Recent
studies have found a correlation between elevated SOCS1 expression and asthma
severity in patients and suggest that SOCS1 may inhibit IFN-gamma dependent
Th1 differentiation, thereby enhancing Th2 mediated pathology. SOCS3 is
expressed in Th2 cells and elevated expression levels are observed in patients
with asthma and atopic dermatitis.
Mark Wheatley
School of Biosciences, University of Birmingham, UK
G-protein-coupled receptors (GPCRs) form a large superfamily of structurally-related proteins which mediate their effects by coupling to G-proteins. They
are one of the largest gene families in the human genome and are a major target
for drug discovery. Indeed, 40-50% of clinically-marketed drugs are active at
this receptor family. A fundamental issue in molecular medicine today is defining, at the molecular level, how GPCRs are activated. Despite being activated by
a wide variety of stimuli from photons to large glycoproteins, these receptors exhibit a conserved protein architecture comprising a bundle of seven transmembrane (TM) helices linked by extracellular loops (ECLs) and intracellular loops.
Defining the binding site of agonists and antagonists to GPCRs, at the molecular
level, is of fundamental importance to understanding their activation by hormones and to rational drug design. For GPCRs in general, the location of the
ligand-binding site and the molecular mechanisms underlying receptor activation
are poorly defined. We systematically investigated the role of residues, which are
highly conserved throughout a sub-family of peptide-GPCRs belonging to Family
A, using a combination of mutagenesis and molecular modelling. In a complementary approach, we synthesised analogues of the peptide hormone which
incorporated residue changes predicted to recover activity at specific mutant
receptors. As a result of these studies we have identified conserved residues that
are required for ligand binding, intracellular signalling and cell-surface expression. This work was supported by grants from the Wellcome Trust and the
BBSRC.
S3-5
S3-3
LIGAND BINDING AND ACTIVATION OF THE CGRP RECEPTOR
James Barwell1, Denise Wootten1, Alex Conner2, John Simms3,
Mark Wheatley4, and David Poyner1
1
School of Life and Health Sciences, Aston University, UK,
2
Medical School, Warwick University, UK; 3Department of Pharmacology,
Monash University, Australia, 4School of Biosciences, University of
Birmingham, UK
G-protein coupled receptors (GPCRs) are divided into several families.
Family A includes rhodopsin and the beta-adrenergic receptor; it is the largest
family and the most studied. However, family B GPCRs include receptors for a
number of important peptides such as glucagon, secretin and calcitonin. They
have virtually no sequence homology with family A GPCRs but recognise the
same G-proteins. Family B GPCRs also show a noticeable tendency to associate with single pass transmembrane accessory proteins known as receptor activity modifying proteins (RAMPs) which can influence their pharmacology and
signal transduction. The CGRP receptor is a family B GPCR. It is a heterodimer formed between a GPCR, calcitonin receptor-like receptor (CLR) and
RAMP1. We have combined extensive mutagenesis with molecular modelling
to study this complex. Ligand binding requires the N-terminus of RAMP1; the
recent publication of a crystal structure for this protein confirms our model and
demonstrates that it forms a helical bundle, probably with the N-terminus of
CLR to recognise the C-terminus of CGRP. The N-terminus of CGRP interacts
with the extracellular loops and possibly transmembrane regions of CLR; the
second and third extracellular loops are of particular significance. Upon ligand
FROM SIGNALING PATHWAY TO THERAPY, A CONCEPT IN
MOLECULAR MEDICINE: THE PHENOMENON OF
INTRACELLULAR CALCIUM
Mehmet Isbir
Department of Pharmacology, Medical Faculty of University of
Mediterranean, Antalya, Turkey
Calcium, as intracellular calcium, is known as an important second messenger, controls almost all cellular functions such as fertilization, secretion, contraction, proliferation, and signaling. Also because of these functions, it is beginning to be considered as an important molecule for therapy of some diseases. It differs significantly from other signals due to its several features. It
serves as an intracellular second messenger as whole class of extracellular stimuli and couple them to cellular responses. Especially in drug therapy, intracellular calcium has an important role as a signaling pathway. In Nifedipine therapy, increase of calcium level in cell is a significant factor in the mechanism
of action of this drug. Also cyclosporine-caused erythrocyte deformability and
the antioxidant effect of carnitine have a relation with intracellular calcium signaling; this affects metabolism as well and, in the process of wound healing,
increased levels of intracellular calcium in fibroblasts act as a signal for the beginning of chemotaxis of these cell towards the wound. Maintaining constant
Ca11 intracellular levels is becoming very convenient, either by controlling
calcium channels or by controlling intracellular Ca11 deposits. Another beneficial side aspects of this idea, since Ca11 measurements are becoming easier
every day, it will be possible in the near future to measure intracellular calcium
as important parameter, either as bio marker or to evaluate the therapy of various diseases.
290
S3-6
GENETIC AND EPIGENETIC CONTROL OF MHC2TA
TRANSCRIPTION
Peter J. Van den Elsen
Department of Immunohematology and Blood Transfusion,
Leiden University Medical Center, The Netherlands
Tight control of major histocompatibility complex (MHC)-class II and class I
gene expression is crucial for an adequate immune response. The class II transactivator (CIITA), encoded by the MHC2TA gene, is essential for transcriptional
activation of all MHC-II genes. The co-activator CIITA exerts its function in
transactivation of MHC genes by interacting with a highly stable multimeric protein ensemble that binds to the SXY regulatory module in the promoters of MHCII genes. This multiprotein complex is comprised of RFX, CREB/ATF and NF-Y,
and together with the coactivator CIITA forms an enhanceosome that drives transcription of both classes of MHC genes. The specificity of this enhanceosome for
MHC-II genes is determined by the unique composition of the SXY module as
well as the unique components and assembly of the multiprotein-DNA complex. In
the enhanceosome, CIITA provides the link with the basal transcription initiation
apparatus and acts as a platform for recruitment of common coactivators for chromatin remodeling activities. Coinciding with MHC-II molecule expression, CIITA
is constitutively expressed in antigen presenting cells of the immune system (e.g.
dendritic cells and B cells), while in other cells types its expression can be induced
(e.g. fibroblasts, epithelial cells and human activated T cells). Constitutive and
induced transcription of MHC2TA is governed through the employment of at least
three independent promoters (CIITA-PI, -PIII and -PIV), which are activated following cell type-specific signaling pathways or in the response to inflammatory
cytokines of which interferon-gamma is the most potent. In cancer, lack of constitutive and inducible MHC-II molecule expression is frequently observed, which is
due to lack of CIITA expression. Our studies have shown that distinct epigenetic
DNA and histone modifications are associated with lack of CIITA expression in
MHC-II deficient cancer cells. For these studies we have employed bisulfite
sequencing, to determine the level of DNA methylation at CpG dinucleotides, and
chromatin immunoprecipitation (ChIP) for the evaluation of post-translational histone modifications and recruitment of non-histone proteins to promoter chromatin.
Our studies have revealed that high levels of tri-methylated histone H3-lysine 27
(3Me-K27-H3), in CIITA promoter chromatin, are associated with transcriptional
silencing of MHC2TA in cancer cells and provide a link with components of the
Polycomb Group proteins.
S4. Thrombosis
S4-1
MOLECULAR BASIS OF THE PLEIOTROPIC EFFECTS OF
ANKAFERD BLOOD STOPPER
Ibrahim C. Haznedaroglu
Department of Hematology, Hacettepe University Medical School,
Ankara, Turkey
Ankaferd Blood Stopper (ABS) comprises a standardized mixture of the
plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum
and Urtica dioica. The basic mechanism of action for ABS is the formation of
an encapsulated protein network that provides focal points for vital erythrocyte
aggregation. ABS–induced protein network formation with blood cells particularly erythrocytes covers the primary and secondary haemostatic system without
disturbing individual coagulation factors (Figure 1). Proteins of plant origin in
Ankaferd were NADP-dependent malic enzyme, Ribulose bisphosphate carboxylase large chain, Mturase K, ATP synthase subunit beta, ATP synthase subunit
alpha, Chalcone-flavonone isomerase 1, Chalcone-flavonone isomerase 2, and
Actin-depolymerizing factor. Furthermore, functional proteomics studies revealed
that proteins, resembling human peptides, have been detected within Ankaferd.
They include ATP synthase, mucin 16 (CD164 sialomucin-like 2 protein),
coiled-coil domain containing 141 hypothetical protein LOC283638 isoform 1,
hypothetical protein LOC283638 isoform 2, dynactin 5, Complex I intermediateassociated protein 30, mitochondrial, NADH dehydrogenase (Ubiquinone) 1
alpha subcomplex, TP synthase, H1 transporting, mitochondrial actin binding 1
isoform, LIM domain and actin binding 1 isoform a, LIM domain and actin binding 1 isoform b, Spectrin alpha non erythrocytic 1, Prolactin releasing hormone
Figure. [Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]
receptor, Utrophin, tet oncogene family member 2 isoform b, Protein phosphatase
1 regulatory subunit 12A, NIMA (never in mitosis gene a)-related kinas, ATPbinding cassette protein C12, Homo sapiens malic enzyme 1, Mitochondrial
NADP(1)-dependent malic enzyme 3, ME2 protein, Nuclear factor 1 B-type,
Abhydrolase domain-containing protein 12B, E3 SUMO-protein ligase PIAS2,
Alpha-1,2-glucosyltransferase ALG10-A, Cofilin, non-muscle isoform, 18 kDa
phosphoprotein, p18, Actin-depolymerizing factor, ADF, Twinfilin-1, Ankyrin
repeat and FYVE domain-containing protein 1, Usherin Precursor, Urotensin II
receptor. Ankaferd upregulates numerous transcription factors including AP2,
AR, CRE/ATF1, CREB, E2F1-5, E2F6, EGR, GATA, HNF-1, ISRE, Myc-Max,
NF-1, NFkB, p53, PPAR, SMAD 2/3, SP1, TRE/AP1, YY1 for its pleiotropic
effects. Those molecular investigations represent a true basis for the upcoming
Ankaferd studies focusing on its wound healing, hemostatic, anti-infective, antineoplastic, preservative biological actions.
S4-2
ENDOTHELIAL CELL PROTEIN C RECEPTOR (EPCR):
WHAT’S NEW?
Nejat Akar
Pediatric Molecular Genetics Department of Ankara University, Turkey
The protein C anticoagulant pathway is the major control mechanism of blood
coagulation. Mutations in the genes of protein C (PC), protein S and thrombomodulin have been identified in patients with venous and/or arterial thrombosis. An endothelial cell-specific transmembrane protein has been identified that binds protein C
and activated protein C (APC) on the cell surface, which is named as endothelial
cell protein C/APC receptor (EPCR). The activation rate of protein C is increased
when protein C is bound to EPCR, a process that is likely to be most important on
larger vessels where EPCR is found primarily. The cloned human EPCR gene is
consist of four exons. Several polymorphisms and different haplotypes were
reported including a 23 bp insertion in the exon III (nt4031) of the EPCR. Another
mechanism leading to dysfunction of the EPCR-mediated coagulation is the
increased levels of soluble protein C receptor (sEPCR) in plasma. sEPCR levels
vary among healthy subjects and the bimodal distribution has been reported several
times. In this report, we will present our data on sEPCR levels in newborns, children and adults; A3 haplotype and relation to sEPCR in pediatric stroke; sEPCR
levels in sepsis, and a 3’ UTR polymorphism related to sEPCR levels and its effect
on thrombosis; EPCR 23 bp deletion patients and effect of TNF-alpha on sEPCR.
S4-3
THROMBOSIS AND THALASSEMIA
Tansu Sipahi
Ufuk University, Faculty of Medicine and Dr. Rıdvan Ege Hospital,
Department of Pediatrics, Ankara, Turkey
Currently used therapeutic approaches such as regular blood transfusions and
iron chelation have prolonged life expectancy in patients with thalassaemia. A
consequence of this brings new complications. There have been numerous reports
of thromboembolic complications associated with thalassaemia, recently. Venous
thromboembolic events such as deep venous thrombosis, portal venous thrombosis, pulmonary embolism, postsplenectomy thrombosis and cerebral thromboembolism have been observed in thalassemic patients. At the end of the 1980’s, a
study concerning survival and causes of death in thalassaemia, major (TM)
patients showed thromboembolism represented the primary cause of death in about
2.5% of the cases. Michaeli et al. described 4% of their b- thalassaemia patients
with rare thromboembolic manifestations. Pignatti et al. identified the overall prevalence of thromboembolic episodes in beta thalassaemia major patients as 2.3%
and 9.6% in thalassaemia intermedia patients in a survey involving nine ltalian
thalassaemia centers. Recently the same group reported thrombotic events in 1.1%
of the thalassemia patients and caused 4.1% of the deaths. In our country the data
compiled from the Turkish Thalassemia Study Group from eleven centers revealed
thromboembolism in 3.27% of the patients (thalassaemia major and intermedia).
Red blood cell membrane phospholipid structure was damaged and the procoagulant phosphatidyl-serine translocated in the outer surface of the red blood cell
membrane. The membrane changes may explain the enhanced aggregation of
RBC’s and their capacity to enhance thrombin generation. At the end platelets and
endothelial cells was activated and tissue factor released. All of these factors
enhance the thrombotic process. The low levels of the anticoagulant factors such
as protein C, protein S and AT III contribute this hypercoagulable condition. Clinical findings and haemostatic anomalies found in thalassaemia patients suggest the
existence of a chronic hypercoagulable state. There are signs of in vivo platelet
activation, shortened platelet survival, decreased levels of naturally occurring anticoagulants such as protein C, protein S, and antithrombin III. The possibility of a
genetic basis for the hypercoagulable state in thalassaemia patients is not clear.
Eldor et al. found no increased prevalence of congenital thrombophilic mutations,
including the FVL, PT G20210A and MTHFR C677T mutations. Finkelstein et al.
found five patients (5/23) had specific mutations indicating hereditary thrombophilia in their thalassaemia major patients group. Further three different studies
revealed controversial data, one from Lebanon in beta thalassaemia intermedia
patients with high FVL mutation, one from Turkey after splenectomy. Even if
thromboembolic complications could be explained by hypercoagulable state found
in thalassaemia major patients; following the first thrombotic event, such as deep
venous thrombosis, portal venous thrombosis, pulmonary embolism, strokes, peripheral arterial and venous thromboses, pulmonary hypertension and development
of cor-pulmonale, should be investigated for congenital thrombophilia. When they
are exposed to thrombotic risk factors such as immobilisation, surgery and delivery, prophylactic antithrombotic agents may be recommended.
S5. Targeted Therapies in Ovarian Cancer
291
the behavior of epithelial ovarian carcinomas. For this reason, targeting neo-angiogenesis has been one of the most promising fields of investigation. Bevacizumab
(Avastin1) is a recombinant humanized anti-VEGF monoclonal antibody which
binds to all the isoforms of VEGF-A. Pre-clinical experiments demonstrated its
ability to block the growth of a number of human cancer cell lines grown in nude
mice, including ovarian cancer. To date Bevacizumab has been evaluated in ovarian carcinoma and primary peritoneal carcinoma (PPC) in phase II trials for relapsing patients. One study from the GOG (Gynaecologic Oncology Group) administered bevacizumab 15 mg/kg every 3 weeks in 62 patients with recurrent or refractory epithelial ovarian carcinoma or PPC. The investigators observed a response
rate of 21%. The median PFS was only 4.7 months but the rate of patients free of
progression at 6 months was 41%, which is higher than normally observed. In
another multicentric phase II trial, 44 platinum resistant patients were treated with
the same dose of bevacizumab obtaining a response rate of 17% and a PFS of 4.4
months. These encouraging data in a platinum-resistant population prompted the
activation of two international phase III trial exploring the addition of bevacizumab to the first line of chemotherapy in previously untreated ovarian cancer. The
results of these two trials will answer the role of bevacizumab in the first line.
S5-2
TARGETED THERAPIES- WHERE ARE WE TODAY? - ROLE OF
ABOGOMAVAB (ANTI-CA125)
Christian Kurzeder
University of Ulm Medical School, Department of Obstetrics and
Gynecology, Ulm, Germany
Abagovomab is a murine anti-idiotypic antibody (anti-id) against the antigen
CA-125 to be used as a therapeutic vaccine for the prevention or delay of recurrent
ovarian cancer. The anti-idiotypic approach has been used to generate immune
responses in a variety of clinical studies including patients with colon cancer, melanoma, and small cell lung cancer. According to the ‘immune network hypothesis’
immunization with a given tumor antigen such as CA-125 will generate the production of antibodies termed Ab1. Abagovomab is an anti-id antibody (Ab2) which
was induced as a response to Ab1 and expresses an internal image of the antigen
CA-125. As shown in a phase I study comparing two s.c. vaccination schedules
Abagovomab can induce the production of anti-anti-id antibodies (Ab3) which recognize the original antigen CA-125. Clinical efficacy of Abagovomab is currently
evaluated in a placebo controlled phase III study in which Abagovomab is applied
as maintenance therapy in patients without evidence of residual disease after completion of primary surgery and postoperative platinum based chemotherapy.
S5-3
S5-1
PREDICTION OF SURGICAL OUTCOME AND LONG-TERM
PROGNOSIS – MOLECULAR PARAMETERS
ROLE OF BEVACIZUMAB IN OVARIAN CANCER
Carsten Denkert
Institute of Pathologie, Charité-Universitätsmedizin Berlin, Campus Mitte,
Charitéplatz 1, 10117 Berlin, Germany
Antonio Gonzalez Martin
Head of Medical Oncology Service, MD Anderson International Spain,
Madrid, Spain
In epithelial ovarian cancer, angiogenesis has been shown to have a central
role in both disease progression and prognosis. Angiogenesis (the development of
new blood vessels) is essential for the process of solid tumor growth and metastasis. Tumoral neo-angiogenesis is stimulated by an increased delivery of proangiogenic cytokines from the tumor cell in response to hypoxia and other factors.
VEGF (vascular endothelial growth factor) is considered the most important proangiogenic factor in the initiation of tumoral angiogenesis. The VEGF family is
composed by 7 related proteins, VEGF A, B, C, D and E and the placental growth
factor (PLGF) 1 and 2. VEGF-A is the key mediator of the family. The secretion
of VEGF induces the mobilization of endothelial progenitor cells from the bone
marrow, which migrate to the tumoral bed. The VEGF stimulates VEGF receptors
(VEGFR-1, and VEGFR-2) in the endothelial surface cell (the main receptor is
VEGFR-2). The VEGFR-2 is a tyrosine kinase receptor, which activation initiates
a cascade of intracellular signaling events, initially causing autophosphorylation of
both receptor tyrosine kinases followed by activation of a number of downstream
proteins, leading to proliferation and survival of the endothelial cell. In humans, a
direct relationship has been demonstrated between the expression of biomarkers
for angiogenesis such as VEGF and VEGF-R, in tumor samples and serum, and
Among gynecological malignancies, ovarian carcinoma has the highest mortality rate. Currently most patients with high stage ovarian cancer are treated with
extensive cytoreductive surgery and platinum-taxane based chemotherapy. As the
current chemotherapeutic treatment options are limited, there is a need for new
therapeutic strategies. For the planning of an individualized therapy in ovarian
cancer, the identification of tissue-based biomarkers is a major task. As ovarian
cancer is a malignancy with a particular poor prognosis, new diagnostic tests are
needed for the planning of an individualized therapy, assessment of response to
surgical treatment and long-term outcome. These molecular markers for prediction
of outcome of ovarian cancer should be independent of classical clinical parameters. Diagnostic biomarkers are currently investigated on different biological levels
using genomics, transcriptomics, proteomics or metabolomics as a major
approach. Protein biomarkers as well as RNA biomarkers can be measured in formalin-fixed tissue using immunohistochemistry as well as new techniques for isolation of nucleic acids. Furthermore, gene expression as well as metabolic signatures can be determined using frozen tissue. In a systems pathology approach the
results from the different biological levels are integrated to a combined signature
that reflects the biological behaviour of the tumor. In particular, the combined
analysis of gene-array data as well as clinical data on intraoperative residual tumor
292
can be used for assessment of long-term prognosis. The combination of different
prognostic parameters to a combined score provides an advanced system for
assessment of prognosis, suggesting that the information obtained form gene
expression analysis should be used in integration with relevant clinical parameters.
Our investigations have the primary goal to provide a basis for identification of
those individual patients that may benefit from new experimental therapeutic
approaches. This analysis might lead to an optimized clinical management.
to these conclusions will be discussed in my lecture, and I will put the understanding of these data in the framework of evolving needs based around concepts
relating to neoadjuvant chemotherapy and delayed surgery, and the use of targeted therapies to modulate or reverse chemotherapy resistance.
S5-6
S5-4
TUMOR BANK OVARIAN CANCER (TOC): EXPERIENCE OF AN
INTERNATIONAL PROJECT: 5-YEAR EXPERIENCE
TARGETED THERAPIES- WHERE ARE WE TODAY?: ROLE OF
SORAFENIB (MULTIKINASE-INHIBITORS)
J. Sehouli, R. Chekerov, G. Oskay-Ozcelik, R. Zeilinger, S. Leodolter,
C. Denkert, E. Stickeler, H. Tulusan, D. Konsgen, W. Mustea, I. Braicu,
N. Costin, and D. Sofroni, W. Lichtenegger
Charité, Department of Gynecology, European Competence Center for
Ovarian Cancer, University of Berlin (Coordination center for the Tumor
bank Ovarian Cancer Network)
Gulten Oskay-Ozcelik and Jalid Sehouli
Department of Obstetrics and Gynecology, Charité University Hospital,
Campus Virchow-Clinic, Berlin, Germany
Despite advances in surgery and chemotherapy, less than 20% of patients
with stage III or IV ovarian cancer survive long-term. In the past, cytotoxic regimens have been developed empirically; combining active agents at maximally
tolerated doses, often without any significant benefit in survival rates. The development of targeted therapies has provided new options for the management of
patients with advanced solid tumors. Advances in understanding the biology of
ovarian cancer have identified multiple molecular targets that differ in normal
and malignant cells. The combination of conventional chemotherapies with targeted therapies presents the most promising strategy to improve the clinical outcome. Sorafenib is a novel oral multi-targeted tyrosine kinase inhibitor that targets the RAF/MEK/ERK signaling pathway, vascular endothelial growth factor
(VEGF) receptor, platelet derived growth factor receptor and flt-3 that has been
proven effective as a single-agent therapy in renal cell carcinoma. There is a
strong rationale for investigating its use in combination with other agents in
ovarian cancer patients. In particular, targeting multiple Raf isoforms with sorafenib may help to overcome resistance to other agents, while the ability of sorafenib to induce apoptosis may increase the cytotoxicity of chemotherapeutic
agents. Based on positive results in preclinical studies, further investigation in
phase I and II studies has shown potential antitumor activity when sorafenib is
combined with cytotoxic agents in different solid tumors. Promising results have
been reported in phase I and II studies of sorafenib combined with paclitaxel and
carboplatin, with oxaliplatin in gastric and colorectal cancer, with docetaxel in
breast cancer and with gemcitabine in ovarian cancer. Phase II and III studies
are currently investigating the use of sorafenib in combination with different
agents in a variety of solid tumors. Translational research programmes will help
to identify the best target population for such innovative therapies.
S5-5
CAN WE PREDICT CHEMOTHERAPY RESISTANCE? WHAT ARE
THE CLINICAL PARAMETERS?
Hani Gabra
Imperial College, London, UK
Ovarian cancer is a highly chemoresponsive tumour with 70-80% objective
response rates using current combination chemotherapy. Platinum-based chemotherapy is a proven cornerstone of therapy and it has significant impact on disease free and overall survival. However, the 5 year overall survival of patients is
only 40%, with disease free survival even lower overall at that point. Relapse of
ovarian cancer occurs because cancer cells have survived their chemotherapy,
and as such, these patients will ultimately die from platinum resistant disease.
Over many years, we have developed imperfect but useful predictors of efficacy
of and resistance to chemotherapy. These predictors can be utilized both in front
line management (closely related to intrinsic resistance, also known as the platinum refractory state) and particularly also the management of relapsed ovarian
cancer. In this latter setting, clinical predictors are routinely used in management
of the disease. The predictors of front line sensitivity/resistance mainly include
adverse histological features (mucinous/clear cell histology), low tumour grade,
poor patient performance status, and the use of platinum-based chemotherapy.
The predictors of resistance in relapsed disease, include the platinum and/or
treatment free interval since last chemotherapy, number and size of recurrent
deposits of tumour and favourable histology (serous), the evidence base relating
The Tumor Bank Ovarian Cancer (TOC) project was started primary in September 2000 in the Charité, Campus Virchow-Klinikum, Department of Gynecology and Obstetrics. Between September 2000 and July 2004, overall 420 patients
with primary and recurrent ovarian cancer were prospectively documented and
recruited into TOC. Tumor, ascites, serum and blood were collected from each
patient with ovarian cancer after given their informed consent. The tumors were
collected at the time of surgery, shock-frozen and stored at -180 C0 in a Dewar
Tank containing liquid nitrogen, the blood and ascites specimens respectively at 80 C0. In median, 10 (range 1-25) samples of each patient are available from each
donor. The following specimen requirements were defined: Each tumor is classified
by the Pathology department upon resection, and anonymous basic data about each
tumor is kept in our database with all relevant clinical, histo-pathological and follow-up data. Furthermore, we have developed and validated a systematic surgical
and histo-pathological tumor documentation system (Intraoperative Mapping of
Ovarian Cancer 5 IMO). IMO represents a new instrument for detailed and objective documentation of tumor spread and helps hereby to provide a more specific tumor staging. This prospective documentation represents a valid instrument for
standard operating procedures and quality control. On January 2004 we initiated
the project TOC-Network, a tumor bank with a multi-centric setting. Overall, seven
European Hospitals are involved in this project. Each university hospital uses the
same SOP’s and online documentation tool. For the statistical analysis we developed an online documentation tool, where are registered all clinical, surgical and
follow-up data from all patients with ovarian cancer. Since January 2004 we enrolled now more than 1100 consecutive patients into TOC Network. The prospective tumor bank allows assessing and verifying the clinical relevance of basic science of ovarian cancer and provides an essential link between basic research and
applied clinical research. Based on the experience the logistic and the established
infrastructure will be used for further clinical and preclinical projects. The scientific
board of TOC is very open to all suggestions for collaborative research projects.
S5-7
CAN WE PREDICT CHEMOTHERAPY RESISTANCE? WHAT ARE
THE MOLECULAR PARAMETERS?
M. Dietel
Institute of Pathology, Charité, University Hospital Berlin, Germany
Introduction: Drug resistance remains a major problem in therapy of systemic
cancer diseases. Due to the high potency to adapt to therapeutic approaches, malignant
tumour cells, develop frequentl escape mechanisms in response to anti-cancer drugs.
Cell culture experiments: To define resistance associated signatures for
common cytostatics, we tested 30 cancer cell lines for sensitivity to 5-FU, cisplatin, cyclophosphamide, doxorubicin, etoposide, methotrexate, mitomycin C,
mitoxantrone, paclitaxel, topotecan and vinblastine at drug concentrations achievable in vivo. The resistance index defined previously was applied to designate
the cell lines as sensitive or resistant, then, the subset of resistant versus sensitive
lines for each drug was compared. Expression signatures were obtained by interrogating Affymetrix U133A arrays.
Preclinical studies targeted to ovarian cancer: We tested 96 ovarian cancer specimens with known clinical follow up to predict platinum-resistance.
Based on a pattern with 45-90 altered gene expressions preliminary results on
the clinical specimens gave a positive predictive value of >82% regarding
response to platinum/paclitaxel therapy.
Results: Prediction profile for the resistance against each chemotherapy
agent was constructed, containing 42-297 genes. The overall accuracy of the predictions in a leave-one-out cross validation was 86%. A list of the top 67 MDR
candidate genes associated with resistance against at least 4 anticancer agents
was identified. Moreover, the differential expressions of 46 selected genes were
also measured by quantitative RT-PCR using a TaqMan micro fluidic card system. Association with resistance was detected for 76 genes.
Conclusion: The gene expression profiling thus can be helpful for the pretreatment assessment of anti-cancer therapy of ovarian carcinomas and others.
Although clinical studies still have to be conducted, it might become possible to
discriminate between responders and non-responders prior to therapy allowing an
individualized strategy with a personalized combination of drugs.
S5-8
FUNCTIONAL EXPRESSION OF ION CHANNELS IN CANCER:
A NEW POSSIBILITY IN CLINICAL MANAGEMENT OF
OVARIAN CANCER?
Mustafa B. A. Djamgoz
Imperial College London, UK
A ‘neuroscience’ approach to understanding the pathophysiology of cancer
has revealed that acquisition of metastatic potential in several carcinomas,
including cancers of prostate, breast and lung, involves up-regulation of functional voltage-gated sodium channels. Primary tumourigenesis has been associated with potassium channels. More recently, ion channels have also been studied in human ovarian cancer and several channels have been detected in vitro
and in vivo. Most work in ovarian cancer has been done on potassium channels.
In particular, voltage-gated potassium channels (VGPCs) have been associated
with cell cycle progression, as in the other carcinomas studied. A general blocker
of VGPCs, 4-aminopyridine, inhibited proliferation of A2780 cells. Rather surprisingly, NS1619, an ‘opener’ of calcium-activated potassium channels (CAPCs)
also inhibited proliferation and induced apoptosis. These results would suggest
(i) that activities of VGPCs and CAPCs (members of the same gene family)
could control ovarian cancer cell proliferation in opposite ways and/or (ii) that
any disruption (increase or decrease) of potassium channels can inhibit the concerted activity within the cell cycle. Inhibitors of potassium channels gated
(closed) by ATP had no effect. The chloride channel blocker NPBB also inhibited proliferation and arrested the cell cycle in S-phase. Finally, aquaporins
(water channels) have also been found to be expressed (upregulated) in ovarian
cancer and could play a role in angiogenesis and ascites formation. Importantly,
ion channel expression generally is controlled by growth factors, especially epidermal and nerve growth factors, which have both been suggested to be involved
in ovarian cancer. Thus, membrane ion channel expression can make a significant contribution to ovarian cancer development/progression and may be controlled by the same mechanisms that drive the overall cancer process.
S5-9
ROLE OF CATUMAXOMAB (ANTI-EpCAM AND ANTI-CD3) IN
OVARIAN CANCER
Radoslav Chekerov, Gulten Oskay-Ozcelik, Werner Lichtenegger,
and Jalid Sehouli
Charité-Campus Virchow-Klinikum, European Competence Center for
Ovarian Cancer, University of Berlin
Ovarian cancer is the most common causes of cancer death in gynaecological malignancies. About 70% of patients are diagnosed with widespread tumor
dissemination into the peritoneal cavity, which associate with malignant ascites
and results in a poor clinical outcome. Ovarian cancer patients presents in
nearly 90% overexpression of the epithelial cell adhesion molecule (EpCAM)
on tumor cells in the ascites fluid. The targeting of epithelial tumor cells within
the peritoneal cavity using EpCAM-specific antibodies is a promising therapeutic approach for patients with malignant ascites. Catumaxomab (anti-EpCAM x
anti-CD3) is a trifunctional monoclonal antibody (trMAb) presenting simultaneous dual antigen binding specificities to the epithelial cell adhesion molecule
(EpCAM) on tumor cells, to the CD3-antigen on T cells and binds selectively
293
to Fcgamma-receptor-positive accessory cells. Catumaxomab is able to induce
a simultaneous activation of different immune cell types at the tumor site,
which results in an effective tumor cells destruction, where different mechanisms, i.e., perforin-mediated lysis, antibody-mediated phagocytosis, and cytokine release are involved. Additionally, results from two mouse models immunocompetent assays indicate a possible role in the generation of a long-lasting
humoral and cellular antitumor response. Following the successful completion
of a phase II/III clinical trial in patients with malignant ascites catumaxomab
(anti-EpCAM x anti-CD3) is approved for the intravenous (i.v.) application and
also for the intraperitoneal (i.p.) treatment of malignant ascites for ovarian cancer, gastric cancer and patients with malignant pleural effusion. The advantage
of the targeted immunotherapy with i.p. application of catumaxomab based on
the direct and specific attack on the ascites-causing tumor cells in the peritoneum. Also most of the effector cells essential for the antitumor activity of
catumaxomab, like T cells or accessory immune cells are located in the peritoneal cavity. The administration of catumaxomab leads to a considerable reduction of ascites production presenting a powerful alternative to conventional
therapies. Drug application schedule of subsequently increasing doses of catumaxomab was found to be safe and feasible for the clinical practice. The most
frequently observed AE’s during treatment were fever, nausea, and vomiting,
probably resulting from a systemic treatment-associated cytokine release as a
consequence of the complex immunoreaction against tumor cells.
A recent study of 258 epithelial tumor patients (recurrent ovarian cancer and
non-ovarian cancer) with EpCAM-positive malignant ascites showed a clinically
relevant prolongation of puncture-free survival and puncture-free time, and a
reduction of ascites symptoms. Currently, a phase I/II study was completed on
catumaxomab administered intraoperatively, immediately after radical surgery to
evaluate the role of this new agent in the primary therapy of advanced ovarian
cancer.
S5-10
CAN WE PREDICT SURGICAL OUTCOME IN ADVANCED
OVARIAN CANCER?
W. Lichtenegger and J. Sehouli
Charité-Campus Virchow-Klinikum, European Competence Center for
Ovarian Cancer, University of Berlin
Surgery is the cornerstone in the clinical management of advanced ovarian
cancer and postoperative residual tumor mass is the most important prognostic
factor. Patients without any macroscopically residuals will have the best clinical
outcome. Various factors have been evaluated to predict optimal surgical results
including serum markers such as CA-125 or CASA and clinical parameters, such
as ascites, age, histological subtype and tumor pattern. For the preoperative evaluation different approaches including radiological methods (eg. ultrasound, CT,
MRI, PET-CT) and laparoscopy are used but non of these are able to predict
optimal surgical results sufficiently. Surgical skills and adequate systemic therapy
have significant impact on long term survival of patients with advanced ovarian
cancer. Various studies indicate that specialist gynaecologists improve survival
for women with advanced ovarian cancer. Newer studies apply modern molecular
biological techniques (eg micro array, proteomics and metabolomics) to identify
specific signatures to predict surgical outcome. Based on the current literature
and own clinical experience these topics will be discussed in detail.
S6. Molecular Mechanisms of Heart Preconditioning
S6-1
SEXUAL DIMORPHISM IN DOCA-SALT INDUCED CARDIO-RENAL
DAMAGE IN MICE
Aysun Karatas
Department of Nephrology and Intensive Care Medicine, Charité Campus
Virchow Clinic, Augustenburger Platz 1, 13353 Berlin, Germany
We tested the hypothesis that female and male mice differ in terms of cardiac hypertrophy following deoxycorticosterone acetate (DOCA) 1 salt hypertension (uninephrectomy and 1% saline in drinking water) and focused on calcineurin signaling. We excluded confounding effects of blood pressure elevation or
294
sex-related blood pressure differences by treating DOCA 1 salt mice with hydralazine (250 mg/L in drinking water). We found that directly measured mean
arterial blood pressure was lowered to control values with hydralazine and corroborated this finding in separate mouse groups with radiotelemetry. Male mice
were more responsive to DOCA 1 salt-related effects. They developed more left
ventricular hypertrophy and more renal hypertrophy after 6 weeks of DOCA 1
salt 1 hydralazine, compared to female mice. In hearts, transcripts for calcineurin Ab and for myocyte-enriched calcineurin interacting protein 1 (MCIP1)
were up-regulated in male, but not in female mice. Enhanced activity of calcineurin Ab, as indicated by diminished phosphorylation of NFATc2 in male
mice, accounted for this sex-specific difference. Stretch-related, inflammatory
and pro-fibrotic responses were also accentuated in male mice, as shown by
higher transcript levels of atrial natriuretic peptide, monocyte chemoattractant
protein-1, and transforming growth factor-b. Our results support sex-specific regulation of calcineurin pathway in response to largely blood pressure-independent
mineralocorticoid action. We suggest that sex-specific calcineurin activation
determines the maladaptive cardiac and renal hypertrophic response and accompanying organ injury in male mice.
study we investigated sex and age related differences and the effect of dietary
administration of phytoestrogens to male and female mice on the heart and kidney proteome utilizing two-dimensional SDS-PAGE and mass spectrometry. The
differential proteome analysis of murine hearts showed a higher influence of dietary phytoestrogen intake for male than female animals in all age groups, resulting in a higher number of differential protein amounts. Sex related comparisons
showed an enhanced quantity of varying protein levels for males versus females
when receiving phytoestrogens in the age of 5 and 13 weeks and the castrated
animals but not for the 24 weeks old animals. For the five and 13 weeks old animals a total number of differentially expressed proteins of 71 and 222 have been
found respectively. Around 80% of the proteins have been successfully identified
by nanoLC-ESI-MS/MS, whereas most of them have been categorized to be
involved in metabolic processes. For the 24 weeks and gonadectomised animals
230 and 121 protein spots showed differential abundance, the MS based identification is in progress. The identified differences in the kidney proteome within
the 5 and 13 weeks groups of animals revealed a predominance of proteins
involved in energy, amino acid and nucleotide metabolisms. In addition to investigations of the total soluble proteome of murine heart and kidney another aspect
of the current project is the selection and analysis of phosphoproteins within the
kidney proteome.
S6-2
ENDOTHELIN-1 AND NITRIC OXIDE IN CARDIOVASCULAR
DISEASE: A PROTEOMIC APPROACH
S6-4
Franz Theuring, Nicolas Vignon-Zellweger, Karima Schwab,
and Berthold Hocher
CCR Institute of Pharmacology, Germany
IMMUNE MECHANISMS OF ALLOGRAFT REJECTION IN CARDIAC
TRANSPLANTATION
The fundamental role of the endothelium in controlling blood pressure has
been brought to light in the mid 1980s with the discovery of Endothelin-1 (ET1) and Nitric Oxide (NO). These both compounds are produced by endothelial
cells, have very strong vasoconstrictor and vasodilator properties, respectively,
and belong to a complex regulatory system inside the vessel wall. ET-1 has also
mitogenic, proinflammatory and fibrotic effects. NO has plethoric protective
effects by relaxing vascular smooth muscle cells (VSMC), inhibiting platelet adhesion and VSMC proliferation. The generation of ET-1 is inhibited by NO
when the activation of endothelial endothelin receptors activates the endothelial
NO synthase. ET-1 overexpressing (ET1/1) mice are normotensive but ET1/1
mice lacking the endothelial NO Synthase (ET1/1eNOS-/-) are hypertensive,
and even more than eNOS-/- mice. This means that a chronic overexpression of
ET-1 alone is not able to promote hypertension but that it is rather an imbalance
between ET-1 and NO expression and activity, which is responsible for the elevation of blood pressure. Furthermore, an activated ET system and a concomitant
reduction of NO production have been observed in many cardiovascular diseases
such as pulmonary hypertension, diabetic nephropathy, congestive heart failure
or preeclampsia. Meanwhile, some powerful treatments, such as endothelin antagonist and NO donor, have been developed. Interestingly, targeting pharmacologically one of these systems affects the other. The underlying mechanisms controlling these observations are not clear though and the real clinical relevance of
the delicate interplay between ET-1 and NO remains controversy. These questions will be, at least in part, answered in our group by the current analysis of
the ET1/1 and ET1/1eNOS-/- mice using the hypothesis free technique proteomics, and by development of new active compounds in clinical research.
S6-3
INVESTIGATIONS OF SEX, AGE AND DIETARY PHYTOESTROGEN
RELATED DIFFERENTIAL PROTEOME PATTERNS OF MURINE
HEART AND KIDNEY
Karima Schwab1, Boris Neumann2, Nicolas Vignon-Zellweger1,
and Christian Scheler2, and Franz Theuring1
1
CCR Institute of Pharmacology, Germany, 2Proeome Factory, Institute of
Protein Chemistry Germany
Cardiovascular disease is one of the most frequent causes of mortality both
for men and women, but have greater prevalence in men compared with premenopausal women. The sharp increase of the cardiovascular risk in women after
menopause and beneficial effects of estrogen submission both in human and
rodents fortify the perception of protective actions of female hormones. In this
Malek Kamoun
Pathology and Laboratory Medicine, University of Pennsylvania, PA,
United States
Multiple factors acting synergistically contribute to chronic organ damage in
heart transplant recipients: 1) alloantigen-dependent factors, 2) innate defense
reaction to tissue damage that is present before transplantation or is a result of
the ischemic injury at the time of transplantation, and 3) non-immunologic factors including viral infections (e.g., CMV). An allograft is rejected as a result of
an immune response directed against major histocompatibility antigens alloantigens expressed on the graft that are absent from the host. Recipient CD41 T
cells are the major cell type responsible for producing the coordinated immune
response to allografts. Interaction of antigen presenting cells with CD4 cells is
regulated by a number of activating and inhibitory costimulatory molecules that
belong to the B7 family molecules. Experimental and clinical studies demonstrated an important role for thymic-derived naturally occurring CD251, CD41,
Foxp31 T regulatory cells (Tregs) that suppress immune responses in various
systems including transplantation. Clinically, "operational tolerance" may be due
to a maintained phenomenon of natural tolerance that is lacking in patients with
chronic rejection. Prior sensitization and/or de novo donor-anti-HLA antibodies
warn of a poor prognosis for allograft survival, even in the presence of chronic
T cell specific immunosuppression. Memory alloreactive B cells and long-lived
plasma cells producing anti-HLA antibodies persist years after the sensitizing
event. The endothelium is thought to be the predominant target of humoral injury
in antibody-mediated rejection (AMR). Long-term survival of allografted hearts
is limited by a progressive fibroproliferative disease, known as accelerated transplant coronary artery disease (TCAD) or cardiac graft vasculopathy. The evolution of TCAD involves AMR, cell-mediated events, and tissue remodeling which
is dependent on cytokines and growth factors. Damage to donor endothelium is
thought to be an important initiating factor in this disease; its effect is in part
mediated by exposing a thrombogenic subendothelial matrix.
S6-5
CARDIAC STEM CELL APPLICATIONS
Selim Isbir
Marmara University, Faculty of Medicine, Department of Cardiovascular
Surgery, Istanbul, Turkey
The most important basis of heart failure is the damage of cardiomyocytes
and the lack of repair mechanisms for them. Fibrosis occurs after the damage to
the cardiomyocytes causing dysfunctional ventricules. The most efficient treatment of advanced cardiac heart failure is heart transplantation, but it has the dis-
advantage of donor problems. Stem cells, differentiating into cardiomyocytes and
endothelial cells, are thought to be an alternative option. However, there are still
several problems related to cardiac stem cell transplantation such as the confusions in deciding the cell types, the necessities about the differentiation status of
the cells before and after transplantation and in the procedures of the operation.
295
main components of a heart substitute starting with pericardium, vascular grafts
and heart valves towards the production of contractile myocardial patch structures.
The vision of complete heart substitutes comes closer. Nevertheless, there still
exists an extensive need of research activities on all levels of tissue engineering.
The presentation overview the current state of cardiovascular tissue engineering
towards the development of heart substitutes and the limitations which have to be
overcome to bridge the rift from bench to bedside.
S6-6
CLINICAL IMPACT AND BIOMATERIAL EVALUATION OF BONE
MARROW ASPIRATE AND PLATELET GEL IN CARDIAC SURGERY
Serdar Gunaydin
Department of Cardiovascular Surgery, University of Kirikkale, Turkey
Perioperative bleeding leads to increased operating room time, blood product
transfusions, pulmonary hypertension, and potentially to mortality. The coagulopathy induced by cardiopulmonary bypass and the multiple high pressure anastomoses created during cardiac surgery often result in bleeding, which is more
effectively controlled with autologous platelet gel (APG) and topical hemostatic
agents than with sutures or electrocoagulation. Several topical hemostatic agents
are available which provide either clotting components (e.g. fibrin sealants and
thrombin glues) or a surface for clotting to be stimulated (e.g. microfibrillar collagen, gelatin sponge, oxidized cellulose). The main advantage of a topical sealant is its tensile strength, which helps in cessation of hemorrhage. Platelet
derived glues are somehow complementary since they have a poor tensile
strength but promote tissue regeneration due to their content of growth factors.
In addition, sealants have varying degrees of efficacy, some carrying a potential
risk of infection or allergic reaction, adhesion formation, prolonged preparation
time and considerable expense. APG was developed in the early 1990s as a
byproduct of platelet-rich plasma sequestration during cardiac surgery. APG has
been approved for postoperative healing. However, there have been few studies
that evaluate the hemostatic effects and biomaterial aspects of APG in open-heart
surgery. We have published comparative clinical trials to document the efficacy
and safety of clinical regimens of APG and several hemostatic agents. Patient’s
own blood and bone marrow aspirate were employed for wound healing or stopping bleeding. We have also harvested endothelial cell (EC) culture and evaluated agents on biomaterial aspect with respect to cytotoxicity and foreign body
reaction, effects on wound healing, inflammatory reaction-resistance to infection
and resorption-clearance time from the tissue. Our pilot work is a start to induce
following clinical trials required to study the potential of the use of APG and to
provide material for sound clinical decision-making in the near future.
S6-8
ACUTE CORONARY SYNDROME
Zehra Bugra
Istanbul University, Istanbul Medical Faculty, Department of Cardiology,
Capa, Istanbul, Turkey
Acute chest pain is one of the most common reasons for presentation to the
Emergency Department. The most serious cause of acute chest pain is myocardial
ischemia or myocardial infarction which occurs when the myocardial oxygen supply is inadequate compared to myocardial oxygen demand. Myocardial ischemia
usually occurs in the setting of coronary atherosclerosis. Acute chest pain suggests
an acute coronary syndrome only 15 to 25 percent of patients after diagnostic evaluation. An acute coronary syndrome develops when the vulnerable or high-risk
atherosclerotic plaque undergoes disruption of the fibrous cap and plaque rupture
is the stimulus for thrombogenesis. The flow reduction may be caused by a completely or subtotally occlusive thrombus. As a result, the spectrum of clinical presentation ranging from unstable angina pectoris through Non-ST segment elevation myocardial infarction and ST segment elevation myocardial infarction are
referred to as the acute coronary syndromes. Of patients with ST segment elevation on the ECG, most develop a Q-wave myocardial infarction, whereas a few develop a non-Q wave myocardial infarction and all present with cardiac markers
such as CK-MB or a cardiac Troponin (Troponin T and I) detected in the blood.
Patients who present without ST segment elevation are suffering from either unstable angina pectoris or a non-ST segment elevation myocardial infarction, and a
distinction is made on the presence or absence of a serum cardiac marker in the
blood. Patients presenting with persistent ST-segment elevation are candidates for
reperfusion therapy (either pharmacological or catheter based) to restore flow in
the occluded epicardial infarct-related artery. Acute coronary syndrome patients
presenting without ST-segment elevation should receive antiischemic therapy followed by catheter based coronary intervention. All patients with acute coronary
syndrome should receive antithrombin and antiplatelet therapy regardless of the
presence or absence of ST-segment elevation.
S6-7
S7. Molecular Markers in Tumor Diagnosis and Therapy
FROM CELL TO HEART SUBSTITUTES: THE ENGINEERED
HEART - FUTURE AND FICTION
S7-1
Stefan Jockenhoevel
Department of Applied Medical Engineering, Helmholtz Institute for
Biomedical Engineering, RWTH Aachen University, Aachen, Germany
ENVIRONMENTAL AND DIETARY FACTORS ON CANCER WITH
REGARD TO CYP450
Cardiovascular tissue engineering is a young discipline, which has grown
rapidly during the last decades. The aim of tissue engineering is to develop a
viable implant or organ substitute with the mechanisms of remodeling and selfrepair and a complete immunological integrity. Tissue engineering approaches to
the construction of indeed any tissue or organ rely on three essential components:
cells, which will ultimately form the new tissue; scaffolds, designed to maintain
the cells in a three-dimensional environment at the implantation site, and signals
that guide the gene expression and ECM production of the cells during tissue development. The production process begins with the harvesting of suitable cell
sources which will ultimately control the development and function of the tissue.
The cells are cultured after harvesting, expanded exponentially to obtain sufficient
cell numbers, and are subsequently seeded onto a supporting scaffold material.
These ‘simple’ steps have already a high impact on the fate of the later tissue engineered substitutes. The cultivation of the tissue-engineered construct following the
seeding process is an important step towards a mechanical stable tissue. Beneath
the biomolecular signalling, a number of different studies have demonstrated the
benefit of dynamic cultivation in bioreactor systems for the development the
extracellular matrix, which is mainly responsible for the mechanical properties of
the neo-tissue. Currently several groups have demonstrated the production of the
Alaattin Sen
Biology Department, Faculty of Arts & Sciences, Pamukkale University,
Kinikli Campus, Kinikli, 20070, Denizli, Turkey
Cytochrome P450 (CYP450) dependent monooxygenases are essential in
conversion of lipophilic xenobiotics into more hydrophilic metabolites, whereby
reducing duration of exposure to xenobiotics and preventing accumulation of the
parent compounds. However, they frequently produce highly reactive intermediary metabolites that can form DNA adducts or cause oxidative stress and pose a
risk for cancer. Local activation of procarcinogens in target tissues by CYP450s
is believed to be an important factor in the etiology of cancer. Therefore, induction or inhibition of these enzymes is one of the biochemical mechanisms by
which diets and environmental factors may alter cancer risk. The members of the
CYP1A subfamily whose expressions were associated with some dietary constituents are involved in metabolism and activation of procarcinogens and other toxic
chemicals. We have conducted various studies to determine the effect of some
diets on hepatic, colonic, pulmonary and nephritic expression of these enzymes.
Our results demonstrated that microsomal CYP1A and associated enzyme activities such as EROD and MROD were significantly reduced by dwarf nettle
(Urtica urens) and/or bitter melon (Momordica charantia) intake in rats. On that
account, substantial reduction in CYP1A expression levels and related activities
296
primary tumors that subsequently metastasized in the mouse model. In the second group, relative to the original patient tumors, 205 genes were found to be
significantly altered in tumors that grew in first human bone implants. 183 of
these were also found in tumors that metastasized to second initially tumor free
human bone implants and in lung metastases. Expression of some of those genes
were validated by qRT-PCR and include; CSF1R which is up regulated and
IGFBP7 that is down regulated. These genes are important in mammary gland
development and/or differentiation and have been implicated in breast cancer
metastasis and associated with poor outcome in breast cancer patients.
Conclusions: By using the ‘‘all human’’ NOD/SCID mouse model, we have
identified many potential targets that are up or down regulated in bone metastatic
breast tumor samples. We anticipate that our study will help in developing breast
cancer biomarkers for patients that are at high risk for bone metastasis and will be
valuable for clinical diagnosis and treatment. ‘‘This work was supported by the
Canadian Breast Cancer Research Alliance special program grant on metastasis’’.
S7-3
METRONOMIC RELEASE OF GEMCITABINE FROM A
POLY-N-ACETYL GLUCOSAMINE (PGLCNAC) GEL INHIBITS
GROWTH OF MURINE PANCREATIC TUMOR CELLS
with diets could be considered as a potential chemoprotective ability of these
diets due to anticipated decrease in the activation of environmental chemical procarcinogens through modulation of the CYP1A enzymes. Association between
consumption of various vegetables and biotransformation enzyme activities in
observational studies will be discussed in detail.
S7-2
MOLECULAR ANALYSIS OF BREAST CANCER METASTASIS TO
BONE IN AN ‘‘ALL HUMAN’’ NOD/SCID MOUSE MODEL
W. Yang, P. Lam, Y. Amemiya, H. Kahn, A. Yee, C. Holloway
and A. Seth
Sunnybrook Research Institute, University of Toronto, Toronto, ON, Canada
Background: Bone is the most common site of metastasis in breast cancer,
affecting up to 80% of women with advanced disease, leading to bone complications or skeletal-related events. Current studies on the bone metastasis process
have been hampered by the lack of preclinical models to evaluate novel therapeutics and to study the biology of the disease.
Methods: To create an ‘‘all human’’ mouse model that explicitly investigate
the bone metastatic behavior of human breast tumors, we first engrafted human
bone fragments into the both flanks of NOD/SCID mice and subsequently transplanted primary human breast tumor under only left flanks of the hu-bone NOD/
SCID mice.
Results: We found that engrafted human bone remains functional for more
than 20 weeks of implantation and that approximately 30 percent of primary
breast tumors survived and generated tumors when placed into hu-bone NOD/
SCID mice. After performing serial re-transplantation experiments using primary
breast tumors, we observed metastasis to the opposite tumor free human bone
fragments and some host tissues including liver, lung and lymph nodes. Interestingly, none of the human breast tumors metastasized to the mouse skeleton, providing evidence that osteo-tropism is essential for bone metastasis. Gene expression profiles using whole genome microarrays were generated from engrafted
patient breast tumors to identify genes that correlate with growth and metastasis.
Two groups of clinically significant genes are emerging from our ongoing
results. One group represents genes associated with the future biological behavior
of the original patient tumors. They are found by comparing gene levels between
patient breast tumors that did and did not metastasize in the hu-bone NOD/SCID
model. A second set of genes associated with the metastatic phenotype, but not
necessarily predictive of that state, are revealed by comparing gene expression
levels between the original patient tumors and the tumors that form in the human
bone implants and mouse tissues. In the first group, relative to the non-metastatic
tumor, 36 genes with significantly altered expression pattern were found in the
John N. Vournakis1, Marina Demcheva1, Carlton Barnett2, Josh Hubbard2,
and Arun Seth3
1
Marine Polymer Technologies, Danvers, Massachusetts, USA, 2Medical
University of South Carolina, Charleston, South Carolina, USA,
3
Sunnybrook Research Institute, Toronto, Canada
Background: There are over 30,000 new cases of pancreatic cancer in the
US annually with a five-year survival rate of 4% and a mean survival time of 6
months following diagnosis. Currently, the primary therapy is surgical removal
of tumor if it is still respectable in about 20% of patients, radiation therapy and
chemotherapeutic treatment with Gemcitabine, a nucleotide analog (deoxycytidine). Most commonly used schedule in clinical practice is 1000mg/m2 intravenously administered weekly for 3 three weeks, followed by a 1 week rest.
Metronomic Dosing: Lower doses of drug, given more frequently, often via
sustained release vehicles. Generally, decreased side effects are observed since
doses do not approach the MTD. The metronmic dosage pproach has been shown
to be effective in prostate and colon cancer.
Hypothesis: Prolonged delivery of Gemcitabine will improve the growth inhibitory effects of Gemcitabine on pancreatic cancer cells in-vivo.
Methods: Gemcitabine is formulated in a poly-N-acetyl glucosamine polymer derived from a marine microalga which has been modified chemically to be
non-immunogenic. In-vitro studies with Panc02 tumor cells are performed with
gel-Gem combinations and MTT proliferation assays are measured. Mice are
injected with Panc02 tumor cells and are treated with gel-Gem. Tumor volumes
are measured as a function of time.
Conclusions: Poly-N-acetyl glucoamine gel is able to deliver Gemcitabine
over a prolonged period. Prolonged dosages of Gemcitabine decrease mPanc02
tumor cell growth in vitro. Systemic and local treatments of mPAnc02 tumors in
vivo are improved by metronomic dosing of Gemcitabine.
S7-4
DNA REPAIR AND SIGNALING IN ALKYLATING
DRUG-INDUCED APOPTOSIS: IMPLICATIONS FOR GLIOMA
AND MELANOMA THERAPY
Bernd Kaina, Luis Z. Batista, Steffen Naumann, Michael Goldstein,
Markus Eich, and Wynand P. Roos
Department of Toxicology, University of Mainz, Obere Zahlbacher Str. 67,
D-55131 Mainz, Germany
Alkylating agents are powerful mutagens and carcinogens. Moreover, methylating and chloroethylating agents are being used in first line therapy of gliomas
and malignant melanomas, and second-line therapy of different other cancers.
These agents induce a dozen of DNA lesions, some of them have been identified
to be carcinogenic, genotoxic and cytotoxic. In MGMT lacking cells O6-methylguanine (O6MeG) and O6-chloroethylguanine (O6ChlG) are the major trigger of
genotoxicity and apoptosis. In the case of O6MeG lesions, this requires MSH2/
MSH6 dependent mismatch repair. O6MeG induces mainly apoptosis, whereas
necrosis is only marginally induced. In contrast, O6ChlG is able to induce both
apoptosis and necrosis. O6MeG triggered cell death is regulated in a cell type
specific manner, utilizing both the mitochondrial damage and the death receptor
pathway. In lymphocytes, O6MeG triggers apoptosis at a low dose range of
methylating agent in proliferating cells only. In contrast, ionizing radiation is
able to induce apoptosis at high frequency in resting lymphocytes. This demonstrates that DNA replication, which is absolutely essential for O6MeG triggered
apoptosis, is not required for apoptosis induced by various other DNA damaging
agents. O6MeG triggered apoptosis is preceded by DNA double-strand break
(DSB) formation, H2AX phosphorylation and ATM/ATR activation. In turn, in
p53 wt expressing glioma cells the Fas/CD95/Apo-1 receptor pathway becomes
activated, whereas in p53 mutated glioma cells Bcl-2 declines, which is followed
by caspase-9, -7 and -3 activation. The efficiency of O6MeG to trigger the p53
dependent Fas pathway is much higher than the p53 independent endogenous mitochondrial pathway, which explains why p53 wt glioma cells are more sensitive
to methylating agents (temozolomide) than p53 mutated cells. Interestingly, p53
wt glioma cells are more resistant than p53 mutant glioma cells to chloroethylating agents, such as BCNU and ACNU, indicating p53 to protect against
O6ChlG-induced apoptosis and necrosis. O6MeG and O6ClG are also able to
induce apoptosis in malignant melanoma cells, which is accompanied by the formation of DSBs. Cells defective in XRCC2 or BRCA-2 are hypersensitive to
O6MeG triggered cell death and chromosomal aberrations while DNA-PKCS
mutated cells display only slightly enhanced sensitivity. The data supports a role
for DSBs as most critical downstream apoptotic lesions and DSB repair by homologous recombination as a new determinator of cellular resistance to monofunctional alkylating agents. Data will also be presented on apoptosis induction
by the cyclophosphamide analogue mafosfamide, and the role of replication and
transcription inhibition in triggering apoptosis will be discussed. Work was supported by DFG KA724/13-1,2 and 13-2.
S7-5
DNA POLYMORPHISMS AND GENETIC PREDISPOSITION FOR
ORAL CANCER
Christos Yapijakis1, Friedreich W. Neukam2, and Eleftherios Vairaktaris1
1
Oral and Maxillofacial Surgery, University of Athens Medical School,
Greece, 2Oral and Maxillofacial Surgery University of Erlangen, Germany
Introduction: In recent years, genetic association studies have provided evidence that inherited functional DNA polymorphisms in genes of factors related
to angiogenesis, inflammation and thrombosis are associated with increased risk
for oral squamous cell carcinoma (OSCC). Here we present the combinatory
effect of 31 such gene polymorphisms in predicting the occurrence of OSCC in
Europeans using multivariate logistic regression analysis.
Material and Methods: A total of 330 individuals of Greek and German origin were studied, consisting of 162 OSCC cases and 168 healthy controls of
comparable age, gender, and ethnicity. DNA was isolated from blood samples of
studied individuals. Functional DNA polymorphisms were investigated by allele
specific PCR or PCR/RFLP methodology in genes that encode: a) cytokines and
their receptors (IL1b, IL-4, IL-6, IL-8, IL-10, IL–18, TNF-a, TNF-b, VEGF,
Leptin, Leptin Receptor), b) matrix metalloproteinases and their inhibitors
(MMP-1,-3,-7,-9,-13, TIMP-2, c) platelet glycoproteins and coagulation factors
(GPIa, GPIba,PAI-1, AGT, ACE, TAFI, Thrombomodulin, Protein Z, SDF1,Factor II, Factor V, Factor XII, Factor XIII, and MTHFR). A series of regression
models (adjusted for age and gender) was constructed in order to assess the contribution of homozygous or heterozygous variant polymorphic genotypes upon
overall, early and advanced stages of OSCC development.
Results: In almost all multivariate logistic regression models, the contribution of TNF-a and IL-6 polymorphisms was consistent and robust. Furthermore,
when the mode of inheritance of each variant allele was taken into account in a
model, five polymorphisms emerged as primary predictors for all OSCC stages:
TIMP-2 (OR526.33; 95% CI512.39–55.95), TNF-a (OR515.27; 95%
CI57.30–31.96), IL-6 (OR58.33; 95% CI53.95–17.58), IL-8 (OR53.54; 95%
CI51.69–7.43) and IL-10 (OR52.65; 95% CI51.28–5.46).
Conclusions: The present regression analysis revealed a highly significant contribution of 5 out of 31 studied factors in the occurrence of OSCC. Based on these
findings and previous reports, possible interactions of the implicated factors leading
to OSCC development, as well as an algorithm of risk estimation will be discussed.
297
S7-6
CONTRASTING MAMMARY EPITHELIAL BRANCHING
MORPHOGENESIS AND METASTASIS USING MOUSE
GENETIC MODELS
Demetri Spyropoulos
Department of Pathology and Laboratory Medicine, Medical University of
South Carolina, United States
With the onset of puberty, mammary epithelial branching morphogenesis is
transformed from simple allometric growth to a highly proliferative and highly
expansive process involving extensive extracellular matrix remodeling and stromal invasion. It is therefore not surprising that similarities are often drawn
between branching morphogenesis and the metastatic spread of breast cancer
cells. One key difference is that branching morphogenesis normally remains limited to the architectural boundaries set in the fat pad. Understanding the molecular mechanisms underlying these differences hold promise for therapies, which
limit breast cancer metastases. Genetically and hormonally manipulated in vivo
mouse models (HoxC gene targeted disruptions, mammary epithelial-specific inducible HoxC genes, MMTV-rtTA TRE-HoxC-IRES-EGFP, and constitutive
breast cancer models, MMTV-cNeu) and in vitro human breast cancer cell lines
(MCF10A, MCF7 and MDA-MB-231) were used to assess impacts on mammary
epithelial growth, differentiation and expression of HoxC and direct target genes.
Cell lines were manipulated using HoxC6 shRNA and cDNA lentivirus and activated Akt isoform retroviral expression vectors. Gene expression and signaling
were measured by quantitative RT-qPCR, IHC, Western blot and the BioPlex200
Multiplex System. HoxC5 & C6 are expressed in mammary epithelium and
stroma and therein differentially repressed by ovarian hormones. This repression
can be modulated in vivo by phytoestrogen exposure. Thoracic mammary glands
fail to develop postnatally in corresponding knockout mice. Embryonic and neonatal induction of epithelial-specific HoxC6 expression in transgenic mice leads
to precocious terminal end bud formation, suggesting distinct cell-autonomous
and non-autonomous functions. Epithelial-stromal reconstitution experiments
(surgical and genetic) show that epithelial-specific, activated Her2/Neu expression is sufficient to rescue ductal elongation defects in the knockout, but that
stromal Hox gene expression is required for ductal side-branching. In contrast,
Her2/Neu-derived mammary tumor metastasis is accelerated in Hox-defective
mammary glands. These results will be discussed in the context of changes in
epithelial and stromal compartments (cellular phenotypes and genetic signatures).
Molecular models will be presented to account for the results presented, especially as they pertain to therapeutic modalities.
S7-7
MOLECULAR CHARACTERIZATION OF GLIOBLASTOMA
MULTIFORME AND PERITUMOR TISSUE
Gigliola Sica
Institute of Histology and Embryology, Faculty of Medicine,
Catholic University of the Sacred Heart, Rome, Italy
Objective: Molecular characterization of glioblastoma multiforme (GBM)
and its surroundings may have relevant clinical implications due to the peculiar
growth pattern of the neoplasia. With the intention to clarify the role played by
peritumor tissue in GBM progression, we evaluated a series of markers (ERK1/2,
JNK, nestin and CD105) in GBM and in tissue located at a distance <1cm up to
3.5 cm from the tumor border.
Methods: Marker expression has been studied by immunohistochemistry
using a series of monoclonal and polyclonal antibodies. To quantify the degree
of neoangiogenesis, both nestin and CD105 microvessel density (MVD) has been
determined.
Results: Our findings show that activated ERK1/2 and JNK were expressed
both in the tumor and in the peritumor tissue with no statistically significant difference. The immunoreactivity was present not only in neoplastic cells but in reactive astrocytes and in apparently normal glial cells also. Nestin was strongly
expressed in GBM and less present in peritumor tissue, but its localization was
identical to that of MAP kinases. All the markers decorated the endothelium that
did express CD105. No statistically significant difference was found in nestin- or
CD105-MVD in peritumor tissue depending on the distance from the tumor mar-
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gin or on the presence of tumor cells, indicating that in this tissue there is an endothelial activation linked to neoangiogenesis and an increase in blood supply,
critical to tumor progression. The presence of stem-like cells in the peritumor tissue might be linked to a migration from the tumor or to the induction of a premalignant state in apparently normal glial cells.
Conclusions: All these data suggest that peritumor areas, apart from the
alterations induced by the space demanding growth of the tumor and the vasculogenic edema, show signs of transformation which can be mainly due to factors
produced by the tumor mass.
S7-8
metastasis. Upregulation of VGSC mRNA and protein have also been demonstrated in human BCa biopsies, and expression in primary tumour has been correlated with lymph node metastasis in vivo. VGSC expression in vitro is controlled by both estrogen and epidermal growth factor, and its biology is consistent with expression occurring early in progression to metastasis. The
predominant channel in metastatic BCa, in vitro and in vivo, is Nav1.5, in its
newly discovered neonatal splice form, i.e. this is an epigenetic oncofoetal phenomenon. Thus, firstly, functional VGSC expression can serve as a novel prognostic marker of metastatic disease. Secondly, since neonatal Nav1.5 differs from
the adult variant by several amino acids in an extracellular part of the protein
and can readily be approached by antibody and/or small-molecule drugs, it also
represents a novel therapeutic target for controlling metastatic BCa.
ISOLATION AND CHARACTERIZATION OF CANCER STEM CELLS
FROM HUMAN GLIOMA CELL LINES
S7-10
Silvia Ferracuti, Cristiana Angelucci, Fortunata Iacopino, and Gigliola Sica
Faculty of Medicine, Catholic University of the Sacred Heart, Institute of
Histology and Embryology, Italy
THE ROLE OF MICRORNA (miRNA)s AS A NOVEL TOOL IN
CANCER DIAGNOSIS AND THERAPY
Objective: Cancer stem cells (CSC) are thought to be responsible for initiation and progression of most human tumors, including gliomas. Established cell
lines may represent a more useful tool than tissue samples to isolate CSC, avoiding the risk of normal stem cell contaminations. The few data present in literature mostly concern rat/murine models. With the aim of gaining a deeper insight
into the glioma biology, we isolated and initially characterized CSC from three
human established glioma cell lines (U373-MG, U87-MG, LI) with increasing
malignancy grade.
Methods: Cells were cultured in serum-free neural stem cell medium to
obtain primary neurospheres (NS). Limiting dilution analysis and clonal assay
were performed to evaluate the frequency of tumor spheres-initiating cells (TSICs) and the self-renewal capability of NS, respectively. At present, the expression of the neural stem cell marker Nestin was evaluated by immunocytochemistry on LI parental cells and cryosections of clonal assay-obtained secondary NS.
Results: Primary NS were derived from the parental cell lines maintained in
the above-mentioned medium. Frequency of TS-ICs was 2.74% for LI cells,
1.56% for U373-MG cells and 1.45% for U87-MG cells. LI cells showed the
highest potential for clone formation (33.3%); compared to the other two cell
lines (9.5% for U373-MG cells; 10.5% for U87-MG cells). The percentage of
Nestin-immunoreactive cells in LI parental cell line and secondary NS were 57%
and 88.7%, respectively.
Conclusions: Our results demonstrate that long-term established human glioma cell lines, with increasing degree of malignancy, harbor a rare subset of
CSC. In particular, isolated CSC from LI glioblastoma cell line, which has never
been previously described, showed self-renewal properties in accordance with
their high grade of malignancy and Nestin immunoreactivity. Work is in progress
to evaluate the expression of other currently studied stemness markers in these
CSC.
Abdullah Uzumcu1, Murat Tekguc1, Serhat Sevli1, O.F. Bayrak1,
and Mustafa Ozen1,2
1
Department of Medical Genetics, Yeditepe University Medical School and
Yeditepe University Hospital System, Istanbul, Turkey; 2Department of
Pathology, Baylor College of Medicine, Houston, TX 77030
miRNAs are non-coding single-stranded RNAs comprised of 20-22 nucleotides and considered as a novel class of gene regulators. It has been suggested
that miRNA expression correlates with classification of various cancers. About
half of the annotated human miRNAs are located in fragile sites of the genome
suggesting that these small molecules might have a vital function in cancer progression. miRNAs can regulate gene expression transcriptionally or translationally. There is an emerging evidence that miRNAs are involved in cancer pathogenesis. A number of studies have detected frequent alterations of miRNA
expression in a variety of human malignancies including prostate cancer. We
have recently demonstrated a widespread deregulation of miRNA expression in
human prostate cancer. The status of the selected miRNAs, in human prostate
cancer cell lines DU-145, LNCaP, LAPC4, PC3 and prostate cancer specimens
have been analyzed. We have transfected these cell lines with precursor miRand anti miR- molecules and compared the gene expression profiles. Data analysis is carried out by the help of Agilent Gene Spring GX software package. We
have also compared the targets for these selected miRNAs identified biologically
in prostate cancer cells and in three different databases. The expression status of
select-targets have been verified and the biological effects of these targets on
prostate cancer cell lines are studied. The results obtained in these studies demonstrate that microRNA(s) can be used to find new markers for prostate cancer
progression and to define promising targets for prostate cancer therapy.
S7-9
S7-11
VOLTAGE-GATED SODIUM CHANNEL UPREGULATION AS A
NOVEL MARKER OF METASTATIC DISEASE: THE CASE FOR
HUMAN BREAST CANCER
DISRUPTED FUNCTION OF PROSTATIC ACID PHOSPHATASE
LEADS TO DISEASES
Mustafa B. A. Djamgoz
Imperial College, London, UK
Cell membrane potential gradients, of the order of 10 million volts/metre,
and associated ion channels, can have profound effects upon cellular activities
but have not been studied extensively in cancer cells. Electrophysiological
recordings from cell lines of several human carcinomas - prostate cancer, breast
cancer (BCa), lung cancers (small-cell, non-small-cell and mesothelioma), cervical cancer and melanoma - have shown that strongly metastatic cells express
functional voltage-gated sodium channels (VGSCs). Where studied, this was
found to be accompanied by downregulation of voltage-gated potassium channels. Accordingly, metastatic cell membranes are electrically excitable and,
indeed, these cells generate regenerative activity (action potentials) when stimulated with small depolarizing stimuli. Treatment with the highly specific tetrodotoxin (TTX), siRNA or polyclonal antibody suppresses the cells’ directional motility and invasiveness in vitro, suggesting that VGSC activity could potentiate
Mark Zylka1, Nathaniel A. Sowa1, Bonnie Taylor-Blake1,
Margaret A. Twomey1, Annakaisa Herrala2, Vootele Voikar3,
and Pirkko Vihko2
1
Department of Cell and Molecular Physiology, UNC Neuroscience Center,
University of North Carolina, USA, 2Department of Biological and
Environmental Sciences, Division of Biochemistry, University of Helsinki,
Finland, 3Neuroscience Center, University of Helsinki, Finland
Our recent finding, a type I transmembrane (TM) isoform of Prostatic Acid
Phosphatase (PAP, EC 3.1.3.2.), is 5’-ectonucleotidase and produces adenosine,
and functions in vivo in neurons via adenosine A1-receptor, which is inhibitory
(Gi) GPCR. TM-PAP encodes the classic histochemical marker of small-diameter
dorsal root ganglia neurons known as thiamine monophosphatase/fluoride-resistant acid phosphatase. PAP knockout in mice leads to inflammatory and neuropathic pain perception. In nerve injury PAP expression is decreased. PAP suppresses pain eight times as effective as morphine. In addition, to dorsal root ganglia neurons PAP is widely expressed i.e. in prostate, brain, neutrophils. PAP-
KO mice have also prostate cancer and anxiety. Molecular mechanisms of PAP
function will be described in this presentation.
S7-12
GENES PREDICTING OUTCOME IN OVARIAN CARCINOMA
L. Rojas-Espaillat, A. Nickles-Fader, N. Rasool, S. Vaziri,
T. Kozuki, P. Elson, M. K. Ganapathi, and R. Ganapathi
Clinical Pharmacology, Taussig Cancer Center, R40 (Room R4-041),
Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland,
Ohio 44195, USA
Objective: The high incidence and mortality of ovarian cancer coupled with
the difficulty in detecting the disease in the early stages poses a major clinical
challenge. We sought to determine novel genes involved in early relapse to first
line treatment.
Methods: Gene expression profiling studies of stage III ovarian or peritoneal
carcinoma was carried out to identify genes associated with treatment outcome.
The functional role of the identified genes in predicting chemosensitivity was
tested in ovarian cancer cell lines.
Results: We identified three genes that were independent predictors of
response to chemotherapy and clinical outcome. Two genes, cisplatin resistanceassociated over-expressed protein (CROP) and proteosome subunit alpha type 3
(PSMA3) were found to be significantly up-regulated in ovarian tumors of
patients who underwent primary surgical cytoreduction and responded poorly to
first line therapy, whereas the third gene, chemokine C-C motif ligand 2 (CCL2),
was significantly down-regulated in this same patient cohort. Reduced expression
of CCL2 in ovarian cancer cell lines was correlated with resistance to cisplatin
and/or paclitaxel. Ectopic overexpression of CCL2 in an ovarian cancer cell line
led to enhanced sensitivity to paclitaxel but not cisplatin and also reduced tumor
cell invasion. Overexpression of CROP and PSMA3 was correlated with resistance to cisplatin in several cell culture models of ovarian cancer. Cisplatin treatment in a dose dependent manner led to 2 to 3-fold increase in accumulation of
cells in G21M phase (indicative of enhanced DNA damage) in CROP siRNA
transfected cells in which CROP mRNA was down-regulated ~70% compared to
cells transfected with a scrambled siRNA.
Conclusions: In the long term, establishing the functional role of CCL2,
CROP or PSMA3 as predictors of response to chemotherapy and outcome in
patients with advanced stage ovarian cancer could allow for the development of
novel strategies for detection and treatment.
S7-13
FROM INCEPTION TO MARKETING OF
LIPOSOMALLY-ENCAPSULATED CISPLATIN (LIPOPLATIN):
A SUCCESS STORY IN NANOTECHNOLOGY
Stavros Kottaridis
Member BOD & Clinical Director, Regulon Alimos, Athens,
Mt. View, CA, Zug Switzerland
LipoplatinTM is a liposomal cisplatin, designed to reduce cisplatin toxicities
(nephrotoxicity, neuropathy, myelotoxicity, nausea/vomiting) without reducing efficacy. Its nanoparticles evade immune surveillance, extravasate preferentially into
tumors and metastases and target endothelial cells of tumor vasculature inducing
cell apoptosis and antiangiogenesis. Human studies have shown a 40-200 fold
higher concentration of platinum in tumor specimens after a single infusion compared to adjacent normal tissue, a beak-through in the industry. Lipoplatin is
administered without pre- or post-hydration on an outpatient basis and offers clear
pharmacoeconomic benefits to the patient and to the health insurance system. This
is the initial report of a randomized phase III trial.
Methods: Eligibility criteria include inoperable/metastatic NSCLC, no previous chemotherapy, WHO PS 0-1, adequate end-organ function. Patients receive
Lipoplatin 120 mg/m2 D1,8,15 (Arm A) or cisplatin 100 mg/m2 D1 (Arm B)
with gemcitabine 1 g/m2 D1,8, in 3-week cycles, with disease evaluation after 3
and 6 cycles. Primary endpoints are OS; secondary endpoints are ORR, DCR,
PFS, and toxicity.
Results: Response rates were (Arm A vs Arm B): PR 37% vs 28%, SD 34%
vs 31%, PD 29% vs 41%. ORR was 37 vs 28%, and DCR was 71% vs 59%.
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PFS range is currently 0.7-16.81 vs 0.2-9.81, and duration of response is 2.814.81 vs 2.1-10.41 (months).
Toxicities were (Arm A vs Arm B): anemia I-III (94% vs 100%), leucopenia
III-IV (9% vs 17%), neutropenia III-IV (11% vs 29%), thrombocytopenia III-IV
(9% vs 22%), nephrotoxicity III (0% vs 5%), nausea/vomiting III-IV (2% vs
12%), neurotoxicity II-III (0% vs 7%), asthenia III (4% vs 17%), anorexia III
(2% vs 15%). Additionally, less antiemetics and G-CSF were administered in
Arm A. The only grade IV adverse event in LipoGem was neutropenia in 2% of
patients.
Conclusion: Lipoplatin appears to have lower toxicity, mainly nephrotoxicity, as well as higher efficacy than cisplatin, when combined with gemcitabine in
advanced NSCLC. Particularly relevant is that Lipoplatin is administered without
pre- or post-hydration, on an outpatient basis. Lipoplatin has received the orphan
drug status by EMEA (European FDA) in 2007 against pancreatic cancer. Regulon is recruiting EU oncology centers with a reputation for excellence to finish a
pivotal trial leading to the registration of the drug in all EU countries.
S7-14
FUNCTIONAL TESTING OF TUMOR SUPPRESSOR
CHROMOSOME REGIONS
Stefan Imreh
Karolinska Institute, MTC, Stockholm, Sweden
The biological complexity of neoplasms requires multiple targets in antitumor drug design. One category of the complexity sources are the deletions that
provide selective growth advantage and are found in virtually all carcinomas. It
was a general consensus that recurrent deletions contain gene/s that inhibit tumor
growth: tumor suppressor genes. Loss of heterozygosity (LOH) analyses and the
analysis of infrequent homozygous deletions offered cumbersome and rarely successful routes in tumor suppressor gene identification. We have developed a
functional model system that mimics homozygous deletions experimentally. It is
based on the transfer of single human chromosomes (via microcell mediated
chromosome transfer or microcell fusion) into tumor cells that either belong to
another species or if human contains already a hemyzygous deletion. When the
resultant microcell hybrids generate tumors in immunsuppressed animals the
transferred chromosome is analysed (by cytogenetic and molecular methods) for
overlapping deletions, candidate genes are identified and further tested functionally for any activity that modifies tumor growth. This model system was first
proposed in 1994 (Imreh et al, Genes Chromosomes and Cancer, 11, 237-245).
During the following years it was developed into a system called ‘‘elimination
test’’(Et) that indeed can deliver tumor suppressor genes (see Kost-Alimova M &
Imreh S, Modeling non-random deletions in cancer, Seminars in Cancer Biology,
17, 19-31, 2007, for a recent rev). On human chromosome 3, the Et, gradually
narrowed down a common eliminated region on 3p21.3 (CER1 or according to
the EMBL Nomenclature Committee C3CER1) that contains 33 genes, six of
them discovered and cloned by the group. Seven may be considered as tumor inhibitory gene candidates and we have published results that confirm the suppressor function for 3 of them: LF (Yang et al, Cancer Lett, 191, 155-64, 2003)
LIMD1 (Sharp et al, Proc.Natl.Acad.Sci 101, 16531-16536), and TMEM7 (Zhou
et al, Cancer Genet Cytogenet,177, 51-59, 2007). M Serrano demonstrated the
role of RIS1 (another CER1 gene) in the Ras induced senescence pathway. All
four, evolutionarily sorted in close proximity, interfere with E2F meditated RB
(LIMD1, RIS1) and NFkB mediated p53 (LF, TMEM7) senescence pathways.
Analysis of eliminated regions on human chr 1, 3, 8, 13 and 17 may provide further suppressor segments with multiple anticancer candidates.
S7-15
MICRO-FOCI INDUCING VIRUS (MFV), A MARKER OF PAEDIATRIC
CANCERS, BEHAVES AS A STEM CELL VIRUS
Ugo Rovigatti
Retrovirus Center and University of Pisa Medical School, Via Abetone e
Brennero 2, 56127, Pisa, Italy
Molecular markers in oncology are very helpful for cancer diagnosis, prognosis and for monitoring patient treatment, as documented by the brilliant presenta-
300
tions in this panel. Molecular markers can also provide interesting/important
insights to cancer aetiology-pathogenesis, particularly in the area of associated
infections. Relevant examples are Helicobacter pylori and HPV, where presence
of a bacterium/virus is important not only for diagnosis but also for their aetiological implications (Nobel Prizes for Physiology and Medicine, 2005 and 2008).
In childhood cancer and especially in childhood leukaemia, the possibility of an
infecting agent being a triggering factor for the actual onset of the disease has
been proposed in 3 –not exclusive- models: 1. Melvin Greaves has clearly documented that the same genetic translocations may be already present at birth in
children who eventually develop frank leukaemia: subsequent infections (immunological stress) could be triggering; 2. Malcolm Smith has hypothesized that a
relevant carcinogenic event may already occur in utero; and 3. Epidemiological
work by Leo Kinlen has documented dramatic increases in situations of population mixings, in which specific infections (a virus) could be triggering (best
reviewed by Greaves, 2006). Since 1988, we have studied a novel virus isolated
from a cancer cluster (i.e., space-time association) of paediatric neuroblastoma
(Micro-Foci inducing Virus or MFV). Similar viruses were subsequently isolated
from paediatric lymphomas (MFRVs). Experimental models have supported an
important role of MFV/MFRVs for inducing genomic instability, specific aberrations/translocations and lymphoma/neuroblastoma in children (Children with
Leukaemia Conference, 2008). Recently, different patterns of infection were
documented in differentiated vs. stem cells of haemopoietic or neuroblastic origin. While MFV causes 100% apoptosis in differentiated cells, HSC/NSC are capable of surviving its infection by rearranging their genomes (gene amplification/
translocation). These findings and compatibility with different aspects of the
mentioned models will be discussed.
S8. Phenotype, Response to Therapy and Molecular
Genetics in Various Fields of Pediatric Endocrinology
S8-1
GENETICS OF INTRAUTERINE AND POSTNATAL GROWTH
David B. Dunger
Department of Paediatrics, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdom
Extremes of birth weight and rapid postnatal weight gain have been associated with childhood risk for obesity and adult risk for type 2 diabetes (T2D).
The extent to which these associations are related to genetic factors or prenatal
and postnatal programming of metabolism has been the subject of considerable
debate. Size at birth is a complex trait reflecting interaction between fetal genes
and the maternal uterine environment. There is a continuous relationship between
maternal glucose levels during late pregnancy, size at birth and the problems
associated with pregnancies complicated by diabetes. Thus, maternal T2D
genetic susceptibility genes will affect size at birth and, through transmission to
the offspring, subsequent disease susceptibility. There are some data to suggest
paternal transmission of T2D genes associated with impaired insulin secretion
may lead to reductions in size at birth. There has also been considerable interest
in inheritance of imprinted genes, particularly those regulating IGF2 expression.
H19, IGF2 and the adjacent INS variable number of tandem repeats (VNTR)
have been related to size at birth demonstrating parent of origin effects. DNA
methylation of the IGF2 gene may be affected by the early life peri-conceptional
and early intra-uterine environmental conditions leading to epigenetic changes,
which persist throughout life. Such epigenetic changes in imprinted and nonimprinted genes, which alter gene expression, could underpin some of the remarkable transgenerational associations between size at birth, early weight gain
and T2D risk. Rates of postnatal weight gain partly reflect the reversal of these
prenatal environmental exposures but they are also strongly related to feeding
practice and infant satiety. Recently genes associated with adult risk of obesity,
such as FTO and MC4R and which probably act through satiety, have been
shown to have an important impact on very early postnatal weight gain. As in
prenatal life, imprinted genes and the INS VNTR may also be important determinants of early weight gain, but data from large population studies are still
awaited.
S8-2
GENETIC ASPECTS OF HYPERINSULINISM IN INFANCY AND
RESPONSE TO THERAPY
Feyza Darendeliler and Firdevs Bas
Department of Pediatrics, Pediatric Endocrinology Unit, Istanbul University,
Istanbul Faculty of Medicine, Istanbul, Turkey
In 50–60% of cases with hyperinsulinism in infancy (HI), genetic etiology is
unraveled. In the analysis of our Turkish patients, out of 18 patients with HI, 6
had SUR1 mutations and one had a mutation in GLUD1 gene. Altogether 38.9%
of the patients had an identifiable genetic defect. The six patients with SUR.1
defect had 8 mutations. These were point mutations N32K in exon 1, I89M in
exon 2, E1141X in exon 28, Q1373X in exon 33, R1494Q in exon 37, splice site
mutations 4310g to a and 4612-2 a to g and a deletion, 155 del in exon 1. Two
patients were compound heterozygous and had I89M/4612-2Ato G and R1494Q/
4310 g to a. One of the patients had homozygous for 155 deletion with a frameshift mutation. 4310 g to a mutation has been reported in other Turkish patients
as well and may have a founder effect in our population. One of the patients had
homozygous for N32K, this mutation was a novel mutation. S445L in exon 11 is
considered as a hot spot for GLUD1 mutations as was also found in our patient.
Regarding the therapeutic response of the patients, the patient homozygous for
the 155del did not respond to diazoxide treatment and underwent surgery. The
patient heterozygous for I89M/4612-2AtoG did not respond to diazoxide and nifedipine treatment and underwent surgery. There was a relapse after surgery for
the patient, which was placed on nifedipine again and is still well on nifedipine
at follow up. The patient heterozygous for R1494Q/4310 g did not respond to
diazoxide as the initial therapeutic agent and was started on nifedipine. She
responded to nifedipine and is still well at follow up. The patient homozygous
for N32K did not respond to any medical therapy and underwent surgery.
Type1DM developed after surgery. The patient with a paternally inherited nonsense mutation in the ABCC8 gene - Q1373X did not respond to diazoxide and
nifedipine and underwent surgery. She had focal lesion. She is still being followed and is doing well. The patient heterozygous for E1141X responded to
diazoxide and nifedipine initially. She showed spontaneous remission and therapy was stopped 3 months after the diagnosis. The patient in our series with the
GLUD1 mutation responded well to diazoxide as expected. In conclusion,
although limited, genetic analysis makes an impact on clinical management.
Acknowledgement: We would like to express our gratitude to K Hussain, Dr.
Sian Ellard, J Fournet and C Stanley for doing the genetic studies.
S8-3
MANAGEMENT OF CHILDREN AND ADOLESCENTS WITH
PRADER-WILLI SYNDROME
Maithe Tauber
Children’s Hospital, Centre Hospitalier Universitaire,
31059 Toulouse, France
Prader-Willi syndrome (PWS) is a complex neurodevelopmental genetic disorder that arises from lack of expression of paternally inherited imprinted genes
on chromosome 15q11-q13. The syndrome has characteristic phenotypes including severe neonatal hypotonia, early onset of severe obesity, short stature, hypogonadism, learning disabilities, behavioral problems and psychiatric phenotypes
with severe consequences and difficult management issues for patients, families
and carers. The lower limit of birth incidence is around 1 in 20,000 to 1 in
30,000 and population prevalence is about 1 in 50,000. PWS is a model showing
that early diagnosis and comprehensive multidisciplinary approach including GH
treatment prevent complications, optimise quality of life, and prolong life-expectancy. At this time we can assume that in these circumstances, we had completely
modified the clinical presentation of most of the children with PWS. Evolving
phenotype from birth to adulthood means that the clinical features that should
lead to a suspicion of the diagnosis depend on the age of the patient and the diagnosis of PWS should be evocated in all infants with severe and unexplained
hypotonia. DNA methylation analysis is the only technique, which can both confirm and reject the diagnosis of PWS. Early diagnosis offers the opportunity for
education of parents (ie parental guidance), caregivers and other healthcare professionals to receive and give social, psychological and educational support. The
strong efficacy of Growth Hormone (GH) treatment in these children explained
the fact that paediatric endocrinologists are often in the first place to coordinate
the care of these infants, children and adolescents. In adult patients, the endocrinologists are also in the first row due to the complications of morbid obesity. At
any age, psychiatrists and psychologists are also needed. We like to say that the
care of these patients is based on the core trio constituted by the paediatric endocrinologist, the endocrinologist and the psychiatrist.
S8-4
EARLY PUBERTY: INSIGHT INTO THE GENETIC
Moshe Phillip
Institute for Endocrinology and Diabetes, National Center for Childhood
Diabetes, Schneider Children’s Medical Center of Israel and Sackler Faculty
of Medicine, Tel-Aviv University, Petah Tikva, Israel
Normal puberty usually begins at the end of the first decade or at the beginning of the second decade of life and is characterized with the appearance of
secondary sexual signs and growth acceleration. Physiological variation of age of
onset of puberty is observed among normal individuals all over the world. Pubertal changes are influenced by both genetic factors and environmental changes.
While several genes were found to be associated with delayed puberty and hypogonadotropic hypogonadism, little is known about the genetic control of normal
puberty and the molecular basis of central gonadotropin–dependent precocious
and early puberty. The present work will describe the accumulating evidence of
the influence genetic factors have on early pubertal development and the attempts
to better understand the molecular basis of precocious puberty.
S8-5
CYP21A2 GENE MUTATIONS IN CONGENITAL ADRENAL
HYPERPLASIA: GENOTYPE–PHENOTYPE CORRELATION
Nurcin Saka
Istanbul University, Child Health Institute, Istanbul, Turkey
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is a common autosomal recessive disorder and is caused by CYP21A2
gene. The enzyme deficiency leads to various degrees of impaired cortisol and
aldosterone synthesis and increase in androgen synthesis. Depending on the residual enzyme activity, the clinical phenotype ranges from severe or classical
which includes salt- wasting (SW) and simple virilising (SV) forms, to mild or
nonclassical (NC) form. CYP21A2 gene is located near its pseudogene
CYP21A1P. To date, over 100 mutations have been described in the human
CYP21A2 gene. Approximately 95% of all disease-causing mutations are large
deletions, large conversions or one of the eight point mutations [P30L, I2G,
G110D8nt, I172L, exon 6 cluster (I236N, V237E, M239K), V281L, Q318X,
R356W]. Large deletions or large conversions are typically associated with SW,
I2G mutation may be associated with SW or SV forms. The I172N mutation is
usually associated with the SV form, and V281L, P30L, R339H and P453S
mutations with the NC form. In this study, we evaluated the frequency of
CYP21A2 gene mutations and the genotype-phenotype correlation in 56 Turkish
patients from 52 families including 15 consanguineous families.
Disease-causing mutations were identified in 77 out of 91 alleles (84.6%).
Mutations were found in 34 of 43 alleles (79.1%) in SW, 32 of 36 alleles
(88.8%) in SV and 11 of 12 alleles (91.6%) in NC forms. The most frequent
mutations were I2G/I2G (22.0%), large conversion (14.3%), I172N (9.9%),
R356W (8.8%), and large deletion (6.6%). The most frequent genotypes were
I2G/I2G (11.5%) and large conversion/large conversion (11.5%) in the SW form,
I2G/I2G (20%) and I172N/G110d8nt (10%) in the SV form, and V281L/V281L
(16.7 %) in NC form. In 21 OHD, there was largely a good correlation of genotype with phenotype. In the SW and NC forms, genotypes of all the patients
were found to be correlated with their phenotypes. However, there are few
patients of genotype/phenotype non-correlation; in the SV form, 5 out of 24
patients predicted the NC form and, two sibs carrying severe mutation on both
alleles that would predict the SW form had the SV form. In conclusion; with the
exception of a few cases, genotype-phenotype correlation has been observed in
patients with CAH. These data will assist in giving enhanced patient care,
genetic counseling and prenatal diagnosis and treatment to patients with CAH
and carriers.
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S9. Public Health Genomics and Nutrigenetics
S9-1
INTRODUCTION TO PUBLIC HEALTH GENOMICS AND ITS
FUTURE IMPLICATIONS
Angela M. Brand and T. Schulte in den Bäumen
German Center for Public Health Genomics (DZPHG), University of
Applied Sciences, Bielefeld, Germany
Objectives: Healthcare systems are currently facing fundamental challenges.
The integration of genome-based health information and technologies requires an
innovative governance model. New ways of organizing these systems based on
personalized health information and stakeholders’ different needs are essential to
meet the challenges in time.
Methods: The task of public health genomics (PHG) has become a specific
challenge having major implications for future research and policy strategies.
The various stakeholders in public health play a key role in translating the implications of genome-based research derived from e.g. systems biology, epigenomics, the systematic analysis of genome-environmental interactions and population-based biobanking. Whereas medicine is currently undergoing remarkable
developments from its morphological and phenotype orientation to a molecular
and genotype orientation, the discussion about the role of genome-based knowledge and technologies for public health still is at the beginning. The Public
Health Genomics European Network (PHGEN) aims to fulfil this task in Europe
(www.phgen.eu).
Results: Recent advances in systems and network biology indicate that specific cellular functions are infrequently carried out by single genes, but rather by
groups of cellular components, including genes, proteins, and metabolites. This
network-based research is already starting to change nosology. Seemingly dissimilar diseases and health outcomes are being lumped together. What were thought
to be single diseases are being split into separate ailments (‘‘diseasomes’’). Just
as they once mapped the human genome, scientists are currently trying to map
these diseasomes. The approach offers a novel method for human disease classification. It defines disease expression based on its molecular and environmental
elements in a holistic way. This knowledge will not only enable clinical interventions but also health promotion messages and disease prevention programmes
to be targeted at susceptible individuals as well as subgroups of the population
(personalized healthcare).
Conclusions: So far, there has been no systematic integration of genomebased knowledge and technologies into public health. Thus, current strategies are
lacking one important aspect of evidence-based. With regard to genomics, the
public health agenda demands a novel vision that reaches beyond the research
horizon to arrive at application and public health impact assessment of this novel
technology.
S9-2
THE ROLE OF NUTRITIONAL SUPPLEMENTS IN
PREVENTIVE GENETICS
Gulden Pekcan
Hacettepe University, Faculty of Health Sciences, Department of Nutrition
and Dietetics, Division of Community Nutrition, 06100 Sihhiye,
Ankara, Turkey
Diet is a key factor in determining genomic stability. Several micronutrients;
vitamins and minerals including calcium, vitamin E, retinol, folate, vitamin B12,
nicotinic acid, zinc and riboflavin are required as cofactors for enzymes or as
part of the structure of proteins (metalloenzymes) involved in DNA synthesis
and repair, prevention of oxidative damage to DNA as well as maintenance of
methylation of DNA and in maintenance of genome stability. The individual
genetic makeup, that is, the presence of polymorphisms in nutrient-regulated
genes, affects individual risk of disease and due to differences in genetic
makeup, supplements may be needed to cover gaps in micronutrient requirements. Supplementation of diet with appropriate minerals and vitamins could, in
some cases, help overcome inherited metabolic blocks in key DNA maintenance
pathways; DNA repair in diseases. The best characterized example is probably
that of methylenetetrahydrofolate reductase (MTHFR) gene variants and folate
supplementation in the prevention of cardiovascular disease risk, cancer and
302
neural tube defects. Folate supplementation was indeed shown to compensate for
decreased activity of MTHFR in T/T individuals, with a parallel decrease in
plasma homocysteine levels. So, in disease prevention the genome damage
caused by micronutrient deficiency is preventable. It is certainly possible that
some individuals, because of their diets or genetic polymorphisms, have unusual
vitamin needs. Personalized diets with nutritional supplementation, which take
into account individual genotype, can lower disease risk in genetically predisposed individuals or population groups.
S9-3
GENETICS AND CANCER PREVENTION
Keith Grimaldi
Research & Development, Sciona Ltd., United States
Most cancers are the result of acquired genetic abnormalities leading to
transformed cells which grow uncontrollably. The genetic changes are usually
base mutations which affect the activity of specific genes such as tumour promoting oncogenes and tumour suppressor genes. Mutations are initiated by random DNA damage caused by carcinogens which bind to bases on the double helix, and radiation which causes strand breaks – the misrepair of these lesions
leads to sequence changes which can result in a modified protein amino acid
sequence, alterations in protein activity and the initiation of tumorigenesis. The
cancer development process is the result of serial mutations in several genes,
usually over many years. Most common cancers are caused by environmental exposure to various DNA damaging agents, such as UV radiation and carcinogens
present in smoke, exhaust fumes, food, etc. There is also a genetic component
where different people react in different ways to the same environmental insults
– not every heavy smoker will develop lung cancer for example. The body has
developed several mechanisms to protect against the development of cancer
including the two phase detoxification process where ingested carcinogens are
made water soluble and expelled in the urine, anti-oxidant enzymes which reduce
DNA damage by oxidative stress, and DNA repair proteins which fix the damage
caused by carcinogens and radiation. Most if not all the enzymes involved in
these processes are polymorphic, that is there are several common versions of
the same genes/proteins which differ by a few nucleotides and which have
altered activity. It is well established by now that these polymorphisms can influence the way individuals responds to environmental toxins, nutrition, physical activity and response to medications – ultimately affecting the development of malignant tumours and their treatment. Knowledge of these ‘‘gene-environment’’
interactions, especially where diet can affect the activity of a gene, will help in
developing more personalised preventive healthcare measures and some examples will be discussed.
S9-4
THE ROLE OF GENOME BASED INFORMATION FOR
PERSONALIZED HORMONE REPLACEMENT THERAPY
Mithat Yilmazturk
Yeditepe University, Faculty of Medicine, Department of Obstetrics and
Gynecology, Istanbul, Turkey
It is known that menopausal hormone replacement therapy (HRT) can
increase the risk of breast cancer. However, it has been understood that this does
not cover all women. Cancer occurs because of hormone therapies, which are
not appropriate for the individual. Climacteric complaints do not occur in every
woman during perimenopausal period, because some women continue to produce
estrogens in various organs (breasts, adipose tissue, bones, etc.) independent
form ovaries. Therefore, cessation of menstruation is not a sole indication for
HRT. If the climacteric complaints continue despite a healthy life style, optimum
body weight and regular exercise, HRT can be necessary. For HRT without complications, it is an important criterion in which way the women feels comfortable. However, scientific information to determine the dose in a personalized way
can be achieved with a genetic investigation. Personal status of genes that are responsible for hormone synthesis and elimination should be taken into account,
because no hormone level testing reflects the 100% actual status for all endocrine
organs. For example, estrogen production of some organs (breast, bones, etc)
cannot be detected in the blood. Blood estrogen levels should be considered to-
gether with symptoms related to estrogen deficiency in whole female organism,
especially with the density of breast tissue. A genetic analysis carried out on a
sample taken with a buccal swab can be used to detect: 1) If the women are predisposed to thrombosis, 2) If the estrogens are eliminated rapidly or slowly, 3)
Indication of the amount of estrogens produced outside ovaries. All these are
related to common genetic variations, called polymorphisms. Thus, we have a
method to predict the necessary dose for each individual case. For example, if a
woman has a special form of one of the two genes on coagulation (Factor V and
Factor II), the risk of thrombosis increases. Another gene (PAI1) that plays a
role in coagulation cascade can show an indication for estrogen use, since estrogens reduce predisposition to thrombosis in a special form of this gene. There
are several other examples on estrogen metabolism related genes and breast cancer. Polymorphisms explain an important part of why same therapy is very successful in one woman, whereas causing side effects in another. Even if the
woman has menopausal complaints, genetic investigation can show us if the therapy will bring more harm than benefits.
S9-5
ROLE OF NUTRITION IN DIFFERENT GENETIC CHARACTERISTICS
Sema Erge
GENAR Institute, Istanbul, Turkey
In recent years, it has been a well established fact that chronic and complex
diseases results from the interaction between our genes, environment and life
style. The interaction between nutrition and genetics has opened new vistas leading to new disciplines in science. One of them, nutrigenetics (the genetics of
nutrition), is an area that studies the response of a subject to a specific diet due
to the variations (polymorphisms) in his/her genetic structure. The results
obtained from these studies are used to develop nutritional recommendations,
which are appropriate for subjects who have specific genetic structures. The
effect of a food on an individual’s health depends on his/her genetic structure.
Several genes and the variations of these genes, which are related to nutrition,
may affect the prevalence, onset, progression and severity of chronic diseases.
Furthermore, making regulations in an individual nutrition based on his/her nutritional requirements; nutritional status and genetic structure may help to reduce
the severity of disease and may treat it. The nutritional recommendations, which
are prepared in the light of your genetic structure, are separated from the traditional ones. In the traditional perspective, it is supposed that every individual are
similar in genetics. For example, it is recommended that salt should be limited in
all hypertension patients, traditionally. However, in clinical practice we know
that not everybody responds to salt restriction in terms of decrease in blood pressure. It was shown that salt sensitivity can be a result of genetic polymorphisms.
One of these polymorphisms are AGT gene G(-6)A polymorphism of AGT gene.
People with GG genotype are less sensitive to salt limitation in terms of blood
pressure, than carriers of GA or AA. Such interesting results are also seen in
other areas of nutrition research, such as fatty acid intake. Contrary to the opinion that poly-unsaturated fatty acids decrease the level of HDL, it is found that
in women who carry A allele in -75G/A polymorphism of APOA1 gene, higher
intake of poly-unsaturated fatty acids is associated with an increase in HDL cholesterol level. Several new gene-nutrient interactions are being investigated and
published. Every research which intends to explain the relationship between
genetic structure, foods/life-style factors and health/disease is a candidate for
making new developments in application of nutrigenetics.
S9-6
GENETICS OF AGING
Serif Akman
Department of Biochemistry, Gulhane School of Medicine, Ankara, Turkey
Longevity has a strong genetic component that has been studied with a variety of model organisms, such as yeast, fruit flies, worms, mice and humans.
These studies showed that multiple genetic pathways are involved in the regulation of longevity and life span. These pathways are mostly related to metabolism
and the resistance to oxidative stress. Longevity genes also include the genes
involved in the regulation of DNA repair and nuclear structure, which cause the
progeroid syndromes when mutated. Other gerontogenes like sirtuins are
involved in the cellular stress response, including caloric restriction. The genes
that regulate telomere length are also longevity assurance genes because telomere
length is associated with cell senescence. Other candidate genes include mitochondrial DNA and the genes that regulate the inflammatory response. Recent
results indicate that the longevity of model organisms can be altered through
genetic manipulation and pharmacological intervention. Most of these interventions involve alterations of one or more of the genes regulating insulin/IGF-I signaling pathway, caloric intake, stress resistance and nuclear structure. The mutations in the genes of insulin and insulin-like growth factor 1 (insulin/IGF-1) signaling pathway have been shown to extend lifespan in model organisms. Klotho
is another longevity genes and its protein has glucuronidase activity, which has
been shown to decline with age. Mutated klotho mice show premature aging.
However, how longevity genes determined from model organisms will translate
to primate aging and longevity remains to be shown. The identification of genetic
pathways that regulate longevity may suggest potential targets for therapy.
S9-7
CHRONIC COMPLEX DISEASES AND GENETICS
Tomris Cesuroglu
R&D Coordinator, GENAR Institute for Public Health and Genomics
Research
Current status of populations with lower fertility status with extended life expectancy is a result of the fourth phase of epidemiological and demographic transition. Chronic and complex diseases are dominating this phase and they stem
from the complex interaction of human genome with lifestyle and environmental
factors. These diseases are serious and costly. Cardiovascular and cerebrovascular diseases, cancers, diabetes, obesity, osteoporosis, psychiatric and neurological
conditions are among major complex diseases and they account for approximately two third deaths in the world. One of the main topics of today’s science
is enlightening the interactions between genome and lifestyle/environmental factors. There are variations in individuals’ genetic make-up -polymorphisms- that
makes each individual response differently when they are exposed to the same
lifestyle or environmental factor. GENAR Institute for Public Health and
Genomics Research was established in 2004 in Hacettepe University Science
Park, Ankara, Turkey. It is the third public health genomics center in Europe and
a cooperating institute of Public Health Genomics European Network. GENAR
aims to transform scientific developments in the area of biotechnology, especially
genetics and genomics, into products and services that improves human health,
quality of life and performance, and extends life span. GENAR Institute has
been working on a preventive health care model to combat with the complex diseases, utilizing public health genomics and personalized medicine approach. It is
an integrative preventive model utilizes individuals’ clinical information, detailed
lifestyle analysis, biomarkers and the genetic make-up in order to prevent, early
detect and treat complex diseases in a targeted way. Based on the results of the
aforementioned components, an optimum lifestyle plan is drawn, including personal menu plans and exchange lists, exercise plans, smoking cessation recommendations and medical follow up plan. The mission of this model is changing
the behavior of individuals. It creates awareness by informing individuals about
their current lifestyle and genetic predispositions. Further, it causes an attitude
change by creating a vulnerability perception. Finally, behavior change is
achieved with the follow-up programme and the trainings.
S10. Round Table Discussions
The Treatment of Unresectable Sarcomas and Melanomas
of the Extremities with Hyperthermic Isolated Limb
Perfusion (HILP) with Melphalan plus TNF
INTRODUCTION
This session will provide speeches describing all aspects of hyperthermic isolated limb perfusion (HILP) with Melphalan and TNF in patients with sarcomas
and in transit melanomas or bulky disease.
This round table will consist of four speeches. First, a description of the natural history of sarcomas and melanomas will be presented. Second, a presentation on vascular access surgical techniques for HILP and technical tips and hints
during surgery will be displayed. The third speech will reveal details on how to
detect and how to avoid TNF leaks, which can be disastrous for patient outcome
303
if they occur. Finally, the session will also include specialist personal experience
with HILP as well as a review of the results reported from other centers in the
literature. Following are the detailed descriptions of the presentations.
S10-1
I. NATURAL HISTORY OF SARCOMAS AND MELANOMAS
K. Lasithiotakis
General Surgery Department, University Hospital of Heraklion, Greece
The natural course of cutaneous melanoma (CM) is determined by its metastatic spread and depends on tumor thickness, ulceration, gender, localization,
and the histologic subtype of the primary tumor. Between 30% and 50% of CMs
arise on the extremities, commonly on the lower limbs. Fifty percent of the
patients with tumor progression firstly develop regional lymph node metastases,
20% satellite or in-transit metastases and about 30% immediately distant metastases. Overall survival of CM patients without clinical evidence of metastasis
exceeds 90% at 10years. In the presence of satellite/in-transit metastasis with
and without regional lymph node involvement, 5-year overall survival rates do
not exceed 60% and 30% respectively. Soft tissue sarcomas are a diverse group
of neoplasms that arise in the connective tissues throughout the body. About
50% to 60% of sarcomas occur in the extremities, commonly on the thigh and
buttock. Size is one of the most important clinical features of soft tissue sarcomas and it is associated with their metastatic potential. Soft tissue malignancies
are usually painless until they encroach on sensitive adjacent structures and this
is related with their late diagnosis. Overall survival of patients with soft tissue
sarcomas has been reported to be between 50-60% at five years and local recurrence rates between 10 and 20% depending of tumor histology and margins of
excision. In this presentation we attempt to explore the factors that influence the
clinical course of a patient with soft tissue sarcoma or cutaneous melanoma of
the extremity in a manner relevant to the application of isolated limb perfusion.
S10-2
II. VASCULAR ACCESS FOR HILP
C. Ioannou
Lecturer of Vascular Surgery, University of Crete Medical School, Greece
Both sarcomas and melanomas (Stage IV and In Transit) are accompanied by
poor survival rates and high amputation rates if left untreated. HILP offers a few
advantages, such as, high dosage concentrations constrained within the affected
limb while excludes the drug from entering the systematic circulation. On the other
hand, HILP offers regional therapy to a potentially systemic disease and requires a
surgically obtained vascular access through which the limb will be isolated and the
drugs will be administered. This talk will cover both the basic principles governing
surgical technique of vascular access, as well as all specifics about vascular access
through the iliac and femoral approach for the lower limbs. Additionally, vascular
access for the upper limbs through the subclavian, axillary and brachial approach
will also be displayed and analyzed. Moreover, radical groin lymphadenectomy and
wound closure utilizing a sartorial muscle flap transposition will also be discussed.
Finally, a number of technical tips and hints for optimal surgical preparation and
vascular access technique will also be presented based on personal experience.
S10-3
III. LEAKAGE DETECTION AND CONTROL DURING HILP
K. Perisinakis
Medical Physics, University of Crete Medical School, Greece
Monitoring of blood leakage from the extracorporeal circuit to the systemic
circulation is obligatory during HILP therapy with a combination of tumor necrosis factor (TNF) and other cytostatic agents, to avoid the extremely morbid or
even lethal effects caused by the high toxicity of these agents. The ideal method
for leakage monitoring during HILP should provide real-time, continuous and
accurate leakage data to allow for rapid intervention if leakage exceeds acceptable levels. Leakage monitoring through pre-cordial radioactivity measurements
with an external scintillation probe is the most popular technique for leakage
monitoring during HILP.
304
The aim of this presentation is a) to briefly describe available techniques for
leakage monitoring during HILP, b) to illustrate the basic physical principles of
leakage monitoring using radionuclides and pre-cordial activity monitoring, b) to
discuss several factors moderating the efficacy of such intra-operative monitoring
of leakage during regional chemotherapy and c) to demonstrate the radiation burden and discuss on the associated radiation risks for the patient and the medical
stuff involved in such treatments.
S10-4
IV. RESULTS AND PERSPECTIVES
O. Zoras
University of Crete Medical School, Greece
We have treated eight cases in our Department (Department of Surgery, University of Crete Medical School, Greece). Three sarcomas of which one leiomysarcoma located just above the left external malleolus, one leiomysarcoma in the
right tibia and the last, a liposarcoma, in the right femorus. All of the pervious
cases were initially treated elsewhere and were referred to us after recurrence.
Furthermore, five intransit melanomas, one located in the left mid tibial area,
two located in the right knee area and two in the right lower tibial area. Chemotherapy dosages for Melphalan was according to common protocol (10 mg/liters
of limb volume) and TNF was 2 mg in total dose. All patients were recovered in
the ICU for 24 hours in accordance to protocol. Although statistic comparison is
not applicable, we observed a notable shrinkage of the tumor and a very good
partial response of the sarcomas and complete response of the melanomas. No
systemic toxicity was observed. Limb toxicity, as measured by the wieberdink
scale, was 0 in five of the cases and 2 in one case. Functional disability was
minimal in all cases. Our experience verifies that the use of Melphalan and TNF
in HILP is safe when guidelines and safety rules.
S10-5
VASCULAR RECONSTRUCTION AND LIMB SALVAGE
IN OSTEOSARCOMAS
Murat Aksoy
Peripheral Vascular Surgery Unit, Department of Surgery, Medical Faculty
of Istanbul, Istanbul University, Turkey
The main goals in the treatment of osteosarcomas of the extremities include
salvage of the limb as well as obtaining disease-free margins during the tumor
excision. In order to achieve these goals, vascular reconstruction may be necessary in patients, whose tumors invade the vascular structures. Although high percentage of the interventions is carried out without sacrifice of the vascular bundle, arterial and/or venous repair may be necessary in some. When necessary, arterial and venous reconstruction is usually performed concomitantly. In patients
with previous deep vein thrombosis, solely arterial reconstruction may be needed.
It is also interesting that not all patients without a venous reconstruction experience edema of the limb. High rates of limb salvage may be achieved in the majority of patients who have lower extremity sarcomas even when en bloc resection includes the artery and vein. The long-term patency rate of the reconstructed
vessels is high. Saphenous vein is the preferred conduit when it is of adequate
caliber and multidisciplinary approach is warranted.
S10-6
prognostic factors were assessed, including age, gender, localization, tumor size,
primary metastasis on presentation, the presence of pathologic fractures, necrosis
rate, and infection. All the patients received chemotherapy before and after surgery. The mean follow-up period was 49.7 months (range 6 to 185) months.
Results: Sixty-nine patients were below 16 years of age. The most frequent
involvement was in the distal femur (47.2%), followed by the proximal tibia
(25%). Sixteen patients presented with a pathologic fracture, and 12 patients
with metastasis. The median tumor size was 10 cm. The overall five- and 10year survival rates were 68% and 60%, and disease-free survival rates were 50%
and 44%, respectively. Only the presence of a pathologic fracture and primary
metastasis on presentation were found to affect prognosis.
Conclusion: The two conditions, primary metastasis and a pathologic fracture, found as the most important prognostic factors in our study are mainly
associated with late presentation. As in every malignant disease, early admission
would provide better survival rates.
S11. Drug Development, From Idea to Clinic
S11-1
CONFORMATIONAL ANALYSIS OF HEPARAN SULFATE
OLIGOSACCHARIDES: ‘‘NEW INSIGHTS INTO STRUCTUREFUNCTION RELATIONSHIPS AND THERAPEUTIC APPLICATION’’
Kevin Murphy, Simone Swift, and David A. Pye
Centre for Molecular Drug Design, Cockcroft Building, University of
Salford, Manchester, M5 4WT, United Kingdom
Three-dimensional molecular structures are one of the fundamental tools in
structure based drug design. The aim of our research is to determine the 3Dstructure of biologically active heparan sulfate (HS) oligosaccharides, using a
combination of molecular modeling and NMR structural refinement and to computationally dock them into a variety of protein ligands. We are using this data
to develop a database of HS-oligosaccharide 3D-structures, so that we may determine rules for the conformational behavior of HS oligosaccharides as a function
of their sequence. The overall objectives being the ability to predict 3D-structure
and binding information, for any conceivable HS oligosaccharide sequence, without the need for its purification. HS has now become a prime candidate for exploitation in rational therapeutic design. There is a growing opinion that the conformational dynamics within HS chains is critical to their observed biological
activities. Investigations into HS conformational dynamics are problematic, given
the structural complexity and heterogeneity of HS chains. However, this goal
will be more obtainable once we understand the important roles HS sequence/
sulfation patterns play in determining the conformational dynamics of iduronate
units and the geometry of the glycosidic linkages. We have produced a library of
highly pure HS oligosaccharides, in amounts that can be utilised for biophysical
studies. A series of purified oligosaccharides have been structurally evaluated by
NMR spectroscopy and their structures modeled. The 1H and 13C spectra of the
oligosaccharides have been fully assigned, to generate sequence information, and
2D-NOESY experiments performed for 3D-structural refinement. By fitting experimental coupling constant data to a new set of theoretical coupling constants,
calculated using explicit water molecular dynamic simulations, we are able to
offer new insights into the role sequence/sulfation patterns play in influencing
iduronate conformational behavior. Our results suggest that flanking 6-O-sulfate
groups alter the balance of the IdoUA(2S) conformational equilibrium. There is
also the suggestion that a cooperative effect may exist for N- and 6-O sulfation.
These observations could be vital to our understanding of the important regulatory functions attributed to 6-O-sulfation within HS chains and will, no doubt,
aid in the structure-based design of new carbohydrate therapeutics.
OSTEOSARCOMA AND OUR CLINICAL EXPERIENCE
Levent Eralp and Harzem Ozger
University of Istanbul, Istanbul Medical School, Department of Orthopedic
Surgery and Traumatology, Turkey
Objectives: We evaluated long-term treatment results of patients with primary osteosarcoma and the effect of prognostic factors on overall survival and
disease-free survival.
Methods: Between 1995 and 2005, 180 patients (111 males, 69 females;
mean age 211/-10 years; range 7 to 64 years) were treated for primary osteosarcoma. Overall and disease-free survival rates were analyzed for 165 patients with
high-grade osteosarcoma with the Kaplan-Meier method. The effects of potential
S11-2
THE POTENTIAL OF SPECIFIC KINASE INHIBITORS FOR THE
CHEMOPREVENTION OF MALIGNANT DISEASE
L. Hampson1, X.T. He1, A.W. Oliver1, J. Hadfield2, T. Kemp2, J. Butler2,
A. McGown2, H.C. Kitchener2 and I.N. Hampson1
1
University of Manchester School of Cancer Studies and Imaging Science,
Gynaecological Oncology Laboratories, St Mary’s Hospital, Hathersage
Road, Manchester M13 OJH, UK, 2Centre for Molecular Drug Design,
Kidscan Laboratories, Cockcroft Building, University of Salford,
Manchester, M5 4WT
S11-4
Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK)
family signalling has been shown to be a requirement for the establishment and
maintenance of transformation by constitutive activation of RhoA. The pharmacological ROCK inhibitor Y-27632 is known to suppress focus formation of
RhoA transformed NIH3T3 cells and has been studied as a potential cancer therapeutic. Here we report the effects of novel structural analogues of Y27632 on
NIH3T3 cells transformed by the Rho guanidine exchange factor (ARHGEF16).
Of the sixty-four compounds synthesised, four demonstrated inhibitory effects on
focus formation of GEF16 transformed cells yet paradoxically with no observed
toxicity against either transformed or non-transformed cells. Most significant was
the observation that transformed foci did not reform when the compounds were
withdrawn from treated cultures. Surprisingly, in vitro kinase inhibitor profiling
of the three most effective inhibitors indicated that they had reduced activity
against ROCK when compared to Y27632 but instead targeted aurora A
(AURKA), p38 (MAPK14) and Hgk (MAP4K4). Unexpectedly, none of these
compounds had any growth inhibitory effects on homogenous cultures of transformed NIH3T3 cells, only showing the ability to suppress the growth of transformed cells when these were co-cultured with non-transformed cells. A novel
Lucifer yellow and PKH67 dye transfer method was used to show that these
inhibitors induced a quantitative increase in the extent of gap junction intercellular communication (GJIC) between transformed and non-transformed cells. In
summary our results are the first to show that blockade of different, yet specific,
kinases can: I) Prevent the formation of non-contact inhibited transformed
NIH3T3 colonies, II) Continue to suppress the re-growth of transformed foci
even when the inhibitor is withdrawn, III) Eliminate transformed foci already
present in heterogenous cultures of transformed and non-transformed cells, IV)
Increase the extent of GJIC between transformed and non-transformed cells.
Selection of kinase inhibitors with these properties may provide a novel strategy
for the chemoprevention of cancer and/or metastases. Moreover, since the inhibitory effects persist after the compounds are withdrawn they further suggest that
therapeutics of this type may not need to be administered continuously.
PAX3 ISOFORMS REGULATE WNT AND NOTCH SIGNALLING
PATHWAYS IN EMBRYONIC STEM CELLS IN VITRO
305
Qiuyu Wang, Jamie Upton, Wen-Hui Fang, Mark Slevin,
and Patricia Kumar
School of Biology, Chemistry and Health Science, Manchester Metropolitan
University, UK
The transcription factor PAX3 is an intrinsic factor involved in lineage commitment and differentiation during myogenesis, melanogenesis and neurogenesis
in neural crest development. Abnormal expression of PAX3 has been found in
different types of tumour of neural crest origin. PAX3 occurs in seven isoforms,
PAX3a-h. Previous studies in our laboratory have shown that these isoforms
have different transcriptional specificities, activities and functions. In this study,
the mouse embryonic stem cell line, E14, was transfected with PAX3c, PAX3e
and PAX3g. Transfectants with pcDNA4 empty vector were used as controls.
PAX3 isoforms showed differential effects on E14 cell proliferation and migration. Furthermore, Affymetrix GeneChip microarray analyses revealed that PAX3
isoforms differentially regulate the expression of genes involved in the Wnt and
Notch signalling pathways, including Wnt5a, Ck1, Dvl2, Fzd2, Notch3, Dll1,
Jag1, Ncstn and Tle3. The changes in expression of these genes were confirmed
by RT-PCR and Western blotting. Both the Wnt and Notch pathways control cell
fate decision during embryogenesis and Notch is a major mediator of growth and
survival in several cancer types. Our results indicate that the effects of PAX3
isoforms in stem cell development in vitro involve the activation of Wnt and
Notch signalling pathways. These findings have advanced our understanding of
the roles of PAX3 isoforms in embryogenesis and their potential contribution to
tumourigenesis.
S12. Biochemical Genetics
S12-1
S11-3
A NOVEL INVESTIGATION OF PAX3 FUNCTION IN EMBRYONIC
CELL LINES UNDERGOING DIFFERENT DIFFERENTIATION
PATHWAYS AND THE CORRESPONDING TUMOURS
Patricia Kumar1, Qiuyu Wang1, Hongmei Li1,2, Wen-hui Fang1,2,
Mark Slevin1, and Shant Kumar2
1
Department of Biology, Chemistry and Health Science, Manchester
Metropolitan University, Chester St, Manchester, M15GD, UK., 2Division
of Laboratory and Regenerative Medicine, Faculty of Medicine and Human
Sciences, Manchester University, Oxford Road, Manchester, M13 9PT, UK.
PAX3 is a transcription factor expressed for only a few days during embryonic development of the neural crest and dorsal dermomyotome, but re-expressed
in tumours of cells derived from these embryonic populations. We have upregulated PAX3 expression by transfection into murine melanocytes, myocytes and
embryonic stem cells for comparison with PAX3 down-regulation using siRNA
in the corresponding tumours: melanoma, rhabdomyosarcoma and neuroblastoma.
We have studied three of the seven PAX3 isoforms which we isolated previously- namely PAX3 c, e and g. The effects of PAX3c, e or g, singly, versus an
empty vector control were determined in transfected cells using assays for cell
proliferation, migration, apoptosis and adhesion. Affymetrix microarrays identified genes up- or down regulated by each of the three isoforms. The relevance of
these genes of interest was confirmed using RT-PCR and western blotting.
Microarray results are being compared between 1) an embryonic cell line and its
corresponding tumour type 2) different lines of cell differentiation-melanogenesis, myogenesis and embryonic stem cell differentiation into sympathetic neurones. Genes up-regulated in embryonic cells while being down-regulated in the
corresponding tumours are likely to be important downstream targets of PAX3.
Genes regulated in the same way by three different isoforms might also be important. PAX3 might use different target genes in cells following different differentiation pathways. It is hoped that this study will illuminate the role of PAX3
in cancer and identify important molecular targets to be used in therapy.
RETROGRADELY TRANSPORTED siRNA TARGETING HUMAN
MUTANT SOD1 EFFECTIVE IN SPINAL CORD MOTOR NEURONS
OF ALS MICE
Albert Rizvanov1, Marat Mukhamedyarov2, Natalya Lannik1,
Ilnur Salafutdinov1, and Rustem Islamov3
1
Department of Genetics, Kazan State University, Kazan, Tatarstan, Russian
Federation, 2Department of Physiology, Kazan State Medical University,
Kazan, Tatarstan, Russian Federation, 3Department of Histology, Kazan
State Medical University, Kazan, Tatarstan, Russian Federation
The transgenic mice model of familial amyotrophic lateral sclerosis (ALS),
expressing human mutant copper/zinc superoxide dismutase (SOD1), is one of
the attractive models for studying therapeutic effect of RNA interference because
of the specific silencing of the mutant gene expression. We studied siRNA mediated down regulation of human mutant G93A SOD1 gene in lumbar spinal cord
of ALS mice. siRNA was applied onto the proximal nerve stump of severed sciatic nerves. The left sciatic nerve of anesthetized animals was cut in the midthigh and the proximal nerve stump was inserted into 7-mm Silastic tube with a
1.47 mm inner diameter as previously described (Murashov et al. 2007). Afterward, the siRNA against human SOD1 mRNA (n53) or corresponding mismatched control siRNA (n53) was pipetted into the tube and the lower end of
the tube was sealed with a petroleum jelly (Vaseline). The tube was glued to the
surrounding skeletal muscles with tissue adhesive (3M Vetbond) and the wound
was closed with surgical clips. Twenty-four hours after surgery the mice were
euthanized and lumbar spinal cords were removed for real-time RT-PCR examination. Treatment with specific siRNA resulted in 48% decrease in human SOD1
mRNA level in lumbar spinal cord (Figure 1) but had no effect on the level of
mouse ChAT and SNAP25 mRNAs. Taken together, our previous data and the
present RT-PCR results clearly proved that siRNA targeting mutant SOD1
mRNA can be taken up by the sciatic nerve, retrogradely transported to the perikarya of motor neurons and inhibit mutant SOD1 mRNA in G93A mice. In
transgenic G93A mice expression of mutant SOD1 gene occurs in every cell but
it is detrimental only to the CNS motor neurons and the treatment should be
directed to this cell type. Our results demonstrated targeted delivery of the
siRNA molecules specific against human mutant SOD1 mRNA into the lumbar
306
S12-3
THE EFFECT OF SEMINAL PLASMA ZINC ON SPERM
PARAMETERS IN HUMAN
Alireza Kahani
Department of Biology, Azad University, Iran
Figure. Effect of siRNA treatment on hSOD1 mRNA level in G93A mice.
motor neurons via peripheral spinal nerve. For the first time, we showed that
local application of siRNA to the nerve may be directly transferred to the spinal
motor neurons and result in siRNA-mediated gene silencing. Further experiments
are needed to trace siRNA retrograde transportation into perikarya of motor neurons and to study effect of retrogradely transported hSOD1 specific siRNA on
protein level in spinal cord.
Zinc in seminal plasma stabilizes the cell membrane and chromatin of spermatozoa. Perhaps, zinc is the most critical mineral for male sexual function.
However, extremely high concentrations of Zn may inhibit sperm head. Zn plays
an essential role in many enzymes on nucleic acid metabolism; proteins and thus
in cellular homeostasis. Zn may affect spermatogenesis with regard to production, maturation, motility and fertilizing capacity of the spermatozoa. To investigate the impact of zinc in seminal plasma and semen parameters in men, we
selected two groups of men as control or fertile and infertile groups. Semen analysis was performed according to WHO including pH, motility (into 4 categories
including none-motile, slow motile, flagella motile and rapid motile), vitality,
morphology (into 3 categories including head, neck and tail defects) and sperm
count. Zinc concentration in seminal plasma and sperm fraction were determined
by atomic absorption spectrometer. The results showed that the Zn concentration
in seminal plasma was negatively correlated with rapid motility (P\0.05) and
positively correlated with flagella motility. There was also a significant correlation between age and sperm motility. Zn concentration in seminal plasma was
positively correlated with sperm concentration (P\0.05).
S12-2
MOLECULAR BIOLOGY AND GENETICS OF INTRACRANIAL
MENINGIOMAS
Ali Kafadar
Department of Neurosurgery, Cerrahpasa Medical Faculty, University of
Istanbul, Turkey
Meningiomas constitute approximately 20% of all brain tumors in males and
up to 38% in females in some series. Meningiomas arise from arachnoid cap
cells, but not all meningiomas have the same clinical course although they are
pathologically classified in the same group. World Health Organization (WHO)
has divided meningiomas in three grades, whereas 90% are categorized as Grade
I, 5% as Grade II and 3-5% as Grade III. Grade I meningiomas generally have a
benign clinical course if treated properly. Grade II and III meningiomas are pathologies difficult to treat, although there have been extensive advances regarding
neurosurgical and other therapeutic modalities, like radiotherapy, chemotherapy
and radiosurgery. These treatment methods only help to deal with the tip of the
iceberg, because the genetics and molecular alterations of high grade meningiomas play a crucial role in the prognosis of these pathologies. A subgroup of
Grade I and Grade II meningiomas show primary genetic changes and are prone
to upgrade to Grade II and Grade III meningiomas respectively. Recent research
has shown that meningiomas similarly progress from the benign to the malignant
state by the accumulation of genetic alterations such as 1p LOH and p73/
RASSF1A promoter methylation. Other reports in the literature indicate a stepwise genetic progression, with the deletion of chromosome 22 as the fundamental
alteration and deletions in other chromosomes like 1p, 14q, and 10q among
others in the progression of these tumors toward a more malignant type. Defining
this subgroup may help to modify the treatment modalities in Grade I and II meningiomas. Therefore, initial genetic and molecular analysis of meningiomas may
change our understanding of meningioma treatment. Our aim is to define this
subgroup especially in young meningioma patients. Another unique point to
raise, is the fact that meningiomas arising from coverings around the spinal cord
and from the skull base are diagnosed as grade I meningiomas. The meningiomas
of this part of central nervous system are very rarely diagnosed as high grade
meningiomas. Most of the high grade meningiomas take their origin from calvarial brain coverings and falx cerebri. This clinico-pathological based observations
need to be further investigated to find out the basic difference between different
parts of coverings of the central nervous system. Whether the basic genetic fingerprints, the presence of a complex karyotype or the intriguing nature of coverings of the human nervous system defines the prognosis and natural history of
meningiomas is the main topic of our research.
S12-4
INFLUENCES OF GENE POLYMORPHISMS OF FOLATE
METABOLISM ENZYMES ON METHOTREXATE TOXICITY
Aslı Tetik1, Vildan Bozok-Cetintas1, Ali Sahin Kucukaslan1, Gokhan
Keser2, Vedat Inal2, Nejat Topcuoglu1, and Zuhal Eroglu1
1
Department of Medical Biology, Medical School, Ege University, Izmir,
Turkey, 2Department of Internal Medicine, Division of Rhematology,
Medical Faculty, Ege University, Izmir, Turkey
Objective: Rheumatoid arthritis (RA) is a chronic disease, affects about 0.51% of the population and develops complications such as pain in joints, limitations
in function and increased risk for cardiovascular disease. RA treatment begins
with methotrexate (MTX) which is one of the most widely used disease-modifying
antirheumatic drug. It is announced that in treatment with MTX, behind the environmental factors, variability in the genetic background of individuals can contribute to efficacy and toxicity. It is possible that alterations in the genes of folate metabolism enzymes could be related to these differential effects. Methylenetetrahydrofolate reductase (MTHFR) and Methionine Synthase (MS) enzymes are strong
candidates for mediating the effects of MTX. We investigated whether single nucleotide polymorphisms in the MTHFR and MS genes are associated with the toxicity of MTX in a group of Turkish patients with RA.
Material and Methods: We studied 54 consecutive RA patients treating
with MTX, and 50 healthy controls. MTX toxicity was developed at 18 patients
(17.3%) in the RA group during treatment. MTHFR C677T, A1298C and MS
A2756G polymorphisms were analyzed by real-time PCR & melting curve analysis and RFLP methods, respectively.
Results: Patients with RA had higher frequency of the mutant MTHFR
C677T genotype TT (14.8% vs 2%, P< 0.05). However we did not find any
association between MTX toxicity and genotypes of the C677T, A1298C and
A2756G polymorphisms.
Conclusions: We could not show any significant association between MTX
toxicity and MTHFR C677T, A1298C and MS A2756G gene polymorphisms in
the small group of Turkish RA patients. Genetic factors and limited number of
patients in each subgroup may explain the lack of association in this study. Cardiovascular disease is one of the major risks of early death in RA patients and
C677T gene polymorphism is a genetic risk factor for this complication. Thus,
we suggest that RA patients with TT genotype may have a risk of early death
due to the cardiovascular disease higher than RA patients with CC or CT genotype and general population.
S12-5
STRESS PROTEINS AND HEME OXYGENASE
Aysel Aricioglu
Department of Medical Biochemistry, Gazi University, Ankara, Turkey
Heat shock proteins (HSP) are a group of molecules whose expression is
increased when cells are exposed to elevated temperatures or other stress conditions
such as; infection, inflammation, exercise, exposure to toxic compounds, starvation,
hypoxia or water deprivation. Although HSPs are upregulated under stress, they
also occur under physiologic conditions. The function of heat-shock proteins is similar in virtually all living organisms. HSPs are involved in protein folding and most
of them are described as molecular chaperons. These proteins are named according
to their molecular weights such as; Hsp70 for the 70 kDa. HO enzyme system is a
microsomal enzyme which has three known isoforms (HO-l, HO-2, HO-3). Heme
oxygenase (HO, EC 1.14.99.3) catalyzes the first and rate-limiting step in heme
degradation. HO degrades heme to carbonmonoxide, iron and biliverdin. Heme oxygenase-l (HO-I), also been classified as heat shock protein 32, has recently seen an
explosion of research interest due to its newly discovered physiological effects.
HO-l is an inducible isoform in response to stress such as oxidative stress, hypoxia,
heavy metals and cytokines. HO-2 is a constitutive isoform that is expressed under
homeostatic conditions. HO-3 displays a high sequence homology with HO-2 but
has a little enzymatic activity. HSPs appear to play significant roles in many of
human diseases such as Alzheimer’s, cardiovascular disorders and lung diseases.
Ultimately, the challenge remains of applying the therapeutic potentials of HO-l to
the treatment of human diseases.
S12-6
3’-UTR HAPLOTYPES IN FMF PATIENTS
Aris Cakiris, Fulya Cosar, Neslihan Abaci, and Duran Ustek
Department of Genetics, DETAE, Istanbul, Turkey
The MEFV mutations can be detected in the majority of familial Mediterranean fever (FMF) patients, but there is an important proportion of patients with
FMF phenotype who carry no coding region mutation. This study aimed to
investigate the promoter region and 3’-UTR polymorphisms of the MEFV gene
in a group of FMF patients with no coding region mutations. The study group
consisted of 289 FMF patients FMF and 103 ethnically-matched healthy individuals. All individuals were first genotyped for five most commonly observed
mutations. All exons of the MEFV gene were screened in patients carrying none
of the 5 mutations. The promoter and 3’-UTR regions of the MEFV gene were
investigated in the remaining patients with no mutation, and we identifed 36
patients (12.5%) with no coding region mutation. Analysis of the 3’-UTR region
showed two Alu repeats, which were located in the 3’-UTR of the reference
mRNA sequence. Sequencing of the 3’-UTR of the MEFV gene showed several
SNPs that were clustered in 2 haplotypes. With the genotyping of all study group
for two of the SNPs, we observed a significant increase in the frequency of heterozygotes for 3’-UTR haplotypes in FMF patients with no mutation compared
to healthy controls (75% versus 48.5%, P50.006, OR53.2). This study showed
a group of 3’-UTR polymorphisms in the MEFV gene, which are clustered in
two haplotypes; a genetic association was observed between the 3’-UTR polymorphisms and FMF patients with no coding region mutations.
S12-7
PHYSIOLOGICAL AND MOLECULAR RESPONSES TO CALORIC
RESTRICTION IN NON-OBESE HUMANS
Eric Ravussin, Anthony E. Civitarese, Leanne M Redman, and the
Pennington CALERIE Group
Pennington Biomedical Research Center, Baton Rouge, LA, 70808,
United States
Caloric restriction (CR) extends life span and retards age-related diseases in
rodents and lower species. Several biomarkers of longevity (glucose, insulin, core
body temperature and DHEAS) have been identified in rodents and monkeys but
whether CR improves these in humans is unknown. Furthermore, the mechanism
through which CR increases life span is unclear. One hypothesis is that metabolic
rate is reduced beyond that expected for the reduction in metabolically active
307
mass leading to reduced oxidative damage and possibly improvement in mitochondrial function. 48 healthy (36.861.0 y), nonsmoking, overweight (BMI 27.860.7)
men and women were randomized into one of four groups for 6 months; Control
5 100% of energy requirements; CR 5 25% restriction; CR1EX 5 12.5% CR
112.5% increase in energy expenditure by structured exercise; LCD 5 low calorie diet until 15% weight reduction followed by weight maintenance. Individual
diets were provided to subjects for 2 weeks before baseline tests, during the first 3
months of intervention and again for 2 weeks before tests at month 6. At baseline
and month 6, body composition was measured by DEXA, 12-h fasting blood samples were collected, sedentary 24h energy expenditure (24h-EE) and 24h core
body temperature were determined in a metabolic chamber and a muscle biopsy
was taken. Weight change at M6 was –1.061.1% (Control), –10.460.9% (CR), –
10.06 0.8% (CR1EX) and -13.960.8% (LCD). At M6, fasting insulin levels
were significantly reduced from baseline in the CR, CR1EX and LCD groups (all,
p\0.01), whereas DHEAS and glucose levels were unchanged. Core temperature
was reduced in CR group by 0.260.05 8C and by 0.360.08 8C in the CR1EX
group (both, p\0.05). At baseline, fat-free mass accounted for 86% of the variance in sedentary 24h-EE. After adjustment for changes in body composition,
sedentary 24h-EE was unchanged in controls (-18652 kcal/d; p[0.05), but
decreased in the CR (-135642 kcal/d), CR1EX (-117652 kcal/d) and LCD
(-125635 kcal/d, groups (all, p\0.008). These ‘‘metabolic adaptations’’ (6%
more than expected based on loss of metabolic mass) were statistically different
from controls (p\0.05). Serum protein carbonyl concentrations and urinary isoprostanes excretion rates were not changed from baseline to month 6 in any group,
whereas DNA damage was reduced from baseline in the CR, CR1EX and LCD
groups at month 6 (p 0.005) but not in the Control group. CR and CR1EX was
associated with an increase in the muscle expression of genes involved in mitochondrial biogenesis and mitochondrial fusion including PGC1-a, mitochondrial
transcription factor A, endothelial nitric oxide, SIRT1 and PSARL (all, p\0.05).
In parallel, mitochondrial content increased by 3565% in the CR group (p\0.05)
and 2164% in the CR1EX group (p\0.05), with no change in the control group
(262%). However, the activity of key mitochondrial enzyme of the TCA cycle
(citrate synthase), b-oxidation (b-HAD) and electron transport chain (COX II)
were all unchanged. This study suggests that 6 months of CR in non-obese humans
was sufficient to improve biomarkers of aging and supports the theory that 24h-EE
is reduced beyond that expected due to reduced metabolic size. Whether this metabolic adaptation translates into overall reduced oxidative damage remains to be
determined. The increased mitochondrial content in association with a decrease in
whole body DNA damage is however, an important indication that CR improves
mitochondrial function in human skeletal muscle.
S12-8
CONTRACTILE TENSION OF ENDOTHELIAL CELLS: AN LPS
BASED IN-VITRO SEPSIS MODEL
Eylem Kurulgan-Demirci1, Peter Linder1, Taylan Demirci2,
Jürgen Trzewik3, Ilja Digel1, Gerhard M. Artmann1,
and Aysegul Temiz-Artmann1
1
Laboratory of Cell Biophysics, Medicine and Molecular Biology, Aachen
University of Applied Sciences, Institute of Bioengineering, Ginsterweg 1,
52428, Juelich, Germany, 2Department of Medical Biology and Genetics,
Turkey Health Science Institute of Dokuz Eylül University, Izmir, Turkey,
3
Research and Development, Johnson & Johnson Medical GmbH, 22851
Norderstedt, Germany
Cellular force is the mechanical tension generated by the cell and is crucial
for control of cell shape and function (e.g. endothelial cell barrier). CellDrum
technology, which was developed in our laboratory, was used to study force, produced in-vitro by HAoEC (Human Aortic Endothelial Cell). This method permits
quantitative measurement of the force generated by the defined type cells under
the influence of lipopolysaccharide (LPS), an endotoxin, causing endothelial barrier dysfunction. Loss of endothelial barrier function is one of the hallmarks of
sepsis. Sepsis, a devastating disease, is the systemic inflammatory response to
infection. It is the most common cause of shock. Gram-negative septic shock
comprises 50% of total cases of sepsis. It is well known that sepsis causes multiple organ dysfunctions. Here we show that exposure of confluent human endothelial cells to LPS induces Interleukin 6 (IL- 6) formation. It is used to prove
the in-vitro LPS induced endothelial model, and causes increasing of actin stress
fibers, leading to an increase in the cellular tension. IL 6 level was shown by
308
ELISA method, actin stress fiber formation was showed by phalloidin staining
and cellular tension results were obtained by CellDrum technology. Results of IL
6 levels and cellular tension of the samples treated with LPS were significantly
different when compared to control. Time course and dose response effects of
thrombin were used as a positive control to confirm the results of CellDrum system. Therefore, the system can facilitate analysis of the experiments to study the
cellular tension and its role in the diseases. It is planned to use high-throughput
of the CellDrum system in drug screening.
S12-9
THE VALUE OF THE GENETICS OF DEMENTIA IN PRIMARY CARE
Guldal Izbirak
Yeditepe University Medical School, Istanbul, Turkey
Dementia is an acquired, generalized, neurodegenerative disorder and characterized with progressive impairment of cognitive function which has effect on the
content of consciousness but not on the level. There are a wide variety of diseases
which can cause dementia. Alzheimer’s disease is responsible for most of the
cases. The etiology for Alzheimer’s disease is multifactorial and increasing age
and positive family history are the two most important risk factors. Two categories
of genetic defects have been defined; those that cause early-onset of Alzheimer’s
disease and those involved in late-onset of Alzheimer’s disease. Early onset is rare
and accounts for \5% of cases. Three rare forms of autosomal-dominant earlyonset Familial Alzheimer’s disease have been identified and are associated with
mutations in amyloid precursor protein, presenilin 1, and presenilin 2 genes on
chromosomes 14, 1, 21. The late-onset form of Alzheimer’s disease is more common. The only clearly identified genetic risk factor for late-onset of Alzheimer’s
disease is Apo lipoprotein E (APO E). The epsilon4 allele of Apo lipoprotein E
influences age at onset of Alzheimer’s disease, but is neither necessary nor sufficient for the disease, whereas epsilon2 allele of Apo lipoprotein E may be protective. There are ongoing studies related to additional genetic influences. The families may ask their family physicians for Alzheimer’s blood test. Testing for genetic
mutations in patients with early-onset of Alzheimer’s disease is not clinically useful because it does not change the medical approach; also in patients with lateonset of Alzheimer’s disease, use of apolipoprotein E genotyping as a tool for
investigating epidemiologic risk factors for AD will be useful for scientific studies
in that area. Preventive strategies may be arranged for patients at risk and, in primary care, a multidisciplinary approach should be aimed at involving medical,
social, psychological support and genetic counseling.
U.S.A). Our results represent the first gene network analysis In Turkey. Here we
define the importance of bringing samples to the microarray laboratory in safe
conditions and value of RNA integrity number. This technology is very useful to
suggest new pathognomonic- prognostic markers and new therapeutic targets.
S12-11
EXPRESSION OF GROWTH ARREST-AND DNA DAMAGE
INDUCIBLE GENE IS SIGNIFICANTLY DIFFERENT IN METABOLIC
SYNDROME
Hoorieh Saghafi1, Mohamad Jafar Mahmoodi2, Arash Hossein-Nezhad1,
and Bagher Larijani3
1
Department of Bio & Nano Technology Unit, Endocrinology and
Metabolism Research Center of Tehran University of Medical Sciences,
Iran, 2Department of Cardiology, Tehran University of Medical Sciences,
Iran, 3Endocrinology and Metabolism Research Center of Tehran University
of Medical Sciences, Iran
Aim: We evaluated the expression of growth arrest-and DNA damage inducible gene (Gadd45) and MnSOD in metabolic syndrome and its components.
Methods: Totally 90 participants in two groups including metabolic syndrome and healthy controls were recruited from outpatient clinic. RNA was
extracted from fresh peripheral blood samples. After synthesis of cDNA in
reverse transcriptase reaction quantitative real-time PCR were performed to evaluate expression of target genes including Gadd45, MnSOD genes. For internal
control, the expression of b-actin gene was evaluated.
Results: There was no significant difference in the expression of MNSOD
gene between two groups. In metabolic syndrome, the expression of Gadd45
gene was significantly higher compared to healthy controls (P50.02). Among
metabolic syndrome components, only hypertension and hyperlipidemia were
associated with expression of the Gadd45 gene. No relation was found between
expression of MNSOD and Gadd45 genes with impaired fasting glucose and
other metabolic syndrome components.
Conclusions: Our finding shows that expression of the Gadd45 gene is significantly different in metabolic syndrome. It is consistent with the previous findings showed that Gadd45 gene transcription is induced by both FOXO3 defending against oxidative stress and SIRTI protecting metabolic consequences of
chronic exposure to high-fat-diet.
S12-12
S12-10
GENE NETWORK AND CANONICAL PATHWAY ANALYSIS IN
HEMATOPOIETIC AND SOFT TISSUE ORIGINATED MALIGNANCIES
Hakan Savli1, Naci Cine1, Deniz Sunnetci1, Nilufer Uzulmez1,
Balint Nagy2, Sara Galimberti3, Kemal Baysal4, Temduang Limpaiboon5,
and Pornngarm Limtrakul6
1
Medical Genetics Department and Clinical Research Unit, University of
Kocaeli, Turkey, 2Laboratory, 1st Department of Obstetrics and
Gynecology, Semmelweis University, Hungary, 3Department of
Hematology, Catania University, Italy, 4Gen Muhendisligi ve Biyoteknoloji
Enstitusu TUBiTAK, Turkey, 5Faculty of Associated Medical Sciences,
Khon Kaen University, Thailand, 6Biochemistry Department, Chiang Mai
University, Thailand
We performed gene expression analysis in hematopoietic tissue, ovarian cancer, prostate cancer, cervical cancer; breast cancer, endothelial cell lines, preeclampsia and HELLP syndrome, using microarray technology in University of
Kocaeli. ABI (Applied Biosystems, Foster City, CA, US) and Aglient (Aglient
Technologies, Palo Alto, CA) platforms were used as microarray chips. Obtained
data were analysed by using Gene Spring (GeneSpring 6.1, Silicon Genetics, Redwood City, CA) and Ingenuity Pathway Analysis (IPA) (Ingenuity Systems,
Mountain View, CA, USA) software programs for gene network and canonical
pathway analysis. Array results were confirmed using Quantitative Real Time
PCR (LightCyder, Roche Diagnostics GmbH, Mannheim, Germany) and TaqMan1
Low Density Array Human Apoptosis Panel (TaqMan1, Applera, Norwalk,
EFFECTIVE HYPERICIN CONCENTRATIONS DIFFER IN
ANTIANGIOGENIC VERSUS ANTITUMORAL CYTOTOXIC EFFECTS
IN VITRO
Ladislav Mirossay, Viktoria Stupakova, Lenka Varinska, Andrej Mirossay,
and Jan Mojzis
Department of Pharmacology, Faculty of Medicine, P.J. Safarik University,
Slovakia
Hypericin is the most powerful, naturally occurring photosensitizer and, as
such, there is renewed interest in the potential of this compound for anticancer
photodynamic therapy (PDT). The purpose of this study was to investigate the hypericin-mediated PDT effects on normal human umbilical endothelial cells
(HUVECs) in comparison with cancer human glioma cell lines U-87 MG and U373 MG, in in vitro conditions. Our data suggest that endothelial cells as well as
glioma cell lines are sensitive only to photoactivated hypericin. The inhibitory
effects of photoactivated hypericin did not differ in endothelial compared to tumor
cells in cytotoxicity and DNA fragmentation. However, the important difference
in sensitivity have been found between the above mentioned cell types in migration and metalloproteinases inhibition assays performed as cell function tests. The
findings in both functional tests and in additional angiogenesis test of tubular formation in vitro were indicative of a higher sensitivity of endothelial cell. In summary, inhibition of basic HUVEC’s angiogenic functions was found at significantly lower (non cytotoxic) concentrations of hypericin compared to direct antitumoral (cytotoxic) effects observed in tumoral cell lines. This work was supported
by the Slovak Research and Development Agency under the contract No. APVV0325-07 and by the Slovak Grant Agency for Science (grant No. 1/4236/07).
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S12-13
S12-15
THE ROLE OF HLA TYPING IN THE DIAGNOSIS OF
AUTOIMMUNE DISEASES
MICROSATELLITE INSTABILITY MARKERS PROFILE IN PATIENTS
WITH COLORECTAL CANCERS CAUSED BY GERM-LINE
MUTATIONS IN DNA MISMATCH REPAIR GENES
Luca Mascaretti
Immunogenetics Laboratory, San Gerardo Hospital, Italy
Since autoimmune diseases (AD) are multifactorial in that they are influenced by both genetic and environmental factors, it has been quite difficult to
assign to the Human Leukocyte Antigen System (HLA) a precise role in these
diseases. In the over 2600 papers published in the past 3 decades on this subject,
there are many conflicting data. The aim of the present paper is to review the
practical importance of HLA in AD; what can HLA add to other diagnostic
tests? Under what circumstances can HLA guide clinicians in the choice of further (perhaps more invasive) tests? At first, mechanisms will be discussed,
namely linkage disequilibrium and presentation of autoantigens to autoreactive T
cells. Evidence for HLA association in the following AD will be briefly reported:
celiac disease, type I diabetes, ankylosing spondylitis, seronegative spondyloarthritis, Behcet disease, uveitis, rheumatoid arthritis, Juvenile arthritis, narcolepsy,
multiple sclerosis and psoriasis. In consideration of the most recent genome-wide
association scan studies, a broader view of the genetics of AD will be illustrated
in which in addition to HLA genes, other immune response genes appear to be
important. Finally, results of a survey conducted on 68 Italian HLA typing laboratories, which perform HLA-AO association studies, will be reported. Data
show that although the number of Italian patients who underwent HLA typing
for disease association studies in 2007 was impressive (23,490), typing
approaches lacked homogeneity and were sometimes redundant. A guideline is
therefore warranted In order to optimize resources and to support HLA laboratories and clinicians in the choice of the best typing strategy for the final benefit of
our patients.
S12-14
DOES MANNOSE-BINDING LECTIN PLAY A CRITICAL ROLE IN
MYOCARDIAL ISCHAEMIA?
Lulufer Tamer-Gumus
Mersin University, Medical Faculty, Deparment of Biochemistry,
Mersin, Turkey
Coronary artery disease (CAD) is an important cause of mortality and morbidity. About 200.000 new coronary events are happening every year in our
country. It is estimated that approximately 130.000 people die because of acute
myocardial infarction (AMI) every year. The CAD incidence in our country has
been determined by TEKHARF study that the incidence in high risk age group
60-69 is 14% where it is 3.8% in adults. Cause of Death in Turkey has been
determined by ‘Türk Kalp Report’ that had been carried out by Turkish Cardiology Society and found that CAD is responsible from 35% of all deaths. CAD is
a multifactorial disease, defined as disease process generally caused by presence
of an atheromatous plaque that limit or block blood stream in coronary arteries.
Gender, age and hereditary reasons are cardiovascular risks that cannot be modified where other risk factors like tobacco consumption, hypertension, insulin resistance, diabetes and hyperlipidemia are modifiable risk factors. Additionally
some candidate risk factors like CRP, homocysteine and lipoprotein(a) are
defined. Recently with determination of the role of infectious agents in formation
and development of atherosclerosis, scientists focus on infection as a cause of
atherosclerosis. There are many infectious agents determined in atherosclerotic
plaques. Within those infectious agents C. pneumoniae and Helicobacter pylori
are well investigated. After determination of C. pneumoniae in atherosclerotic
plaques, many studies looked for a relation between C. pneumoniae, CAD and
atherosclerosis. In hyperlipidemic animal models, it has been shown that C.
pneumoniae triggered lesion formation and inflammation. Additionally C. pneumoniae can survive in atherosclerotic plaques. An ancestral innate immunity
member, mannose-binding lectin (MBL), acts on carbohydrate residues on microorganisms resulting in immune response. These immune responses are antibody
independent opsonisation and complement activation. C. pneumoniae is one that
is recognized by MBL. In vivo studies showed that MBL reduces C. pneumoniae
infection.
Mahdi Montazer Haghighi1, Seyed Reza Mohebbi1, Mahsa Molaei2,
Somaye Ghiasi3, Reza Fatemi1, and MohammadReza Zali1
1
Department of Cancer, Research Center for Gastroenterology and Liver
Diseases, Iran, 2Department of Pathology, Research Center for
Gastroenterology and Liver Diseases, Iran, 3Department of Registry,
Research Center for Gastroenterology and Liver Diseases, Iran
Background: Hereditary non-polyposis colorectal cancer (HNPCC also
known as Lynch syndrome) is the most common cause of early onset hereditary
colorectal cancer. In the majority of HNPCC families, microsatellite instability
(MSI) and germline mutation in one of the DNA mismatch repair genes (MSH2,
MLH1, PMS2, MSH6) are found. Tumors developing through this pathway have
alterations in the length of short, repeated (usually mononucleotide or dinucleotide) sequences of DNA, i.e., microsatellites. The aim of the study was to examine microsatellite markers stability in order to find the most effective marker to
predict early-onset in patients with colorectal cancer caused by germ-line mutations in MMR genes.
Methods: Patients (n534) from 592 patients who were referred between
2002 and 2007 to Taleghani Hospital Tehran. They were selected for screening
to detect mutations in MMR genes based on family history, Immuno-histochemistry (IHC) and microsatellite instability (MSI). The MSI was performed using a
pentaplex marker panel (BAT-26, BAT-25, NR-21, NR-24 and NR-27). MSI
were investigated by PCR and fragments analysis to find other instabilities. Furthermore, in order to detect mutations, the entire coding sequence of each gene
was analyzed using direct sequencing.
Results: We could find 34 mutations in the patients. The MSI result of
related to the patients with the mutations revealed that in BAT26 marker 28 vs 6
cases have instability, in BAT 25 marker there were 21 vs13 patients with instability NR21 marker showed 22 unstable vs. 12 stable, NR24 marker revealed 26
unstable vs. 8 stable and finally in the case of NR27 marker the number of people with instability was equal with patients 17/17.
Conclusion: We could find NR27 marker, a quasimonomorphic polyA
stretch located in 5, UTR inhibitor of apoptosis 1 gene, is considered the most
sensitive and specific marker of MSI to prediction and screening HNPCC in Iranian population.
S12-16
IDENTIFICATION OF AMADORI-MODIFIED IMMUNOGLOBULIN
G IN TYPE 2 DIABETES PATIENTS WITH SECONDARY
COMPLICATIONS
Nadeem A. Ansari and Moin Uddin
Department of Biochemistry, JN Medical College, Aligarh Muslim
University, India
Hyperglycemia induced nonenzymatic glycation products are increased in diabetes and complications. The glycation of proteins mostly occur at intra-chain lysine residues. Many studies have focused on the role of advanced glycation endproducts (AGEs) in diabetes complications but the role of early glycation Amadori-modifed proteins has not been extensively investigated. In the present study,
we have obtained Amadori-rich glycated poly-L-lysine and developed an antiserum in experimental animals. Serum antibodies have been used to identify early
glycation products on in vitro glycated IgG and on the IgG purified from type 2 diabetic patients with secondary complications. UV, CD and NMR Spectroscopy
were applied to characterize the glycated poly-L-lysine. High content of Amadori
products was evaluated by fructosamine assay. Patients with chronic hyperglycemia and long-term history of type 2 diabetes mellitus with diabetic nephropathy,
retinopathy and atherosclerosis have been analyzed in the study. Normal healthy
subjects were taken as control. IgG from sera of the subjects was purified by affinity chromatography. IgG, obtained from the normal subjects, was glycated with
the procedure used for glycation of poly-L-lysine. The binding characteristics of
the induced antibodies have been assessed by ELISA. We have observed an
increase in UV absorbance and significant changes in ellipticity in the CD spec-
310
trum of glycated poly-L-lysine when compared to the native molecule. Resonance
signals corresponding to glucose were detected in 1H-NMR spectrum of the glycated poly-L-lysine. Anti-glycated poly-L-lysine antibodies showed appreciable
recognition of the IgGs obtained from type 2 diabetes patients having secondary
complications as well as the in vitro glycated IgG while the recognition of IgG
from the normal subjects was insignificant. Our results indicate the presence of
glycation induced damage caused by Amadori-modified lysine residues on the IgG
molecule in type 2 diabetes patients having associated secondary complications.
Thus, Amadori-modified IgG might serve as a potential biomarker and a target for
controlling progression of the disease complications.
S12-17
THE FIRST IDENTIFIED ENDOGENOUS ENDOPLASMIC
RETICULUM (ER)-ASSOCIATED DEGRADATION (ERAD)
INHIBITOR: SVIP
Petek Ballar1, Yongwang Zhong2, Yuxian Shen2, and Shengyun Fang2
1
Department of Biochemistry, Faculty of Pharmacy, Ege University, Izmir,
Turkey, 2Medical Biotechnology Center, University of Maryland,
Biotechnology Institute, Baltimore, United States
Objective: ERAD plays a major role in protein quality control system,
ensuring that misfolded proteins cannot transit through the secretory pathway. It
involves misfolded protein recognition, ubiquitination, retrotranslocation, deglycosylation, and proteasomal degradation. We have previously reported that direct
interaction between gp78, an ER-resisdent ubiquitin ligase, and p97/VCP, an
ATPase, couples the ubiquitination with retrotranslocation during mammalian
ERAD1. Furthermore, gp78 interacts with and recruits p97/VCP to the ER membrane via a novel p97/VCP interacting motif (VIM). Interestingly, a highly conserved VIM is also found in the small p97/VCP interacting protein (SVIP). Here,
we focused on characterization of SVIP and its role in ERAD.
Methods: 293 cells stably expressing substrates are established. Immunoblotting and immunoprecipitations are performed as reported. Silencer1 predesigned
siRNAs are purchased from Ambion. Polyclonal anti-SVIP antibodies were generated by immunizing rabbit with purified GST-SVIP. Cells were transfected
with Lipofectamine-2000 or calcium phosphate precipitation. Microsomes were
isolated as described and alkaline extraction; gradient fractionation and proteinase K digestion were performed as reported. 1 mM 2-OHM (2-hydroxymyristic
add) was delivered to 293 cells and cells were incubated for 24h.
Results: SVIP a membrane anchored protein by myristoylation. It is localized to ER membrane and a member of ERAD complex. We hypothesized that
regulation through VIM-binding proteins is a mechanism by which ERAD activity is regulated. Indeed, VIM in gp78 is essential for degradation of ERAD substrates CD3d and the Z variant of a-1-antitrypsin; however, SVIP via its VIM
inhibits the degradation of these proteins. We also demonstrated that SVIP inhibits ERAD via negatively regulating the formation or multisubunit gp78 complex.
Conclusion: By sharing VIM, SVIP compete with gp78 to interact with p97/
VCP and Derlin1, thereby negatively regulating the assembly of gp78-p97/VCPDerlin1 complex. Thus, SVIP is an endogenous inhibitor of ERAD.
S12-18
UBIQUITIN LIGASES AS TARGETS FOR DRUG DISCOVERY
Shengyun Fang1 and Petek Ballar2
1
Medical Biotechnology Center, University of Maryland Biotechnology
Institute, Baltimore, MD, USA; 2Ege University, School of Pharmacy,
Biochemistry Department, Izmir, Turkey
Breakthroughs in our understanding of the ubiquitin-proteasome system provide enormous potentials for drug discovery. Many human diseases are associated with disturbances of this system. However, only one drug, namely VelcadeTM that targets this system is commercialized for the treatment of patients with
multiple myeloma. VelcadeTM is an inhibitor of proteasome activity and blocks
degradation of many proteins. Therefore, ubiquitin ligases (E3s) that regulate
only one or a few substrates are expected to be more specific drug targets. However, the functional roles of many E3s in physiology and pathology are still not
fully understood. Hundreds of human proteins in database are predicted to be
E3, but only a small fraction of them has been studied. Therefore, identification
and validation of novel E3s as drug targets are among the current focuses in
drug discovery. Despite our incomplete understanding of the biological roles and
mechanisms of the ubiquitin system, researches have identified direct links
between some E3s and disease development. We have characterized two E3s,
mdm2 (Hdm2) and gp78, which play critical roles in tumorigenesis and metastasis. We discovered that mdm2, overexpressed in about 5-10% of all tumors, is a
RING finger E3 for the tumor suppressor protein p53. Overexpressed mdm2 suppresses the activation of p53, thereby abrogating the tumor suppressor function
of p53 and causing tumors. gp78 is an endoplasmic reticulum-localized RING
finger E3. Its expression is upregulated in various cancers. A recent study suggests that gp78 promotes sarcoma metastases by targeting the metastasis suppressor, KAI1/CD82, for proteasomal degradation. Using mdm2 as an example, we
have established a high throughput assay for screening inhibitors of mdm2 ubiquitin ligase activity. In preliminary screens, a family of three closely related 7nitro-5-deazaflavin compounds were identified as specific inhibitors of mdm2
with IC50 values of about 20 lM. These studies provide a proof of principle for
E3-targeted drug discovery.
S12-19
THE EFFECTS OF SOME PROBIOTICS ON GTFB AND GTFC GENE
EXPRESSION LEVELS IN BIOFILM PRODUCING S. MUTANS BY
REAL-TIME RT-PCR
Rasoul Salehi, Arezo Tahmourespour, and Gilda Eslami
Department of Genetics and Molecular Biology, Isfahan University of
Medical Sciences, Iran
Dental caries and periodontal diseases are major public health problems in
the world that occur in nearly 95% of the general public. Although fluoride and
other preventive efforts have led to a dramatic decline in dental caries, the ability
to control the actual infection has been limited and dental disease remains a
‘‘silent epidemic’’ in the world. Streptococci are the pioneer strains in plaque formation and mutans Streptococci are the main etiological agent of dental plaque
and caries. In general, biofilm formation is a step-wise process, which begins by
adhesion of planktonic cells to the surfaces. Evidence shows that expression of
required genes, such as glucosyltransferase B and C (gtfB and gtfC) is well-regulated after initial adhesion and results in formation of dental plaque, caries and
other periodontal disease. The aims of this study was to determine the effect of
some probiotic strains such as Lactobacillus fermentum ATCC9338, L. acidophilus DSM 20079, L. rhamnosus ATCC 7469 and their biosurfactants on the Streptococcal adhesion specially S. mutans as the main infectious agent of dental plaque and caries. Oral streptococci from dental plaque and caries were isolated and
identified. Also their biofilm formation potential (by slide and microtiter plate
test) was assessed. Also assessment of antimicrobial and anti-adhesive properties
of the probiotics (with 4 methods) and their biosurfactans (after production,
extraction, and partial characterization by drop collapse test and FTIR) have
been performed. Then the effect of biosurfactants on gtfB & gtfC gene expression level was quantified by using Real-time RT-PCR. The results showed that
about 47.5% of isolated Streptococci were mutans Streptococci. In the presence
of probiotic strains, the adhesion of Streptococci were reduced and this reduction
was significantly more when the whole culture of probiotic strain was inoculated
to the system 30 minutes before the oral Streptococci (25% adhesion reduction).
No antimicrobial effects were observed. In the biofilm environment, the studied
biosurfactants significantly decreased gtfB and gtfC expression in the S. mutans
ATCC35668 and some of the clinical isolate S. mutans with different profile. It
is concluded that the reduction caused by biosurfactants in the adhesion process
could be due to decreased in gtfs gene expression profile.
S12-20
TARGETING GENE DELIVERY IN GENE CELL AND STEM
CELL THERAPY
Renad Zhdanov1, Ahmet Arslan2, and Fikrettin Sahin1
1
Department of Genetics and Bioengineering, Yeditepe University, Istanbul,
Turkey, 2Department of Molecular Biology and Genetics, Gaziantep
University, Turkey
Delivery of therapeutic genes to specific cells and tissues remains one of the
most important problem of gene and gene cell therapy, because of genetically
modified cells are used in many clinical trials for autologous or heterologous cell
transplantation both in vivo and ex vivo to cure neurological disorders, cancer, and
some other diseases. The talk will cover the state-of-art research in the field of
gene targeting, taking examples from our own research. Targeted viral gene delivery is widely used in gene therapy to cure, e.g. neurological diseases (herpes simplex viruses demonstrate an affinity to neural tissues) or diseases involving epithelium/endothelium location (adenoviral vector constructs of five generations). Targeting non-viral vectors for effective gene delivery could be managed using any
addressing groupings, e.g. folic acid conjugated lipoplexes for transformed cells in
cancer gene cell therapy. Great advantage in gene cell therapy could be attained by
use of transduction peptide (e.g. Pen1) or oligosaccharide addressing moieties specific to surface receptors or certain lectins of certain tissues. Use of nuclear localization signal in lipoplex formulations can dramatically improve efficiency of nonviral formulations. Varying lipid structure is another method to improve biodistribution of lipoplexes. Specific expression of therapeutic genes in certain cells and
tissues can be also reached using tissue-specific promoters while corresponding
genetic constructs are prepared. Use of radiation inducible promoters (e.g. in
Ad.EGR-TNFa vector) can up-regulate gene expression; only in the case those
cells will be given radiation dose. Thus, the effect of radiation could be potentiated
by antivascular TNFa gene therapy. Special attention is paid to gene cell and stem
cell therapy of neurodegenerative diseases and to achievement of efficient transfection of therapeutic genes including genes of defined transcription factors against
umbilical cord blood stem cells and embryonic like and adult stem cells as well.
311
been eliminate after starting clinical studies as a result of inadequate pharmacokinetics. Recently, in vitro bioavailability and bioequivalence studies have been
come into prominence because of their importance for prediction of in vivo bioavailability of drugs. Orally administered drugs must cross the gastrointestinal barrier, if they are to reach their systematic therapeutic target sites. Therefore, highthroughput assay are needed to meet the increased demand for screening of permeability characteristics. Especially, for estimation of orally administered drug
permeability, some in vitro intestinal permeability testing system have been
developed for last decade. One of these systems PAMPA (Parallel Artificial
Membrane Permeability Assay) is a non-cell based assay designed to predict passive trans-cellular permeability. The other common HTS permeability assay system is Caco-2 permeability cell based drug transport assay system. Both of these
cell and non-cell based permeability assays, are critical for the early drug discovery and development process. They are also very important assay protocols to
prove bioequivalence of generic drug bioavailability to original drug. Both of
these assay systems and p-glycoprotein (calcein AM assay) studies, were used
for testing permeability characteristics of oxicam derivates in current study. As
oxicam derivates, meloxicam, tenoxicam and lornoxicam were selected for testing their permeability studies. Previously, effects of administrated doses of oxicams on cell viability were determined by using MTT and lactate dehydrogenase
(LDH) assays. According to PAMPA and Caco-2 permeability assays, permeation of oxicam derivates occurred by cellular transport systems and it is possible
that oxicam derivatives are p-glycoprotein substrates. Acknowledgement: This
study was granted by the 3070669 numbered Tubitak TEYDEP project.
S12-23
S12-21
POSSIBLE ROLE OF DIOXIN AND PAH IN MALE INFERTILITY:
A CASE CONTROL IMMUNOHISTOCHEMICAL STUDY ON AHR,
MMP9 AND SEX STEROID RECEPTORS
Sepideh Arbabi Bidgoli1, Mona Karimi1, Zahra Asami1,
Mansour Djamali Zavarhei2, and Hoda Baher1
1
Department of Toxicology & Pharmacology, Pharmaceutical Sciences
Branch, Islamic Azad University, Iran, 2Department of Pathology,
School of Medicine, Tehran University of Medical Sciences (TUMS), Iran
Infertility is one of the major health problems, which affect more than 15% of
couples by unknown mechanisms. Role of Poly Aromatic Hydrocarbons (PAH)
and Dioxin on male idiopathic infertility phenomenon as well as their effects on
angiogenic pathways and sex steroid receptors remained unclear, which was considered as the main goals of present study. Aryl hydrocarbon receptor (AhR) is the
exposure biomarker of mentioned toxicants and its role in male infertility, sex steroid receptors (ER, PgR, AR) and MMP9, as one of the major biomarkers of
angiogenesis were studied in our experimental and case-control study. We compared the differential expression of above receptors in PAH exposed and PAH
unexposed mice as well as 30 infertile and 10 fertile testicular tissue specimens of
males using immunohistochemical analysis. Significant differences between AhR
expression in Leydig, Sertoli, spermatid cells as well as significant differences
between MMP9 expression in Leydig and Sertoli cells were found in the present
study (p\0.05). A significant association between MMP9 and AhR in Leydig cells
was one the major findings of present study which could be interpreted as a possible mechanism of idiopathic male infertility. ER overexpression in Leygid cells,
lack of AR expression in fertile unexposed testicular cells, and significant association between ER and PgR in Leydig cells are other important findings of present
research. These findings could be applied as future diagnostic or therapeutic targets of male infertility by confirming in larger studies.
S12-22
ADIPONECTIN AND C-REACTIVE PROTEIN RELATIONSHIP IN
PLASMA AND ADIPOSE TISSUE (STUDY AMONG HEALTHY OBESE
EGYPTIAN FEMALES)
Somaya Eltabei Mohamed Soliman
Radioisotopes Application, Nuclear Research Center AEA, Egypt
Increasing evidence indicates that adipose tissue is both a dynamic endocrine
organ, as well as, a highly active metabolic tissue, and that adiposity contributes
to a pro-inflammatory milieu (Berg and Scherer 2005). Adipose tissue secretes
adipokines that have been proved to play important roles in the atherosclerotic
process. These include tumor necrosis factor (TNF), leptin, plasminogen activator
inhibitor (PAI-1), interleukin-6 (IL-6), resistin and angiotinsinogen. Serum adipokine levels are elevated in humans with excess adiposity, and visceral fat appears
to produce several of these adipokines more actively than subcutaneous (Sc) adipose tissue. (Fain et al, 2004). Adiponectin (Acrp30), a recently described adipokine of emerging importance is distinct from other known adipokines in that, alone
among them, it appears to improve insulin sensitivity, it inhibits vascular inflammation and it acts as an endogenous modulator of obesity-related diseases. C- reactive protein (CRP) is an acute phase reactant that serves as a pattern–recognition
molecule in the innate immune system and it is a well known systemic marker for
inflammation. Previous prospective studies indicated that a chronic low-grade
inflammation is involved in the pathogenesis of atherosclerosis, and elevated highsensitive CRP (hs- CRP) level was a risk factor for coronary artery disease , peripheral vascular disease and cerebrovascular disease. Elevated plasma hs- CRP
levels were also strongly associated with obesity and obesity – related diseases:
including insulin resistance, diabetes mellitus and hyperlipdaemia . Although, a
recent report indicated that plasma hs – CRP level decreased during weight reduction, the precise interaction of CRP with obesity has not been fully elucidated.
The principal source of CRP production is the liver, however, some data showed
that the arterial tissue can produce CRP as well as complement proteins. These
products and their associated m RNA are substantially up regulated in atherosclerotic plaque, with smooth muscle cells and macrophages the main producers. This
supports the concept that CRP may be an endogenous activator.
IN VITRO INTESTINAL PERMEABILITY STUDIES OF OXICAM
DERIVATIVES
S12-24
Seval Korkmaz and Banu Barutca
Department of Cell Culture, FARGEM (Pharmaceutical Research and
Development Center Inc.), Turkey
GENE EXPRESSION PROFILE ANALYSIS OF 3T3/NIH FIBROBLASTS
AFTER ONE HOUR MECHANICAL STRESS
Taylan Demirci1, Eylem Kurulgan-Demirci2, Trzewik Jürgen2,
Peter Linder2, Ilya Digel2, Gerhard Artmann2, Meral Sakizli1,
and Aysegül Temiz-Artmann2
Pharmacokinetics are widely recognized as an important factor in the drug
discovery and development process, because many candidate compounds have
312
1
Department of Medical Biology and Genetics, Turkey Health Science
Institute of Dokuz Eylül University, Izmir, Turkey, 2Department of Cell
Biophysics, Aachen University of Applied Sciences, Juelich, Germany
Mechanical stretching can induce a response in 3T3 fibroblast cell. A method
based on CellDrum1 Technology provides the mechanical stretching environment to 3T3/NIH cells in-vitro used to define transcriptional profiling of mechanical stretched NIH 3T3 cell on top of a flexible silicone membrane. A time
course between 5 min to 12 hour was applied to decide which time period is
more suitable for evaluating transcriptional profiling. mRNA expression levels of
the genes Egr-1, Fgfr2, P53, Itgb3 and Itgb5 were evaluated by real time PCR
with time course. Most of the mRNA expression levels were significantly
changed after one hour of mechanical stretching. One hour stretched mRNA
samples were hybridized to define expression profile on a whole mouse genome
oligo microarray slide. We found that the early response genes which play a role
in stretch activation of intracellular signal transduction pathways, showed significant increase mRNA of Egr1, Egr2 and Egr3. Fgfr2 mRNA expression were also
significantly up-regulated.
Nuclear proteins such as Oct4, Sox2 and nucleostemin have been shown to play
important roles. As hES cells differentiate, the nuclear proteome undergoes significant changes. Understanding these changes would allow us to develop novel
methods to differentiate hES cells more efficiently. We have compared the hES
cell nuclear proteome with hES cell-derived neuroprogenitor cells using twodimensional difference gel electrophoresis (2-DIGE). We detected 1521 protein
spots matched across three gels. In silico statistical analysis revealed that only
2.1% of the densitometric signal was significantly changed. The ranges of average ratios varied from 1.2 to 11-fold at a statistically significant p-value \0.05.
Tandem mass spectrometry identified fifteen proteins previously shown to be
involved in chromatin remodeling, mRNA processing and gene expression regulation. Therefore, nuclear proteomic analysis of differentiation enabled the detection of proteins affecting gene regulation at different levels. Notably, three members of the heterogeneous nuclear ribonucleoprotein family (AUF-1 and FBP-1
and 2) register a 54%, 70% and 99% increased expression, highlighting a potential role for neurodifferentiation of hES cells. By contrast Cpsf-6 disappears during differentiation with an 11-fold decrease in neuroprogenitor cells, highlighting
this protein as a novel marker for undifferentiated hES cells.
S12-25
S12-27
CLINICAL AND CYTOGENETICS ASSESSMENT OF PATIENTS WITH
CHROMOSOME DISORDERS
STUDY OF GRAPE SEED EXTRACT ON EXPERIMENTAL DIABETIC
NEPHROPATHY IN RAT
Teresa Mattina1, Concetta Simona Perrotta1, Paul D.Grossfeld2,
and Peter Hammond3
1
Medical Genetics, University Catania, Italy, 2Division of Cardiology,
Department of Pediatrics, UCSD School of Medicine, La Jolla, CA, USA,
3
Molecular Medicine Unit, Institute of Child Health UCL, London, UK
We have defined a protocol for the clinical and cytogenetic evaluation of
patients with chromosome 11q disorders. Our protocol can be extended to clinical research concerning any chromosome region. The most detailed information
concerning a chromosome region is obtained by analyzing as many patients as
possible with abnormalities involving different subregions of the same chromosome segment, and by using the same procedures in all cases. We analyzed 126
patients with balanced or unbalanced rearrangements involving the 11q region.
Biannual meetings organized by the European and US 11q family networks
allowed us to obtain a large number of observations with follow up. Collaborations in Europe and in USA covered a number of subspecialties. For homogeneous evaluation, each patient was examined by two clinical geneticists (TM,
CSP). Results were recorded in a 289 items checklist, prepared using definitions
from the OMD. Photographic documentation of face, hands, feet and total body
was always obtained. 3D scanning of facies was performed (PH) in 30 cases and
a dense surface model of facial morphology in 11q terminal deletion was constructed for discrimination testing. The medical history was obtained from each
family by direct interview; translators were available when necessary. All families were invited to fill in a questionnaire, presented in different languages. Definition of the rearrangements obtained by traditional cytogenetics and FISH, was
refined by high resolution array CGH. Combining data from patients having different chromosome abnormalities within the 11q region was rewarding. Our dysmorphological checklist and questionnaire can be used for standardized assessment of children with any chromosome disorder. 3D scanning of facies can be
used to record the facial morphology of any case of rare disorder. If enough
patients with the same condition are available, a 3D surface model of facial dysmorphology of the syndrome can be used to aid phenotype-genotype analysis.
S12-26
COMPARATIVE NUCLEAR PROTEOMICS OF HUMAN EMBRYONIC
STEM CELLS
Ugur Salli, Miguel Barthelery, Amritha Jaishankar, and Kent Vrana
Department of Pharmacology, Pennsylvania State University, PA, USA
Human embryonic stem (hES) cells can replicate indefinitely (self-renewal)
and differentiate into any type of cells (pluripotency). The underlying molecular
mechanisms that account for self-renewal and pluripotency are poorly understood. In addition, hES differentiation methods require further research to generate homogenous populations of desired cell types such as dopaminergic neurons.
Yousef Doustar, Saeed Sedig-Etegad, Siavash Gavami,
and Amir Sefid-Moy-Azar
Veterinary Pathology, Islamic Azad University of Tabriz Branch, Iran
Diabetes mellitus and its complications are major public health problems.
Antioxidants are frequently recommended in the treatment of type I and type II
diabetes mellitus and can be used to decrease oxidative stress. The purpose of
this study was to investigate the effect of grape seed extract on apoptosis in diabetic nephropathy. In this study, 12 weeks old (200-300gr) 56 rats were selected,
and divided into two groups; treatment and control groups. Diabetes was induced
by intraperitoneal injection of streptozotocin (50 mg/kg) in these two groups. All
rats were kept under normal room and food conditions. Grape seed extract was
administered to the treatment group for 12 weeks per day. After 12 weeks renal
tissues were sampled in both groups and 5-6 micron tissue section were prepared
by H&E and TUNEL assay staining method. Histopathological analysis of tissue
sections demonstrated that histopathological changes and apoptosis were significantly different in both groups. We demonstrated that grape seed extract is able
to reduce oxidative stress and apoptotic changes. In addition, grape seed extracts
improve diabetic nephropathy disease in patients by reducing HbA1c, oxidative
stress, hyperglycemia, VLDL, expression of apoptosis regulatory gene and TGFb and increase insulin sensitivity, HSPG, HS, HDL, IGF and EGF.
S12-28
TRANSCRIPTIONAL AND STRUCTURAL ANALYSIS OF AMSACTA
MOOREI ENTOMOPOXVIRUS PROTEIN KINASE GENE
Hacer Muratoglu, Remziye Nalcacioglu, and Zihni Demirbag
Department of Biology, Karadeniz Technical University, Turkey
Amsacta moorei Entomopoxvirus (AmEPV) has 279 unique open reading
frames (ORF). Among these, AMV197 has 900 nt coding for a protein of 299
amino acids. Sequence derived amino acid analysis of AMV197 suggested it to
be a serine/threonine protein kinase (pk) having conserved pk and serine/threonine pk domains. For transcriptional analysis of pk gene, mRNA was isolated
from AmEPV infected Ld652 cells at different time intervals after infection. RTPCR analysis with specific primers for pk indicated that the transcription of the
pk gene started at 4 h post infection, reached to maximum level at 12 h.p.i., and
continued to be transcribed up to 24 h.p.i. Infection of Ld cells in the presence
of Ara-C (inhibits DNA replication), followed by RT-PCR on isolated mRNA
using specific primers for this gene showed that pk is transcribed as early gene.
5’RACE analysis on RNA isolated from AmEPV-infected Ld cells showed that
transcription of pk initiated at position-54 relative to translation start site of the
gene. Rapid amplification of the 30 cCNA ends of the pk transcript showed that
there were two polyadenylation start points. They are located at 22 and 32 nucleotides downstream of the poly(A) signal. Also the translational stop site and
poly(A) signal of pk are overlapped. We also investigated the positions of the
protein kinase subdomains in AMEPV pk amino acid sequence by comparing
with other pk family members. In AMEV pk sequence we clearly identified the
subdomains I, II, VI, VII, VIII and predicted the subdomains III, IV, IX and X.
However, the subdomains V and XI were hard to locate.
S13. Metabolic Syndrome
S13-1
THE ROLE OF CIRCULATORY ADIPOKINASE IN POST PARTUM
METABOLIC SYNDROME
Hoorieh Saghafi1, Arash Hossein-Nezhad1, Zhila Maghbooli1,
and Bagher Larijani2
1
Department of Bio & Nano Technology Unit, Endocrinology and
Metabolism Research Center of Tehran University of Medical Sciences,
Iran, 2Department of Endocrinology, Endocrinology and Metabolism
Research Center of Tehran University of Medical Sciences, Iran
Aim: The aim of this study was to determine the role of adipokinase concentration in early post partum metabolic syndrome.
Methods: In a cohort study, 146 pregnant women were recruited from prenatal care clinic and followed up until 6-12 weeks after delivery. Serum concentration of adipokinase including visfatin, resistin and adiponectin were evaluated by
ELISA method during universal screening for gestational diabetes mellitus.
Results: Postpartum follow up showed that visfatin concentration has no
relation with postpartum developed metabolic syndrome. But adiponectin concentration during pregnancy was lower in women with postpartum metabolic syndrome (6.9662.22 vs. 9.8165.32 mg/ml, P50.02). Resistin concentration during
pregnancy was also lower in women with postpartum metabolic syndrome
(4.4062.22 vs. 5.5561.563 ng/ml, P50.03). Indeed, in logistic regression analysis we did not detected a relation between postpartum metabolic syndrome and
adipokinase independent of age and pre-pregnancy BMI.
Conclusion: Our finding shows that the possible role of obesity in development of postpartum metabolic syndrome may be explained by the adipocytes
secreted adipokinase.
S13-2
RELATIONSHIPS BETWEEN FREE LEPTIN AND INSULIN
RESISTANCE IN WOMEN WITH POLYCYSTIC OVARY SYNDROME
Javad Mohiti-Ardekani1, Nasim Taarof1, and Abbas Aflatonian2
1
Department of Biochemistry, Yazd University of Medical Science, Iran,
2
Department of IVF, Yazd University of Medical Science, Iran
Objective: Patients with polycystic ovarian syndrome have increased insulin
resistance and high incidence of obesity. The obese gene product, leptin play a
central role in food intake and obesity and circulate in both free and bound
forms. The free form is the biologically active leptin.
Design: We assessed the correlation of metabolic parameters with free
and bound form levels in 27 polycystic ovarian syndrome (PCOS) women
(aged 2665.6 years) and 27 healthy controls (aged 25 64 years).
Methods: Total leptin, insulin levels were measured using ELISA-kits. Free
leptin form was purified by Gel filtration chromatography and their collected
fractions were measured by ELISA-kit. Insulin resistance was calculated by
homeostasis model assessment (HOMA).
Results: In PCOS patients and control groups was found a correlation
between leptin and body mass index (BMI). A significant difference was found
between leptin and free leptin in PCOS subject and control (P<0.05). A significant correlations were found between free leptin and leptin with Insulin resistant
in PCOS subject (r50.53 p50.004, r50.69 p50.00) and control groups respectively (r50.57, P50.003, r50.71, p50.002).
Conclusion: The biological active free form of leptin is responsible for insulin resistant and leptin action in body.
313
S13-3
METABOLIC SYNDROME
Kubilay Karsidag
Istanbul University, Department of Internal Medicine, Istanbul, Turkey
Metabolic syndrome (syndrome X) is a complicated syndrome with insulin resistance, hyperinsulinemia, central obesity, dyslipidemia, atherosclerosis, inflammation, polycystic ovarian syndrome. It is necessary to understand the mechanism
of insulin resistance to be able to understand this syndrome. Peripheral insulin resistance (PIR) is the insufficient response to endogenous and exogenous insulin.
Glucose is the main energy source for the organism and it enters the cells through
the action of insulin. Insulin receptor is a complex structure consisting of 2 beta
and 2 alpha subunits connected to each other by disulphide bonds. The alpha subunit is the extracellular part, directly interacting with insulin. The beta subunit is the
biggest part passing throughout the cell membrane. After insulin binding, tyrosine
phosphorylation occurs at the intracellular part of the receptor and activates the insulin receptor substrate proteins (IRS). There are 4 types of IRS proteins as IRS-1,2,-3 and -4.-1 and IRS-2 are the main proteins having roles in intracellular signal
transduction. After their activation, the subsequent step is the translocation of glucose transporters (GLUT) to the cell membrane by the help of phosphoinositide-3
kinase [PI(3)K]. There are 13 types of GLUT. After cessation of the insulin stimulation, the clathrin-coated vesicles turn back to the inside of the cell to be used
again in another cycle. Insulin resistance occurs because of the defects in this pathway and clinically it is diagnosed as metabolic syndrome. The main problem in insulin resistance was previously thought to be the down-regulation of the insulin
receptors but afterwards it was found that this was not a cause but the effect. The
problem in metabolic syndrome is known to be at the post-receptor level. The theories describing the defects in the post-receptor systems are the endoplasmic reticulum stress and the defects in mitochondrial fatty acid binding protein (FAB’s).
S13-4
DIETARY FACTORS RELATED TO TYPE II DIABETES MELLITUS
AND HEART DISEASE
Peter J. Butterworth
AKC Nutritional Sciences King’s College, London, UK
In healthy individuals, appetite and energy expenditure are balanced and body
weight remains relatively constant. Regulation of food intake and energy expenditure by the brain is in response to various peptides (e.g. leptin, ghrelin, GLP-1
etc.) released from adipose tissue and the gastro-intestinal tract under different
conditions of satiety. Disorders of the regulatory system can result in obesity and
lead to type II diabetes and cardiovascular disease. With globalisation of food and
dietary preferences, obesity is reaching epidemic proportions worldwide. This is
now a major concern for human health. Increases in knowledge of how diet affects
the regulatory systems is important for prevention and treatment of obesity. The
main source of carbohydrate for humans is starch from cereal products (bread,
pasta, cakes etc.), together with starch from root vegetables such as potato and
yam. Consumption of starchy foodstuffs occurs on an enormous scale and contributes to the obesity epidemic. The focus of our studies1 using enzymology, DSC,
X-ray diffraction and NMR spectroscopy has been on how the first stage of starch
digestion, involving pancreatic a-amylase, is influenced by the structure of starch
after processing in conditions that mimic those encountered in domestic and commercial production of food. Better understanding of this early stage of digestion
should help in the establishment of dietary regimes and functional foods that avoid
rapid and large increases of blood glucose and insulin concentrations following
intake of starch-rich nutrients. Such increases have long been associated with the
development of diabetes and the metabolic syndrome.
S13-5
INTERACTION OF LIFESTYLE AND GENETIC FACTORS IN
EXPRESSION OF THE METABOLIC SYNDROME
Philip Barter
Lipid Research, Heart Research Institute, Sydney, Australia
The prevalence of overweight and obesity are increasing worldwide and setting
the scene for a major new epidemic of premature cardiovascular disease. Some (but
not all) overweight people have a cluster of abnormalities that include impaired fast-
314
ing glucose, hyperinsulinemia, elevated blood pressure, an increased level of inflammatory markers and an atherogenic dyslipidemia characterized by low high density
lipoprotein cholesterol (HDL-C) and high plasma triglyceride. This cluster is
referred to as the metabolic syndrome. Genetic analysis has revealed four distinct
clusters: an adiposity-related cluster (including CRP), a blood pressure-related cluster and two lipid-related clusters (one including HDL-C, triglyceride, adiponectin
and LDL particle size and the other apo B and non-HDL-C). These genetic clusters
are paralleled by phenotype clustering, suggesting that common genes with pleiotropic effects may contribute to observed phenotypes. It has also been observed that
overweight people with atherogenic dyslipidemia tend to be insulin resistant, hypertensive and pro-inflammatory, while overweight people whose plasma lipids are normal do not have these other abnormalities, suggesting that an interaction between
lifestyle and genes is necessary for the full expression of the metabolic syndrome.
Thus, identification of people solely based on an elevated plasma triglyceride and a
low HDL-C uncovers an overweight group of people who have a generalized metabolic disorder and who appear to be genetically different from overweight people
with normal plasma lipids who may be at relatively low risk of developing diabetes
and cardiovascular disease despite being overweight. The challenge now is to identify the genetic variations that determine whether increased adiposity leads to the
cluster of metabolic abnormalities referred to as the metabolic syndrome.
S13-6
CELL AND GENE THERAPY APPROACHES TO PROLONG
ISLET-GRAFT SURVIVAL IN PATIENTS WITH TYPE 1 DIABETES
Ercument Dirice1, Ahter D. Sanlioglu1, Sevim Kahraman1,
Mustafa K. Balci2, Abdulkadir Omer3, and Salih Sanlioglu1
1
Human Gene Therapy Unit and the Department of Medical Biology and
Genetics, Faculty of Medicine, Akdeniz University, Turkey, 2The Division
of Endocrinology and Metabolism, Akdeniz University, Faculty of
Medicine, Antalya, Turkey; 3Section on Islet Transplantation and Cell
Biology, Joslin Diabetes Center, Harvard Medical School, Boston,
Massachusetts, USA
Diabetes is the third most common disease and the fourth leading cause of
death in North America. Currently, insulin injection and pancreas transplantation
are the two treatment modalities for patients with type 1 diabetes (T1DM). As an
alternative, transplantation of pancreatic islets has been suggested to avoid major
surgery and the complications associated with lifelong enzyme injections. Even
though intra-hepatic islet transplantation has been a promising approach for the
treatment of patients with T1D, the success of the approach was challenged due to
the high frequency of non-functioning grafts and secondary graft failure. The main
goal of islet transplantation is to completely correct the diabetic syndrome and
abolish the need for chronic immunosuppressive drug therapy. In order to maintain
long term graft function, both alloimmune and autoimmune barriers have to be
considered. Accordingly, tolerance induction is one of the objectives in islet transplantation. Initial studies investigating the protection of islets from the immune
system involved the transplantation of islets into immune privileged sites. It
quickly became obvious that these sites did not protect the grafts through sequestration, but relied on the activation of apoptotic pathways, such as Fas ligand
(FasL)-induced apoptosis. Another approach was to encapsulate the islets prior to
transplantation. However, this procedure was restricted by an intense fibrous reaction to the foreign material used for encapsulation and the induced cytokine
response, which eventually led to destruction of the grafted islets. Despite all the
methods devised to protect the beta cells from the immune mediated destruction,
the agents could only delay, not prevent the eventual failure of the transplanted
beta cells. Thus, gene therapy arose as an alternative treatment modality for the
treatment of T1D patients. This talk concerns recent progress in gene and cell therapy approaches to prolong islet/graft survival in patients with type 1 diabetes.
S14. Biochemistry
S14-1
Pediatric Infectious Research Center, Faculty of medicine, Shahid Beheshti
University (M.C), Tehran, Iran
Infections are the major cause of morbidity and mortality in febrile neutropenic patients with malignancy. Rapid diagnostic tests are needed for prompt diagnosis and early treatment, which is crucial for optimal management. We assessed
the utility of sTREM-1 in the diagnosis of bacteremia and fungemia in febrile neutropenic patients. Febrile neutropenic children with malignancy hospitalized in
Mofid Children hospital during a period of one year from January 2007 were
recruited for this cross sectional study. Simultaneous blood samples were collected
for measurement of serum sTREM-1 levels and for blood cultures, which were
grown in BACTEC media. Gold standard for the presence of infection was a positive BACTEC culture. Sixty-five children between the ages of 15 months and 15
years (mean age of 66.26 37 months), entered this study. 35 patients (53.8%)
were female, and 30 (46.2%) male. 30 patients (46.2%) had ALL, 2 (3.1%) AML,
1(1.5%) lymphoma and 32(49.2%) were under treatment for solid tumors. The
mean level of sTREM-1 was 250.68 6 66.93 pg/ml (range 3-2000 pg/ml) and
blood cultures with BACTEC system were positive in 13(20%) patients (12 bacterial and one fungal culture). The cut-off point of sTREM-1 in patients with positive bacteremia was 185 pg/ml, with a sensitivity and specificity of 84.62% and
86.54%, respectively. Our study revealed a significant association between serum
sTREM-1 level and bacteremia and fungemia in febrile neutropenic patients with
malignancy with acceptable sensitivity and specificity; however further investigations are needed for verification of our findings.
S14-2
DISCREPANCY BETWEEN WHOLE BLOOD INTERFERON GAMMA
ASSAY AND TUBERCULIN SKIN TEST FOR DIAGNOSIS OF
LATENT TB INFECTION IN BCG VACCINATED CHILDREN
Abdollah Karimi1, Shirin Sayyahfar2, Seyyed Alireza Fahimzad1,
Fatemeh Fallah1, Akram Golnabi1, and Shahnaz Armin1
1
Pediatric Infectious Research Center, Faculty of Medicine, Shahid Beheshti
University (M.C), Tehran, Iran, 2Iran University of Medical Sciences,
Tehran, Iran
Tuberculosis is one of the major health problems in the world. For more
than a century latent tuberculosis infection (LTBI) has been diagnosed by tuberculin skin test with many false positive & negative results. So we have been
looking for a better alternative. Interferon gamma release assay (IGRA) is a
novel tool for Mycobacterium tuberculosis infection diagnosis which has not
been evaluated yet in Iran. There has been limited experience with human PPD
based IGRA in pediatric range especially in BCG vaccinated children. So the
aim of our study was comparing the TST and human PPD based IGRA for diagnosing Latent tuberculosis infection (LTBI) in BCG vaccinated children.
Between august 2006 and may 2007 a total of 242 children aged 1 month to 168
months (mean 21 months) admitted to different wards of Mofid children hospital
and met the inclusion criteria were enrolled in a cross sectional study with sequential manner. Each patient was tested with both TST and human PPD based
IGRA. 28 (11.6%) had indeterminant, 123 (50.8%) had positive and 91(37.6%)
had negative results with IGRA. 12 (5%) had positive and 130 (95%) had negative results with TST. The overall agreement between TST and Quantiferon was
43.5% (kappa5 0.004, P.V.50.87, SE50.03). With increasing age, the probability of TST positivity decreased but was not statistically significant (PV: 0.25).
Also with increasing age, the probability of IGRA positivity was increased but
was not statistically significant. It seems that in low-income countries TST is still
a reliable diagnostic tool for diagnosing LTBI in children. Although different
versions of IGRA are available, it is important to choose a proper one considering history of BCG vaccination.
S14-3
FORENSIC ASPECTS OF ALCOHOL
EVALUATION OF SERUM STREM-1 LEVELS IN FEBRILE
NEUTROPENIC PATIENTS WITH MALIGNANCY
Ahmet Belce
Council of Forensics Medicine, Turkey
Abdollah Karimi, Babak Soltani, Mohammad-Taghi Arzanian, Alireza
Fahimzad, and Farideh Shiva
Over-consumption of alcohol and drunkenness are involved in many traffic
accidents, homicides and suicides. Alcoholmeters, which measures breathe alco-
hol, provide fast and non-invasive monitoring of blood alcohol level indirectly. If
necessary, positive report of alcoholmeter must be confirmed by blood alcohol levels. In Turkey, the legal blood alcohol limit is 50 mg/dL. Retrospective calculation
of blood alcohol is only applicable in alcohol elimination phase. Alcohol impairment show great differences for individuals. In many forensic cases, only alcohol
levels are determined but signs and symptoms of alcohol influence on person,
which are very important for legal evaluation of safe drive, are neglected. In emergency situations, fast and reliable methods to differentiate ethanol intoxication
from impairment caused by drinking more lethal alcohols such as methanol are
needed. Distinguishing between alcohol ingestion in life and microbial production
after death is a common problem. During the putrefactive process alcohol may be;
lost due to evaporation or, produced by microbial activity, primarily on glucose.
High environmental temperature, terminal hyperglycemia, terminal septicemia,
and abdominal trauma, all provide fertile conditions for ethanol synthesis.
Advanced putrefaction of corpses may give production to alcohol levels as high as
200 mg/dL. Ethanol levels in postmortem blood must be corroborated by analysis
of other body fluids such as vitreous humor and urine. After death, vitreous humor
is well protected from bacterial infiltration. Similarly, urine is useful because it
normally contain little or none substrate for bacterial conversion to ethanol. Consequently, presence of ethanol of ante-mortem alcohol intake and its absence is an
indicator of artifact in blood sample. Evaluation of post-mortem alcohol concentrations in body fluids must always take place with extreme caution and taken into
account autopsy findings and circumstances of death.
S14-4
COMPARISON OF PARALLEL ASSAYS OF TOLUENE RESULTS IN
PLAIN SERUM TUBES, AND SERUM SEPARATOR GEL TUBES
315
phisms in patients with spina bifida and their mothers revealed that neither of the
common risk mutations alone increased the risk for the occurrence of spina
bifida. Severe, first pointed out, from epidemiological evidence, a possible relationship between zinc deficiency and central nervous system malformation. In
fact, the essential role of zinc in embryonic development and a relationship
between maternal zinc deficiency and NTDs have been the subject of several
studies. Furthermore, in a rare autosomal recessive disorder characterized by a
severe nutritional zinc deficiency, acrodermatitis enteropathica, there is severe intestinal mucosal atrophy that can be reversed by effective oral zinc supplementation. In seven pregnancies in patients with acrodermatitis enteropathica, there
was one spontaneous abortion and two major congenital malformations. Conversely, pregnancy outcome was good when a patient with acrodermatitis enteropathica was given supplemental zinc throughout her pregnancy. A case of a
nutritionally zinc deficient young Turkish woman was reported who had previously delivered two anencephalic stillborn infants. After zinc supplementation,
she delivered a normal full term child. Our recent report concerning maternal
plasma zinc concentrations after an oral zinc tolerance test in pregnancies associated with NTDs in Turkey showed that some of the affected women had defective zinc absorption due to chronic zinc deficiency, which returned to normal after zinc supplementation. Previous data and findings in human studies are evidence for the possible role of zinc metabolism at least in some of the mothers of
babies with NTDs. Therefore, it is logical that genes that mediate absorption of
ingested zinc could be a candidate for teratogenic loci. Zn T4 gene (also known
as SLC3A4), encodes one of the zinc transport proteins. However, previous studies were unable to disclose mutations in AE families. We analysed the effect of
a common polymorphism in the exon 5 of the SLC30A4 ZNT4 (AF025409)
gene 915 T-C on zinc absorption. Carrying TT in homozygote state brought a
two-fold risk for the mother. Our data revealed that at least in some of the mothers with NTD babies, especially in regions with low diet zinc content, zinc deficiency should be considered.
Yilmaz Bozkurt, Senol Korkut, Faruk Bicer, Müfit Iris, and Ahmet Belce
Council of Forensic Medicine, Istanbul, Turkey
Toluene has been frequently abused by teenagers and adults who inhale
vapors of paint and glue solvents. In this study, we collected one milliliter of
blood specimens from fifteen healthy volunteers simultaneously into each plain
(non-gel) tubes and serum separator gel (SSG) tubes. Whole blood samples were
stored at 4 C8 for forensic purposes for ten days. Toluene analysis was performed by using headspace gas chromatographic technique. No toluene was
found in plain tubes. However mean blood toluene concentration in SSG tubes
was 51.47 (SD68.12) mg/L, a level which is associated with comatose-fatal
dose. Investigators reported that absorption of some therapeutic drugs by barrier
gels in serum separator blood collection tubes cause volume- and- time dependent reduction in total and free drug concentrations. However, our results show
that barrier gels cause falsely elevated blood toluene concentrations. Toluene as
a solvent may cause diffusion of gel contents into serum medium. Prolonged
storage of blood in contact with gel may result in this false increase. According
to the above findings, we suggest that evaluation for forensic purposes of blood
analysis results in SSG tubes requires extreme caution.
S14-5
NEURAL TUBE DEFECTS AND ZINC
Ece Akar and Nejat Akar
Pediatric Molecular Genetics Department, Ankara University, Turkey
Meningomyelocele, encephalocele and anencephaly are the most common
severe congenital malformations of the central nervous system (CNS). These
malformations arise due to failure of closure of the neural tube and are referred
to as neural tube defects (NTD) with high incidences in some parts of the world,
including Turkey. NTD causation is believed to be multifactorial, with genetic
and environmental components. The relationship between periconceptional dietary folate supplementation and the prevention of up to 75% of NTD’s is well
recognized, although the mechanisms underlying the beneficial effect of folate
are only partly understood. There is a growing interest in the relationship
between variation in genes that are involved in the folate-homocysteine metabolic pathway and the risk of spina bifida. The evaluation of this relationship is,
however, complicated by the potential involvement of both the maternal and the
embryonic genotype in determination of disease risk. Although the data varies
among study groups, Turkish data on homocysteine metabolism related polymor-
S14-6
PROTEIN GLYCOSYLATION IN BACTERIA
Hamdi Uysal
Department of Biochemistry, Ankara University, Faculty of Veterinary
Medicine, Ankara, Turkey
Glycosylation is a significant covalent modification of proteins. Protein glycosylation is an essential and highly conserved process in eukaryotic cells and it
appears to be rare in prokaryotic organisms. In the past two decades a change
has taken place in the convincement that glycosylation of proteins is restricted to
eukaryotic organisms. Today, we can assume from different observations that
prokaryotic glycoconjugates may well be as common as glycoproteins in higher
organisms or plants. In prokaryotes, the earliest examples of protein glycosylation have been found with Archea, which are able to present glycosylated surface
layer (S-layer) proteins. Most studied prokaryotic glycoproteins are the S-layer
glycoproteins of Archeabacteria. Moreover, membrane-associated, surface-associated, secreted glycoproteins and exoenzymes glycoproteins are also well studied
in both, Archea and Eubacteria. It is now clear that prokaryotes are capable of
glycosylating proteins. In prokaryotes, many different glycoprotein structures
have been observed that display much more variation than those observed in
eukaryotes. Up to date, the knowledge on the functional aspects of prokaryotic
glycoproteins is rather scarce. Here, the biosynthesis, structure and functions of
bacterial glycoproteins will be discussed on the light of current literature. Moreover, I will focus on the glycosylated proteins of Helicobacter pylori that have
been associated with gastric diseases such as ulcer and cancer. I will also discuss
the glycosylated protein profiles of Enterococcus faecalis strains (aggregation
substance (AS), gelatinase, cytolysine).
S14-7
MITOCHONDRIAL DISORDERS IN NEURODEGENERATIVE
DISEASES
Makbule Aydin
Department of Neuroscience, Istanbul University DETAE, Istanbul, Turkey
316
There is now a growing body of evidence that neurodegenerative diseases
share various pathological features, such as accumulation of aberrant protein
aggregates, microglial activation, and mitochondrial dysfunction. These pathological processes are associated with generation of reactive oxygen species (ROS),
which cause oxidative stress and subsequent damage to essential molecules, such
as lipids, proteins, and DNA. The brain is particularly vulnerable to oxidative
damage because of its high oxygen utilization, its high content of oxidizable polyunsaturated fatty acids, and the presence of redox-active metals (Cu, Fe). Mitochondria represent not only a major source of ROS generation, but also a major
target of ROS induced damage and could therefore play a central role in oxidative stress in neurodegenerative diseases. The oxidative phosphorylation system
(OXPHOS) consists of five multi-enzyme complexes, Complexes I-V, and is a
key component of mitochondrial function relating to energy production, oxidative
stress, cell signaling and apoptosis. Mitochondrial ROS are generated by the respiratory chain during ATP synthesis due to the leakage of electrons primarily
from complex I, complex III and more recently complex IV to molecular oxygen. Defects or a reduction in activity in various components that make up the
OXPHOS enzymes can cause serious diseases, including neurodegenerative disease. Oxidative stress can results from the imbalance between antioxidants and
ROS. Organism has evolved the antioxidant system against oxidative stress damage. It is now clear that different brain cell types, namely astrocytes and neurones, can have differing antioxidant reserves. In conclusion, enhanced ROS production and oxidative injury play a major role in the onset and progression of
neurodegenerative disorders.
S14-8
INHIBITION OF UVA-INDUCED AP–1 ACTIVATION MAY PREVENT
PHOTOAGING IN HUMAN DERMAL FIBROBLASTS: AN IN-VITRO
STUDY
Mehtap Yuksel-Egrilmez1, Semra Kocturk2, Gulgun Oktay2,
Sebnem Aktan3, Halil Resmi2, Sebnem Ozkan3, and Gul Guner2
1
Research Laboratory, Dokuz Eylul University, School of Medicine, Turkey,
2
Department of Biochemistry, Dokuz Eylul University School of Medicine,
Turkey, 3Department of Dermatology, Dokuz Eylul University School Of
Medicine, Turkey
Objective: Human skin is exposed to solar UV radiation. It was previously
reported that UV causes photoaging by activation of growth factor receptors in
human dermal cells. They lead to downstream signal transduction through phos-
phorylation of mitogen-activated protein kinase pathways, such as c-Jun N-terminal kinase (JNK) resulting in activation of c-Jun and c-Fos, subunits of AP-1
transcription factor. These pathways induce matrix metalloproteinases (MMPs)
that degrade skin connective tissue. Generation of reactive oxygen species by
UV is also critical in triggering MAPK pathways. Melatonin, the product of pineal gland, is the most effective free radical scavenger. We examined whether
melatonin ameliorates UVA-induced responses in vitro.
Methods: Human Dermal Fibroblasts (HDFs) were isolated from punch
biopsies of healthy individuals. HDFs were pretreated with melatonin (10-6 M)
for one hour and exposed to UVA and then incubated for various time points for
every parameter. Epidermal growth factor receptor (EGFR) activation, MMP-1
and MMP-3 activities, levels of nitrotyrosine and tissue inhibitor of metalloproteinase (TIMP-1) were measured by ELISA. Procollagen type I C-peptide (PIPC)
levels were determined by EIA. JNK activation was analyzed by Western blotting. c-Jun and c-Fos activities were measured by DNA binding activity assay.
Levels of malondialdehyde (MDA) and oxidized/reduced glutathione were analyzed by HPLC.
Results: UVA significantly increased EGFR activation, nitrotyrosine levels
and MMP-1 and MMP-3 activities. UVA also induced phosphorylation of JNK
and c-Jun and c-Fos activities. MDA levels and oxidized/reduced glutathione ratio were also increased by UVA exposure (p<0.05). Pretreatment with melatonin
significantly decreased EGFR activation, nitrotyrosine levels and MMP-1 and
MMP-3 activities in HDFs. Melatonin inhibited UVA-induced JNK activation
and c-Jun and c-Fos activities. MDA levels and oxidized/reduced glutathione ratio were reduced by melatonin (p<0.05). Pretreatment with melatonin increased
UVA-induced TIMP-1 levels (p<0.05) but had no effect on PIPC levels
(p>0.05).
Conclusions: Our results show that melatonin has protective effects on
extracellular matrix metabolism and oxidative damage induced by UVA irradiation. These data also indicate that melatonin with antioxidant activities may prevent photo-aging by inhibiting JNK activation and AP-1 activity. This study was
supported by TUBITAK (SBAG-2708).
S14-9
SELECTIVE CYTOTOXICITY AND ANTICANCER PROPERTIES OF
SOUR TEA (HIBISCUS GOSSYPIFOLIUS MILL) EXTRACT ON
HUMAN BREAST ADENOCARCINOMA CELL LINE
Mohsen Mohammadian-Yajloo1, Parvin Pasalar1, Shahnaz Khaghani1,
Malihe Paknejad1, Massoud Amanlou2, and Shahla Rezaei1
Figure. Effect of Hibiscus gossypifolins Mill extract at various concentrations on human breast adenocarcinoma cell line (MCF-7) and human fetal foreskin fibroblast
(HFFF) (Black and White bars respectively) following 24, 48 and 72 hours incubation. Each concentration was assayed in 8 wells and repeated 3 times. Mean and
Standard Deviation (SD) of three independent experiments done in triplicate are shown. P \ 0.05.
1
Department of Clinical Biochemistry, Faculty of Medicine, Medical
Sciences, University of Tehran, Tehran, Iran, 2 Department of Medicinal
Chemistry School of Pharmacy, Medical Sciences, University of Tehran,
Tehran, Iran
Objectives: Majority of the currently available anticancer drugs are designed
to have selective toxicity towards tumor cells. Among these, the focus of many
studies is on natural compounds, which inhibit the growth of cancer cells more
selectively than normal cells and have high therapeutic index. In present study,
the cytotoxic effect of sour tea (Makke tea or Hibiscus gossypifolius Mill) aqueous extract on human breast adenocarcinoma cell line (MCF-7) and normal fetal
foreskin fibroblast (HFFF) was investigated.
Methods: The plant calyces were extracted by maceration method with distilled water, and then evaporated to dryness using rotary evaporator. The extract
was prepared as a stock solution, sterilized and further diluted to final concentrations. The cells were grown in completed RPMI-1640 and seeded in 96-well
micro plates at concentration of 2.53104 cells/well. After 12 hours incubation,
different concentrations of the extract were added and cells further incubated for
24, 48 and 72 hours. Cell survival percent was determined at 540 nm using MTT
assay.
Results: At concentration of 0.5 mg/ml of the extract, following 72 hours
incubation, the number of viable MCF-7 cells was less than 50%. Cytotoxicity
was considered whenever cell survival percent was less than 50. The extract was
not cytotoxic towards normal HFFF cell line in all tested concentrations.
Conclusions: These results suggest that the aqueous extract, in a concentration and time dependent manner, inhibits the growth of MCF-7 more selectively
than HFFF cells.
317
Molecular oxygen plays an essential role in many metabolic processes associated with aerobic life. Although the production of intracellular reactive oxygen
species (ROS) represents a tightly regulated process, a partial reaction of oxygen
with electrons leads to formation of ROS. However, while excess reactive oxygen
species (ROS) are toxic, regulated ROS play an important role in cell signaling.
Proteins, nucleic acids, and lipids can undergo various forms of oxidative modification. In numerous instances, these modifications result in irreversible loss of
function. The age-dependent accumulation of oxidatively modified and dysfunctional macromolecules provides the basis for the theory of aging. It is increasingly
apparent that these reactions participate in redox dependent regulation of cell metabolism. A number of biochemical and physiologic stimuli, such as perturbation
in redox status, expression of misfolded proteins, altered glyc(osyl)ation and glucose deprivation, overloading of products of polyunsaturated fatty acid peroxidation (Hydroxynonenals, HNE) or cholesterol oxidation and decomposition, can
disrupt redox homeostasis, impose stress and subsequently lead to accumulation of
unfolded or misfolded proteins in the cells. Alzheimer’s (AD), Parkinson’s and
Huntington’s disease, amyotrophic lateral sclerosis and Friedreich’s ataxia are
major neurological disorders associated with production of abnormally aggregated.
It has been indicated that the ROS generated by skeletal muscle play an important
role in influencing redox-regulated processes that control, at least some of, the
adaptive responses to contractile activity. These processes are also recognized to
be modified during ageing and in some disease states, providing the potential that
interventions affecting ROS activity may influence muscle function or viability in
these situations. The studies indicate a cellular mechanism for redox regulation of
cell signaling in ageing and disease will be discussed.
S15.3
S15. Redox Regulation of Cell Signaling in
Ageing and Disease
S15-1
AGE-RELATED LOSS OF SKELETAL MUSCLE MASS AND
FUNCTION: THE ROLE OF DYSREGULATION OF
REDOX HOMEOSTASIS
Malcolm J. Jackson
School of Clinical Sciences, University of Liverpool,
Liverpool L69 3GA, U.K.
Loss of skeletal muscle mass and force generation is an inevitable consequence of ageing such that there is an inevitable loss of ~40% of muscle mass
between the ages of 40 and 70 years of age. The consequences of this loss of
muscle mass are important factors in the increasing frailty seen in elderly subjects. Reactive oxygen species (ROS) have been implicated in the processes
underlying ageing for a considerable period of time, but their precise role
remains unclear. As part of an integrated programme of studies to examine this
area, we have determined whether single skeletal muscle fibres from aged mice
have an increased activity of specific ROS, whether manipulations to increase
ROS activities cause premature loss of muscle mass, and whether modifications
that reduce oxidative damage to muscle proteins reduce the loss of muscle mass
and function that occurs in ageing mice. Our current data support a role for ROS
in age-related loss of muscle mass and function since skeletal muscle fibres from
aged mice show increased ROS activities, a lack of the key regulatory enzyme
Cu, Zn superoxide dismutase causes premature age-related loss of muscle mass
and overexpression of the heat shock protein HSP70 protects against some
aspects of age-related muscle function. This work is supported by the Wellcome
Trust, Medical Research Council and U.S. National Institute on Aging.
S15-2
REDOX REGULATION OF CELL SIGNALING IN AGEING
AND DISEASE
Nesrin Kartal-Ozer1 and Efstathios Gonos2
1
Department of Biochemistry, Faculty of Medicine, Marmara University,
Istanbul, Turkey, 2Institute of Biological Research and Biotechnology,
National Hellenic Research Foundation, Athens, Greece
AGE-RELATED PROTEIN AGGREGATES – ACTIVE MODULATORS
OF CELLULAR SIGNALING
Tilman Grune
University of Hohenheim, Institute of Biological Chemistry and Nutrition,
Biofunctionality and Food Safety (140f), Germany
One of the highlights of postmitotic aging is the intracellular accumulation
of highly oxidized and cross-linked proteins, known as lipofuscin. Lipofuscin is
insoluble and not degradable by lysosomal enzymes or the proteasomal system,
which is responsible for the recognition and degradation of misfolded and oxidatively damaged proteins. These aggregates have been found in various cell types,
including heart, liver, kidney, neuronal tissue, and dermal tissue, and are associated with the life span of a single postmitotic cell and, consequently, of the
whole organism. It is not always clear why protein aggregation takes place, but a
disturbance in the homeostasis between protein synthesis and protein degradation
seems to be important. The result is the accumulation of modified proteins,
which tend to form high molecular weight aggregates. Such formed lipofuscin is
not an inert waste product of the cell metabolism, but plays an active role in the
cage-related changes of cell function. Affected by the lipofuscin presences are
key metobolic processes as gene expression, redox status and protein turnover.
Therefore, the physiological changes during aging or in the pathophysiological
changes during premature aging in postmitotic cells seem to be partially mediated by lipofuscin.
S16. Emerging Vector-Borne Diseases in Istanbul
and Surrounding Areas
S16-1
STUDIES ON THE PREVALENCE OF CRIMEAN CONGO
HEMORRHAGIC FEVER VIRUS IN TICKS OF DOMESTICATED
AND WILD ANIMALS
Aysen Gargili
Department of Microbiology and Clinical Microbiology, Cerrahpasa Medical
Faculty, Istanbul University, Istanbul, Turkey
Crimean-Congo haemorrhagic fever (CCHF) is an epidemic in Turkey since
2003. During the first known epidemic in Crimea, in 1944-1945, relations of the
ticks and the disease have been established. Identification of the vector ticks and
318
the prevalence of the virus in ticks are important for epidemiology of the infection. Aiming to gain data on the vectors and related epidemiology of the infection, a total of 3236 tick samples were collected from domestic and game animals and from field in endemic and non-endemic areas in 2006-2008. Ticks were
identified at sub-species level. Pools were constructed including 5-20 ticks and
RNA was extracted using commercial extraction kits. Engorged females of various species were allowed to lay eggs and RNA extracted from egg batches separately. Following reverse transcription, a 593 bp part of the S segment of virus
genome was amplified with the specific primer sets using a nested protocol. Positive samples were sequenced with an automatic sequencer and obtained
sequenced were edited and aligned using software packages. Phylogenetic analyses were carried out by distance method using neighbor-joining algorithm. Viral
RNA was amplified in 27 tick pools, out of ticks collected from domestic animals and in 13 out of collected from game animals. Positive samples were
belonging to Hyalomma m. marginatum, Rhipicephalus bursa, Boophilus annulatus and Dermacentor marginatus species. Viral RNA was also found in egg
batches of Hyalomma m. marginatum species for the first time and vector role of
this species confirmed in Turkey. Presence of the virus in game animals showed
the importance of the domestic animal-tick-wild life cycle in distribution of the
infection. Viral RNA presence in ticks such as Dermacentor marginatus suggests
that these ticks may play vector role in winter season when the dominant vector
of the infection, H. m. marginatum, is inactive, and thus, may play an important
role in the continuation of the infection in wildlife in endemic areas. Although
relatively few sequences are available since the disease outbreaks were recorded
in Turkey for the first time in 2003, those which are available are related to viral
lineages found in East Europe and South-western Russia (Europe II clade). The
sequences obtained from positive samples in this study were grouped with the
Europe/Turkey strains and Greece strain clustered within the Europe I and
EuropeII clades respectively.
S16-2
MOLECULAR DETECTION AND PREVALENCE OF BORRELIA
SENSU LATO GENOTYPES IN HUMAN BITING TICKS IN
ISTANBUL, TURKEY
Sevgi Ergin
Istanbul University, Cerrahpasa Medical Faculty, Department of
Microbiology and Clinical Microbiology, Turkey
Lyme borreliosis is the most common vector–borne human infection in
Europe, North America, Asia and North Africa. The disease is a multi-system
disorder involving the nervous system, joints, skin, and heart and its early clinical symptom is erythema migrans. It is caused by Borrelia burgdorferi sensu
lato and transmitted to humans by the bite of an infected tick of the genus Ixodes. The Borrelia burgdorferi sensu lato complex has 11 different genospecies.
The spread of borreliosis depends on geographical, environmental and climatic
factors and pathogenicity of Borrelia burgdorferi sensu lato strains. Borrelia
burgdorferi sensu stricto, Borrelia andersonii, and Borrelia bisettii have been
reported in North America. Borrelia burgdorferi sensu stricto, Borrelia garinii,
Borrelia afzelii, Borrelia valasiana, and Borrelia lusitaniae have been identified
in Europe. Borrelia garinii, Borrelia afzelii, Borrelia valasiana, Borrelia japonica, Borrelia turdi, Borrelia tanukii and Borrelia sinica have been identified in
Asian countries. Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia
garinii are recognized as pathogenic for humans.
The main vector for Borrelia burgdorferi sensu lato strains is Ixodes ricinus
in Europe. Advances in polymerase chain reaction (PCR) technology have provided sensitive and fast methods for the diagnosis of Borrelia. The technique has
been applied to detection of Borrelia burgdorferi sensu lato DNA in infected
ticks, as well as in human specimens, such as cerebrospinal fluid or synovial
fluid, urine and skin. In the PCR protocols different segments of Borrelial genes
(eg. the flagellin genes, the outer surface protein A) have been used. A passive
surveillance system was developed in the urban area of Istanbul, Turkey, to
understand the most prominent species of ticks biting humans and their seasonality patterns. This study reports on the most important findings in the years 2006
and 2007. A total of 3550 cases were reported in this period. Ticks (adults,
nymphs and larval) were collected from human patients admitted to hospitals after reporting tick bites in Istanbul. Parasitism by adult Ixodes ricinus extended
over a wide time frame (March–October), most cases concentrated in June–July.
In this study, Ixodes spp were observed in the 54% of patients. A total of 368
Ixodes spp. of the samples collected in 2006 were screened by using highly sensitive semi- nested PCR for the presence of Borrelia sensu lato genotypes. The
first and second PCR [an initial step at 948C for 2 min, (45 cycles of PCR amplification 948C for 1 min, 508C for 1 min, 728C for 2 min), final extension at
728C for 10 min] were performed with the ospA primers (OSPAFw1: ttg gga ata
ggt cta ata tta gc, OSPAFw2: atg yaa gca aaa tgt tag c, BorR: act aat gtt ttv cca
tct tc) to detect 3 genospecies of Borrelia burgdorferi sensu lato. Nine out of
110 nymphs (8.18%) and 31 out of 237 females (13.08%) were found to be
infected with Borrelia sensu lato species. Borrelia valaisiana, Borrelia affzelii,
Borrelia garinii and Borrelia spielmani were detected in samples according to
the sequencing results. It should be noted that the high prevalence of Ixodes spp.
bites in the city and the existence of Borrelia sensu lato strains in the human biting ticks might increase the Borreliosis risk.
S16-3
EMERGENCE OF A NEW CRIMEAN CONGO HAEMORHAGIC FEVER
VIRUS STRAIN IN BALCANIAN PENINSULA OF TURKEY
Kenan Midilli
Istanbul University, Cerrahpasa Medical Faculty, Department of
Microbiology and Clinical Microbiology, Turkey
A mild Crimean Congo Hemorrhagic Fever (CCHF) case due to an AP-92
like strain was defined during the summer season of 2007 in rural Balkanian part
of Istanbul. Ap92 strain, which had been isolated from tick in Greece in 1970s
and was the sole member of a different clade among CCHFv strains and this
strain had not been related with any infection in human beings. Therefore, we
aimed to perform a serum survey and a field tick survey in the region, where the
index case acquired the infection, to present the risk factor analysis for serologic
CCHF positivity. RNA of CCCFv was found in the blood sample of the index
case by nested PCR following RT PCR procedure. The serum surveillance studies were performed in four major districts. IgM and IgG type antibodies for
CCHF were detected by a commercial ELISA kit. The individuals, who were
positive for IgM and/or IgG were prospectively surveyed, and four months later,
the second sera from the same individuals were collected and re-studied for IgM
and IgG. In total 56 ticks were collected from cattle from the same residential
areas, 38 of them were identified as Boophilus annulatus and 18 as Rhipicephalus bursa. The phylogenetic analysis of the obtained sequence of CCHFv
revealed that, the strain was closely related to AP92 strain, which was reported
from Greece. The sequence was deposited in the Genbank under accession number EU057975. Seven hundred forty one subjects, who live in the region, were
tested for CCHF IgM and IgG. In the first run, 39 (5.26%) subjects were found
to be IgM positive, and 35 (4.72%) subjects were IgG positive. Thirty four out
of 35 (97.4%) IgG positive patients were also IgM positive. Only one patient
was found to have IgG positive but IgM negative (0.14%). The second serologic
analysis among IgM and/or IgG positives for CCHF was performed 4 months
later. Baseline sera were re-tested simultaneously for both IgG and IgM. All the
IgM positivities except one, converted to IgG positivity. One IgG positive individual was found to be IgG negative. The first positive result of this individual
was accepted as false, and this individual was considered as sero-negative.
Accordingly, the analysis was performed among 38 IgM positive individuals versus 703 IgM negative individuals. Tick samples were screened for CCHFv RNA.
The virus was not detected among the ticks. The mean age was higher among
IgM positive individuals (54 vs 37, p\0.001). Among the IgM seropositive individuals, being older (54 vs 37, p\0.001), male gender (76.3%, p50.001), performing agricultural activity (39.5%, p50.036), and having history of tick bite
(15.8%, p50.014) were found to be significantly higher than IgM negative individuals. In the region, 5.26% of the subjects had IgM positivity, and four months
later, all these IgM positive subjects, except one, developed IgG positivity. The
IgM positivity implies a recent infection. Except for the presented case, the
others did not seek medical advice. We can thus conclude that they were asymptomatic or mild cases. The attack rate that was defined as the proportion of diseased subjects among the infected ones, found to be one out of 38 (2.6%). Index
case with AP92 like strain was mild according to severity criteria. On the other
hand, the infection with AP92 strain was also reported to be almost asymptomatic. This was the first naturally acquired human infection due to an AP92 like
strain. We concluded that a new strain of CCHFv was emerged in the Balcanian
part of Turkey and differences in the clinical presentation can be explained by
different virulence among the circulating strains.
319
S17. Young Investigators
S17-1
NUTRACEUTICALS & CANCER
Aslihan Avci
Department of Biochemistry, Ankara University, Faculty of Medicine,
Turkey
Nutraceuticals are extracts of foods claimed to have a medicinal effect on
human health. In other words, nutraceutical implies that the extract or food is
demonstrated to have a physiological benefit or provide protection against some
diseases like hypercholesterolemia, cancer, atherosclerosis etc. Researches over
the last decade have shown that several micronutrients in fruits and vegetables
reduce cancer. The active components of dietary phytochemicals that most often
appear to be protective against cancer are: curcumin, genistein, resveratrol, diallyl sulfide, S-allyl cysteine, allicin, lycopene, capsaicin, diosgenin, 6-gingerol,
ellagic acid, ursolic acid, silymarin, catechins, eugenol, isoeugenol, dithiolthiones, isothiocyanates, isoflavones, protease inhibitors, saponins, phytosterols,
vitamin C, limonene, lutein, folic acid, beta carotene, selenium, vitamin E, flavonoids, and dietary fiber. Flavonoids are water-soluble polyphenolic molecules
containing 15 carbon atoms. Some flavonoids have apoptotic effect on cancer
cells such as resveratrol, lupeol, quercetin, genistein, curcumin, delphinidin, capsaicin, slymarin, epigallocatechine, lycopene. Adenosine deaminase plays an important part in the purine metabolism and DNA turnover. There are two isoforms
of ADA, which are ADA1 and ADA2. The plasma AD2 isoform is also
increased in most cancers. In our study possible effects of aqueous extract of
Urtica dioica leaves on adenosine deaminase activity were investigated in prostate tissue from patients with prostate cancer. It was found that aqueous extract
of Urtica dioica produces significant inhibition of adenosine deaminase (ADA)
activity in prostate tissue. In another study, we investigated possible effects of
tomato juice on ADA activity in prostate tissue from cancer patients. We have
seen that tomato juice causes significant inhibition on the ADA activity in prostate tissues. Another study, designed by our group, was aimed to investigate possible effects of aqueous extract of wheatgrass (Triticum aestivum L.) on oxidant/
antioxidant status in Baf3p210-E255K (imatinib-resistance) Chronic Myeloid
Leukemia (CML) cell line. In this cell line, it was observed that the extract
caused inhibition of ADA activity but increase of SOD and CAT activities
(p\ 0.05).
S17-2
THE STUDY OF HMGCR PROMOTER POLYMORPHISM IN
CORONARY HEART DISEASE
Aysegül Basak Akadam1, Hülya Yılmaz-Aydogan1, Turgay Isbir1,
Selim Isbir2, and Atike Tekeli2
1
Department of Molecular Medicine, Institute for Experimental Medicine,
Istanbul, Turkey, 2Cardiovascular Surgery, Marmara University School of
Medicine, Istanbul, Turkey
Background: Coronary heart disease (CHD), which is multifactorial, is the
leading cause of death in developed countries. Genes of lipoprotein synthesis and
metabolism are very important genes in tendency to atherosclerosis and CHD,
because plasma lipids are main risk factors in CHD. 3-hydroxy-3-methyl Glutaryl
CoenzymeA (HMG-CoA) reductase (HMGCR) is one of the most important
enzymes in endogenous cholesterol biosynthesis. Statins, which are used for lowering cholesterol level, are inhibitors of this enzyme. These drugs prevent atherosclerotic plaques or stabilize them by lowering intrahepatic cholesterol biosynthesis. However, there are some individual differences in response to the drugs. One
of the reasons of that may be variations of gene of this enzyme. Consequently,
the gene coding for HMGCR is thought to be responsible for the tendency to develop CHD. In our study, our aim is that to investigate effects of HMG-CoA reductase promoter (-911 C>A) polymorphism on serum lipoprotein level and development of CHD in Turkish patient with CHD.
Material and Methods: HMGCR-911 gene polymorphism was studied in 61
patients with CHD (19 (31.1%) women, 42 (68.9%) men). Healthy persons 54
(29 (53.7%) women, 25 (46.3%) men) without any symptoms of CHD were
selected for the control group. Gene polymorphism was studied by the polymerase chain reaction followed by agarose electrophoresis. Statistical analyses were
Figure 1. The structure of the HMGCR homotetramer. [Color figure can be
viewed in the online issue, which is available at www.interscience.wiley.com.]
performed by SPSS for windows version 11.0. Genotypes and allele frequencies
were compared by chi-square test. Lipid and the other parametric analysis were
compared by Student’s t test.
Results: In the patient group, frequencies of HMGCR mutant C allele
(p50,010) and CC genotype (p50,000) are higher than controls. In addition to
HMGCR mutant C allele is associated with high serum LDL-cholesterol levels
in the patients group (P50.01). Our findings suggested that these variants might
be independent risk factors in development of CHD.
Conclusion: We observed that HMGCR-911 mutant C allele has frequency
in CHD patient higher than normal A allele and it causes an increase serum
LDL-cholesterol levels. Therefore, we thought that HMGCR variant is influencing CHD by its detrimental effects on serum lipid profile.
S17-3
COMPARISON OF LIPID PROFILES WITH APO A-I MSPI
POLYMORPHISM ON OBESE CHILDREN WITH HYPERLIPIDEMIA
BY USING DGGE
Bahar Toptas1, Feyza Darendeliler2, Firdevs Bas2, and Turgay Isbir1
1
Medicine, Istanbul University, Capa, Istanbul, Turkey, 2Department of
Pediatric Endocrinology, Istanbul University, Istanbul Faculty of Medicine,
Istanbul, Turkey
Childhood obesity appears to lead to the appearance of a number of cardiovascular risk factors, such as altered lipid levels, impaired glucose tolerance,
which could contribute to atheroma plaque development and lead to the development of coronary heart disease in adult life. Apo A-I is a lipid binding protein
which plays an important role in cholesterol transportation and the transport of
lipids in plasma. Defects or variations of these apolipoprotein genes are also
associated with altered plasma concentrations of lipids and lipoproteins. For our
study, we built three separate groups; one of them was the group of obese children with hyperlipidemia, second one was the group of obese children without
hyperlipidemia and the last group comprised healthy children with neither hyperlipidemia nor obesity. In our study, we aimed to evaluate Apo A-I MspI polymorphism and plasma lipid parameters altogether in childhood and adolescence
cases and this study in Turkey is to determine possible relationships of all discussed parameters. We amplified the related gene segments by using polymerase
chain reaction (PCR) and determined different patterns by using denaturating
320
gradient gel electrophoresis (DGGE) and applied automatic sequence analysis.
This analysis results that two polymorphisms exist at the promotor region of
gene. There were two or three nucleotide polymorphisms on promotor region of
Apo A-I gene, known as MspI polymorphism. These polymorphisms are; (G75A), C183/T and G183A. We observed that the obese control group -75GA
genotype had significantly higher than the -75GG genotype. Our investigations
on the obese control group showed that individuals carrying -75 GA genotype
had higher plasma total-cholesterol levels than the carries of GG genotypes. Our
investigations on the healthy control group showed that individuals carrying GG
genotype had higher plasma VLDL-cholesterol levels than the carries of GA
genotypes. No relationship was found between MspI polymorphism and HDLcholesterol levels in the patients and control groups.
Results: Survivin genotypes distribution among our patients was not significantly different from that among controls (p>0.05). We found that serum survivin levels in patients (11.7363.57 pg/ml) were found significantly lower
(26.3763.58 pg/ml) than in controls (p50.017).
Conclusion: Our results suggest that the decreased survivin serum levels
may be an important factor in the development of intracranial aneurysms by
effecting apoptotic pathways. However, larger studies are needed to verify this
suggestion and the role of survivin polymorphism needs to be further investigated.
S17-6
DOES DNA COPY VARIATION PREDISPOSE TO SCHIZOPHRENIA?
S17-4
ASSOCIATION BETWEEN P21, P27 GENE POLYMORPHISMS AND
COLORECTAL CANCER RISK AND PROGNOSIS
Canan Cacina1, Ilhan Yaylim-Eraltan1, Soykan Arikan2,
Esra Kaytan-Saglam3, and Turgay Isbir1
1
Department of Molecular Medicine, Institute of Experimental Medicine,
Istanbul University, Capa, Istanbul, Turkey, 2Surgery Clinic, Istanbul
Research and Education Hospital, Istanbul, Turkey, 3Radiation Oncology,
University of Istanbul Institute of Oncology, Istanbul, Turkey
Colorectal cancer is important health problem in industrialized countries.
Most cases of colorectal cancer are sporadic, and genetic and environmental factors play important roles. Cyclin Dependent Kinases form complexes with
cyclins to modulate cell proliferation through cell-cycle control, whereas CDK
inhibitors inhibit the kinase activities of the complexes and block cell-cycle transitions. p21Cip1 (CDKN1A) and p27Kip1 (CDKN1B) proteins belong to the
Cip/Kip family are two putative tumor suppressors and CDK inhibitors. We have
examined CDKN1A Ser/Arg, C120T and CDKN1B C-79T, Val109Gly polymorphisms as a marker of tumour risk and progression in sporadic colorectal cancer.
50 colorectal cancer cases and 60 healthy controls were enrolled in this study.
The distribution of CDKN1A Ser31Arg genotypes were different between colorectal cancer patients and controls (p50.036). There were no associations between
CDKN1A C120T and CDKN1B Val109Gly, C-79T genotypes and susceptibility
to colorectal cancer. G allele of Val109Gly was associated with decreased
tumour differentiation (p50.037). In the analyses stratified by the age, we have
observed that the frequency of G allel had 8-fold increased risk of colorectal
cancer. If these findings were confirmed, they would have clinical value in helping to assess the genetic risk for colorectal cancer thus opening new perspectives
for the study of molecular factors underlying the mechanisms of colorectal
cancer.
S17-5
A POSSIBLE ROLE OF SURVIVIN IN THE INTRACRANIAL
ANEURYSMS
Didem Kafadar1, Ali Metin Kafadar2, Ilhan Yaylim-Eraltan1,
H. Arzu Ergen1, Mehmet Yasar Kaynar2, and Turgay Isbir1
1
University of Istanbul, Turkey, 2Department of Neurosurgery, Cerrahpasa
Medical Faculty, University of Istanbul, Turkey
Background: Apoptosis, commonly observed under a wide range of physiological conditions, occurs in the various pathological situations including some
vascular diseases. Both congenital and acquired factors are believed to contribute
to the formation and progressive development of intracranial aneurysms. A considerable body of literature has implicated survivin in the maladaptive pathways
following vascular injury. In this study, we investigate whether there is any relationship between survivin serum levels and survivin polymorphism in the developing intracranial aneurysms.
Methods: Survivin levels were assessed in the circulation of 21 patients with
intracranial aneurysm and 49 controls, by Enzyme Immunometric Assay (EIA).
Survivin promoter polymorphism was genotyped by a polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) assay.
Dilihan Gumus
School of Medicine, Psychiatric Genetics Unit, Cardiff University, Cardiff,
United Kingdom
Genetic aberrations at the DNA copy-number level, such as deletions, amplifications, and unbalanced translocations, are common causes of human genetic
disorders. I had undertaken a high-resolution genome-wide analysis of DNA
sequence copy number changes using 97 Schizophrenia patients to detect aberrations and identification of candidate genes by of chromosomal breakpoints.
Briefly, I had compared my findings to a reference dataset of 372 control individuals analysed in our laboratory, and checked them by the Database of
Genomic Variants to distinguish frequent and rare variations. Rare aberrations
were tested for inheritance from the parents, and validated with Affymetrix 250K
SNP arrays or 244K Agilent oligo-arrays. Thirteen aberrations passed our criteria. Two of them were very interesting and might be pathogenic. A deletion on
2p16.3 disrupts NRXN1 and was present in an affected sibling. A de novo duplication on 15q13.1 spans APBA2. The proteins of these two genes play a role in
synaptic development and function and interact directly. Both regions have been
affected by CNVs in other neurodevelopmental disorders. They remind possible
overlapping mechanisms of pathogenesis and suggest two strong positional and
functional candidate genes for schizophrenia. The paper regarding this study published in ‘‘Human Molecular Genetics’’ February issue as the cover paper. After
this, a follow-up study designed for NRXN1 gene; briefly, we aimed to resequence individuals for this gene to find out variants. In the first instance started
with a small group including 15 schizophrenics and than continued with 96 cases
and controls of exons. Interesting findings came up in this study, which I will
present in my talk.
S17-7
THE STUDY OF GLY82SER POLYMORPHISM OF RECEPTOR
FOR ADVANCED GLYCATION END PRODUCTS (RAGE) IN
DIABETIC AND NON DIABETIC PATIENTS WITH CORONARY
ARTERY DISEASE
Ozlem Kucukhuseyin1, Hulya Yilmaz-Aydogan1, Selim Isbir2,
Atike Tekeli2, and Turgay Isbir1
1
Istanbul University, Capa, Istanbul, Turkey, 2Department of Cardiovascular
Surgery, Marmara University School of Medicine, Istanbul, Turkey
Atherosclerotic coronary artery disease (CAD) is a multifactorial disease leading cause of morbidity and mortality. RAGE protein is a multiligand receptor of
immunoglobulin superfamily expressed at low levels in adult tissues in homeostasis, but highly overexpressed at sites of vascular pathology. The hyperglycemic
state of diabetes mellitus is associated with micro and macrovascular disease development. RAGE complexes, identified on different cell types, mediate AGE removal, trigger proinflammatory and procoagulatory genes and cellular migration
and proliferation. Gly-Ser change at position 82 occurs in ligand-binding domain
of RAGE. The aim of this study was determination of possible risks in the development of CAD in DM(1) and DM(-) patients and associations with RAGE.
Material and Method: We genotyped 52 DM(-),62 DM(1) patients with
CAD&55 CAD-free subjects. Genomic DNA was isolated from leukocytes by
use of salting-out procedure. The Gly82Ser change in the third exon was analyzed by PCR-RFLP method. All computations were performed by using the
SPSS 11.
Result: GlyGly genotype frequency is statistically higher in DM(-) patients
when compared with controls (p<0.001). GlySer frequency is higher in controls
when compared with DM (-) group (p<0.001). GlySer frequency is statistically
higher in DM(1) group when compared with both control and DM(-) group
(p<0.001). Ser allele frequency is respectively increased in the order of
DM(1)CAD>Control group >DM(-)CAD. These results reveals none association between Gly82Ser polymorphism and the development of CAD in DM(-)
patients. In DM(1) with Ser allele, higher prevalence of left ventricule hypertrophy (LVH) was observed. But the significant difference between Gly82Ser and
LVH was only found in the whole patient group. This study reveals that Ser allele has much more importance in the development of LVH than the other cardiovascular risk factors.
Conclusion: The AGE-RAGE engagement results in activation of proinflammatory and prothrombotic pathways. This study reveals in DM(-) patients presence of Gly allele contributes to CAD with receptor-ligand interaction. However
change of Gly to Ser in DM(1) patients alters receptor-ligand interactions,
results in higher blood levels of toxic AGE compounds (eg. HbA1c, glycated
LDL and collagen). In vascular diseases, binding of AGEs to endothelial proteins
increases vascular permeability dysfunction and basal membrane thickness. In
conclusion, we found that Ser allele contributes to CAD in DM(1) patients by
triggering macrophages consequent to increased levels of AGEs.
S17-8
THE STUDY OF -374T/A POLYMORPHISM OF RECEPTOR FOR
ADVANCED GLYCATION END PRODUCTS (RAGE) IN DIABETIC
AND NON DIABETIC PATIENTS WITH CORONARY ARTERY
DISEASE
Ozlem Kucukhuseyin1, Hulya Yilmaz-Aydogan1, Selim Isbir2 Atike Tekeli2,
and Turgay Isbir1
1
Istanbul University, Capa, Istanbul, Turkey, 2Department of Cardiovascular
Atherosclerotic coronary artery disease (CAD), due to the interaction of multiple genetic and environmental risk factors, is a complex disease and a major
cause of morbidity and mortality in Western population. However, diabetes mellitus is the most prevalent metabolic syndrome, which is characterized by hyperglycemia resulting in various short-term changes in lipid and protein metabolism
and long-term irreversible vascular changes. Chronic hyperglycemia results in
formation and accumulation of irreversible advanced glycation end products
(AGEs) that cause a plethora of adverse effects, resulting in dysfunction of the
vasculature and effects shown to be mediated by specific AGE binding receptors
(RAGE). The engagement of AGE with RAGE results in the activation of proinflammatory and prothrombotic pathways. A functional -374T/A polymorphism in
the promoter of RAGE gene effects on transcriptional activity. The aim of this
study was the determination of the possible risks in the development of CAD in
diabetic [DM(1)] and non-diabetic [DM(-)] patients and its association with
RAGE gene.
Material and Method: We genotyped the -374T/A RAGE polymorphism in
52 DM(-), 62 DM(1) with CAD and 55 CAD-free subjects. Venous blood samples were collected in Vacutainer tubes containing EDTA. Genomic DNA was
isolated from leukocytes by use of salting-out procedure. The A-T transversion
polymorphism at position -374 in the promoter region of the RAGE gene was
analyzed by PCR-RFLP method. All computations were performed by using the
SPSS11.0 statistical package.
Result: AA genotype frequency is statistically higher in the whole patient
group when compared with control group (28.3%-13.2%). AA genotype frequency is statistically higher in the patient group with DM(1) when compared
with control group (38.9%-13.2%). Homozygosity for the A allele was found
higher, but not statistically significant in DM(-) patients (17.3%) when compared
with control group (13.2%). In this study, conversely with other studies, we
found A allel as an independent risk factor in CAD with DM(1).
Conclusion: The repression of RAGE expression by A allele results in
higher blood levels of toxic AGE compounds such as HbA1c, glycated LDL and
collagen. In vascular diseases, binding of AGEs to endothelial proteins increases
vascular permeability dysfunction and basal membrane thickness. Accordingly,
we found that A allele contributes to CAD in DM (1) patients by triggering
macrophages with consequent increased AGEs levels.
321
S17-9
IS LOX-1 K167N POLYMORPHISM PROTECTIVE FOR CORONARY
ARTERY DISEASE?
Ozlem Kurnaz1, Hulya Yilmaz-Aydogan1, Selim Isbir2, Atike Tekeli2
and Turgay Isbir1
1
Cardiovascular Surgery, Marmara University School of Medicine,
Istanbul, Turkey
Background: Human lectin-like oxidized low-density lipoprotein receptor 1
(LOX-1), encoded by the OLR1 gene, was identified as a cell surface endocytosis receptor for oxidized low-density lipoprotein (Ox-LDL) in vascular endothelial cells. OxLDLs are avidly ingested by macrophages, resulting in foam cell
formation. OxLDLs are also involved in inducing smooth muscle cell (SMC)
migration, proliferation, and transformation. Previous studies have identified 7
novel polymorphisms in the LOX-1 gene. A single nucleotide polymorphism
K167N (G501C) of the LOX-1 gene that results in an amino acid dimorphism
(Lys/Asn) at residue 167 was found. Replacement of this Lys residue causes
reduced binding and internalization of Ox-LDL. The purpose of this study was
to investigate the effect of the LOX–1 K167N gene polymorphism in Turkish
patients with CAD.
Methods: K167N polymorphisms were studied in 116 patients with CAD
(32 (27.59%) women, 84 (72.41%) men. Healthy persons (45 (42.86%) women,
60 (57.14%) men) without any symptoms of CAD were selected for the control
group. Gene polymorphism was studied by the polymerase chain reaction followed by the agarose electrophoresis. Genotypes and allele frequencies were
compared by chi-square test. Clinical, nonclinical parameters and alleles were
compared by Kruskal Wallis test, while genotypes and clinical and nonclinical
parameters were compared by Student’s t test.
Results: The frequency of KK genotype and K allele were higher in the
CAD group than control subjects (p<0.05), while frequency of NN genotype
was higher in the control group than CAD subjects (p<0.05). It was observed
that the decreasing CAD risk in patients who had N allele, increased with male
sex (OR: 0.400? 1.534) and smoking (OR: 0.400 ? 0.917).
Conclusion: We suggest that male sex and smoking decrease the protective
effects of K167N mutant N allele. Although male sex and smoking were lower
than other cardiovascular risk factors in patients with N allele and were higher
than other cardiovascular risk factors in patients with K allele, we conclude that
the adverse effects of K allele on the CAD risk, resulting from LOX-1 K167N
polymorphism, is a risk factor independent from the other cardiovascular risk
factors. Finally, LOX-1 variants are effective on CAD by gene-environment
interactions more than gene-gene interactions.
S17-10
THE EFFECTS OF LOX-1 3’UTR188 CT POLYMORPHISM ON
CORONARY ARTERY DISEASE IN TURKISH PATIENTS
Ozlem Kurnaz1, Hulya Yilmaz-Aydogan1, Selim Isbir2, Atike Tekeli2,
and Turgay Isbir1
1
Istanbul, Turkey
Introduction: LOX-1 (lectin-like oxidized LDL receptor-1) was identified as
the major receptor for Ox-LDL in endothelial cells. Besides, this receptor is considered a critical molecule responsible for the binding, internalization and degradation of Ox-LDL in endothelial cells. Other studies showed that LOX-1 is also
expressed in macrophages, vascular smooth muscle cells (SMC), monocytes, platelets and fibroblasts. Over the last decade, LOX-1 has emerged as a promising
candidate gene for the development of cardiovascular disease, as the chromosomal region encoding the LOX-1 gene is associated with both atherosclerosis
and hypertension. Seven polymorphisms have been identified in the LOX-1 gene,
including a C-to-T substitution in the 3’-untranslated region (UTR). This polymorphism affects the binding of nuclear proteins in an allele-specific manner,
and the 3’UTR/T allele has been associated with an increased risk for coronary
artery disease (CAD) and acute myocardial infarction. The purpose of this study
322
was to investigate the effect of the LOX–1 3’UTR188 CT gene polymorphism in
Turkish patients with CAD.
Methods: LOX-1 gene 3’UTR188 CT polymorphisms were studied in 116
patients with CAD (32 (27.59%) women, 84 (72.41%) men). Healthy persons
(44 (44.4%) women, 55 (55.6%) men) without any symptoms of CAD were
selected for the control group. Gene polymorphism was studied by the polymerase chain reaction followed by the agarose electrophoresis. Genotypes and allele
frequencies were compared by chi- square test. Clinical, nonclinical parameters
and alleles were compared by Kruskal Wallis test, while genotypes and clinical
and nonclinical parameters were compared by Student’s t test.
Results: Distribution of genotypes and alleles of the LOX-1 3’UTR188 CT
polymorphism was not significantly different between controls and patients
(p>0.05). It was observed that in patients with 3’UTR T allele, high frequency
of hypertension was associated with TT genotype and high frequency of LVH. It
was found that male sex and smoking with 3’UTR-188T allele increase the risk
of CAD (respectively OR: 1.288 ? 2.889; OR: 1.288 ? 3.472).
Conclusion: We suggested that male sex and smoking increase the CAD risk
together with 3’UTR T allele. Finally, LOX-1 3’UTR188 variants are effective
on CAD by gene-gene interactions.
S17-11
COMPARISON OF LIPID PROFILE WITH APO B-100 ECORI
POLYMORPHISM ON OBESE CHILDREN WITH HYPERLIPIDEMIA
BY USING DENATURATING GRADIENT GEL ELECTROPHORESIS
Ozlem Timirci1, Feyza Darendeliler2, Firdevs Bas2, and Turgay Isbir1
1
Pediatric Endocrinology, Istanbul University, Istanbul Faculty of Medicine,
Istanbul, Turkey
Obesity, especially the cases starting from childhood, causes high morbidity
and mortality. In childhood obesity, the environmental factors are not assumed to
be effective as genetic factors, so genetic factors should be taken care of much
more than in adult cases. Apo B-100 is a glycoprotein that is one of the important factors in cholesterol homeostasis. Dyslipidemia is important in the pathogenesis of atherosclerosis; so genes related to lipoprotein metabolism are thought
to be responsible for the development of coronary artery diseases. For our study,
we built three separate groups; one of them was the group of obese children
with hyperlipidemia, second one was the group of obese children without hyperlipidemia and the last group contained of healthy children with neither hyperlipidemia nor obesity. In our study, we aimed to evaluate Apo B-100 EcoRI polymorphism and plasma lipid parameters altogether in childhood and adolescence
cases and this will be the first study in Turkey to determine possible relationships
among all discussed parameters. We amplified the related gene segment by using
polymerase chain reaction (PCR) and determined different patterns by using
denaturing gradient gel electrophoresis (DGGE) and applied automatic sequence
analysis. All results were examined by Proseq, BioEdit computer programme.
There was a single nucleotide polymorphism on 29th exon of ApoB-100 gene,
known as EcoRI polymorphism (G4154A). When we examined genotypic
changes for this single gene known as EcoR1 (G4154A) polymorphism in the
study groups, we could not find mutant A allele in the healthy control group. On
the other hand, we found mutant A allele in the patient group and a correlation
between total cholesterol and LDL-cholesterol levels, and GA genotype. The outcomes of this study, lead us to think that, obese children patients having A allele
could have a higher risk to have hyperlipidemia in future.
Ovarian cancer is the leading cause of death among gynecological malignancies. Oxidative stress is potentially harmful to cells and reactive oxygen species
are known to be induced in the initiation and progression of cancer. Paraoxonase
(PON) is an antioxidant enzyme, which eliminates lipid peroxides. M/L55 is one
of the common polymorphisms of PON that influence its concentration and activity. We studied whether the M/L55 polymorphism was related with PON activity
or not. There were 50 subjects in patient group and 52 healthy control subjects.
Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP), and agarose gel electrophoresis were used and PON activity was
measured by spectrophotometric assay of p-nitrophenol production following
addition of paraoxone. We found that smoker cases are significantly higher in
patients than controls (26.9% vs 7%; X2:7.81, p: 0.005; OR: 4.88 95% CI: 1.4915.99). Frequencies of PON 55 LL, MM, and LM genotypes among the patients
were 0.53, 0.10, 0.37; among the control subjects, there were 0.46, 0.04, 0.50,
respectively. Consequently, MM genotype frequencies were higher in patients
than control groups, but this was not statistically significant (p[0.05). Our findings showed that this polymorphism was associated with age onset of ovarian
cancer, which increased in age order of the LM \ LL \ MM genotype in the
PON1 55 polymorphism. In conclusion, our results suggested that the PON1 192
AA genotype may play an important role as a risk factor for ovarian cancer in
Turkish population and L allele may be associated with early onset of disease.
S17-13
INVESTIGATION OF TNF-RELATED APOPTOSIS INDUCING LIGAND
AND BCL-2 ASSOCIATED X PROTEIN GENE POLYMORPHISMS IN
BREAST CANCER PATIENTS
Yemliha Yildiz1, Ilhan Yaylim-Eraltan1, Soykan Arikan2, and Turgay Isbir1
1
Medicine, Istanbul University, Capa, Istanbul, Turkey, 2Surgery Clinic,
Istanbul Education and Research Hospital, Istanbul, Turkey
Apoptosis is a fundamental biochemical cell-death pathway essential for normal tissue homeostasis, cellular differentiation, and development. TNF-related
apoptosis inducing ligand (TRAIL) and Bcl-2 associated X-protein (BAX) are
two critical proteins in extrinsic and intrinsic pathways of apoptosis. The aim of
this study was to investigate the susceptibility and prognostic implications of the
TRAIL and BAX gene polymorphisms in breast cancer. We investigated a single
nucleotide polymorphism (SNP) in the 3-untranslated region of the TRAIL gene
at position 1595 exon 5, and the G(-248)A polymorphism in the promoter region
of the BAX gene. We used PCR, RFLP and gel electrophoresis techniques to
detect these polymorphisms in 67 breast cancer patients and 57 healthy controls.
We found no association between TRAIL and BAX genotypes and susceptibility
of breast cancer. There were no association between tumor stages, lymph node
metastasis and TRAIL or BAX genotypes. However, a significant association
was found between the TRAIL C allele and perineural invasion (p50.049, OR: 8
95% CI 0.883-72.499). Patients with the T3/T4 stage breast carcinoma showed a
lower prevalence of TRAIL C allele when compared to the patients with T1/T2
stage breast cancer (P5 0.05; OR: 0.157 95% CI 0.025-0.977). TRAIL CC genotype was seen more commonly in our breast cancer patients with angiolymphatic
invasion compared to other genotypes (p50.05). In addition, an association was
found between BAX gene polymorphism and angiolymphatic invasion (p50.036,
OR: 0.133, 95% CI 0.022-0.805). These results suggested that TRAIL and BAX
gene polymorphism might accompany poor prognostic factors and influence the
tumor course.
S18. School of Molecular Medicine
S17-12
INVESTIGATION OF PON1 192 AND PON1 55 POLYMORPHISMS IN
OVARIAN CANCER PATIENTS
Aytac Arpaci, Burak Dalan, Bedia Agachan, Uzay Gormus, Yaman Tekant,
Rukset Attar, and Turgay Isbir
Istanbul University, Capa, Istanbul, Turkey
S18-1. MOLECULAR MEDICINE AND
DIAGNOSTIC APPLICATIONS
MOLECULAR MEDICINE AND DIAGNOSTIC APPLICATIONS
Turgay Isbir1, Ilhan Yaylim-Eraltan1, Oguz Ozturk1, Bedia Agachan1,
Hulya Yilmaz-Aydogan1, Umit Zeybek1, H. Arzu Ergen1, Uzay Gormus1,
and Michael Klentze2
1
Istanbul University, Institute for Experimental Medical Research,
Department of Molecular Medicine, Istanbul, Turkey, 2Germany
Developments in the study of eukaryotic molecular biology have given rise
to a new era in the understanding of the pathology of human disease. Advances
in recombinant nucleic acid technology have enabled researchers to identify
genes responsible for inherited disease, to dissect the pathological mechanism of
infectious organisms, and to define the regulators of cell differentiation and proliferation that act aberrantly during neoplasia. Diagnostic application of the molecular technology is a logical consequence of this new level of understanding
disease. The new molecular assays are primarily based on the principles of
nucleic acid hybridization and rely on a thorough knowledge of the DNA and
RNA sequences relevant to each diagnostic system. Because DNA sequences
vary from person to person, nucleic acid hybridization assays are also relevant to
the science of individual identification, as in forensic and parentage testing, and
to our ability to enhance tissue matching for transplanter. In this session, we discuss the basic concepts of molecular medicine in its connection to the clinical
analyses of nucleic acid with an introduction to nucleic acid chemistry and biochemistry.
323
MDTPs’’, and ‘‘Findings from QA Surveys in MDTPs and Recommendations’’
will be presented by Diler Aslan, Tamer Inal, and Francois Rousseau, respectively. The second will be case-based and interactive, and aims to find the
answers of these questions: How can I establish the IQC program? How can I
find the EQASs for the steps of the procedures? How can I evaluate and interpret
the results of IQC and EQASs’ results, and improve performances according to
them? What are the performance characteristics of MDMs?
The session responsibility will be shared by the experts in the QA for CLs
and MDTPs in several extents, Gulderen Yanikkaya Demirel, Yesim Ozarda
Ilcol, Tamer Inal, Francois Rousseau, Diler Aslan, and Gul Guner.
Conclusion: The following issues will be reviewed: OECD Guidelines for
QA in Molecular Genetic Testing, 2007; European Molecular Genetics Quality
Network (EMQN) (http://www.emqn.org/emqn/), Harmonizing Genetic Testing
across Europe (EuroGenTest) (http://www.eurogentest.org/); Standards and
Guidelines for Clinical Genetics Laboratories (http://www.acmg.net/); ISO
15189; accreditation standards; some national legislation on MDTPs. The general
review on what have been done, and how they have been done will be summarized in the individual laboratory level by international view.
S18.2. GENETICS AND STATISTICS,
QUALITY CONTROL
EDUCATIONAL PROGRAMME: GENETICS AND STATISTICS,
QUALITY CONTROL AND ACCREDITATION IN CLINICAL
LABORATORIES
Diler Aslan1, Gul Guner2, Tamer Inal3, Francois Rousseau4,
Yesim Ozarda Ilcol5, and GulderenYanikkaya Demirel6
1
Department of Biochemistry, Pamukkale University, Faculty of Medicine,
Turkey, 2Department of Biochemistry, Dokuz Eylul University, Faculty of
Medicine, Turkey, 3Department of Biochemistry, Cukurova University,
Faculty of Medicine, Turkey, 4Human genetics and Molecular Research,
Laval University, Faculty of Medicine, Canada, 5Department of
Biochemistry, Uludag University, Faculty of Medicine, Turkey,
6
Immunology Centro Labs, Turkey
Background: Molecular diagnostic methods (MDMs) have been implemented in clinical laboratories (CLs) by the rate increasing exponentially. Guidelines and standards are being established for best practices, quality, and accreditation to get clinically useful and reliable results in timely and cost-effective
manner. Every CL should establish a quality assurance (QA) system by constructing internal quality control (IQC), and participating in the external quality
assessment schemes (EQASs) for MD testing procedures (MDTPs). The accreditation is necessary for substantiating the technical and clinical competency.
Aim and Objectives: This educational programme aims to introduce what
have been done to assure the quality of MDTPs, and how they have been implemented in order not to harm the patients. The expected outcomes for the participants are: 1) to learn how to establish their IQC, and how to evaluate the IQC
results; 2) to choose the EQAS providers; 3) to learn about the guidelines, standards and several national legislations.
Method: There will be two sessions. In the first session ‘‘Guidelines and
Standards for QA in MDTPs in CLs’’, ‘‘Accreditation of CLs in the Context of
S18.3. REPRODUCTIVE MEDICINE
PGD/PGS IN REPRODUCTIVE MEDICINE
Nesrin Ercelen
VKV Amerikan Hospital, Department of Genetics, Istanbul, Turkey
Preimplantation Genetic Diagnosis (PGD) describes procedures involving the
removal of polar bodies from oocytes or blastomeres from embryos in order to
test for mutations in gene sequence or chromosomal aneuploidy before transfer.
PGD is considered the earliest form of prenatal diagnosis. It is used to detect
genetic diseases, its significant aim is to improve pregnancy rates and decrease
miscarriage rates with IVF. Routinely up to 9 to 12 chromosomes can be analyzed on a single fixed nucleus by fluorescent in situ hybridization (FISH), in
which DNA probes labeled with fluorochromes hybridize to their respective
chromosomes, allowing for the identification of ploidy status. Indications for
PGD include advanced maternal age (AMA), recurrent implantation failures
(RIF), recurrent spontaneous abortions (RSA), severe male factor (SMF) and
translocations. Blastomeres biopsied from 6-8 cells embryos on day 3 are fixed
on a slide. Slides are pretreated with 2XSSC, formaldehyde and pepsin solution
to remove residual cytoplasm around nucleus. MultiVysion PB (Vysis) probe is
used for screening of chromosomes 13, 16, 18, 21, 22 at the first round of
hybridization. Four Color Custom (Vysis) probe is used for screening of chromosomes 15, 17, X, Y at the second round of hybridization. Hybridization is
accomplished in HyBrite instrument by a denaturation step at 738C for 5 min
and hybridization step at 378C for 4 hours. Slides are posthybridized in
0.4XSSC, 0.3% NP-40 for 5 min and in 2XSSC, 0.1% NP-40 for 1 min. Antifade is applied as counterstain on the slides. Each nucleus is analyzed on a fluorescent microscope with appropriate filters. At least two geneticists will evaluate
the data. Related IVF center will be informed, as the results are ready.
324
POSTER SESSION
Posters
P-01
of the asymptomatic students with positive PCR for P. vivax will be necessary at
given time intervals in order to identify successive symptomatic malaria. In this
sense the PCR method is recommended.
THE EFFECT OF CARNOSINE ON OXIDATIVE STRESS
PARAMETERS IN DIFFERENT TISSUES OF AGED RATS
P-03
A. Fatih Aydin, Canan Kucukgergin, Gul Ozdemirler-Erata,
Necla Kocak-Toker, and Mujdat Uysal
Istanbul University, Medical Faculty, Department of Biochemistry, Istanbul,
Turkey
MOLECULAR PROFILE OF TRAIL AND ITS RECEPTOR
EXPRESSION ON PANCREAS OF STREPTOZOTOCIN-INDUCED
DIABETES IN NOD MICE
Sevim Kahraman1, Ercument Dirice1, Mustafa K. Balci2, Abdulkadir Omer3,
Salih Sanlioglu1, and Ahter D. Sanlioglu1
1
Medical Biology and Genetics, and Gene Therapy Unit, Akdeniz
University, Faculty of Medicine, Antalya, Turkey, 2The Division of
Endocrinology and Metabolism, Akdeniz University, Faculty of Medicine,
Antalya, Turkey, 3Section on Islet Transplantation and Cell Biology Joslin
Diabetes Center, Harvard Medical School, USA
Carnosine (b-alanyl-L-histidine) is a dipeptide with antioxidant properties.
One of the mechanisms underlying the aging process is proposed to be oxidative
damage by free radicals. This study was done to investigate the effects of carnosine treatment on lipid peroxidation and antioxidant status of liver, heart, brain
and testis of aged (22 months) and young (5 months) rats. Carnosine (200 mg/
kg, i.p.) was administered to young and aged rats for 1 month. Malondialdehyde
and diene conjugate levels, non-enzymatic (glutathione, vitamin E and vitamin
C) and enzymatic (superoxide dismutase, glutathione peroxidase and glutathione
transferase) antioxidant status were determined in tissues of young and old rats
treated with and without carnosine. Carnosine treatment caused significant
decreases in lipid peroxidation without any apparent changes in antioxidant status. Our findings indicate that carnosine supplementation seems to be useful for
decreasing age-induced oxidative stress in tissues.
P-02
COMPARISON OF 2 METHODS OF DIAGNOSIS OF PERIPHERAL
BLOOD SMEAR AND POLYMERASE CHAIN REACTION AT
ASYMPTOMATIC MALARIA IN STUDENTS AGED 7-11 YEARS OLD
Abdollah Karimi, Hassan Pourmoshtagh, Alireza Fahimzad, Fatemeh Fallah,
Shahnaz Armin, and Saeed Maham
Pediatric Infectious Research Center, Faculty of medicine, Shahid Beheshti
University (M.C), Iran
The incidence of malaria in the east and southeast areas of Iran is high and
that area’s are considered as hypoendemic. WHO recommends that the standard
method for diagnosis of symptomatic malaria is thick and thin peripheral blood
smears (PBS), which has a low cost; however, for asymptomatic malaria and in
patients that have low plasmodium parasitemia and in mixed infections with two
or more types of plasmodium, the PCR method can be more effective in diagnosis than the PBS method. On the other hand, people that have asymptomatic
malaria with positive PCR for plasmodium can be considered as sources of
malaria and cause increased transmission of plasmodium to other people in that
area. This indicates the importance of detection of people who have asymptomatic malaria in an endemic area. This is a diagnostic study with cross sectional
form. Because students where more accessible than children younger than 7
years old, the study population was chosen among malaria asymptomatic students
between 7 and 11 years, in the Ghale Ganj area of Kerman in Iran. The goal of
this study was comparison of two methods PBS and PCR for diagnosis of
malaria in this population. 900 asymptomatic students between 7-11 years old in
the Ghale Ganj area of Kerman were entered in this study. 469 students (52.1%)
were female and 431 (47.9%) were male. All of 900 students were negative for
P. vivax and P. falciparum by thick and thin blood smear; PCR method detected
DNA P. vivax in 10 blood samples (1.1%). 871 students (96.8%) had negative
history of malaria in the last year and 29 (3.2%) had positive history of malaria
but P. vivax DNA was not detected in any of their blood samples by the PCR
method. In this study specificity and negative predictive value (NPV) of the PCR
method were respectively 98.89% and 100%, compared to PBS. It was impossible to determine sensitivity, positive predictive value (PPV) and likelihood ratio
(LR) of the PCR method, because all 900 students had negative PBS. In the
hypoendemic area of Ghale Ganj of Kerman, for the determination of malaria
infection in children, because of the high cost of the PCR method and the time
needed with this process for the identification of malaria parasite, the PCR
method is not suitable in comparison of the PBS method. Long term follow up
Background: The effector molecules playing a role in the development of
Type 1 diabetes (T1D) should be clearly defined for its molecular mechanism to
be elucidated. Non-Obese Diabetic (NOD) mice are the most frequently preferred
animal models used in T1D research. They develop spontaneous disease with
very similar characteristics to that in humans. TNF-Related Apoptosis Inducing
Ligand (TRAIL) is a recently defined TNF family member. Its blockage was
shown to create sensitivity for development of T1D, yet the exact role of TRAIL
in T1D development is not known. We aimed to detect the differences in TRAIL
ligand and receptor expression patterns in the course of T1D development in
NOD mice, to help unravel the role of this molecule in the disease process. We
used Streptozotocin, which destructs pancreatic beta cells mainly through DNA
fragmentation, to accelerate disease manifestation.
Materials and Methods: The optimal dose of Streptozotocin was 150 mg/dl,
which was used to accelerate diabetes in NOD mice, and in Non-Obese Diabetes
Resistant (NOR) mice as controls. Pancreatic tissues were collected at certain
time intervals reflecting sequential steps of disease development at days 0, 1, 2,
4, 7, 14, and 21. All mice were followed for diabetes development by blood
sugar measurements. Immunohistochemical analyses were performed for the
detection of alterations in TRAIL ligand and receptor expression patterns.
Results: The majority of the NOD mice developed diabetes by day 4 after
STZ administration, reflected by blood sugar of 250 mg/dl and over. Immunohistochemical analysis of pancreatic tissues collected from Streptozotocin-induced
pre-diabetic to severely diabetic mice revealed significant alterations in TRAIL
ligand and receptor expression patterns.
Conclusion: NOD mice with Streptozotocin administration have been shown
to be a suitable disease model to study the molecular pathogenesis of T1D.
Alteration in TRAIL ligand and receptor composition during the course of disease development sheds light on the mechanism of T1D.
P-04
HIGHLIGHTS IN THE MOLECULAR PATHOGENESIS OF
TYPE 1 DIABETES
Sevim Kahraman1, Ercument Dirice1, Mustafa K. Balci2, Abdulkadir Omer3,
1
Genetics, 2The Division of Endocrinology and Metabolism, Akdeniz
University, Faculty of Medicine, 07070, Antalya, Turkey; 3Section on Islet
Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical
School, Boston, Massachusetts, USA
Type 1 diabetes (T1D) is caused by autoimmune destruction of the pancreatic
beta cells through the action of pro-inflammatory cytokines such as TNF-alpha
and FasL produced by the invading, activated mononuclear cells. These cytokines
trigger apoptotic death of the islets, which in the advanced state lead to clinical
manifestation of the disease with well-known diabetes symptoms such as hyperglycemia and related complications. Over 90% of the pancreatic islets are usually
destroyed at this stage. Despite this clear scene, the effecter molecules playing a
role in the development of T1D and their related mechanism are not yet well char-
acterized. Elucidation of the molecular pathogenesis of T1D is crucial for a better
understanding of the disease, which will lead to novel complementary or alternative modes of treatment. Although there is a clear need for further research in the
area, collecting human pancreatic tissue for scientific study is not considered ethical, nor medically suitable, as every bit of the intact tissue is crucial in humans for
diabetes-free survival. Thus much of what is known today on T1D pathogenesis is
provided by research on animal models, particularly the Non-Obese Diabetic
(NOD) mice. These mice were found to share many characteristic histo-pathological features of T1D development with humans, including the various sequential
stages of insulitis, eventually leading to diabetes. Besides developing spontaneous
T1D in approximately 24 to 30 months when kept in suitable environmental conditions, NOD mice can be induced for a much faster development of the disease,
through agents such as Cyclophosphamide and Streptozotocin, for application of
various research designs. This presentation will focus on the molecular pathogenesis of T1D development based on studies mainly, but not strictly, in NOD mice.
Various aspects, particularly the cytokine approach, will be discussed in the light
of related reports and ongoing research.
P-05
MOLECULAR CHARACTERIZATION OF TRAIL LIGAND AND
RECEPTOR EXPRESSIONS IN CYCLOPHOSPHAMIDE-INDUCED
TYPE 1 DIABETES IN NOD MICE
Ercument Dirice1, Sevim Kahraman1, Mustafa K. Balci2, Abdulkadir Omer3,
1
Medical Biology and Genetics and Gene Therapy Unit, Akdeniz University,
Faculty of Medicine, Antalya, Turkey, 2The Division of Endocrinology and
Metabolism, Akdeniz University, Faculty of Medicine, Antalya, Turkey,
3
Section on Islet Transplantation and Cell Biology Joslin Diabetes Center,
Harvard Medical School, USA
Background: Type 1 Diabetes (T1D) is an autoimmune inflammatory disease affecting predominantly children. The mechanism of T1D is not yet well
understood. Collection of human pancreatic tissue for scientific research is not
considered ethical, nor medically suitable. Thus, animal models are widely used
in investigations to further understand the pathogenesis of T1D. Non-Obese Diabetic (NOD) mice are the most frequently preferred animal models in T1D
research. TNF-Related Apoptosis Inducing Ligand (TRAIL) has recently been
related to T1D pathogenesis. However, its role in disease development is not yet
clear, which requires further research. We aimed to detect the differential pattern
of TRAIL ligand and receptor expressions in pre-diabetic to severely diabetic
stages in NOD mice. Cyclophosphamide (CY) was used to accelerate diabetes
development. It mainly acts on suppressor T cells to ease destruction of pancreatic beta cells by cytotoxic T lymphocytes.
Materials and Methods: Diabetes development was accelerated in NOD and
Non-Obese Diabetes Resistant (NOR) mice by CY application at an optimal
dose of 200 mg/kg. Development of diabetes was monitored in all mice by blood
sugar measurements. Pancreatic tissue collection was performed at days 0, 1, 4,
1, 14, 21, and 28. Changes in TRAIL ligand and receptor expression patterns
were detected by immunohistochemistry.
Results: No rise was observed in blood sugars of CY-induced NOD mice
until after day 10. From this time on, mice displayed a gradual rise in blood sugars until eventually developing diabetes (with blood sugar levels over 250 mg/
dl). Immunohistochemical analysis revealed substantial changes in TRAIL ligand
and receptor expression levels in the course of T1D development.
Conclusion: Cyclophosphamide-administered NOD mice proved to be a useful model for investigating the molecular pathogenesis of T1D, with relatively
slow-progressing, yet prominent manifestation of the disease. Changes detected
in TRAIL ligand and receptor expression patterns suggested an important role
for these molecules in the molecular pathogenesis of T1D.
325
1
Department of Biochemistry, Dokuz Eylul University, Faculty of Medicine,
Izmir, Turkey, 2Hematology Laboratory, Dokuz Eylul University, Faculty of
Medicine, Izmir, Turkey, 3Department of Oncology, Dokuz Eylul
University, Faculty of Medicine, Izmir, Turkey
Objective: It is known that colon cancer is a type of cancer which has high
incidence and mortality. Recently anti-cancer effect of green tea has become a
popular research subject. Apoptotic effect of epigallocatechin-3-gallate (EGCG),
the major catechin derivative of green tea, has been demonstrated in different
cancer cell lines. The number of studies related with colon carcinoma is quite
limited. In this study, we aimed to investigate the anticancer effects of EGCG on
human colon carcinoma cell line, HCT-116 cells.
Materials and Methods: HCT-116 cells were treated with different doses of
EGCG (0-100 uM) for 24 hours. MTT cell viability assay was carried out to
investigate the effects of EGCG on viability. Sufficient inhibitory dose of EGCG
was decided from cell viability assays data. The type of cell death, induced with
different doses of EGCG, was determined by Annexin-V-FITC analysis with
Flow Cytometry. Induction of Apoptosis was also confirmed by Cell Death
Detection assay and Flow Cytometric DNA Fragmentation analysis. Etoposide
was used as positive control for induction of apoptosis in all experiments.
Results: MTT assays demonstrated that induction of cell death had occurred
in a dose dependent manner. However 50 lM and 100 lM concentrations of
EGCG caused approximately 50% inhibition of cell viability, the lower concentration of EGCG (50 lM) was used as a sufficient inhibitory dose in all experiments. Apoptosis was found to be major cell death type of EGCG in different
doses by both DNA Fragmentation and Annexin-V-FITC analysis with Flow
Cytometry. The results of Annexin-V-FITC analysis showed that sufficient inhibitory dose of EGCG (50 lM) caused 21.8% early apoptosis 2.9% late apoptosis
and 4.8% necrosis. Flow Cytometric DNA Fragmentation analysis confirmed
these results with 31.8% cells in sub G0/G1 phase of cell cycle (Fig. 1) Cell
death data with a sufficient inhibitory dose of EGCG (50 lM) was consistent
with the results with a 7.27 fold enrichment of mono- and oligonucleosomes.
Conclusion: Our data suggests that EGCG induces primarily apoptosis but
not necrosis in HCT-116 cell line in a dose dependent manner. Especially our
findings proved that doses of EGCG lower than 100 lM have the capacity to
induce apoptosis in vitro. For this reason, EGCG can be a candidate as additive
agent in chemotherapy. These results may be the basis for further studies on the
anticancer use of EGCG and other green tea cathechines.
P-06
APOPTOTIC EFFECT OF (-)-EPIGALLOCATECHIN-3-GALLATE ON
HCT-116 COLON CARCINOMA CELLS
1
1
2
3
Ali B. Ozkaya , Birce Akpinar , Halil Ates , Zekiye Altun ,
and Semra Kocturk1
Figure 1. Effects of 50 uM EGCG treatment for 24 hours on cell cycle in HCT116 cells (treated: A, untreated: B), and Annexin-V/PI binding to cells (treated:
C, untreated: D). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
326
P-07
P-09
CXCL12/CXCR4 MODULATES GLIOMA CELL SURVIVAL AND
MOTILITY
A FAST CHI-SQUARE BASED ALGORITHM FOR TEXT
CATEGORIZATION OF MEDLINE CITATIONS
Anália Carmo1,2, Helena Carvalheiro1, Maria Teresa Cruz1,3,
and Maria Celeste Lopes1,2,3
1
Cellular Immunoloy and Oncobiology, Center for Neuroscience and Cell
Biology, University of Coimbra, Portugal, 2Center of Investigation in
Environment, Genetics and Oncobiology (CIMAGO), Portugal, 3Pharmacy
Faculty, University of Coimbra, Portugal
Andrej Kastrin1, Borut Peterlin1, and Dimitar Hristovski2
1
University Medical Centre Ljubljana, Institute of Medical Genetics,
Slovenia, 2University of Ljubljana, Institute of Biomedical Informatics,
Slovenia
The regulation of cell survival and motility in both normal and disease situations depends on a variety of mediators. Among these mediators is the chemokine CXCL12 also named stromal-derived factor 1, which binds mainly to the
cell-surface receptor CXCR4. The axis CXCL12/CXCR4 has been associated
with an increased cell survival and motility in several tumours. Gliomas are brain
tumours that account for more than 50% of the tumours that arise within the central nervous system. They are highly proliferative, angiogenic and locally very
invasive. The mechanism by which malignant glioma develops is not completely
understood. In order to understand the role of CXCL12/CXCR4 in gliomas, we
studied the survival and motility of glioma cells treated with AMD3100, an antagonist of the CXCR4. In this study we used the human glioma cell line U-118.
The assays were performed in the presence and/or absence of CXCL12 and
AMD3100. CXCR4 expression in glioma cells was evaluated by western blot
and immunofluorescence. Cell survival was evaluated by flow cytometry and
confocal microscopy, using Hoechst and propidium iodide. Cell migration studies
were performed using the scratch assay. Cytoskeleton organization was analysed
using phalloidin. Regarding cell survival, our results show that the inhibition of
CXCR4 by AMD3100 increase cell death by apoptosis. The motility studies
showed that CXCL12 increases significantly the migration ability of the U-118
cells, whereas AMD3100 significantly reduces the migration ability and disrupt
cell cytoskeleton. In conclusion, our in vitro studies using the U-118 cell line
indicate that CXCL12/CXCR4 axis induces an increase in cell survival and motility and therefore may play an important role in the development of gliomas.
Semantic ambiguity and ambiguous gene symbols in particular, are important
problems for biomedical text mining systems. When detecting gene symbols in
MEDLINE1 citations using thesaurus-based techniques one of the biggest challenges is the fact that many gene symbols also denote other, more general biomedical concepts. Our approach to this task is first to categorize the MEDLINE citations
into genetic and non-genetic domains and then to extract gene symbols only in the
genetic domain using standard string matching techniques. Therefore, a significant
effort is spent on identifying which MEDLINE citations are relevant for each domain. Our statistical procedure requires a construction of a background and a
genetic domain corpus. We used ontological information provided by MeSH1 for
this categorization task. A MeSH descriptor is considered to be a positive indicator
if its relative observed frequency in the genetic domain corpus is greater than its relative observed frequency in the background domain corpus. The output of scoring
process is a list of scores for all the citations, with the highest total given to those
citations containing MeSH descriptors typical for the genetic domain. Validation
was done on a set of 734 manually annotated MEDLINE citations. It achieves predictive accuracy of 0.84 with 0.63 recall and 0.67 precision. We thoroughly evaluated the method by comparing it to three well-known machine learning algorithms,
namely support vector machines, decision trees, and naı̈ve Bayes. Although the differences are not statistically significantly different, results showed that chi-square
scoring performs better than compared algorithms. We suggest that the chi-square
scoring is an effective solution to help categorize MEDLINE citations. In a real
application the trade-off between precision and recall can be tuned by changing a
single threshold parameter. The algorithm is currently implemented in the BITOLA
literature-based discovery support system (http://www.mf.uni-lj.si/bitola/).
P-10
P-08
IDENTIFICATION OF HAEMOGENOMIC BIOMARKERS IN
NEURODEGENERATIVE DISEASES: META-ANALYSIS APPROACH
Andrej Kastrin and Borut Peterlin
Institute of Medical Genetics, University Medical Centre Ljubljana,
Slovenia
Neurodegenerative diseases are a family of disorders which includes complex
multifactorial diseases (e.g., Parkinson’s disease) and monogenic diseases (e.g.,
Huntington’s disease) among others. Global gene expression studies have provided new insights into the pathogenesis of these diseases. Several studies report
on differentially expressed genes in brains and blood of patients with neurodegenerative disorders. Although experimental results of global gene expression
measuring may identify common genes, each research group will typically produce different list of statistically significant differentially expressed genes, which
calls into question the reliability and validity of each gene list. Combination of
results from research groups using a meta-analysis approach provides an accurate
estimate of the degree of a differential expression for each gene. Precise and reliable measures of gene expression are especially important in biomarker set construction. In this study, we consider Parkinson’s disease and Huntington’s disease
as a model of complex disease. We analyzed microarray datasets of brain tissue
and whole blood to investigate common patterns of gene expression in a given
disease. Raw gene expression data sets were downloaded from the Gene Expression Omnibus (GEO) public repository. We identified one gene (ETV5) in Huntington’s disease was expressed both in brain tissue and blood, in contrast to Parkinson’s disease where three genes (SLC29A1, ZNF710, PRKAR1A) were found
in common. ETV5 is primary involved in regulation of transcription, whereas
other three genes are responsible both for transcription and signal transduction.
Our findings support the idea that haemogenomic approach could identify useful
biomarkers for neurodegenerative disorders. Further genomic studies are necessary to increase the reliability of biomarker identification.
LEIDEN FV, PT G20210A Y MTHFR C677T MUTATIONS
FREQUENCY IN MEXICAN PATIENTS WITH PRIMARY
TROMBOPHILIA
Marı́a de la Luz Arenas-Sordo1, César Zavala-Hernández2,
Edgar Hernández-Zamora1, Carlos Martı́nez-Murillo3,
Abraham Majluf-Cruz4, Elba Reyes-Maldonado5, and Jorge Vela-Ojeda6
1
Department of Genetics, Instituto Nacional de Rehabilitación, Mexico,
2
Clinical Laboratory, Instituto Nacional de Rehabilitación, Mexico,
3
Department of Hemathology, Hospital General de México, 4Hemosthasis
and Thromobiosis, Instituto Mexicano del Seguro Social, Mexico,
5
Department of Morphology, Instituto Politécnico Nacional, Mexico,
6
Department of Hemathology, Instituto Mexicano del Seguro Social, Mexico
Introduction: There are at least 26 genes mutations or polymorphisms
involved in primary trombophilia. For different reasons, it is unknown which is
its real incidence; one of these reasons being racial differences. Worldwide it is
supposed that the incidence is approximately 1:2,500 – 1: 5,000. We suppose
Mexico has the same problem about trombophilia, but we do not know the incidence and prevalence of the different mutations in the coagulation factors and
we can not have the same patients follow programs that already prove their
effectiveness in other countries. We do not have a national register of the
patients and to contact them is very difficult.
Aim: To determine trough PCR-RFLP, Factor V Leiden, PT G20210A y
MTHFR C677T mutations and their frequency, and compare them with a Caucasian group.
Method: We did a descriptive and prospective study with 200 patients with
primary trombophilia and 100 healthy persons. By using PCR-RFLPS method,
we looked for the mutations Leiden Factor V, PT G20210A y MTHFR C677T.
Results: The age of both groups was between 18 and 45 years. Four patients
(2%) were heterozygous for the PT G20210A mutation. None of the controls had
the mutation. For the Factor V Leiden mutation none of the patients neither the
controls had it. The MTHFR C677T mutation, 117 patients were heterozygous,
13 homozygous and 70 did not have it. In the controls the proportion was similar, 46 were heterozygous, 6 homozygous and 48 did not have it.
Discussion: Our results show that we are different relative to the Caucasian
people, at least in this sample, because we did not find the prevalence of the Leiden Factor V mutation and the proportion founded in the MTHFR C677T mutation in its heterozygous form, was greater than reported in the literature. It is
necessary to study more patients and general population, and to define if the
sample we studied is similar, because our admixture is very important. It could
be that our sample corresponds to three or four subpopulation.
P-11
EFFECT OF L-CARNITINE ON NO PRODUCTION IN
LIPOPOLYSACCHARIDE INDUCED RAW 264.7 CELLS
Asli Koc1, Arzu Z. Karabay1, Tulin Ozkan2, Aynur Karadag3,
Asuman Sunguroglu3, Fugen Aktan1, and Zeliha Buyukbingol1
1
Ankara University, Faculty of Pharmacy, Department of Biochemistry,
Ankara, University Graduate School of Health Sciences, Turkey, 2Ankara
University, Institute of Biotechnology, Turkey, 3Ankara University, Faculty
of Medicine, Department of Medical Biology, Ankara University Graduate
School of Health Sciences, Turkey
Objective: L-Carnitine (b-hydroxy-g-trimethylaminobutyrate), a natural quaternary ammonium compound, is a ubiquitous constituent of mammalian plasma
and tissues. It has been shown to exert protection in pathological conditions characterized by increased oxidative stress. Lipopolysaccharide (LPS) activates multiple signaling pathways in macrophages, and induces the production of several
inflammatory mediators including NO. The present study investigates the effect
of L-carnitine on nitric oxide production induced by lipopolysaccharide in RAW
264.7 macrophage cells.
Material and Methods: Cell Culture and stimulation: RAW 264.7 cells were
cultured in RPMI 1640 medium supplemented with 10% heat inactive Fetal Calf Serum (FCS), 2mM L-Glutamine, and antibiotics (1.000.000 U/ml penicillin and 1 g/
mL streptomycin) at 37 0C in a humidified atmosphere of 5% CO2 in air. After
counting with 0.4% trypan blue, cells (4 x 104) were plated in 96-well micro plates
and incubated in the absence or presence of LPS (1 lg/mL) for 20 hours. Different
concentrations of L-carnitine (10-8 M,10-7 M,10-6 M,10-5 M,10-4 M,10-3 M,10-2
M,10-1 M) was added to cultures 1 hour before stimulation with LPS. Determination
of cell viability: To determine the effect of L-carnitine on cell viability, conventional MTT (thiazolyl blue tetrazolium bromide) reduction assay was used according to the instructions of the manufacturer. Nitrite measurements: Samples (100 ll)
of culture media were incubated with an equal amount of Griess reagent at room
temperature for 10 min in a 96-well micro plate before determining the absorbance
at 548 nm. The absorbance at 548 nm was compared with standards of NaNO2. Statistical analysis: Differences among groups were evaluated by One Way Anova
analysis. P values less than 0.05 were considered as statistically significant.
Results: L-carnitine was shown to be cytotoxic to RAW 264.7 cells at 10-1
M concentration. LPS induced NO production was reduced by L-carnitine in a
dose dependent manner. The concentrations of L-carnitine effective for reducing
NO levels were found to be 10-3 M, 10-4 M, and 10-5 M.
Conclusion: Data obtained from this study indicates that L carnitine has an
inhibitory effect on NO production in LPS induced macrophage cells. However
further investigation is needed to unravel the mechanisms underlying this effect.
L-carnitine may have the potential to be used as a therapeutic agent in disorders
characterized by increased NO production.
P-12
VITAMIN-D RECEPTOR (FOK-I) GENE POLYMORPHISM IN
TURKISH PATIENTS WITH MYOCARDIAL INFARCTION
Atike Tekeli1, Uzay Gormus1, Hulya Yilmaz-Aydogan1, Selim Isbir2,
Umit Zeybek1, Canan Cacina1, Ilhan Yaylim-Eraltan1, and Turgay Isbir1
1
Department of Molecular Medicine The Institute of Experimental
Istanbul, Turkey
Background: It is known that the vitamin D receptor (VDR) is involved in
vascular smooth muscle cell growth and could therefore be involved in plaque
327
instability. A single-nucleotide polymorphism (defined by the endonuclease FokI)
located in the start codon of the VDR creates the alleles F and f, resulting in different proteins. The goal of this study was to examine the association of the
FokI polymorphism and myocardial infarction.
Materials and Methods: For this purpose, the distribution of VDR FokI
polymorphism in 16 patients with myocardial infarction was determined by polymerase chain reaction-based restriction fragment length polymorphism and compared with that of 31 healthy control subjects.
Results: There were no significant differences between patients and controls
for FokI polymorphism. Among our patient group, the distribution of the FF, ff,
and Ff genotypes was 62.5% (n510), 31.3% (n55), and 6.3% (n51), respectively, versus 51.6% (n516), 12.9% (n54), and 35.5% (n511) in our control
group. There was no significant difference in the distribution of FokI genotypes
between patients with myocardial infarction and control group. We observed that
F allele carrying subjects have lower serum HDL-cholesterol concentration and
higher body mass index (BMI) levels compared to ff genotype in patients group.
But, this difference was not statistically significant.
Conclusion: Our preliminary results show that VDR (Fok-I) polymorphism
may play a role in development of myocardial infarction affecting serum lipid
levels. However, prospective studies with larger groups are needed to investigate
this issue further.
P-13
MOLECULAR PROFILE OF TUMOR NECROSIS FACTOR-RELATED
APOPTOSIS INDUCING LIGAND (TRAIL) AND ITS RECEPTORS ON
T CELLS IN PATIENTS WITH RHEUMATOID ARTHRITIS
Atil Bisgin1, Veli Yazisiz2, Ender Terzioglu2, and Salih Sanlioglu1
1
Human Gene Therapy Unit and Department of Medical Biology and
Genetics, Akdeniz University, Faculty of Medicine, Antalya, Turkey,
2
Department of Rheumatology, Allergy and Immunology, Akdeniz
University, Faculty of Medicine, Antalya, Turkey
Background: Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disorder. It is known that increased apoptosis by binding of death ligands to
their cognate receptors may induce autoimmune conditions like RA. TNF related
apoptosis inducing ligand (TRAIL), a member of the TNF superfamily, induces
apoptosis in sensitive cells and shown to be essential for the negative selection
of T cells in the thymus. We hypothesized that TRAIL may play a role in the
dysregulated apoptotic pathway which contributes to the pathogenesis of RA.
Methods: In this study, 20 patients with RA, and 20, sex and age matched
healthy controls were enrolled. RA severity was assessed according to the objective DAS28 (disease activity score) scoring system. For every patient and control, the frequency of CD41 T cells and CD81 T cells were determined by flow
cytometry. Fluorescence-activated cell sorting (FACS) was used to sort CD41 T
cells and CD81 T cells. Then the expressions of TRAIL ligand and its receptors
on these peripheral blood lymphocytes were analyzed by flow cytometry.
Results: We previously reported that synoviocytes of RA patients express
various TRAIL receptors on the surface and these surface TRAIL receptor profiles mainly influence whether RA synoviocytes are sensitive or resistant to cell
death by adenovirus mediated delivery of hTRAIL. In this study, we report an
increased expression of TRAIL and its receptors both death and decoy, in peripheral blood T cells from patients with RA compared with control individuals
which usually demonstrated little expression. In particular, CD81 T cells
expressed large amounts of all TRAIL receptors and TRAIL ligand itself.
Conclusion: We conclude that both CD41 and CD81 T cells are essential
for development of RA. TRAIL signaling pathways modulating cell death and
survival might be implicated in RA and thus TRAIL markers might be useful in
evaluating RA patients.
P-14
THE EXPRESSION PROFILE OF CYCLOOXYGENASE-1 AND
CYCLOOXYGENASE-2 GENES AFTER RESVERATROL TREATMENT
IN STREPTOZOTOCIN-INDUCED DIABETIC RATS
Atiye S. Yar1, Sevda Menevse1, Ebru Alp1, and Gulnur Take2
328
1
Department of Medical Biology and Genetics, Gazi University, Ankara,
Turkey, 2Department of Histology and Embryology, Gazi University,
Ankara, Turkey
Cyclooxygenase (COX), which has two isoforms, COX-1 and COX-2, is the
key enzyme of prostaglandins and thromboxanes biosynthesis from the substrate
arachidonic acid (AA). Especially, COX-2 is induced in inflammatory disease
such as Diabetes Mellitus (DM). DM is a group of metabolic diseases characterized by hyperglycemia resulting from defects of insulin secretion or insulin
action. Resveratrol (trans-3,4’,5-trihydroxystilbene; RSV), has a variety of biological and pharmacological effects including antioxidant, anti-inflammatory, and
anticarcinogenic effects. It was found to be a non-selective enzyme inhibitor of
COX-1 and COX-2. Moreover, the most recent data suggest that RSV has a beneficial role in preventing diabetes and alleviating some diabetic complications.
The aim of the present study was to investigate the changes of COX-1 and
COX-2 gene expression, protein level and complications of DM in kidney tissue
after RSV treatment in STZ induced-diabetic rats. This is the first study to clarify
the effect of RSV on mRNA and protein expression of COX-1 and COX-2 in
kidney tissue in STZ-induced diabetic rats. Three months-old, 44 Wistar albino
male rats were used for the study. Rats were divided into 6 groups as following:
control, sodium citrate (sham) control, diabetic, control 1 DMSO, sham control
1 RSV and diabetic 1 RSV group. Experimental diabetes was induced by i.p.
injection of 55 mg/kg STZ to diabetes group. After induction of chronic diabetes,
every day 10 mg/kg RSV was administered intraperitoneally to the diabetic
group and citrate buffer (sham control) group for 4 weeks. At the end of the second month, all rats were sacrificed under anesthesia and kidney tissues were flash
frozen in liquid nitrogen and stored at -80 8C for quantification of mRNA levels
and fixed in formalin for immunohistochemical study. In this study, there were
no statistically significant alterations in COX-1 mRNA levels among groups
(p[0.05). However, immunohistochemical study showed that COX-1 expression
was inhibited by RSV treatment in sham control 1 RSV group. As for COX-2,
no significant differences were found in COX-2 mRNA or protein levels among
groups (p[0.05). Thus, we can conclude that RSV has no significant effect on
COX-1 and COX-2 mRNA expression in STZ induced DM rat kidney tissue. On
the contrary, in our immunohystochemical examination, COX-1 was suppressed
markedly in RSV treated sham control but there was not enough suppression in
RSV treated diabetic group.
P-15
ALTERATIONS IN FREE RADICAL PRODUCTS AND
ANTIOXIDANT CAPACITY IN PATIENTS UNDERGOING
CORONARY ARTERY SURGERY
1
1
2
after ischemia and reperfusion. Significant decreased nitration and impaired antioxidant status were seen after reperfusion in both groups. Moreover, elevated
protein carbonyls were found in CABG group. Off-pump procedure is associated
with lower degree of oxidative stress than on-pump coronary surgery.
P-16
OXIDATIVE STATUS AND SERUM HOMOCYSTEINE LEVELS WITH
CORONARY ARTERY STENOSIS
Aymelek Gonenc1, Sena Irmak1, Yesim Ozkan1, Mustafa Sahingeri2,
and Mehmet Emin Korkmaz2
1
Department of Biochemistry, Gazi University, Ankara, Turkey,
2
Department of Cardiology, Guven Hospital, Ankara, Turkey
It is well known that free radicals contribute to endothelial dysfunction and
are involved in the pathogenesis and development of cardiovascular diseases,
such as atherosclerosis. The aim of this study was to provide evidence for
enhanced oxidative stress in coronary artery disease and to assess an association
with the severity of the disease. In this study, serum malondialdehyde (MDA),
total antioxidant capacity (TAC), nitrite1nitrate, homocysteine, folic acid and
vitamin B12 were measured in 32 healthy control subjects and 69 patients with
angiographically proven CAD. MDA was measured as thiobarbituric acid reactive substances (TBARS). Homocysteine was quantitated by HPLC technique.
TAC and nitrite1nitrate were assayed with spectrophotometric methods. Measurements of vitamin B12 and folic acid were carried out by chemiluminescence
analysis using Elecsys kits. The patients with CAD were divided into categories
according to the number of diseased coronaries; one-vessel (n523), two-vessels
(n526) and three-vessels (n520). Serum malondialdehyde, homocysteine and
nitrite1nitrate levels were significantly elevated in CAD groups compared with
the controls (p\0.01). However, TAC and vitamin B12 levels were lower in
patients compared with healthy controls (p\0.01). The levels of MDA and nitric
oxide were significantly increased in the CAD groups compared with the controls
(p\0.01). But TAC concentrations were significantly diminished in two- and
three-vessels CAD groups compared with the controls (p\0.01). The levels of
homocysteine and vitamin B12 were increased in controls compared with onevessel CAD patients (p\0.01). Serum folic acid levels of patients with CAD
were observed to be lower than controls, this differences were not significant
(p[0.05). According to the results, increased lipid peroxidation, nitric oxide and
decreased antioxidant levels contribute to elevated oxidative stress which in turn
is related to the severity of disease.
P-17
1
Aymelek Gonenc , Aysun Hacisevki , Helen R. Griffiths , Meral Torun ,
Haldun Karagoz3, and Bolkan Simsek1
1
Department of Biochemistry, Gazi University, Ankara, Turkey, 2Life and
Health Science, Aston University, Birmingham, United Kingdom, 3Cardiac
Surgery, Guven Hospital, Ankara, Turkey
Oxidative stress and reactive oxygen species play an important role in the
aetiology and/or progression of a number of human diseases. Some previous
studies have demonstrated oxidative stress during coronary artery bypass surgery
as well as ischemia/reperfusion of the human heart. The aim of this study was to
investigate changes of protein, lipid oxidation and antioxidant status during
bypass operation in blood of patients with off-pump and on-pump coronary artery bypass surgery and to compare oxidative stress parameters in two bypass
method. Serum lipid hydroperoxide (LPx), nitric oxide (NOx), protein carbonyls,
nitrotyrosine (NT), vitamin E, b-carotene levels and total antioxidant capacity
(TAC) were measured in blood of 30 patients undergoing OPCAB (off-pump
coronary artery bypass grafting) and 12 patients undergoing CABG (on-pump
coronary artery bypass grafting). Serum vitamin E, b-carotene and nitrotyrosine
concentrations were determined with the high performance liquid chromatography. Other oxidant and antioxidant parameters were studied spectrophotometrically. In OPCAB group, NOx (p\0.05) and nitrotyrosine (p\0.05) levels
decreased after reperfusion. Similarly, b -carotene ( p\0.01) and TAC ( p\0.01)
levels also decreased after anesthesia and reperfusion. In CABG group, NOx
(p\0.01) and NT (p\0.01) levels decreased after ischemia and reperfusion.
However, protein carbonyl (p\0.01) levels elevated after ischemia and reperfusion. Vitamin E (p\0.01), b-carotene (p\0.01) and TAC (p\0.01) decreased
OXIDATIVE MTDNA DAMAGE IN THE HEART TISSUE OF
CIGARETTE SMOKE EXPOSED MICE AND PROTECTIVE EFFECTS
OF VITAMIN E AND SELENIUM
Ayse G. Mutlu1 and Kayahan Fiskin2
1
Mehmet Akif Ersoy University, Arts and Sciences Faculty, Department of
Biology, Turkey, 2Akdeniz University, Arts and Sciences Faculty,
Department of Biology, Turkey
Oxidative mtDNA damage that have been created by cigarette smoke and
possible protective effects of vitamin E and selenium which are powerful antioxidants were measured in the heart tissue of Mus musculus in this search. The reactive oxygen species (ROS) and their active intermediate metabolites produced
by cigarette smoke may create oxidative damage on mitochondrial DNA.
mtDNA damages may trigger mitochondrial dysfunction. Mitochondria contribute
to cardiac dysfunction and myocyte injury via a loss of metabolic capacity and
by the production and release of toxic products. Reactive oxygen species induce
myocyte hypertrophy, apoptosis, and interstitial fibrosis by activating matrix metalloproteinases. These cellular events play an important role in the development
and progression of maladaptive cardiac remodeling and failure.
Quantitative PCR (QPCR) method was used to measure oxidative damage and
mtDNA copy number in the heart tissue. The lesion present in the DNA blocks the
progression of any thermostable polymerase on the template. Consequently, the
DNA amplification decreases in the damaged template. QPCR method is highly
sensitive for measuring DNA damage and repair. 6 groups were designed for the
Cigarette Smoke, Vitamin E and Selenium applications named as ‘‘CS’’,
‘‘CS1VitE’’, ‘‘CS1VitE1Se’’, ‘‘CS1Se’’, ‘‘Control’’, ‘‘Se1VitE’’. Cigarette
smoke created mtDNA damage in the heart tissue of mice. However our results
have suggested that antioxidant supplementation may prevent this damage. This is
especially important for children whose parents are smokers.
P-18
THE STUDY OF PCSK9 GENE VARIANTS IN CORONARY HEART
DISEASE
1
1
1
Aysegul Basak Akadam , Hulya Yilmaz-Aydogan , Turgay Isbir ,
Atike Tekeli2, and Selim Isbir2
1
Medicine, Istanbul University, Capa, Istanbul, Turkey, 2Cardiovascular
Background: Coronary heart disease (CHD) is a multifactorial disease.
Genes of lipoprotein synthesis and metabolism are very important genes in tendency to atherosclerosis and CHD, because plasma lipids are main risk factors in
CHD. An important parameter for determination of serum lipid/lipoprotein level
is specific receptors on the cell membrane. LDL-receptor (LDLR) is one of the
receptors which provides to take LDL-C into the cell. The activity between this
receptor and Apolipoprotein B (Apo B) ligand of LDL provides to take it into
the cell with endocytosis. Proprotein convertase subtilisin/kexin 9 (PCSK9) gene
causes degradation of LDLR on cell membrane. Some mutations on PCSK9 may
result in increase/decrease in LDLR level and hypercholesterolemia/hypocholesterolemia. Some studies showed that mutation and polymorphism of PCSK9
gene resulting in function-gain/function- loss cause hypercholesterolemia/hypocholesterolemia. In our study, our aim is to investigate PCSK9 R496W, N425S
and E670G gene polymorphism causing function-gain and Y142X gene polymorphism causing function-loss in addition to effects on serum lipoprotein level and
development of CHD.
Materials and Methods: PCSK9 gene polymorphisms were studied in 65
patients with CHD. 52 healthy persons without any symptoms of CHD were
selected for the control group. Gene polymorphism was studied by the polymerase chain reaction followed by the agarose electrophoresis. Statistical analyses
were performed by SPSS for windows version 11.0. Genotypes and allele frequencies were compared by chi-square test. Lipid and the other parametric analysis were compared by Student’s t test.
Results: In the patient group, frequencies of PCSK9 R496W mutant C allele,
E670G mutant T allele, and Y142X mutant X allele and XX genotype are higher
than controls. Our findings suggested that these variants might be independent
risk factors in development of CHD. In the patient group, we observed the
PCSK9 R496W normal T allele is associated with increased serum triglyceride
Figure 1. Cellular trafficking and potential sites of PCSK9 action. [Color figure can
be viewed in the online issue, which is available at www.interscience.wiley.com.]
329
level compared with PCSK9 R496W mutant CC genotype (p<0.05). In the
patients with E670G normal A allele have higher serum total-cholesterol level
than E670G mutant GG genotype (p<0.01). In addition, Y142X mutant X allele
is associated with increased serum cholesterol level (p<0.05).
Conclusion: In conclusion, we suggested that the PCSK9 gene variants may
tend to increase the risk of CHD, because they cause detrimental effects on serum lipid profile.
P-19
AN AZOOSPERMIC CASE WITH Y ISOCHROMOSOME
Aysegul Turkyilmaz, Diclehan Oral, Selda Simsek, and Hilmi Isi
Department of Medical Biology and Genetic, Faculty of Medine, Dicle
University, Diyarbakir, Turkey
Infertility is a common problem affecting approximately 15% of couples. It
is widely accepted that gender determination in man depends on the presence of
a factor or factors on the Y chromosome. These factors may be localized within
the Y chromosome through the study of structural anomalies of the Y. A thorough review of seven different structural anomalies of the Y is presented: dicentric Y chromosomes, Y isochromosomes, ring Y chromosomes, Y; autosome,
Y;X, and Y;Y translocations, and Y chromosome deletions. A few studies indicate that one or more factors on the long arm of the Y chromosome may also
influence testicular development. If such a factor is present on the long arm, then
it must be also very near the centromere. The theory that predicts that separate
genes independently control the initial development and maturation of the testis
(on the long and short arms of the Y, respectively) may be premature. In this
report we present a 41 years old male with infertility. He and his partner had
been trying for a child for nine years. He was phenotypically a normal male and
failure to conceive could be due to his partner. Peripheral blood chromosome
analysis shows that patient has Y isochromosome. This finding was confirmed by
fluorescent in situ hybridization (FISH).
P-20
DETERMINATION OF ASSOCIATION BETWEEN CA VI EXON 2
GENETIC POLYMORPHISM AND DENTAL CARIES AMONG
TURKISH DENTAL STUDENTS
Aysen Yarat1, Leyla Koc-Ozturk1, Korkut Ulucan1, Serap Akyuz2,
and Hayati Atala2
1
Basic Medical Sciences, Marmara University, Istanbul, Turkey, 2Clinical
Sciences, Marmara University, Istanbul, Turkey
Aim: Carbonic anhydrases (CAs) participate in the maintenance of pH homeostasis in various tissues and biological fluids of the human body. Carbonic anhydrase isoenzyme VI (CA VI) is the only secretory isoenzyme of the mammalian
CA gene family. It is exclusively expressed in the serous acinar cells of the parotid
and submandibular glands, from where it is secreted into the saliva. Recent
research has indicated that salivary CA VI has a remarkable role in protecting teeth
from caries. The aim of this study was to investigate the association between CA
VI Exon 2 genetic polymorphism and dental caries among Turkish dental students.
Methods: Forty-four healthy dental students with caries (n524) and without
caries (n520) participated in this study. The occurrence of caries in individuals
was measured by using the DMFT (number of decayed, missing, and filled teeth)
index. Gene polymorphism of CA VI Exon 2 was determined by PCR (polymerase chain reaction) and DNA sequencing. DNA was extracted from peripheral
blood cells by using High PCR Template Preparation Kit (Roche, Germany)
according to the manufacturer’s guide. CA VI gene Exon 2 region was amplified
by using specific primers (5’-TGTCTTAGAAGGGGCACT-3’sense and 5’TACCTGTGTGGCCATTGT -3’ antisense). For salivary analysis, fasting unstimulated whole saliva was collected. Salivary CA activity and salivary buffering
capacity were determined by the methods of Verpoorte, and Ericson, respectively. Furthermore, salivary pH was measured with pH paper (pH Indikatorpapier, Merck Neutrolit-5.5-9.0) and salivary flow rate was calculated. Statistical
analysis was performed by SPSS for Windows version 10.0.
Results: Salivary pH, flow rate, buffering capacity and CA activity were not
significantly different between the students with caries (DMF-T55.6) and without caries (DMF-T50). Two SNPs were detected which are responsible threo-
330
nine to methionine change in codon 55 (T55M) and tyrosine to serine in codon
71 (Y71S). These alterations were found in 58-63% and 75-60% of the groups
with and without caries, respectively.
Conclusion: This is the first study which investigates the gene polymorphism
of CA VI Exon 2 among Turkish dental students. Two SNPs found in this study
may be specific polymorphisms affecting Turkish population. Acknowledgements. This study was financially supported by Grant Project No: SAG.YY.008/
140604 from the Commission of Scientific Investigations Projects of Marmara
University.
P-21
ROLE OF THE MANNOSE-BINDING LECTIN-2 X/Y (MBL-2 X/Y)
POLYMORPHISMS IN PATIENTS WITH COLORECTAL CANCER
Lokman Ayaz1, Bahadir Ercan1, Nurcan Aras-Ates2, Musa Dirlik3,
and Lulufer Tamer-Gumus1
1
Department of Biochemistry, Mersin University, School of Medicine,
Mersin, Turkey, 2Department of Medical Biology and Genetics, Mersin
University Faculty of Medicine, Turkey, 3Department of General Surgery,
Mersin University Faculty of Medicine, Turkey
The mannose-binding lectin (MBL) pathway of innate immunity is part of
the first line of defense against microorganisms. MBL recognizes and binds to
carbohydrate patterns on the surface of microorganisms leading to complement
activation by the MBL-associated serine protease2 (MASP-2). In developed
countries colorectal cancer is the second most common cancer. Epidemiologic
evidence suggests that many of the geographical variations reflect variations in
environmental or lifestyle exposures, perhaps acting with variations in genetic
factors. MBL may play a role as a phase reactant due to inflammatory processes
related to cancer disease. Such inflammatory processes are well known in colorectal cancer tissue and have been shown to be independent of stage of disease.
We aimed to investigate whether profile of MBL-2 X/Y genotyping may be associated with the risk of colorectal cancer.
This study includes all cases of colorectal cancer consecutively hospitalized
in the surgery departments of Mersin University Hospital. The study group consisted of 66 patients were diagnosed with histologically confirmed cancer of the
colon or rectum. The control group consisted of 59 were selected among healthy
people. Genomic DNA of control and patients was extracted from whole blood
using High Pure PCR template preparation kit. Genotyping of MBL-2 polymorphisms were detected by using a LightCycler MBL-2 mutation detection kit in
real-time PCR. Logistic regression analyses showed that the MBL-2 X/Y heterozygote genotype was associated with a significantly increased risk of colorectal
cancer. The odds ratio of colorectal cancer for the MBL-2 X/Y heterozygote genotype was 2.67 (95% CI51.24-5.75, P50.012) compared with the control
group. Our results suggest that MBL-2 X/Y heterozygote genotype have higher
risk of developing colorectal cancer.
In conclusion, our results suggest that MBL-2 X/Y allele gene polymorphisms
may be associated with genetic susceptibility to colorectal cancer. Large-scale
molecular epidemiological studies that investigate the role of MBL-2 X/Y genotype together with other susceptibility gene polymorphism and biomarkers of carcinogen exposure are necessary to expand our current understanding of the MBL2 polymorphism in colorectal cancer.
dinic (AP) endonuclease (APE1) plays a central role in the DNA base excision
repair (BER) pathway. The aim of the present study was to investigate the association between polymorphism in the APE 1 gene and the risk of lung cancer.
Methods: We compared the distribution of the APE1 Asp148Glu polymorphisms in 94 lung cancer patients and 35 controls in males using PCR–RFLP
analysis.
Results: Frequencies of the APE1 genotypes among controls was: Asp/Asp,
0.77; Asp/Glu, 0.11; Glu/Glu, 0.11. The distribution among cases was: Asp/Asp,
0.54; Asp/Glu, 0.35; Glu/Glu, 0.11. Statistical difference was found in the distribution of genotypes of APE1 Asp148Glu (P < 0.05). Individuals with at least
one APE1 148Glu allele showed an increased risk of lung cancer (22.9% vs
45.7%) (p50.018; OR52.84 (95% CI51.17-6.91).
Conclusions: Evidence for association between the Asp148Glu polymorphism in APE1 and lung cancer was found in male Turkish population. The
148Glu allele of APE1 appears to confer a risk for lung cancer.
P-23
VOLUMETRIC EVALUATION OF BRAIN IN DIABETIC AND
OVERECTOMIZED RATS
Deniz Unal1, B. Zuhal Altunkaynak1, Zekai Halici2, O. Nuri Keles1,
Elif Cadirci2, M. Eyup Altunkaynak1, and Bunyami Unal1
1
Department of Histology and Embryology, Ataturk University, Medical
School, Erzurum, Turkey, 2Department of Pharmacology, Ataturk
University, Medical School, Erzurum, Turkey
Objective: Aging is accompanied by a decrease in brain volume and by an
increase in cerebrovascular disease. Significant age-related volumetric loss was
observed in anterior and posterior white matter but not in total grey matter volumes. Patients with diabetes had smaller total brain gray matter volumes when
compared with the control subjects after controlling for age, intracranial volume,
and years of education. In this study we have investigated the effect of both
type-1 diabetes and aging process in brain volume.
Methods: For this reason, we used 20 Wistar albino female rats. They were
divided into 4 groups (Group 1, n5 5; Group 2 n5 5 Group 3, n5 5 Group 4,
n5 5). Ovariectomy was applied to two groups (Group 2, n5 5; Group4 n5 5)
and all of the groups were kept for three months for the aging process. Thereafter alloxan was intraperitoneally injected to group 3 and group 4 for inducing
diabetes. Age and weight matched group 1 was used as control Then, all animals
were killed after 8 weeks under sevorane anesthesia and right hemispheres of
their brains were removed. Volumes of right hemispheres were stereologically
detected with Cavalieri method.
Results: Mean volumes of right hemispheres were 585 mm̊, 482.862 mm̊,
321.816 mm̊ and 475.41 mm̊ in all groups, respectively. All consecutive values
were statistically evaluated and the values were different from each other (p<
0.05).
Conclusions: According to our results; both ovariectomy and diabetes caused
a decrease in hemisphere volume. Diabetes leads to a bigger decrease than ovariectomy. However, in the elderly, diabetes may protect from volume loss.
P-24
P-22
ASSOCIATION OF APURINIC/APYRIMIDINIC ENDONUCLEASE
(APE1) GENE POLYMORPHISMS WITH LUNG CANCER IN MALES
Bedia Agachan1, Ozlem Kucukhuseyin1, Pinar Aksoy2,
Ilhan Yaylim-Eraltan1, Akif Turna3, Uzay Gormus1, and Turgay Isbir1
1
Biochemistry, Pharmacy Faculty, Istanbul University, Istanbul, Turkey,
3
Department of Thoracic Surgery, Yedikule Teaching Hospital for Chesting
Diseases and Thoracic Surgery, Turkey
Aim: Genetic variations in DNA repair genes may alter the functional properties of repair enzymes and consequently the cancer risk. The apurinic/apyrimi-
STEREOLOGICAL AND HISTOLOGICAL ANALYSIS OF THYROID IN
20 DAYS RAT FOETUSES
M. Eyup Altunkaynak1, B. Zuhal Altunkaynak1, Deniz Unal1,
Mehmet Bilici2, Ismail Can1, Ozgen Vuraler1, and Bunyami Unal1
1
Department of Histology and Embryology Ataturk University,
Medical School, Erzurum, Turkey, 2Internal Medicine, Ataturk University,
Medical School, Erzurum, Turkey
Objective: Thyroid hormones have been known to be essential for prenatal
development. Clinical observations of good neurological outcome following early
treatment of congenital hypothyroidism were used to support the view that thyroid hormones are not required early in neurodevelopment. In this study, we
investigated thyroid volume and histological aspects of thyroid in 20 day-old rat
foetuses.
Methods: We studied with quantitative and light microscopy methods the
thyroid of 10 specimens of 20 day-old foetuses. Days of gestation were counted
from the day following an overnight mating that was considered day 1 of gestation. For electron microscopy, the thyroid samples were fixed, sectioned and
stained by toluidine blue. Stereological estimations were performed on thyroid
volume. Volumetric values were estimated via Cavalieri method. In this study,
histological examination was performed at light microscopy levels.
Results: Mean thyroid volume was 0.39 mm3 in right lobe of 20 days foetuses and 0.29 mm3 in left lobe of the animals. Right lobes were significantly
higher than left ones. In histological examination, many follicle cell which not
organized as follicle and C cells were defined. Rarely follicles were observed.
Capillaries were peripherally occupied. Between right and left lobes mesenchymal connective tissue was seen. In this area, mesenchymal cells, fibroblasts and
vessels were found.
Conclusions: It is now clear that thyroid hormones are essential for both foetal and post-natal neurodevelopment and for the regulation of neuropsychological
function in children and adults. This study contributes to our new understanding
of development of thyroid and its structure.
331
matory mediators and a target organ for the effects of the inflammatory mediators. We aimed to examine the effect of sepsis in liver tissue.
Materials and Methods: For this aim, 10 Wistar albino adult rats were
investigated. They were divided into two groups, to the control group saline was
given intraperitoneally, to the experimental group Pseudomonas Auriginosa was
given intraperitoneally to obtain a sepsis model. All rat livers were histologically
examined after staining with haematoxylin and eosin.
Results: In the light microscopy investigation of livers, some pathological
changes were seen in sepsis group such as sinusoidal dilatation and endothelial
irregularity in sinusoidal area, fibrinoid necrosis of vessels, vacuolar degeneration
in both cytoplasm and nuclei of hepatocytes. Also lymphoid cell infiltration was
observed in central and portal areas and intra-sinusoidal inflammation were many
necrotic areas were detected.
Conclusions: Our findings suggest that liver was affected by sepsis and this
condition resulted in hepatic failure.
P-27
P-25
DECREASED LIVER VOLUME AND HEPATIC PATHOLOGY
FOLLOWING RAT SEPSIS: A STEREOLOGICAL AND
HISTOLOGICAL INVESTIGATION
Hamdullah Uyanik1, Abdullah Uyanik2, Deniz Unal3, Zekai Halici4,
B. Zuhal Altunkaynak3, and Bunyami Unal3
1
Department of Microbiology, Ataturk University, Medical School,
Erzurum, Turkey, 2Internal Medicine, Ataturk University, Medical School,
Erzurum, Turkey, 3Department of Histology and Embryology, Ataturk
University, Medical School, Erzurum, Turkey, 4Department of
Pharmacology, Ataturk University, Medical School, Erzurum, Turkey
Objectives: Pseudomonas aeruginosa bacteremia is a serious and possibly
fatal condition. It is important to determine changes which were seen in different
organs of subjects suffering from P. aeruginosa bacteremia. We explored effects
of the opportunistic pathogen, P. aeruginosa on liver volume in adult rats
exposed to Pseudomonas infection.
Methods: After immediate post-burn infection with P. aeruginosa, mortality
and morbidity (daily weight changes) were monitored in rats. After this process
livers removed from all animals and tissues were paraffin embedded, serially sectioned, and stained with haematoxylin and eosin. Volumes of livers were stereologically estimated. Histological images were acquired using an Olympus BX51
compound binocular microscope.
Results: Volumetric values of livers significantly decreased in septic rats
according to those of controls (p<0.05). Light microscopy investigation of livers
in sepsis group, pathological changes such as lymphoid cell infiltration, ballooning degeneration and necrosis were detected.
Conclusions: These observations suggest that liver volume was affected by
sepsis, resulting in hepatic failure. Thus, the capacity of the liver is an important
determinant of the outcome in sepsis.
P-26
THE EFFECT OF CUMIN (CUMINUM CYMINUM) ON RETINAL
STRUCTURE IN EXPERIMENTALLY INDUCED DIABETIC RABBITS:
A HISTOLOGICAL VISION
Selina Aksak1, Deniz Unal1, Ahmet T. Yazici2, M. Eyup Altunkaynak1,
1
Department of Histology and Embryology Ataturk University, Medical
School, Erzurum, Turkey, 2Department of Ophtalmology, Beyoglu State
Hospital, Turkey
Objective: Retinal capillary closure induced by hyperglycemia is the principal pathophysiologic abnormality underlying diabetic retinopathy, but the mechanisms by which this induction occurs are not clear. It was reported that cumin
was effective in preventing glycation of TSP and alpha-crystallin in diabetic
lens. In this study, we have in mind to see the effects of cumin on the retina of
rabbits with streptozotocin induced diabetes.
Materials and Methods: For this aim; 18 female New Zealand white rabbits
of almost uniform age were selected for the study. All animals were randomly
divided to three groups. Negative controls were fed with commercial rabbit
chow. Rabbits in positive control groups were fed with commercial rabbit chow
and received intravenous injection of streptozotocin at 65mg/kg.b.w following
twelve hours fasting whereas negative controls received saline. The antiglycating
potential of cumin was also investigated by feeding streptozotocin (STZ)-induced
diabetic rabbits with diet containing 0.5% cumin powder. Development of diabetes mellitus was confirmed by examining the fasting glucose level along with
blood urea and serum creatinine in the blood taken from marginal ear vein.
Results: Although, cumin did not prevent streptozotocin-induced hyperglycemia, as assessed by blood glucose and insulin levels, light microscopical observations indicated that diet supplement by cumin decreased vessel dilatation and
retinal damage in eye of diabetic rabbits.
Conclusions: Black cumin seed oil has been used since ancient times for
medicinal purposes and as a food flavoring. Today, scientists are evaluating this
and many other botanicals for safety and effectiveness, searching for alternatives
to synthetic drugs that often have devastating side effects. At this point, our
results indicate that cumin can prevent pathological changes resulting from diabetic retinopathy. In the light of these findings, we suggest that cumin may be a
safe and effective alternative to insulin for the treatment of this disorder.
HISTOLOGICAL STRUCTURE OF LIVER IN PSEUDOMONAS
SUBJECTED SEPTIC RATS
Deniz Unal1, Hamidullah Uyanik2, Zekai Halici3, Abdullah Uyanik4,
1
School, Erzurum, Turkey, 2Department of Microbiology, Ataturk University,
Medical School, Erzurum, Turkey, 3Department of Pharmacology, Ataturk
University, Medical School, Erzurum, Turkey, 4Internal Medicine, Ataturk
University, Medical School, Erzurum, Turkey
Objective: Sepsis is the systemic inflammatory response to infection frequently associated with hypoperfusion followed by tissue injury and organ failure. In patients with sepsis, the liver has two opposing roles: a source of inflam-
P-28
ELEVATED PLASMA LEVELS OF HLA-G IN PATIENTS WITH
CRIMEAN-CONGO HEMORRHAGIC FEVER
Bilkay Basturk1, Hurrem Bodur2, Esragul Akinci2, Arzu Aral1,
Yavuz Uyar3, and Ahmet Carhan3
1
Department of Immunology, Gazi University, Faculty of Medicine, Ankara,
Turkey, 2Infectious Diseases and Clinical Microbiology Clinic, Ankara
Numune Education and Research Hospital, Turkey, 3Laboratory of Virology,
Refik Saydam Hygiene Center, Turkey
332
Table 1
Patient Groups and Mean Levels of HLA-G levels (IU/ml)
Group No
HLA-G
p value
1 (n 5 27)
2 (n 5 2)
3 (n 5 42)
12.7
70.3
3.8
p \ 0.05*
p \ 0.05*
p \ 0.05*
Objective: HLA-G is a non-classical MHC Class I type antigen which interact to natural killer cells. It has been detected on trophoblasts in early gestational
placenta, placental chorionic endothelium, activated monocytes and tymic epithelium, and its expression is stimulated in various types of malignancies. HLA-G
suppresses proliferation of CD41 T lymphocytes and inhibits NK cells and also
T-cell dependent cytolysis.
Materials and Methods: 29 patients (two of them were dead) with CCHF,
and 42 healthy volunteers have been included to this study. Plasma HLA-G levels of patients were measured on the first and the third day of hospitalization.
Plasma HLA-G levels for fatal, nonfatal patients and healthy controls were compared. Groups; 1: On the first day of hospitalization (n527), 2: Ex patients
(n52), 3: Healthy volunteers (n542).
Results: HLA-G levels were increased in patients (fatal and nonfatal) with
CCHF and it has been found statistically significant compared to healthy volunteers (Table 1).
Conclusion: This is the first study which shows the association between
plasma HLA-G levels for CCHF and healthy volunteers. Our preliminary results
suggest that HLA-G may be an important marker for CCHF prognosis and evaluate the response to therapy.
P-29
EMPHASIS OF THE EFFICIENT IN VITRO NEUROGENESIS
METHOD FOR BONE MARROW DERIVED HUMAN MESENCHYMAL
STEM CELLS
Busra Mammadov, Nihal Karakas, and Sevim Isik
Department of Biology, Fatih University, Turkey
Objective: We aimed to determine the most efficient in vitro neural differentiation method for hMSCs in terms of neural morphology, neuron specific protein
expression and stable differentiation.
Methods: hMSCs, obtained from Karadeniz Technical University, Department of Hematology, were expanded in DMEM, 10% MSC-qualified FBS and
subjected to neural differentiation after the third passage. Four different methods
were used for differentiation: retinoic acid, retinoic acid under serum reduced
conditions, antioxidants (DMSO, b-mercaptoethanol), chemicals (KCl) and lastly
enriched cytokine combinations (hEGF, bFGF, BDNF, FGF-8, IBMX, dbcAMP)
within serum reduced Neurobasal Media plus B27 supplement. Differentiation
was carried out for fourteen days and morphological changes were followed by
phase-contrast microscopy besides analyzing neural marker expressions (NF-H,
NeuN, NSE, bIII tubulin) by immunofluorescence during this period.
Results: hMSCs induced by retinoic acid showed slow progression of neural
differentiation with low percentage but the neural-like morphology of differentiated cells was stable and supported by high expression of neurofilament. When
hMSCs were induced with retinoic acid under serum reduced conditions, they
were not able to express mature neural proteins although they were shown to
express bIII tubulin -an early neural marker- in very high levels indicating that
although these cells started to differentiate, they were not able to mature. Exposure to antioxidants caused hMSCs to gain neural-like morphologies in a shorter
period of time but these morphological changes reverted back in a few days and
these cells did not show neural marker expression. Differentiation of hMSCs
under cytokine combinations resulted in the highest percentage of stable differentiation with most readily observable neural morphology.
Conclusions: This study revealed that enriched cytokine combinations in a
serum reduced Neurobasal Media is the most efficient method for in vitro neurogenesis of hMSCs when compared to retinoic acid and antioxidant methods. Our
results also further support the idea that neural differentiation with stress-inducing materials like antioxidants cause cytotoxic morphological changes without
neural protein expression rather than a real neural differentiation.
P-30
EFFECTS OF ENDOTHELIAL NITRIC OXIDE SYNTHASE INTRON
4(ENOS4A/B) POLYMORPHISM ON BLADDER CANCER
Canan Kucukgergin1, Oner Sanli2, Akin Soner Amasyali2, Ismet Nane2,
and Sule Seckin1
1
Department of Biochemistry, Faculty of Medicine, Istanbul University,
Istanbul, Turkey, 2Department of Urology, Faculty of Medicine,
Istanbul, Turkey
Aim: It has been demonstrated that the level of nitric oxide may play an important role in bladder cancer formation and/or progress. The aim of this study
was to investigate the association between eNOS4a/b polymorphism and bladder
cancer.
Materials and Methods: The present study includes patients (n575, mean
age563.5) with histopathologically confirmed transitional cell carcinoma of bladder and healthy controls (n5110, mean age561.7). For determination of
eNOS4a/b gene polymorphism, PCR method was used. Statistical analysis for genotype distribution was performed by using Chi-square test.
Results: There were not significant differences in the age and BMI between
patients and controls. In addition, aa genotype distribution of the eNOS4a/b polymorphism was significantly higher in patients with bladder cancer compared with
control group (p<0.05).
Conclusion: According to these findings, the distribution of bb genotype in
eNOS4a/b polymorphism is more frequent in the Turkish population. However,
the aa genotype distribution of the eNOS4a/b polymorphism is more likely to be
seen in bladder cancer patients compared with healthy controls. In conclusion,
we suggest that the aa genotype of the eNOS4a/b polymorphism is a risk factor
for bladder cancer in the Turkish population.
P-31
TNF GENE POLYMORPHISMS IN SARCOIDOSIS
C. Pamukcu1, B. Yalcin1, O. Ates2, A. Topal-Sarikaya1, H. Turker3,
and G. Ongen4
1
Department of Molecular Biology and Genetics, Science Faculty, Istanbul
University 34118, Vezneciler, Istanbul, Turkey, 2Department for Medical
Biology, Medical Faculty, Gaziosmanpasa University, Tokat, Turkey, 3Chest
Disease Department, Sureyyapasa Hospital, Istanbul, Turkey, 4Chest Disease
Department, Cerrahpasa Medical Faculty, Istanbul University,
Istanbul, Turkey
Table 1
P-values of TNF gene polymorphism in sarcoidosis patients
TNF Locus,
Polymorphism
Patients n550
31 women, 19 men
age 43.7613.3
TNF-a 308G/A (rs1800629)
A/A
0
G/A
22 (44%)
G/G
28 (56%)
Allele
A
22
G
78
TNF-a 238G/A (rs361525)
A/A
0
G/A
5 (10%)
G/G
45 (90%)
Allele
A
5
G
95
TNF-a 376G/A (rs1800750)
A/A
0
G/A
4 (8%)
G/G
46 (92%)
Allele
A
4
G
96
252
TNF-b 1 G/A (rs909253)
A/A
5 (10%)
G/A
33 (66%)
G/G
12 (24%)
Allele
A
43
G
57
Controls n5150
60 women, 90 men
age 5864
0
32 (21.3%)
118 (78.6%)
32
268
0
5 (3.3%)
145 (96.6%)
5
295
0
4 (2.6%)
146 (97.3%)
4
296
P (Pc)
0.003 (0.009)
0.005 (0.01)
0.12
0.07
0.10
333
world after breast, colorectal and lung cancer. Tumor necrosis factor–related apoptosis inducing ligand (TRAIL) is a type II transmembrane protein and member
of the TNF-superfamily of ligands. In humans, TRAIL binds to two death-inducing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2/KILLER. TRAIL has also
been shown to bind three decoy receptors, DcR1/TRID/TRAIL-R3, DcR2/
TRAIL-R4 and osteoprotegerin. TRAIL selectively induces apoptosis in cancer
cells but not in normal cells, and a number of clinical trials have recently been
initiated to test the safety and antitumoral potential of TRAIL in cancer patients.
In addition, TRAIL is one of the novel markers tested for its potential to be used
as prognostic indicator in cancer. However, the contribution of TRAIL and
TRAIL receptor expressions to endometrial cancer development is unknown.
Method: We examined the expression of TRAIL and all four TRAIL receptors in 100 patients with endometrioid carcinoma, 80 patients with endometrial
hyperplasia and 20 normal endometrial tissue using immunohistochemistry to
assess their relationship with other prognostic factors. TUNEL assay was performed to detect apoptosis using In Situ Cell Death Detection kit.
Results: Contrary to our previous findings (prominent DcR2 expression in
prostate and lung carcinoma versus to DR4 in breast cancer) TRAIL and its
receptors are differentially expressed in endometrioid carcinoma and endometrial
hyperplasia tissues.
Conclusions: Expression profile of TRAIL and its receptors suggested that
TRAIL signaling pathway may play an essential role during the course of endometrial carcinoma.
0.11
15 (10%)
45 (30%)
90 (60%)
0.000 (0.000)
75
225
0.001 (0.002)
P-33
Objective: Sarcoidosis is a multisystem, granulomatous disorder of unknown
etiology, characterized by lymphocytes and mononuclear phagocytes accumulation and granuloma formation in affected organs. During several studies focused
on the genetic risks of sarcoidosis, the most prominent finding was the linkage to
a section within MHC on the short arm of chromosome 6. This evidence is in
keeping with a series of studies that have shown associations between class II
MHC alleles and sarcoidosis. The TNF (Tumor Necrosis Factor) complex located
adjacent to the MHC class II loci on chromosome 6 might play an important
part in determining severity of disease, together with MHC class II alleles.
Therefore we investigated four TNF single nucleotide polymorphisms, TNF–a
-308G/A, -238G/A, -376G/A and TNF- b 1252 G/A in 50 Turkish sarcoidosis
patients and 150 healthy control subjects.
Methods: TNA-a -308G/A, -238G/A, -376G/A and TNF- b 1252 G/A polymorphisms were analyzed with amplification refractory mutation system-polymerase chain reaction (ARMS-PCR).
Results: The TNF gene polymorphism allele frequencies are shown in Table 1.
Conclusions: The statistics results that observed between patients and control
subjects, showed that there is an association between sarcoidosis and TNF-a-308,
TNF-b 1252 allele polymorphisms (p50.009 and p50.000) in Turkish population. But we found no evidence of association between TNF-a -238, -376 allele
polymorphisms and sarcoidosis (p50.12 and p50.10). Acknowledgement: This
research was supported by The Scientific and Technological Research Council of
Turkey.
P-32
EXPRESSION PROFILES OF TRAIL LIGAND AND RECEPTORS IN
ENDOMETRIAL CARCINOMA
Cigdem Aydin1, Ahter D. Sanlioglu1, Elif Pestereli2, Gulgun Erdogan2,
Irem Hicran Ozbudak2, and Salih Sanlioglu1
1
Department of Medical Biology and Genetics, Akdeniz University, Antalya,
Turkey, 2Department of Pathology, Akdeniz University, Antalya, Turkey
Background: Endometrial cancer is the most common gynecological malignancy and the fourth most common cancer type in women in the developed
GENISTEIN TRIGGERS APOPTOSIS AND DNA
HYPOMETHYLATION IN HUMAN LEUKEMIA CELLS VIA
PHOSPHATIDYLINOSITOL-3-KINASE NEGATIVE REGULATOR
PTEN GENE
Cigir Biray-Avci1, Yavuz Dodurga1, Sinem Numanoglu1, Guray Saydam2,
and Cumhur Gunduz1
1
Department of Medical Biology, Ege University, Izmir, Turkey,
2
Department of Hematology, Ege University, Izmir, Turkey
Genistein is a natural tyrosine kinase (TK) inhibitor found in soy beans. TK
plays an important role in tumorigenesis and can affect many biological processes such as TK inhibition, cell cycle arrest, stimulation of apoptosis and inhibition of tumorigenesis. We aimed to investigate whether there is a relation
between genes found in PI3K signal pathway and apoptosis in HL-60 in time
and dose dependent manner.
Cytotoxicity of genistein in HL–60 cells was assessed by Trypan Blue Dye
and XTT tests at 1 - 150 lM doses. DNA Ladder, Annexin V-EGFP and Comet
methods were used to detect apoptosis and DNA damage. Mitochondrial membrane potential depolarization was evaluated by JC-1 dye method. Total RNA
isolation was assessed to determine PTEN, PIKE and PIK3CA gene expressions.
mRNA expression levels of genes were studied by using Real-time Online RTPCR. DNA isolation was performed to detect gene copy number and copy number variations were detected by Q-PCR using related primer probes that we
designed. To evaluate the hypermethylation of the genes, DNA was treated with
bisulphate and methylation was detected by MS-Q-PCR. IC50 dose of genistein
was found as 50 lM in HL-60 cells. Apoptosis was increased by 20% in 24th
hour, 12% in the 48th hour and 4% in the 72nd by Annexin V-EGFP method in
IC50 dose. Apoptosis was detected up to 40% by MitoProbe JC–1 assay and distinct apoptosis was detected in the 48th hour by DNA Ladder method. According
to the Comet assay results, DNA fragmentation was observed in the 72nd hour
at the IC50 dose. RT-PCR results of PTEN, PIK3CA and PIKE genes mRNA
expression results were: 34% increase significantly, 482% decrease in the first
day, 290% increase in the third day, respectively. No significant change was
detected in the copy numbers of PTEN, PIK3CA and PIKE genes by using gene
copy number variation analysis. According to MS-Q-PCR analysis results of promoter region methylation of PTEN gene, methylation was decreased 20% in the
72nd hour. A significant increase was determined in IC50 dose of genistein in
protein expression of PTEN gene in 72nd hour. In conclusion, by treating leukemia cells with the IC50 dose of genistein, a distinct increase in apoptosis and
reactivation of tumor suppressor genes silenced by hypermethylation with epigenetic regulation were detected. This result suggests that genistein can be used in
the prevention and treatment of leukemia as an effective agent.
334
P-34
P-36
INTRA-ARTERIAL DELIVERY OF MUSCLE PRECURSOR CELLS TO
LIMB SKELETAL MUSCLES IN NONHUMAN PRIMATES
EXOGENOUS MUSCLE PRECURSOR CELLS MIGRATE INTO
THE SKELETAL MUSCLE TISSUE OF NONHUMAN
PRIMATES FOLLOWING INTRAMUSCULAR AND
EXTRAMUSCULAR IMPLANTATION
Daniel Skuk, Martin Paradis, Marlyne Goulet, and Jacques P. Tremblay
Human Genetics Research Unit, Laval University Hospital Center, Canada
A major limitation of cell therapies in the field of myopathies is that muscle precursor cells, when implanted intramuscularly, fuse only with the myofibers reached by the injection trajectories. An intravascular delivery of muscle
precursor cells may be obviously a better strategy than intramuscular injections, but so far this strategy seemed to succeed only with a special cell type
called ‘‘mesoangioblast and’’. Earlier experiments of intravascular delivery of
muscle precursor cells in mice produced limited results. However, this animal
model is quite different from human in several aspects of muscle cell transplantation biology. According to our experience, nonhuman primates are more
appropriate models for human extrapolations in this area: muscle precursor cell
culture and transplantation biology are very similar. Consequently, we performed intra-arterial injections of ß-Gal-labeled allogeneic muscle precursor
cells in tacrolimus-immunosuppressed cynomolgus monkeys. The cell suspension was injected in one femoral artery. Some skeletal muscles were damaged
with a 27G needle at the time of or 3 days previous to the cell infusion. Several organs and muscles were biopsied 1 hour, 1 day and 1 month post-transplantation, and the biopsies were analyzed by histology. We observed that
most intra-arterial delivered ß-Gal1 cells were retained mainly in the capillaries of the skeletal muscles of the leg ipsilateral to the injection. Scarce ßGal1 cells were observed in the lungs only at 1 hour post-transplantation, and
no ß-Gal1 cells were observed in other organs or other muscles. One month
post-transplantation, ß-Gal1 myofibers were observed only in the skeletal
muscles of the ipsilateral leg to the injection, when these muscles were injured
concomitantly to the cell injection.
The most important constraint for the intramuscular transplantation of muscle
precursor cells as a cell therapy for myopathies is that the grafted cells tends to
fuse only with the muscle fibers around the injection sites. This restriction is usually attributed to a ‘‘migration deficiency’’ of the muscle precursor cells. Our
recent observations in nonhuman primates suggest nevertheless that exogenous
muscle precursor cells fuse with the recipient’s muscle fibers after an intra-muscular migration, though this migration seems directed mostly to the muscle fibers
damaged by the injections. To analyze the migration ability of the implanted muscle precursor cells, we perform a study in cynomolgus monkeys. Small regions (1
cm3) of skeletal muscles were damaged by performing percutaneous penetrations
with a 27G needle. One hour later, 0.5 ml of a suspension of ß-galactosidase-labeled muscle precursor cells were implanted subcutaneously over the damaged
regions and over non-damaged regions. The recipients were immunosuppressed
with tacrolimus. One month later, the muscle regions beneath the subcutaneous
cell implantations were sampled and analyzed by histology. In the damaged
regions, ß-galactosidase-positive muscle fibers were observed up to 1 cm distant
from the muscle surface. The pattern of the ß-galactosidase-positive muscle fibers
was coincident with the pattern of the intramuscular needle trajectories. This indicated that the subcutaneously implanted muscle precursor cells were able to
migrate several millimeters into the underlying muscle in nonhuman primates,
although they fused essentially only with damaged muscle fibers. Therefore, the
problem of the intramuscular transplantation of muscle precursor cells is not a
‘‘migration deficiency’’ but rather the incapacity to diffuse through a non-damaged
tissue to fuse indiscriminately and spontaneously with non-damaged muscle fibers.
P-35
P-37
DIAGNOSING ACUTE REJECTION OF MYOFIBERS FOLLOWING
MYOGENIC-CELL ALLOTRANSPLANTATION: A STUDY IN
NONHUMAN PRIMATES
EARLY DEATH AND PROLIFERATION AMONG MUSCLE
PRECURSOR CELLS TRANSPLANTED IN SKELETAL MUSCLES
OF NONHUMAN PRIMATES
Transplantation of cells with myogenic potential has possible future applications in the therapeutic management of myopathies. In the case of cell allotransplantation, the recipients can experience rejection episodes that need to be
diagnosed. However, there are no histological criteria of acute rejection in this
specific context. We thus decided to determine the main histological features
that enable to diagnose acute rejection in skeletal muscles that received allotransplantations of myogenic cells in subjects treated with suboptimal immunosuppression. Using the animal model that allows the best clinical extrapolation
in transplantation research, we allotransplanted myogenic cells expressing ß-galactosidase in cynomolgus monkeys. Immunosuppression was done with tacrolimus, targeting suboptimal blood levels. The cell-grafted sites were biopsied 1,
3 and 5 months after transplantation. Biopsies showed abundant ß-galactosidase
expressing myofibers 1 month post-transplantation, but the graft was progressively lost during the following months. In all the periods there were focal
dense lymphocyte accumulations composed mainly by CD81 and CD41 lymphocytes, including lower amounts of macrophages. MHC-I was expressed in
abundant myofibers. Lymphocyte invasion of myofibers was frequent, but myofiber necrosis and apoptosis were rare. We concluded that the histological feature that may be used to diagnose acute rejection of myofibers is the presence
of focal dense endomysial accumulations of T-lymphocytes (quite equally
CD81 and CD41) with a component of macrophages, partially or completely
surrounding myofibers. Lymphocyte invasion of the myofibers may further support the diagnosis, but it is not clearly observed in all cases of lymphocyte
accumulations. Myofiber MHC-I expression can also further support the diagnosis, but it is difficult to observe it in myofibers surrounded by dense accumulations of immune cells expressing MHC-I. Due to the particular behavior of
myofibers during the immune attack, necrosis or apoptosis should not be
searched for the diagnosis of myofiber rejection.
There were several studies in mice describing the rapid death of a large percentage of the muscle precursor cells transplanted in skeletal muscles. However, there is
no information about the early behavior of muscle precursor cells following transplantation in humans or, at least, in animal models close to humans. Considering that
nonhuman primates are considered the best model for human extrapolation in preclinical transplantation research, we conducted a study in macaque monkeys to clarify several aspects of this subject. Monkey muscle-derived cells, labeled with ß-galactosidase and/or [14C]thymidine were auto- or allo-transplanted (under tacrolimus
immunosuppression in some cases) in skeletal muscles of rhesus and cynomolgus
monkeys. Muscle samples were taken at several post-transplantation periods (1 hour,
1 day, 3 days, 7 days and 3 weeks) and analyzed either by histological techniques or
by quantifying the radiolabel [14C]thymidine. The results showed that the transplanted cells exhibited a cell-death phenomenon approximately similar to that
observed in mice. With different dynamics, cell death was observed in autologous
and allogeneic (immunosuppressed or not) transplantations. Necrosis was detected
among several transplanted cells by the intracellular deposition of complement, as
soon as in post-transplantation day 1. Apoptosis was also detected among several
transplanted cells, by the expression of active caspase 3, as soon as in post-transplantation day 1. On the opposite, many cells were in proliferation at all post-transplantation periods during the 7-day follow-up, as observed by the immunodetection of the
Proliferating Nuclear Cell Antigen (fluorescent immunodetection of Proliferating
Cell Nuclear Antigen at different post-transplantation periods is shown in the figure,
the intramuscular accumulations of implanted cells being indicated between arrowheads). Therefore, necrosis and apoptosis are present among the intramuscularly
transplanted muscle precursor cells in nonhuman primates, and may be responsible
of an early cell death quite similar to that previously reported in mice. The high level
of proliferation among the surviving cells may be responsible for the final survival of
the graft, insuring the transplantation success 3 weeks later.
P-38
P-40
CONSTRUCTION OF BICISTRONIC B-GALACTOSIDASE REPORTER
PLASMIDS FOR EUKARYOTIC EXPRESSION OF CD40 LIGAND
(CD40L) GENE
PROGNOSTIC AND DIAGNOSTIC SIGNIFICANCE OF SURVIVIN
Didem Kafadar
University of Istanbul, Turkey
Yusuf Dolen, Gunes Esendagli, Sevil Oskay, and Dicle Guc
Hacettepe University, Institute of Oncology, Ankara, Turkey
The aim of the study was to construct recombinant bicistronic plasmids carrying rat CD40L cDNA and b-galactosidase (lacZ) reporter gene.
Methods: The coding sequence of lacZ gene was obtained from pcDNA4/
TO/lacZ construct (Invitrogen) by PmeI blunt-end digestion and targeted into
multiple cloning site (MCS)-B of SmaI-digested pIRES vector (Clontech). Following the ligation reaction, clones carrying the inversely oriented lacZ insert
were re-digested from the newly positioned NotI sites giving sticky ends. Following the treatment of NotI-cut pIRES with alkaline phosphatase, the ligation
resulted in pIRES-lacZ (control reporter) vector.
For the insertion of CD40L cDNA into MCS-A of pIRES vector; CD40L
cDNA was amplified with Pfu DNA polymerase from previously constructed
pCD40L-IRES2-EGFP plasmid with the primers carrying XhoI and MluI restriction sites and cloned into MCS-A by directional cloning (pCD40L-IRES). Subsequently, lacZ fragment was inserted into MCS-B region using the same approach
explained above, resulting in pCD40L-IRES-lacZ construct. Determination of
insert positions was carried out with NheI and XhoI re-digestions.
NIH/3T3 mouse fibroblast cell line was transfected with the constructs (2lg
plasmid, 1:3 DNA: liposome ratio) using Lipofectamine2000 (Invitrogen) reagent. The expression of the constructs was observed with staining of b-galactosidase reporter.
Conclusion: The pIRES-lacZ and pCD40L-IRES-lacZ plasmids were
expressed in NIH/3T3 cells indicating the functionality of the constructs. Further
in vivo gene transfer experiments are planned.
335
Survivin belongs to the apoptotic inhibitor proteins family and plays a key
role in the regulation of apoptosis and cell division. It is frequently expressed in
embryonic tissues and in tumors and is almost absent in normally differentiated
tissues. Survivin is expressed in G2/M phase of the cell cycle in order to maintain
the homeostasis of the fast dividing cell. Overexpression of survivin is regarded as
a poor prognostic marker in many malignant tumors since overexpression can
overcome cell cycle control points to ease the abnormal progression of transformed cells through mitosis. Survivin is localized in mitotic apparatus and can
bind to caspases to protect the cells from apoptosis. In a recent study it has been
demonstrated that survivin enhanced FasL expression in colon cancer cells. It is
suggested that survivin enables cancer cells to suppress attack by immune cells by
inhibition of Fas-mediated apoptotic signaling. As a result, survivin helps cancer
cells to get away from the immune system and live longer. Because survivin functions in cell cycle progression and it is expressed in tumor cells associated with
growth so it may be a negative prognostic factor. The presence of survivin is
associated with expression of bcl-2 and with reduced apoptotic index in breast carcinoma, in neuroblastoma, in gastric cancer, in colorectal cancer, and high-grade
non-Hodgkin’s lymphoma. Survivin expression may be regulated by p53 in some
cancer cell lines. A correlation is found between p53 accumulation and survivin
expression in cancers like gastric, pancreatic, prostate, lung and epidermoid carcinoma. Survivin can be detected in biological liquids. In bladder cancer it is
detected in urine and it is a specific and a sensitive prognostic factor. Recent work
on apoptosis allows to develop new drugs to cure cancer. Many drugs kill tumor
cells by stimulating apoptosis but some tumors show resistance to these drugs.
Several studies have demonstrated that survivin expressing tumors are resistant to
cancer drugs which stimulate apoptosis. Survivin signaling pathways may be used
in cancer diagnose and therapy in the future.
P-39
TURNER- DOWN SYNDROME: A CASE REPORT
P-41
Diclehan Oral, Selda Simsek, Aysegul Turkyilmaz, and Mahmut Balkan
Departments of Medical Biology and Genetic, Medical Faculty, University
of Dicle, Diyarbakir, Turkey
OXIDATIVELY INDUCED DNA BASE DAMAGE: POOR
PROGNOSTIC MARKER IN COLORECTAL CARCINOMA
Co-occurrence of two numerical chromosomal abnormalities in the same individual is relatively rare chromosomal abnormality. Our patient is a seven years old
girl; she has six healthy sisters and one brother. Clinically, the patient has most of
the phenotypic features of Turner syndrome as well as some features of Down syndrome. Characteristic findings included pterygium colli, cubitus valgus and short
stature. The characteristic features for Down syndrome low posterior hairline,
inner epicanthic folds, short fingers and prominent ears were evident.
Cytogenetic analysis of peripheral blood lymphocytes revealed a mosaic
karyotype 45,X,inv(9)[37]/47,XX,inv(9),121[8]. Cytogenetic analysis was performed from GTG banded metaphases. This finding was confirmed by fluorescent
in situ hybridization (FISH).
Didem Keles1, Guldal Kirkali1, Aras Emre Canda2, Zahide Cavdar1,
Cem Terzi2, Mehmet Fuzun2, Miral Dizdaroglu3, and Gulgun Oktay1
1
Department of Biochemistry, Faculty of Medicine, Dokuz Eylul University,
Izmir, Turkey, 2Department of Surgery, Faculty of Medicine, Dokuz Eylul
University, Izmir, Turkey, 3Chemical Science and Technology Laboratory,
National Institute of Standards and Technology, Gaithersburg,
MD 20899, USA
Molecular pathways that have roles in the development of colorectal cancer
involve multiple genetic changes that may be caused by overproduction of reactive
oxygen species (ROS) in cancer-related genes. Various types of ROS are formed
336
in vivo and many are powerful oxidizing agents, capable of damaging DNA and
other biomolecules. Increased formation of ROS can promote the development of
malignancy, and the ‘normal’ rates of ROS generation may account for the
increased risk of cancer development in the aged. These facts led us to hypothesize that oxidative damage by free radicals to DNA may accumulate in colorectal
cancer patients. To test this hypothesis, we investigated oxidative DNA damage in
colon and rectum tissues of cancer patients. DNA was isolated from 27 colorectal
cancer tissues obtained any carcinogenic lesions. DNA samples were analyzed by
liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure the levels of various typical oxidatively induced products of
DNA. Our results show that the patients with colorectal carcinoma at the presence
of perineural and lymphatic invasion accumulate statistically significant levels of
these lesions in their DNA. This work suggests that the oxidative stress with
excess production of free radicals in colorectal carcinoma patients may lead to
accumulation of oxidative DNA damage. This, together with the presence of perineural and lymphatic invasion due to mutations of cancer-related genes may be
considered as poor prognosis factor in colorectal carcinoma.
P-42
RELATIONSHIP OF CLASSICAL AND NON-CLASSICAL RISK
FACTORS WITH GENETIC POLYMORPHISM OF PON 1 RELEVANT
TO CORONARY ARTERY DISEASE
Dilara Kaman1, Necip Ilhan1, Kerem Metin1, Mehmet Akbulut2,
and Bilal Ustundag1
1
Firat University, Firat Medical Center, Department of Biochemistry, Elazig,
Turkey, 2Firat University, Firat Medical Center, Department of Cardiology,
Elazig, Turkey
Background: In addition to the well established cardiovascular risk factors,
evidence suggests a possible role of genetic and non-classical risk factors, such
as Lp(a), Oxide LDL, in the development and progression of atherothrombosis.
We aimed to determine the relationship of classical and non-classical cardiovascular risk factors with polymorphisms (PON55 L:M, PON191 Q:R) of paraoxonase gene potentially involved in cardiovascular risk in the Turkish population.
Increased concentrations of lipoprotein(a) [Lp(a)] have been considered a genetically determined risk factor for coronary artery and cerebrovascular disease. Oxidized Low Density Lipoprotein (oxLDL) particles are known to initiate the development of coronary artery disease.
Methods: We have determined the prevalence of classical (lipid profile,
blood pressure, glycemia, diabetes, smoking, body mass index and family history
of coronary heart disease) and non-classical cardiovascular risk factors (lipoprotein (a), PON levels, Oxide LDL) in a population-based study. PON 1 L/M 55
and Q/R 192 genotypes were determined by PCR, RFLP and agarose gel electrophoresis techniques. We analysed the relationship of these risk factors with PON
gene polymorphism potentially involved in cardiovascular risk.
Results: Genotype distributions for PON 1 L/M55 polymorphism were significantly different between controls and Coronary Artery Disease, CAD (1)
patient group (p<0.05), but genotype distribution of PON 1 Q/R192 polymorphism, there was not significantly difference among groups (P>0.05). Serum
PON 1 paraoxonase activity was lower in CAD (1) patients than in controls. In
both groups, the highest paraoxonase activities were detected in LL and RR genotypes, intermediate activities in LM and QR genotypes, and the lowest activities in MM and QQ genotypes. The highest levels of HDL and the lowest level
of oxide –LDL was detected in MM genotype in CAD (1) patients (p<0.05).
Conclusions: PON 1 L/M55 gene polymorphism, but not PON 1 Q/R192,
were associated with non-classical risk factors, specifically Lp(a), Oxide LDL
and PON levels in CAD(1) patients.
Objective: Gossypol is a natural polyphenolic compound extracted from cotton
plant (Gossypium species) and it was reported to have potent anticancer activities
in many types of malignancies, including breast cancer. Because of the low toxicity
profile of gossypol and the differences in mechanism of cytotoxicity, combinations
of doxorubicin, cisplatin, paclitaxel, vinorelbine and gemcitabine with gossypol
were studied in vitro to investigate for possible synergistic/additive effects.
Methods: MCF-7 cells were exposed in vitro for 24, 48 and 72 hours to gossypol in combination with these drugs, either simultaneously or sequentially. Cytotoxicity was determined by XTT Cell Proliferation Kit (Roche). Drug synergy
was assessed by using CalculSyn 2.0 software (Biosoft). For showing apoptosis
for the synergistic combinations, DNA fragmentation and caspase 3/7 enzyme
activity methods were used.
Results: Combination treatment with docetaxel and gossypol and paclitaxel
and gossypol resulted in synergistic cell proliferation inhibition in MCF-7 cell
line, compared to any agent alone. Apoptosis tests by measuring DNA-fragmentation and caspase 3/7 enzyme activity are still running for the synergistic combination of the drugs.
Conclusion: Combining gossypol with other cytotoxic agents known to be
effective in breast cancer may allow a reduction in cytotoxic agent doses and by
this way may diminish adverse effects while maintaining the therapeutic effect.
P-44
METHYLENETETRAHYDROFOLATE REDUCTASE C677T GENE
POLYMORPHISM IN TURKISH FEMALE RECURRENT APHTHOUS
STOMATITIS PATIENTS
Duygu Ofluoglu1, Umit Zeybek2, Ozlem Kucukhuseyin2, Hakki Tanyeri1,
and Turgay Isbir2
1
Oral Medicine and Surgery, Istanbul University, Dentistry Faculty, Istanbul,
Turkey, 2Department of Molecular Medicine, The Institute of Experimental
Medicine, Istanbul University, Capa, Istanbul, Turkey
Recurrent aphthous stomatitis (RAS) is the most common oral mucosal
lesions in general population. A higher prevalence has been found in the higher
socioeconomic groups, in females and individuals with stress. The exact aetiology of RAS is still unknown but different predisposing factors, including iron,
vitamin B12 and folic acid deficiencies have been reported. MTHFR is the
enzyme responsible for converting 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, the primary circulatory form of folic acid. This study was
designed to investigate the association of MTHFR polymorphism and folic acid
status in Turkish female RAS patients.
Our study was carried out in 30 female RAS patients and 30 female controls.
Serum iron, total iron binding capacity, ferritin, vitamin B12 and folic acid levels
were investigated. MTHFR C677T genotypes were determined by polymerase
chain reaction, restriction fragment length polymorphism techniques. Folic acid
levels were low in three RAS patients and one control and were in normal values
in one RAS patient and one control. MTHFR C677T frequencies of the CC, CT,
TT genotypes among female RAS patients were 56.6%, 33.3% and 10% respectively and 66.6%, 26.6% and 6.6% for control patients.
Populations homozygous for the C677T MTHFR variant are established as
having increased risk of cardiovascular disease, neural tube defects in normal population and left ventricular hypertrophy in Type II diabetic patients. Despite the
thermolabile MTHFR variant creates an additional requirement for folate, there
was no study in the literature evaluating the association of MTHFR genotypes and
folate status in RAS patients. According to our data, RAS patients with the TT genotype showed a higher prevalence of low plasma folate levels when compared
with the CC and CT genotypes. We conclude that MTHFR gene mutations may be
a possible risk factor for lower folate status among RAS patients.
P-43
P-45
SYNERGISTIC EFFECT OF GOSSYPOL, A COTTON-PLANT SEED,
WITH CONVENTIONAL CHEMOTHERAPEUTICS IN MCF-7 CELLS
Burcak Karaca1, Harika Atmaca2, Ali Guher Eniseler2,
Duygu Unuvar-Purcu2, Asli Kisim2, Bulent Karabulut1, Selim Uzunoglu2,
and Ruchan Uslu1
1
Department of Medical Oncology, Ege University, Faculty of Medicine,
Izmir, Turkey, 2Department of Molecular Biology, Celal Bayar University,
Manisa, Turkey
PARAOXONASE 55/192 GENE POLYMORPHISMS AND SERUM
PARAOXONASE ACTIVITY IN TURKISH OSTEOPOROTIC PATIENTS
E. Hakan Eraltan1, Bahar Toptas2, Mehmet Uyar3, Bedia Agachan2,
Hulya Yilmaz-Aydogan2, Umit Zeybek2, M. Kerem Ozyavuz2,
and Turgay Isbir2
1
Physical Therapy Department, Azerbaijan Medical University, Baku,
Azerbaijan, 2Department of Molecular Medicine, The Institute of
Experimental Medicine, Istanbul University, Capa, Istanbul, Turkey,
3
Physical Therapy and Rehabilitation Clinic, Uskudar State Hospital,
Istanbul, Turkey
Background: Oxidative stress may affect cellular functions in various pathological conditions, including osteoporosis. Paraoxonase 1 confers antioxidant
properties on high-density lipoprotein, with which it is associated, by reducing
the accumulation of lipid peroxidation products. We have now examined whether
the 584A-->G (Gln192Arg) and 172T-->A (Leu55Met) polymorphisms of the
paraoxonase 1 gene and the 959G-->C (Cys311Ser) polymorphism of the paraoxonase 2 gene are associated with osteoporosis risk in postmenopausal women.
The purpose of this study was to demonstrate any relationship between serum
PON1 activity and 584A-->G (Gln192Arg) and 172T-->A (Leu55Met) polymorphisms of the paraoxonase 1 gene in osteoporosis.
Materials and Methods: The study included 28 osteoporotic and 20 nonosteoporotic postmenopausal women. The distribution of PON 55/192 gene polymorphism was determined by polymerase chain reaction-based restriction fragment length polymorphism. PON activity was measured by spectrophotometric
assay of p-nitrophenol production following addition of paraoxon.
Results: We found that PON 55 and 192 genotype distribution was similar
in osteoporosis patients and controls (p>0.05). Although the serum PON1 activity was lower in osteoporosis patients (58.45 6 36.17 U/L) than the healthy controls (122.65 6 40.60 U/L), the difference was not statistically significant
(p>0.05). PON 192 BB homozygotes had significantly lower PON1 activity than
AA and AB genotypes among the nonosteoporotic subjects and osteoporosis
patients (p50.039).
Conclusion: Our results suggest that the paraoxonase activities are affected
by PON1 genetic variability in Turkish osteoporotic and nonosteoporotic postmenopausal women.
P-46
EFFECTS OF ASTRAGALUS EXTRACT ON OXIDANT/ANTIOXIDANT
STATUS AND MYOCARDIAL CONTRACTILITY IN
STREPTOZOCIN-INDUCED DIABETIC RATS
Mehmet Namuslu1, Bulent Kilicoglu2, Ayca Bilginoglu3, Ebru Gurleyik1,
Esma Zeydanli3, Aslihan Avci1, Belma Turan3, and Ilker Durak1
1
Ankara, Turkey, 2Department of General Surgery, Ankara Educations and
Search Hospital, Ankara, Turkey, 3Department of Biophysics, Ankara
University, Faculty of Medicine, Ankara, Turkey
337
sacrificed and tissues (liver, kidney, and hearth) and blood samples were
obtained. Serum glucose, serum and tissue (liver, kidney) malondialdehyde
(MDA) levels, antioxidant enzyme activities (glutathione peroxidase, catalase,
superoxide dismutase) and xantin oxidase activity were determined. And also
myocardial contractility was examined in rats.
Results: In our study, it was found that Astragalus treatment decreased blood
glucose levels. There was no change in antioxidant enzyme activities and MDA
levels between the groups. However we observed that astragalus increased myocardial contractility. The treatment of diabetic rats with Astragalus improved the
diabetes-induced depression in the left ventricular developed pressure (LVDP)
and the rate changes in developed pressure (1dP/dt). And also this treatment
induced a significant protection against diabetes-induced depressed phenylephrine-stimulated isometric contraction and endothelial-dependent isoproterenol and
Ach stimulated relaxation in thoracic aorta (p<0.05) (Figure 1).
Conclusion: In our study, Astragalus treatment may cause a preventive effect
on myocardial tissue through different mechanisms, but not through antioxidant
system; the mechanism is still unclear and further studies are needed.
P-47
PLASMA ADA AND PON1-ARYLESTERASE ENZYME ACTIVITIES IN
LUNG CANCER PATIENTS
Ebru Gurleyik1, Mehmet Taspinar2, Derya Oztuna3, Cansel Atinkaya4,
Ulku Yazici5, Aslihan Avci1, and Asuman Sunguroglu2
1
Ankara, Turkey, 2Department of Medical Biology Ankara University,
Faculty of Medicine, Ankara, Turkey, 3Department of Biostatistics, Ankara
University, Faculty of Medicine, Ankara, Turkey, 4Department of Thoracic
Surgery, Kirikkale University, Faculty of Medicine, Kirikkale, Turkey,
5
Department of Thoracic Surgery, Ataturk Center for Chest Disease and
Thoracic Surgery, Turkey
Objective: Overproduction of reactive oxygen and nitrogen species may
cause formation of oxidative DNA damage and may also induce oxidative damage in some molecules such as lipids, proteins, etc. The pathogenesis of cancer
has not yet been clarified, but oxidative stress is suspected to contribute to carcinogenesis by different cellular mechanisms. Hence, our study aimed at examining the oxidant and antioxidant parameters in lung cancer patients and reference
individuals and identifying possible correlations between those parameters.
Methods: The study included 24 lung cancer patients and 5 non-cancer subjects as controls. Blood samples were collected and plasma malondialdehyde
Objective: Oxidative stress has been suggested as a contributory factor in
development and complications of diabetes. The aim of the present study was to
determine the protective effects of Astragalus extract on lipid peroxidation, antioxidant status and myocardial contractility in streptozotocin-induced diabetic
rats.
Methods: The study was performed with 20 rats divided into three groups: a
diabetic group treated with a dose of 1mg/kg/day Astragallus, a diabetic group
and a non-diabetic control group fed with a normal diet for four weeks. In this
study, we used streptozocin (STZ) to inducediabetes in rats. The animals were
Figure 1. LVEDP and LVDP levels in rats
Figure 1. PON1-Arylesterase Activity in Groups. [Color figure can be viewed in
the online issue, which is available at www.interscience.wiley.com.]
338
(MDA) level, adenosine deaminase (ADA) enzyme activity and PON1-arylesterase activity were determined by spectrophotometric methods.
Results: In this study, PON1-arylesterase activity was significantly lower
(p<0.01) in lung cancer patients compared to controls (figure 1). However controls presented higher ADA activity than cancer patients, it was not statistically
significant. There was no statistically significant difference in MDA levels
between groups. Also there was a positive correlation between PON1-arylesterase
and ADA activities.
Conclusions: Our results show that low PON1-arylesterase and ADA activities are due to inefficient antioxidant defense system which could be induced by
oxidative damages in cancerous patients. Decreased PON1-arylesterase activity
could also be explained by depletion in the total -SH groups levels. The activity
of ADA in lung cancer patient was found lower than in patients with tuberculosis
and infectious pleural effusions. Our results indicated that ADA activity in cancer patients was found to be decreased compared to controls. Further studies are
needed to clarify the possible mechanisms underlying the decreased enzyme
activities.
P-48
MOLECULAR ANALYSIS OF KERATINOCYTES WHICH DERIVED
FROM MOUSE EMBRYONIC STEM CELLS
H. Seda Vatansever1, Elgin Turkoz-Uluer1, Hasan Aydede2,
and M. Kemal Ozbilgin1
1
Department of Histology and Embryology, Faculty of Medicine, Celal
Bayar University, Manisa, Turkey, 2General Surgery, Faculty of Medicine,
Celal Bayar University, Manisa, Turkey
Objective: The skin plays a crucial role in protecting the integrity of the
body’s internal milieu. Major area of research in regenerative medicine is the utilization of adult and embryonic stem cells in skin grafting and tissue engineering.
Mouse embryonic stem cells (ES), which are pluripotent stem cells derived from
inner cell mass of blastocysts, proliferate or are able to differentiate under suitable conditions. We studied how to differentiate and identify keratinocytes from
mouse embryonic stem cells in order to use them in wound healing models.
Methods: ES were cultured on mitomycin-C treated mouse fibroblast cells in
ES medium including 1000U/mL leukemia Inhibitor factor (LIF). After culturing
ES cells for 4 weeks, embryoid bodies (EBS) were grown in media without LIF.
EBs were transferred onto matrijel coated Petri dishes after 2 days, and cultured
in media with 0.5 nM BMP-4 for differentiating them into keratinocytes. For
identification of keratinocytes, they were fixed in 4% paraformaldehyde solution
for 30 min and stained with anti-keratin-8 and anti-keratin 14 antibodies using
indirect immunohistochemistry method.
Results: When mouse ES cells were cultured with LIF, the ES colonies were
observed after 2 days of culture. The EBs were done by collecting the ES cells
from colonies and when the EBs were formed, the outer and inner layer of EBs
were observed. After incubating the cells in media containing BMP-4 for 10-15
days, they started to differentiate into keratinocytes. The cells were stained positively with both anti-keratin-8 and anti-keratin 14 antibodies.
Conclusions: During the treatment of wound healing, it is a problem to find
autologous skin graft. Therefore, the patients may have to wait for generating the
graft or an allogenic donor should be used. In our study, the keratinocytes, which
differentiated from mouse ES cells, were expressing both keratin-8 and keratin14, which are early and late term keratinocyte specific markers, respectively.
After this preliminary study, these cells may be used to treat experimental surgical wound.
P-49
DETECTION OF THE PREVALENCE OF HELICOBACTER PYLORI IN
DIFFERENT ORGANS USING REAL-TIME PCR
Ahmet Ozbek1, Tuba Demirci2, Elvan Ozbek2, Hakan Dursun3,
Belkis Aylu4, and Vural Fidan5
1
Department of Microbiology and Clinical Microbiology, Medical Faculty,
Ataturk University, Erzurum, Turkey, 2Department of Histology and
Embryology Medical Faculty, Ataturk University, Erzurum, Turkey,
3
Department of Gastroenterology, Medical Faculty, Ataturk University,
Erzurum, Turkey, 4General Surgery, Region Training and Research
Hospital, Erzurum, Turkey, 5Otorhinolaringology, Region Training and
Research Hospital, Erzurum, Turkey
Objective: As a well known gastric pathogen, Helicobacter pylori play a
major role in the pathogenesis of chronic gastritis, duodenal and gastric ulcers,
adenocarcinoma and gastric lymphoma. Also recent studies proposed the oral
cavity and gallbladder as extragastric reservoirs of H. pylori infection. H. pylori
can cause the same immunological changes in the oropharyngeal mucosa as in
gastric mucosa and also can contribute to the progression of oropharyngeal diseases. In this study, the prevalence of H. pylori was investigated in the tonsil,
stomach and gallbladder samples from patients.
Methods: Total 45 samples from different 45 patients were included. Distribution of the samples was as the following: 10 from the stomach with gastritis,
22 from the gallbladder with cholecyctitis, and 13 from the tonsils with chronic
infection. To detect the presence of H. pylori, melting analysis by means of realtime PCR was used. Bacterial DNA was extracted using a QIAamp1DNAminikit. Species specific primers were targeted CagA region. The hypothesis test for
one proportion was used to estimate the highest possible rate of H. pylori in the
samples. Independent sample t test was applied using Microsoft1 SPSS 13.0, to
analyse a statistically significant difference between the different tissues as to the
number of H. pylori-positive samples.
Results: Seventeen of 45 samples were positive. Three, 10 and 4 positivesamples were stomachs, gallbladders and tonsils, respectively. The highest possible rate of H. pylori was 50% for total samples. The rates separately were 55%
for stomach, 62% for gallbladder, and 53% for tonsil. Statistically important difference found between gallbladder and stomach (p<0.01), between gallbladder
and tonsil (p<0.01), and between stomach and tonsil (p<0.05) as to the existence of H.pylori.
Conclusions: Our data suggest H. pylori is a bacterium frequently-found in
extragastric tissue infections apart from the gastric infections.
Acknowledgement: This study was supported by the Scientific Research
Funds (2005/183-184) of our university.
P-50
MYCOBATERIUM TUBERCULOSIS IN ACUTE PERIRADICULAR
ABSCESSES: A MOLECULAR STUDY USING A PCR METHOD
Selcuk Ozbek1, Ahmet Ozbek2, and Elvan Ozbek3
1
Department of Endodontics, Faculty of Dentistry, Ataturk University,
Erzurum, Turkey, 2Department of Microbiology and Clinical Microbiology
Faculty of Medicine, Ataturk University, Erzurum, Turkey, 3Department of
Histology and Embryology Faculty of Medicine, Ataturk University,
Erzurum, Turkey
Objectives: In this study, we aimed to investigate the presence of Mycobacterium tuberculosis in acute periradicular abscesses by real-time PCR method
and to evaluate its statistical significance.
Methods: Pus samples from 55 patients with acute periradicular abscess and
33 saliva samples (as a control group) selected randomly from the patients visiting the Endodontics Clinic of Dentistry Faculty were used. DNA was extracted
from the samples using a QIAamp1DNA mini-kit and analyzed with real-time
PCR. IS 6110 was targeted with species specific primers for amplification. ‘‘The
left-tailed hypothesis test for one proportion’’ was used to estimate the highest
possible rate of M. tuberculosis in the both the acute periradicular abscesses and
saliva, and ‘‘independent samples t test’’ was applied using Microsoft1 SPSS for
Windows Version 13.0, to analyse a statistically significant difference between
two groups as to the number of M. tuberculosis-positive samples.
Results: M. tuberculosis was positively detected in 7 of 55 pus samples, and
in 5 of 33 saliva samples. According to the hypothesis test, the highest possible
rate of M. tuberculosis was 21% in acute periradicular abscesses, and 28% in saliva (z51.645, alpha5 0.05). No important difference was found statistically
between two groups as to the number of M. tuberculosis-positive samples
(p>0.05).
Conclusions: As an agent of tuberculosis, one of the most widespread diseases afflicting human beings, M. tuberculosis has an importance in endodontic
infections, too. This bacterium can use several ways to reach the tooth pulp. In
our study, the data suggest that the bacteria may be spread out from carious
lesions passing through the dentine to the pulp. The results are very important
also for dentists to avoid the horizontal transmission of saliva-borne M. tuberculosis during dental treatment processes.
Acknowledgement: This study was supported by the 2005/184-numbered
Scientific Research Fund of our university.
P-51
PREVALANCE OF TREPONEMA DENTICOLA IN ACUTE
PERIAPICAL ABSCESSES
1
Erzurum, Turkey, 2Department of Microbiology and Clinical Microbiology
Histology and Embryology, Faculty of Medicine, Ataturk University,
Erzurum, Turkey
Objectives: Although Treponema denticola is one of the most prevalent
agents for endodontic infections, it is highly difficult to grow. So, molecular microbial methods are more suitable for identifying this bacterium. In this study,
real-time polymerase chain reactions targeted 16s rRNA were used to detect the
occurrence of T. denticola in acute periapical abscesses.
Methods: Pus samples were collected from 35 patients with acute periapical
abscesses. To obtain the DNA from samples, QIAamp1 DNA Mini Kit
(QIAGEN1 GMBH, GERMANY) was used. DNA extracted from each sample
was amplified by using both a conventional PCR with ubiquitous primers (50 —30
GAT TAG ATA CCC TGG TAG TCC AC and CCC GGG AAC GTA TTC
ACC G —base positions were 796-818 and 1381-1399 respectively) and a realtime PCR with species-specific primers (forward primer; 50 -AGAGCAAGCTCT
CCCTTACCGT-30 , reverse primer; 50 -TAAGGGCGGCTTGAAATAATGA-30
and a probe 50 -FAM-CAGCGTTCGTTCTGAGCCAGGATCA-TAMRA-30 ). The
left-tailed hypothesis test for one proportion was used to estimate the highest
possible rate of T. denticola in the acute periapical abscess.
Results: All of 35 samples were positive with ubiquitous primers by conventional PCR. This result was used as positive control to eliminate false negative
results owing to PCR inhibitors. The results of real-time PCR amplification introduced 26 positive samples for T. denticola. According to the hypothesis test, the
highest possible rate of T. denticola was 84% in the acute periapical abscess (z5
-1.645, alpha5 0.05).
Conclusion: T. denticola is a frequently-found bacterial agent in acute periapical abscesses, and real-time PCR with TaqMan probe system is very convenient method for detection of T. denticola.
339
phatic cells infiltrations and numerous neutrophiles were observed. A prominent
epithelial loss was detected in the bronchioles. Additionally, alveolar collapse
and hyalinization were found. Much more type 2 pneumocytes were present in
the alveolar wall. Particularly, accumulations of type 2 pneumocyte-like cells
were remarkable in the interalveolar septa. Vascular injuries including acute vasculit and fibrinoid necrosis were found.
Conclusions: Our results are consistent with previous reports stating the
morbidity and mortality associated with anthrax infection. But, this is the first
study suggesting that LF can lead alone to these deleterious effects.
Acknowledgement: This study was supported by the 2007/13-numbered Scientific Research Fund of our university.
P-53
LIVER INJURY INDUCED BY ONE-DOSE ANTHRAX LETHAL
FACTOR IN ADULT FEMALE RATS
B. Zuhal Altunkaynak and Elvan Ozbek
School, Erzurum, Turkey
HISTOPATHOLOGICAL CHANGES OF LUNGS AFTER ONE-DOSE
ANTHRAX LETHAL FACTOR INJECTION TO ADULT FEMALE RATS
Objectives: Anthrax infection is usually fatal even with optimal medical
care. Further insights into anthrax pathogenesis are therefore urgently needed to
develop more effective therapies. Animal models that reproduce human disease
will facilitate this research. Lethal factor (LF) and protective antigen (PA) are
secreted by Bacillus anthracis, and their binary combination yields lethal toxin
(LT). Lethal toxin is responsible for many pathologies of anthrax, but the role of
lethal factor alone on the pathology of anthrax is unclear. Here, we describe the
detailed histopathology of liver in adult rats subjected to one-dose lethal factor.
Methods: Six adult female Sprague Dawley rats were used. The animals
were divided into two equal-sized groups as a control and a treatment group.
Rats in the treatment group were administered intraperitoneally Lethal Factor,
100 lg/kg, after dissolving with distilled water (Anthrax Lethal Factor, Recombinant, Bacillus anthracis, Calbiochem, Merck, Germany, Catalog no: 176900). After one-dose LF injection, rats were maintained under standardized conditions
with free access to laboratory pellets and tap water for 24 hours. Control rats
were injected only distilled water. After 24 hours, liver was removed from the
anesthetized animals, and processed for histopathological examination.
Results: One-dose LF application caused hepatic injury including cytoplasmic and nuclear damages in hepatocytes, sinusoidal dilatation and hepatocellular
lysis. Light microscopy of liver sections from LF-injected rats demonstrated
many apoptotic bodies in the parenchyma, ballooning degeneration and cytoplasm loss of hepatocytes and peri-sinusoidal inflammation. Additionally, hypertrophy and hyperplasia of Kupffer’s cells were determined.
Conclusions: There is no previous study reporting on the pathologic situation
derived from lethal factor toxicity alone. In the present study, well-defined pathologies of the liver bear marked similarity to primary pathologies observed during human disease. Therefore, our data will allow researchers to study other
major pathologies observed in humans.
B. Zuhal Altunkaynak, and Elvan Ozbek
Department of Histology and Embryology, Faculty of Medicine, Ataturk
University, Erzurum, Turkey
P-54
P-52
Objectives: Recent events have brought attention to the potential of Bacillus
anthracis as an agent of bioterrorism. Bacillus anthracis secretes toxic proteinsprotective antigen (PA), lethal factor (LF) and edema factor (EF), and cause
shock and mortality. In the present study, we aimed to characterize microscopic
pathology of the lungs in the one dose LF-treated rats.
Materials and Methods: Six adult female Sprague Dawley rats were randomly allocated into two equal-sized groups. Rats in the treatment group were
given intraperitoneally LF (Anthrax Lethal Factor, Recombinant, Bacillus anthracis, Calbiochem, Merck, Germany, Catalog no: 176900) after dissolving with distilled water (100 lg/kg in dosage). After one-dose LF injection, rats were maintained under standardized conditions with free access to laboratory pellets and
tap water for 24 hours. Control rats were injected only distilled water. After 24
hours, lung samples were removed from the anesthetized animals, and processed
for histopathological examination.
Results: In LF-treated animals, pulmonary edema and hemorrhage were seen
by light microscopy. Connective tissue septa among the pulmonary parenchyma
were thickened in comparison with that of the control group. In these septa, lym-
ANTHRAX LETHAL FACTOR CAUSES TO STRUCTURAL CHANGES
OF HEART IN FEMALE RATS: A LIGHT MICROSCOPIC STUDY
B. Zuhal Altunkaynak and Elvan Ozbek
School, Erzurum, Turkey
Objectives: Anthrax infections are frequently associated with severe hypotensive shock and high mortality despite appropriate antibiotics and aggressive
hemodynamic and pulmonary supports. Previous observations suggest that anthrax toxins have direct cardiovascular effects. Herein, we aimed to investigate
possible harmful effects of anthrax lethal factor (LF) on heart in female rats.
Methods: In the present study, adult female Sprague Dawley rats were randomly allocated into two groups as a control (n53) and a treatment group
(n53). Rats in the treatment group were administered intraperitoneally Lethal
Factor, 100 lg/kg, after dissolving with distilled water (Anthrax Lethal Factor,
Recombinant, Bacillus anthracis, Calbiochem, Merck, Germany, Catalog no:
340
176900). After one-dose LF injection, rats were maintained under standardized
conditions with free access to laboratory pellets and tap water for 24 hours. Control rats were injected only distilled water. After 24 hours, heart was removed
from all animals, and processed for light microscopic examination.
Results: In LF-injected rats, the heart was moderately affected with cardiomyocyte degeneration and increased separation of myofibers. The interstitial
space between cardiomyocytes was enlarged. Multifocal necrosis areas of cardiomyocytes were observed and interfiber myxoid edematous accumulations were
detected. Additionally, platelet/fibrin thrombi were found within the coronary
vessels. The wall of these vessels was necrotic and vascular mononuclear cell
accumulation was obvious. Fibroblast infiltrations within the interstitium were
interpreted as an evidence of cardiac muscle damage. Lymphoid cell infiltrations
at the perivascular and/or diffuse forms were extensive in the cardiac muscle.
Conclusions: Previous studies have stated that anthrax lethal factor or edema
factor can produce pathological effects if they are together with the protective
antigen. But, our study has demonstrated for the first time that lethal factor could
cause histopathological changes of the heart even if it was applied alone.
P-55
HISTOPATHOLOGICAL EFFECTS OF BACILLUS ANTHRACIS
LETHAL FACTOR ON KIDNEYS IN ADULT RATS
Elvan Ozbek1, B. Zuhal Altunkaynak1, and Ahmet Ozbek2
1
School, Erzurum, Turkey, 2Department of Microbiology and Clinical
Microbiology, Faculty of Medicine, Ataturk University, Erzurum, Turkey
Objectives: Bacillus anthracis, the causative agent of anthrax infection, relies
on a polyglutamic capsule and anthrax toxin to establish infection and to kill its
host. Anthrax toxin is made up of three separate protein components: the receptor-binding protective antigen (PA), the adenylyl cyclase edema factor (EF), and
the metalloproteinase lethal factor (LF). PA and EF together constitute edema
toxin (ET), while PA and LF constitute lethal toxin (LT). Studies on the activity
of anthrax toxins individually or in combination are crucial for understanding of
their role in anthrax pathogenesis. In the present, we investigated possible hazardous effects of lethal factor on kidneys in rats.
Methods: Six adult female rats were randomly divided into two equal-sized
groups. Rats in the treatment group were given intraperitoneally Lethal Factor,
100 lg/kg in dosage, after dissolving with distilled water (Anthrax Lethal Factor,
Recombinant, Bacillus anthracis, Calbiochem, Merck, Germany, Catalog no:
176900). After one-dose LF injection, rats were maintained under standardized
conditions with free access to laboratory pellets and tap water for 24 hours. Control rats were injected only distilled water. After 24 hours, kidneys were removed
from the anesthetized animals, and processed for light microscopic examination.
Results: Histological investigation showed a prominent dilatation of blood
vessels and Bowman’s space in the renal cortex. Mononuclear cell infiltrations in
the glomeruli and nephronal degenerations including glomerulosclerosis and tubular defects were observed in the kidneys of the treatment group.
Conclusions: Many previous studies have reported that LF or EF has not
any role on pathological mechanism of anthrax although highly purified toxin
was not available for those studies. But herein, our findings indicate that anthrax
lethal factor can cause histopathological changes in rat kidneys. This is the first
study suggesting that LF can lead to hazardous effects on kidneys even if it is
applied alone.
P-56
MICROSCOPIC PATHOLOGY OF SPLEEN IN RATS SUBJECTED
TO ONE-DOSE INJECTION OF BACILLUS ANTHRACIS
LETHAL FACTOR
Elvan Ozbek1, B. Zuhal Altunkaynak1, and Ahmet Ozbek2
1
School, Erzurum, Turkey, 2Department of Microbiology and Clinical
Microbiology, Faculty of Medicine, Ataturk University, Erzurum, Turkey
Objective: Bacillus anthracis, the etiologic agent of anthrax, produces three
primary virulence factors: lethal toxin (lethal factor 1 protective antigen), edema
toxin (edema factor 1 protective antigen), and a capsule. The capsule is absolutely required for dissemination and lethality in a murine model of inhalation
anthrax, yet the roles of the toxins during infection are ill-defined. In this study,
we aimed to determine possible histopathological effects of anthrax lethal factor
(LF) on spleen in a rat model.
Methods: Age-matched adult female rats (n56) were divided into two
equal-sized groups as a control and a treatment groups. Intraperitoneal LF injections (100 lg/kg) were carried out with sterile distilled water for the treatment
group (Anthrax Lethal Factor, Recombinant, Bacillus anthracis, Calbiochem,
Merck, Germany, Catalog no: 176900). After one-dose LF injection, rats were
maintained under standardized conditions with free access to laboratory pellets
and tap water for 24 hours. Control rats were injected only distilled water. After
24 hours, spleen was removed from all animals. Formalin-fixed and paraffin-embedded spleen sections were stained with Hematoxylin and eosin (H-E), and
examined by light microscopy.
Results: LF injection led to dramatic microscopic changes in the architecture
of the spleen. In the white pulp, the periarteriolar lymphoid sheath showed a loss
of lymphocytes, visible at low power as a starry sky pattern. In contrast to controls, the marginal lymphocyte zone was also no longer apparent. In the red
pulp, there was also significant depletion of lymphocytes with relative sparing of
macrophage numbers. In addition, occasional neutrophils were observed. Many
of the splenic macrophages contained hemosiderin suggesting hemorrhage.
Lastly, in red pulp, many giant cells and vascular deficits including vasculitis
and fibrinoid necrosis were also evident.
Conclusions: Our results indicate that LF can cause pathological changes in
spleen as that in the animals subjected to anthrax infection, even if it is applied
alone. Moreover, the detailed description of the splenic pathology can serve as a
basis for further experimentations.
P-57
THE CORRELATION OF SERUM LEPTIN LEVELS WITH GENDER,
OBESITY AND CHRONIC STRESS IN A RAT MODEL
Elvan Ozbek1, Tulin Fidan2, Ahmet Ozbek3, and B. Zuhal Altunkaynak1
1
School, Erzurum, Turkey, 2Department of Child Psychiatry, Faculty of
Medicine, Ataturk University, Erzurum, Turkey, 3Department of
Microbiology and Clinical Microbiology, Faculty of Medicine,
Ataturk University, Erzurum, Turkey
Objective: Leptin is a circulating hormone that plays a key role in regulating
food intake and body weight via its actions on specific hypothalamic nuclei. In
particular, recent studies have demonstrated that chronic unpredictable stress and
chronic social defeat exhibit low leptin levels in plasma. In the present study, we
investigated the relationship between leptin, stress and obesity in obese and nonobese rats subjected to chronic mild stress (CMS).
Methods: Adult male and female Sprague Dawley rats of the obesity group
were fed with a fatty diet (30% fat) for 2 months. Control rats were maintained
on standard rat chow. Following the diet procedure, body mass index (BMI) values of all animals were calculated. Subsequently, half of both control and obesity
groups were exposed to chronic mild stressors for 2 weeks with access to a preferred diet. At the end of the experiment, blood samples were obtained and serum leptin levels (SL) were detected with ELISA method.
Results: Mean BMI values of both fatty diet-fed females and fatty diet-fed
males were significantly higher than those of the control animals, confirming our
obesity model (p<0.05, Independent samples t test). SL was higher in female
controls than that in male controls. SL of obese males and females were significantly higher in comparison with that of the controls (p<0.01, Bonferroni test).
Interestingly, CMS caused a significant increase in SL of non-obese control
males while SL significantly decreased in CMS-exposed obese males (p<0.01,
Bonferroni test). No significant effect of CMS was detected on SL of both obese
and non-obese females.
Conclusion: Our data suggest that there are gender differences as regards serum leptin levels in both healthy life and chronic stress circumstances. In such a
situation, stress may be a contributing factor to obesity in fatty males. Moreover,
it is possible that obese males subjected to stressors may give a better response
to leptin-replacement therapy to fight obesity.
P-58
MOLECULAR IDENTIFICATION OF ENTEROCOCCUS FAECALIS IN
ROOT CANALS VIA REAL-TIME PCR SYBR GREEN
1
Erzurum, Turkey, 2Department of Microbiology and Clinical Microbiology,
Histology and Embryology, Faculty of Medicine, Ataturk University,
Erzurum, Turkey
Objectives: Enterococcus faecalis is a microorganism that can survive
extreme challenges. Reports on the prevalence of E. faecalis in root canals vary
considerably, potentially because of variations in clinical sampling and sample
analysis methods. The aims of this study were to investigate the presence of E.
faecalis in primary and secondary endodontic infections using real-time PCR and
to determine the statistical importance of the presence of E. faecalis in endodontic infections.
Materials and Methods: E. faecalis was investigated in 90 microbial samples collected from patients who were attended at the Endodontic Clinic of Faculty of Dentistry. Medical histories revealed that all patients were in good general health, and had no important systemic diseases. Microbial samples were
taken from 90 patients, 45 (Group A) with failed endodontic treatments (secondary endodontic infections) and 45 (Group B) with chronic apical periodontitis
(primary endodontic infections). DNAs were extracted from the samples by using
a QIAamp1 DNA mini-kit and analyzed with real-time PCR SYBR Green. The
left-tailed hypothesis test for one proportion was used to estimate the highest
possible rate of E. faecalis in primary and secondary endodontic infections.
Results: E. faecalis was detected in 47 of 90 patients. When the hypothesis
test was applied, the highest possible rate of E. faecalis was 60% in all endodontic infections (z5-1.645, alpha5 0.05). Real-time PCR SYBR Green allowed to
the detection of E. faecalis in 35 of 45 patients in Group A, and in 12 of 45
patients in Group B. According to the hypothesis test, the highest possible rates
of E. faecalis were 86% and 38% in secondary and primary endodontic infections, respectively. When the Fisher’s exact test (Microsoft1 SPSS for Windows,
version13.0) was applied, E. faecalis was found significantly more often in
patients with secondary endodontic infections than in patients with primary endodontic infections (p<0.01).
Conclusions: Melting analysis via real-time PCR SYBR Green method is
very convenient method for detection of E. faecalis in endodontic samples. Our
results suggest that E. faecalis is a frequent isolate for endodontic infections, and
is more often associated with secondary than primary endodontic infections.
Acknowledgements: This study was supported by the Scientific Research
Funds (2005/183 and 2005/184) of our university.
P-59
341
4 polymorphic variants. The allele frequencies of exon 4 were not statistically
significantly different in lung cancer and control groups. According to our
results, for two polymorphisms of EPHX, only exon 3 Tyr113His polymorphism
was related to high risk in patients with lung cancer. In conclusion, we suggest
that EPHX polymorphisms may be a risk factor for lung cancer patients.
The present work was supported by the Research Fund of Istanbul University. Project No. T878/02062006.
P-60
GENETICALLY ENGINEERED PANCREATIC ISLETS EXPRESSING
TRAIL EXTENDS SURVIVAL OF TRANSPLANTED PANCREATIC
ISLETS IN STZ-INDUCED DIABETIC RATS
Ercument Dirice1, Ahter D. Sanlioglu1, Sevim Kahraman1,
Abdulkadir Omer2, Mustafa K. Balci3, and Salih Sanlioglu1
1
Human Gene Therapy Unit and Department of Medical Biology and
Genetics, Faculty of Medicine, Akdeniz University, Antalya, Turkey,
2
Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center,
Harvard Medical School, USA, 3Division of Endocrinology and Metabolic
Diseases, Faculty of Medicine, Akdeniz University, Antalya, Turkey
Background: Type 1 Diabetes Mellitus (T1DM), is a chronic T-cell-mediated autoimmune disease characterized by selective destruction/dysfunction of insulin-secreting b-cells. Current therapy for T1DM based on the replacement of
insulin or on islet transplantation and immune suppression has substantial limitations. An alternative approach to curing T1DM might be the reversal of preexisting autoreactivity by ex vivo genetically engineered b-cells to prolong islet/graft
survival. While TNF-related apoptosis-inducing ligand (TRAIL) has been shown
to play a crucial role in inhibiting diabetic inflammation and autoreactive T-cell
activation in vitro and in vivo, we aimed to develop a novel therapeutic approach
by modifying pancreatic islets to over-express TRAIL in order to prevent autoreactive T-cell attack.
Methods: PI/FDA and DTZ stainings were performed to determine cell viability and purity. Both fluorimetric measurements and fluorescent microscopy
were used to determine the transduction efficacy of AdEGFP infected islets.
While immunohistochemical methods were performed to show TRAIL expression after Ad5hTRAIL infection, cytotoxic effects of exogenous TRAIL expression were determined by Annexin-V staining. Ad5hTRAIL and AdLacZ-transduced or untransduced rat pancreatic islets were transplanted under the kidney
capsule of STZ-induced diabetic rats. The diabetic status after islet transplantation was followed up for 90 days.
Results: AdEGFP infections revealed optimum doses of adenovirus vectors
to efficiently transduce pancreatic islets. Blood glucose levels and survival rates
indicated that 40 mg/kg/BW STZ administration was the optimum dose to use.
Histopathology analysis demonstrated that islet/graft of non-infected or AdLacZinfected kidney sections were heavily infiltrated with mononuclear cells. In contrast, Ad5hTRAIL infected islets displayed non/minimal mononuclear cell infiltration. While AdLacZ-infected pancreatic islets did not manifest normoglycemia
during the 90 day post-transplantation period, Ad5hTRAIL-infected islets prolonged normoglycemia.
Conclusion: Modulation of allogeneic immunity through adenovirus mediated
TRAIL gene delivery was crucial to extend the survival of transplanted islets.
EFFECTS OF EPOXIDE HYDROLASE HIS139ARG ANDTYR113HIS
POLYMORPHISMS ON LUNG CANCER RISK
Emel Zelal Erkisi
Department of Molecular Medicine, DETAE, Istanbul, Turkey
P-61
Microsomal epoxide hydrolase (EPHX) plays an important role in both activation and detoxification of carcinogens. Two polymorphisms at exon 3 Tyr113His and exon 4 His139Arg have been reported to be associated on lung cancer
risk. We used PCR, RFLP and gel electrophoresis techniques to detect these two
polymorphisms on 56 lung cancer patients and 40 controls. In our study, we
found statistically mean frequencies and significant difference in the distribution
of exon 3 polymorphic genotypes on lung cancer patients and controls. We found
OR 2.551 for distribution of exon 3 His113/His genotype among lung cancer
patients, p50.002. When patients were grouped according to smoking habits, we
found OR 1,641 for His 113 allele in smoking lung cancer patients p50.381. In
the case of familial cancer background, in lung cancer patients OR increases to
3.5, p50.014. However, we could not find results in the same direction for exon
IDENTIFICATION OF BRM AS A CANDIDATE TUMOR SUPPRESSOR
FROM 9P24 REGION IN HEAD AND NECK SQUAMOUS CELL
CARCINOMAS
Esra Gunduz1, Mehmet Gunduz2,3, Levent Beder3, Kadir Demircan4,
Ryo Tamamura2, Noboru Yamanaka3, and Hitoshi Nagatsuka2
1
Department of Medical Genetics, Faculty of Medicine, Fatih University,
Turkey, 2Department of Oral Pathology and Medicine, Graduate School of
Medicine, Dentistry and Pharmaceutical Sciences, Okayama University,
Okayama, Japan, 3Department of Otolaryngology Head and Neck Surgery,
Wakayama Medical University, Wakayama, Japan, 4Department of
Molecular Biology and Biochemistry, Okayama University, Okayama, Japan
342
Head and neck squamous cell carcinoma (HNSCC) is the fifth most frequently occurring malignancy worldwide, representing a major international
health problem. Despite improved detection, aggressive and multidisciplinary
treatment approaches, including preoperative or postoperative chemotherapy or
radiotherapy with surgery, head and neck cancers are still a great threat to human
life and limited improvement in 5-year survival has been gained in decades.
Patients are often in the advanced stage of the disease and despite combined
therapy, the outcome remains poor. Recent molecular developments have shown
that HNSCC has been associated with genetic alterations either in tumor suppressor genes (TSG) or oncogenes.
TSG are defined as genetic elements whose loss or mutational inactivation
allows cells to acquire neoplastic growth. One of the critical steps in identifying
TSG is loss of heterozygosis (LOH) analysis. In this study, allelic loss of the
short arm of chromosome 9 was examined in 39 head and neck squamous cell
carcinomas by using 13 highly polymorphic microsatellite markers. We found
two deletions of hot spots at 9p21 and 9p24. LOH was detected at least at one
locus in 28 out of 39 (71.8%) cases. p16, a well-known tumor suppressor gene,
was thought to be a target for the deletion of 9p21 region. However a novel frequently deleted chromosomal area, 9p24 region, and a candidate tumor suppressor gene from this region, BRM, was identified. Other possible candidate tumor
suppressors from the region included COCK8, Kank and VLDLR genes. To verify high ratio of deletion at BRM gene locus, a specific marker close to BRM
was designed, and this marker yielded the highest LOH (57.1%), suggesting that
BRM has a tumor suppressive role in head and neck carcinogenesis. Furthermore, comparison of clinical-pathological variables showed that LOH at the
BRM locus was associated with a better prognosis. Further studies for the role of
BRM are warranted.
P-63
IDENTIFICATION OF BIOLOGICAL THEMES IN MICROARRAY
DATA FROM NORMAL HUMAN FIBROBLAST CELLS UNDER
MAGNETIC FIELD THERAPY
Eylem Kurulgan-Demirci1, Taylan Demirci2, Peter Linder1, Peter Kayser1,
Porst Dariusz1, M. Gerhard Artmann1, and Aysegul Temiz-Artmann1
1
Department of Cell Biophysics, Medicine and Molecular Biology, Aachen
52428, Juelich, Germany, 2Department of Medical Biology and Genetics,
Turkey Health Science Institute of Dokuz Eylul University, Izmir, Turkey
Oligonucleotide microarrays were used to study gene expression during magnetic field therapy. Two different magnetic field therapies (Med-Tec, Germany)
such as dynamic and static were examined with the Agilent whole human microarray. Data analysis using GeneSifter was performed to identify differentially
regulated genes and distinct patterns of gene expression. The goal of this study
is to determine the biological significance of the gene lists derived using these
methods. ANOVA analysis identified 214 genes that showed significant differential regulation as a consequence of magnetic field therapies. PAM (partitioning
around medoids) clustering separated these genes into two groups: one cluster
contained genes showing down-regulation after magnetic field therapy, the other
cluster had genes showing up-regulation. According to gene onthology (GO)
reports, up-regulated genes were identified as genes involved in actin cytoskeleton organization and biogenesis, actin filament-based process, and muscle contraction. In the second cluster, representing down-regulated genes, several different gene families were identified, including physiological process, response to
stimulus and response to stress.
P-62
HEPARIN INHIBITS HGF-INDUCED MOTILITY, INVASION AND
PROLIFERATION OF HEPATOCELLULAR CARCINOMA CELLS BY
SUPPRESSING OF EGR-1 EXPRESION
Evin Ozen, Esra Erdal, and Nese Atabey
Department of Medical Biology and Genetics, Dokuz Eylül University,
The Institute of Health Science, Turkey
Background and Aim: HGF-SF/c-MET is the most potent proliferative signaling pathway for hepatocytes and its aberrancies are very important to explain
proliferative and metastatic characteristics of hepato-carcinogenesis. Additionally,
it is known that Heparin Sulphate Proteoglycans (HSPGs) work as a co-receptor
for HGF/SF and that this interaction modulates signaling. In particular carcinomas, heparin has been found to be increased HGF-induced cell invasion, cell motility and cell proliferation. Contradictorily, increased in HSPG expression has
been reported to have negative effect on cell proliferation in HCC. We previously showed that heparin inhibits HGF-induced cell proliferation, cell invasion
and cell motility and further microarray analysis was performed to determine the
molecular mechanism behind heparin induced HGF-SF/c-MET signaling in HCC.
One of the genes that dysregulates heparin and/or HGF-SF induction is Egr-1 in
SK-Hep1. Therefore Egr-1 may contribute to heparin and/or HGF-SF induced
biological consequences in HCC.
Methods: First we investigated the dose dependent effects of heparin on
HGF-SF induced invasion, motility and proliferation of SK-Hep1 cells with Boyden chamber, invasion assay and Trypan Blue dye exclusion methods respectively. Alteration in Egr-1 expression level and its transcriptional activity were
determined by RT-PCR, Western-blotting and luciferase reporter assay with
pGL2-luc-B-Egr1 vector, respectively. In order to define molecular mechanisms
of altered Egr-1 expression, phosphorylated and un-phosphorylated forms of
MAPK, AKT and JNK protein levels were analyzed by western blotting.
Results. We showed that heparin inhibits HGF-induced cell invasion, cell
motility and cell proliferation and also HGF- induced Egr-1 expression was
found to be inhibited by heparin in HCC cell lines in a dose dependent manner.
Moreover, it was found that heparin inhibits HGF-induced transcriptional activity
of Egr-1 and HGF-induced p-MET, p-MAPK and p-AKT levels in a dose dependent manner while it did not affect un-phosphorylated ones.
Conclusion. Our data suggest that heparin induced Egr-1 down-regulation
inhibits cell motility, invasion and proliferation induced by HGF-SF in HCC cell
lines. It may be a new aspect useful for understanding the molecular mechanism
in HCC and good target to improve new drugs.
P-64
INVESTIGATION OF MTHFR C677T GENE POLYMORPHISM IN
PATIENT WITH LUNG CANCER
Eylem Kurulgan-Demirci1, H. Arzu Ergen2, Hulya Yilmaz2, Pinar Aksoy2,
and Turgay Isbir2
1
Department of Cell Biophysics, Medicine and Molecular Biology, Aachen
52428, Juelich, Germany, 2Department of Molecular Medicine, The Institute
of Experimental Medicine, Istanbul University, Capa, Istanbul, Turkey
The relationship between MTHFR C677T (homozygous mutant genotype,
VV) polymorphism and lung cancer pattern were studied prospectively in healthy
adults and patients with lung cancer. The results suggest that the MTHFR C677T
polymorphism by itself plays an important role in the etiology of lung cancer.
PCR (Polymerase Chain Reaction), RFLP (Restriction Fragment Length Polymorphism) and agarose gel electrophoresis techniques were used to determine
the C677T (homozygous mutant genotype, VV) genotypes. In our study, we
found that all lung cancer patients had statistically significantly high frequency
of MTHFR C677T genotype in comparison with non-cancer controls (P \0.001)
MTHFR C677T genotype (homozygous mutant genotype, VV) were statistically
significantly different (P\ 0.001) in the group of patients with lung cancer than
controls. The allele frequencies of V and A (wild type allele) were not statistically different in study groups. According to our results, MTHFR C677T polymorphism was related to high risk in patients with lung cancer by causing lung
cancer. In conclusion, our results suggest that MTHFR C677T polymorphism is
associated with lung cancer diseases in Turkish population.
P-65
THE EFFECT OF LEUKEMIA INHIBITORY FACTOR ON IN VITRO
FERTILIZATION RATE OF MOUSE OVA
Fakher Rahim1, and Ghasem Saki2
1
Pysiology Research Center, Faculty of Medicine, Iran, 2Department of
Anatomical Science, Faculty of Medicine, Iran
Objective: The aim of this study is to examine the effect of human recombinant
leukemia inhibitory factor in different doses on rate of fertilization of mouse oocytes.
Interventions: Mice were sacrificed at 12-14 hours after hCG or 36-38 hours
after hMG injection. Mature oocytes were obtained and divided randomly into 5
groups. Oocytes in group A (n5157) were cultured as the control group in TYH
medium. Oocytes in groups B, C, D, E (n5137, 154, 166 and 159, respectively)
were cultured in the same medium supplemented with recombinant human leukemia inhibitory factor in four different concentrations (5, 7.5, 10, 20ng/ml, respectively) for 1 hour. After that time, 100,000 spermatozoa were added to every
drop and after 24-26 hours, two cell embryos were recorded. Fertilization was
assessed by recording the number of 2-cell embryos and analysed by X2 tests.
Main outcome measurement: Two cell embryos.
Results and Conclusion: No significant difference was detected in the rate of
two cell embryos in the studied experimental groups as compared with the rate of
two cell embryos in control group (Group A). This study showed that, different
concentrations of recombinant human leukemia inhibitory factor in standard medium does not enhance the in vitro fertilization rate of mouse oocytes.
P-66
SERUM GAMMA-GLUTAMYLTRANSFERASE IN
RHEUMATOID ARTHRITIS
Fatih Kara1, Habib Emre2, Abdulkadir Yildirim1, Fatih Akcay1,
and Ebubekir Bakan1
1
Department of Biochemistry, Medical Faculty, Ataturk University,
Erzurum, Turkey, 2Department of Internal Diseases, Ataturk University,
Medical Faculty, Erzurum, Turkey
Objective: Rheumatoid arthritis (RA) is a chronic inflammatory disease, and
oxidative stress plays an important role in its etiopathogenesis. It has been shown
that gamma-glutamyltransferase (GGT) is upregulated after oxidative stress
through the Ras signal transduction pathway and can be used as a marker related
with oxidative stress. The aim of our study was to investigate the serum GGT
levels in RA patients.
Methods: 76 patients with RA (24 men and 52 women) were included in this
study. 64 patients with generalized anxiety disorder (25 men and 39 women) were
used as a control group. Serum GGT levels were determined in all participants.
Results: We found that serum GGT levels were significantly higher in RA
patients (mean 48.0 6 40.7 IU/L) compared to control group (mean 20.4 6 12.7
IU/L) (p<0.0001). There was no significant difference between men (mean 40.9
6 27.5 IU/L) and women (mean 51.3 6 45.4 IU/L) in patients with RA (p>0.05).
Conclusions: These results may be useful in understanding the role of oxidative stress in RA. However, further studies are needed to assess the importance
of GGT increase in RA.
P-67
THE INVESTIGATION OF IL-1 b GENOTYPE ON PATIENTS
TREATED WITH OSTEOINTEGRATED DENTAL IMPLANTS
Ferhat Dizen1, Meral Unur1, Bahar Toptas2, Bedia Agachan2,
and Turgay Isbir2
1
Department of Oral Medicine and Oral Surgery, Istanbul University,
Faculty of Dentistry, Istanbul, Turkey, 2Department of Molecular Medicine,
The Institute of Experimental Medicine, Istanbul University, Capa, Istanbul,
Turkey
In our study, we aimed to investigate the relationship between Interleukin-1b
(IL-1b) 13953 gene polymorphism and smoking in osteointegrated dental
implant losses. 56 implanted cases and 40 healthy control subjects were included;
polymerase chain reaction, restriction fragment length polymorphism and agarose
gel electrophoresis techniques were used and IL-1b13953 polymorphisms were
investigated. IL-1b13953 genotype distributions were similar in both groups.
There were no statistically significant relationships between age, plaque index,
pocket depth size, smoking and level of implant losses in IL-1b positive patient
group (p[0,05). Three of the five patients that were having implant loss, were
smokers, losing the implant earlier and having higher average plaque and bleeding indexes. As a result, we can hypothesize that being IL-1b positive and smok-
343
ing can affect implant loss, but the study group must be increased to obtain statistically significant results.
P-68
PLASMA ACYLATED GHRELIN, OBESTATIN AND LEPTIN IN
DIFFERENT STAGES OF CHRONIC KIDNEY DISEASE PATIENTS
Yildiz Oner-Iyidogan1, Pernur Oner2, Figen Gurdol2, Hikmet Kocak3,
Pinar Cetinalp-Demircan2, Yasar Caliskan4, Taner Kocak5, and
Aydin Turkmen4
1
Medical Services Training School, Medical Laboratory Programme,
Istanbul University, Turkey, 2Department of Biochemistry, Istanbul Faculty
of Medicine Istanbul University, Turkey, 3Department of Biochemistry,
Istanbul Bilim University, Turkey, 4Department of Nephrology, Faculty of
Medicine Istanbul University, Turkey, 5Department of Urology, Faculty of
Medicine Istanbul University, Turkey
Objective: Malnutrition and loss of appetite remain a frequent problem in
patients with chronic kidney disease (CKD). These patients have inflammation
accompanied by high levels of plasma leptin, an appetite-modulating hormone. A
newly described hormone ghrelin is also involved in regulation food intake and
energy balance. In patients with end-stage renal disease and hemodialysis, high
plasma ghrelin concentration has been reported, but the metabolic impact of
ghrelin in CKD is unknown. The aim of this study was to characterize the
changes in circulating levels of ghrelin, obestatin, leptin, interleukin (IL)-6, and
tumor necrosis factor-alpha (TNF-a) at different stages of CKD.
Materials and Methods: 37 hemodialyzed (HD) patients (23 men, 14
women, mean age 37.662.28 years, mean BMI 22.5 6 0.7 kg/m2); 42 peritoneal
dialyzed (PD) (23 men, 19 women; mean age 42.6 6 2.03 years, mean BMI
23.6 6 0.5 kg/m2) and 37 early stage CKD patients (24 men, 13 women;
54.162.5 years of age, mean BMI 24.6 6 0.5 kg/m2) were studied. The control
group consisted of 31 healthy subjects (14 men, 17 women; mean age 38.4 6
1.7 years, mean BMI 24.6 6 0.5 kg/m2). Leptin, acylated ghrelin, obestatin and
cytokine determinations were done by commercially available ELISA kits.
Results: The patients with CKD had significantly higher serum leptin but lower
acylated ghrelin levels than the controls. The level of these hormones in dialysis
patients did not differ from those in early-stage CKD. TNF-a and IL-6 levels were
significantly higher in CKD patients than in controls, the highest levels being present in dialysis patients. Obestatin levels were relatively lower in HD patients.
Conclusion: Low acyl-ghrelin levels observed in CKD patients may be
involved in the loss of appetite and poor nutritional status. The high levels of
TNF-a and IL-6 indicate the presence of chronic inflammatory state.
P-69
FATTY ACID COMPOSITION OF HDL IN RENAL TRANSPLANT
RECIPIENTS TREATED WITH CYCLOSPORIN/TACROLIMUS
Filiz Bakar1, Fugen Aktan1, Kenan Keven2, Bilgehan Dogru1,
Acar Tuzuner3, Bulent Erbay2, and Serpil Nebioglu1
1
Department of Biochemistry, Ankara University, Faculty of Pharmacy,
Turkey, 2Department of Nephrology, Ankara University, Faculty of
Medicine, Turkey, 3Department of Surgery, Ankara University Faculty of
Medicine, Turkey
Objective: Atherosclerotic cardiovascular disease is the leading cause of
morbidity and mortality in patients with end-stage renal disease receiving renal
replacement therapy. It is known that the concentration of cholesterol in HDL is
an inverse predictor of future atherosclerotic cardiovascular disease. The aim of
our study was to investigate the alterations of HDL fatty acid levels to evaluate
oxidative stress in renal transplant recipients receiving Cyclosporin (CsA) and
Tacrolimus (FK506).
Materials and Methods: Renal transplantation patients treated with CsA (n56)
and FK506 (n510) were studied and the samples were collected before transplantation
(BT), at the 60th (AT60) and 120th day (AT120). Six healthy donors formed our control group. Fatty acid composition analysis was performed on Agilent 6890N gas chromatography. Non oxidized fatty acid levels were measured. Therefore, the decrease in
fatty acid levels defines the increase of oxidized amounts.
Results: In FK506 group, the levels of C16:0 increased significantly at AT60
compared to control and BT. C18:3 and C20:4 decreased significantly compared
344
to control. However in CsA group, we did not observe any significant changes
after transplantation except C18:0 level at AT60. In addition to this, C18:1 and
C18:2 levels at AT60 in FK506 group were significantly higher than CsA group.
Conclusions: Although the unsaturated fatty acid levels at AT60 in FK506
group were higher than the pre-transplant ones, the further decrease may be the
indicator that unsaturated fatty acid content of HDL is vulnerable to oxidation.
The increase observed in unsaturated fatty acids of FK506 group may be the
sign of its efficiency. However, there was no difference in CsA group. According
to the results, it may be considered that the lipid profile of patients on FK 506
therapy was more favorable than patients treated with CsA.
The study was supported by Ankara University Research Foundation (200108-03-028), Ankara University Biotechnology Institute (35) and TUBITAK
SBAG-AYD-438.
P-70
ENDOSULFAN-INDUCED NEUROTOXICITY AND
ACETYLCHOLINESTERASE INHIBITION IN RABBITS: THE
PROTECTIVE EFFECT OF VITAMIN C
Firdevs Mor1 and Ozlem Ozmen2
1
Department of Pharmacology and Toxicology, Mehmet Akif Ersoy
University, Veterinary Faculty, Turkey, 2Department of Pathology, Mehmet
Akif Ersoy University, Veterinary Faculty, Turkey
The neurotoxic effect and acetylcholinesterase inhibition of endosulfan and
the ameliorating effect of vitamin C on the brain of New Zealand white rabbits
was studied. Cerebrum and cerebellums of the rabbits were examined grossly and
histopathologically, and caspase-3 activity was detected by immunohistochemical
methods. Twenty-four rabbits were divided into four groups (n56). Rabbits in
Group I (END) were given, daily by oral gavage, a sublethal dose of endosulfan
(1 mg/kg bw) in corn oil for 6 weeks. Group II (END1C) received the same dose
of endosulfan and additionally Vitamin C (20 mg/kg bw) every other day during
this period. Group III (OIL1C) received oral corn oil daily and Vitamin C every
other day for 6 weeks. Group IV (OIL) received corn oil daily by oral gavage
throughout the experiment. Significant reduction of acetylcholinesterase was
observed in the END group and amelioration was seen in the END1C group. Hyperemia and slight hemorrhages in brains and cerebellums were seen in some rabbits in the END group. There were no gross cerebral and cerebellar lesions in the
other groups. Hemorrhages, degenerations and slight gliosis were the marked histopathological findings of some rabbits belonging to the END group. Caspase-3
positive reaction was more severe in this group than in the others. An ameliorating
effect of Vitamin C on gross, histopathological and immunohistochemical findings
was observed in END1C group. The results revealed that endosulfan can cause
neurotoxic effects in rabbits. However, toxicity was decreased by Vitamin C treatment which also increased the acetylcholinesterase activity in serum.
P-71
EPIDEMIOLOGIC SURVEILLANCE OF CHLAMYDIA
TRACHOMATIS INFECTION AND STRAINS GENOTYPING IN
MOROCCAN POPULATION
Fouzia Radouani
Institut Pasteur Du Maroc, Casablanca, Morocco
Objectives: To evaluate the prevalence of C. trachomatis infection among
consultants for genital infection by a direct detection with molecular biology
methods.
Materials and Methods: Our study is focused on 438 subjects consulting
for genital infections. They were divided into two groups: group 1 (n5321) was
constituted from female sex workers (FPS) recruited in the ALCS, they have an
average age of 33 years, the group 2 (n5117) was constituted from women and
men consulting for genital infections at Pasteur Institute, they had an average
age of 36 years. Subjects included benefited from an epidemiological investigation determining the age, sex, and date of contaminant contact. In addition, they
were subjected to a standard clinical examination and through this physical examination, symptoms of infections were searched. After informed consent of the
patients, blood sample was taken, as well as a urethral or endocervical samples.
In this study, several serological methods were used, such as Immunoblotting
and ELISA. A plasmidic C. Trachomatis DNA detection by PCR, followed by a
chromosomic PCR for genotyping were also carried out.
Results: The majority of recruited patients had unprotected sex practice.
Only 19% said that they used the condom. The clinical information collected
showed that vaginal discharge was found in 78% in group 1 subjects against
48% in group2 subjects, cervicitis in 38% against 36%, pruritus in 46% against
21%, burning urination in 40% against 29%, and pelvic pain in 40% against
19%. The results of molecular analysis showed that 11% of the group 1 and 3%
of the group 2 consultants were positive by one or the other of the two PCR
tests. The sequencing was conducted for 16 positive samples, an initial analysis
of the sequences by the BioEdit software followed by the BLAST search tool at
the National Centre for Biotechnology Information showed the predominance of
the Strain I. Serological test conducted for the detection of IgG anti C trachomatis, directed against recombinant proteins showed 84% positive among group 1
subjects, while 15% only among group 2 subjects were positive in C. trachomatis
IgG antibodies. The analysis of the immunological profiles showed that the subjects were infected one or more times with C. trachomatis during their life. We
found ten different immunological profiles in group 1 and seven in group 2.
P-72
HUMAN PAPILOMAVIRUS DNA AND MRNAS - MARKERS FOR
CERVICAL LESIONS PROGRESSION
Gabriela Anton1, Demetra Gabriela Socolov2, Anca Botezatu1,
Cristina Daniela Goia1, Mariana Anton3, Anca Daniela Stanescu2,
Adriana Plesa1, and Sergiu Teleman4
1
Institute of Virology, Bucharest, Romania, 2Department of Gynecology,
University of Medicine and Pharmacy, Iassy, Romania, 3Department of
Genetics, University of Medicine and Pharmacy Bucharest, Romania,
4
Department of Pathology, University of Medicine and Pharmacy,
Iassy, Romania
Persistent HPV infections are responsible for cervical lesions progression.
Type-specific DNA/RNA diagnosis is important for disease prognosis and treatment.
Objectives: Our study used the molecular detection of human papillomavirus
DNA corroborated with HPV mRNA expression as a predictive value for disease
progression.
Methods: Considering HPV infection clearance in young women, from a
cohort of 485 women (age 17-55.) we selected 78 patients over 29 years old
with normal/inflammatory, and ASCUS cytology. HPV typing (InnoLipa) and viral mRNAs presence (PreTect HPV Proofer) were determined in cervical-brush
specimens at base line and in the samples obtained at 6/12 months interval. As
negative controls, cervical specimens from 18 patients (age 29- 43) without HPV
infection (negative in InnoLipa and cytology tests) were used.
Results: At baseline, the selected women were HPV negative (26.9%) or
presented HPV 16, 18, 31, 33, 45, 66 types as single infection (28.1%) or coinfections. The distribution of HPV mRNA type was as follows: HPV16 -29%,
HPV18 -13.8%, HPV31 -13.8%, HPV33 – 3.8%, HPV45 – 3.44%. After 6/12
months of clinical management, 58.9% of the patients were HPV DNA negative.
Three HPV DNA negative women at base-line (no mRNAs detected), became
HPV DNA positive (type 16 as single infection or co-infection with type 33).
Although PreTect HPV Proofer is a qualitative kit, E6/E7 mRNA levels
remained higher in 42% cases with ASCUS which presented persistent infection
with HPV16 in single infection or in co-infections with 31 and 45 types.
Conclusions: Our results indicated that viral persistence, an important factor in
cervical lesions evolution, might be associated with viral type and mRNA presence.
P-73
COMPARISON OF ANTIPROLIFERATIVE AND ANTIANGIOGENIC
EFFECTS OF QUERCETIN AND FLAVONOID MIXTURE IN
IN VITRO CONDITIONS
Gabriela Mojzisova1, Jan Mojzis2, Lenka Varinska2, Martina Pilatova2, and
Alojz Bomba1
1
Department of Experimental Medicine, P.J. Safarik University, Slovakia,
2
Department of Pharmacology, P.J. Safarik University, Slovakia
345
Background: Epidemiological studies have consistently shown that regular
consumption of plant-based diets is strongly associated with reduced risk of developing chronic diseases, such as cancer and cardiovascular disease. It is now widely
believed that the actions of the phytochemicals alone do not explain the observed
health benefits of diets rich in fruits and vegetables, because taken alone, the individual compounds studied in clinical trials do not appear to have consistent preventive effects. In the present work the antiproliferative and antiangiogenic effects
of quercetin (Q) and flavonoid mixture (FM) have been compared.
Material and methods: The present study was conducted to examine the
effects of Q and FM on proliferation and cell death in the Jurkat and HeLa cell
line including cytotoxicity (MTT test), cell cycle analysis, apoptosis detection by
flow cytometry or DNA fragmentation. Furthermore, we have also tested the
effects of the studied compounds on the growth, endothelial cell migration (ECM;
wound healing assay), matrix metalloproteinase activity (MMP; gelatine zymography) and tube formation (CTF) by human umbilical vein endothelial cells
(HUVECs).
Results: Both, Q and FM showed antiproliferative activity in Jurkat as well as
in HeLa cells. It significantly reduced the growth of both cancer cell lines (IC50 for
Q is 75.4 and 82.1 lg/ml and IC50 for FM is 34.3 and 50.0 lg/ml). In FM-treated
Jurkat cells we found a significant increase in the fraction of cells with sub-G0/G1
DNA content, which is considered to be a marker of apoptotic cell death. Apoptosis
was also confirmed by annexin V staining and DNA fragmentation. Similar effects
we observed with Q but only in higher doses. Furthermore, FM at the non-toxic
doses of 20 lg/ml and 4 lg/ml inhibited ECM and CTF what indicates its potential
antiangiogenic properties. FM also inhibited the activity of matrix metalloproteinases (MMPs), preferentially MMP-9, at the doses of 40 lg/ml to 4 lg/ml.
Summary: Our data suggest that FM possesses marked antiproliferative and
antiangiogenic properties in vitro. We believe that the additive and synergistic
effects of flavonoids in FM are responsible for its potent anticancer activities.
This work was supported by the Slovak Research and Development Agency
under the contract No. APVV-0325-07, by the Slovak Grant Agency for Science
(grant No. 1/4236/07) and by the AV grant 4/0028/07.
Background: Leukemia inhibitory factor (LIF) is a group of secreted glycoproteins with molecular weights ranging from 38-67 kD, resulting from differential protein glycosylation. LIF is constitutively expressed at high levels in the
human fallopian tube epithelium and has an important role in the motility and vitality of sperm. In the present study, the effect of human recombinant LIF on
human sperm motility and survival was investigated in vitro.
Materials and Methods: Normal spermatozoa of 30 fertile men were collected
and after preparation were incubated in Ham’s F101FCS 10% medium, containing
various concentrations (0, 3, 5, 10, and 50 ng/mL) of LIF at 37 8C under 5% CO2 for
6, 24 and 48 hours. Sperm motion characteristics were measured using a Makler
chamber. Sperm survival was determined using the hypo-osmotic swelling test. Collected data were analyzed by one-way ANOVA and LSD test using SPSS version
11. The difference in values were considered significant when p<0.05.
Results: Sperm motility was significantly higher after 24 h exposure to 5-10
ng/mL LIF (p<0.05). The survival rate of sperm was significantly prolonged
when exposed to 50 ng/mL LIF (p<0.05). Non-progressive motility and survival
rate of sperm were significantly higher after 48 h exposure to 50 ng/mL and 1050 ng/mL LIF, respectively. (p<0.05). There was no significant difference in
progressive sperm motility during the 48 h exposure of sperm to the various concentrations of LIF.
Conclusion: According to these results, the effect of LIF on sperm motility
and survival was dependent on the dose of LIF supplementation and the length
of incubation.
P-74
Objective: The metabolic syndrome is a term that is often applied to the disorders that exist together in many patients with different types of cardiovascular
disease (CVD), as well as diabetes, in association with insulin resistance, hyperinsulinemia and cellular ionic abnormality. It is an inducing factor, associated
with oxidized low density lipoproteins (LDL), for CVD. In this study, we determined the LDL susceptibility to lipid peroxidation (LDLox) in 67 elderly
patients (aged 69 6 7 years).
Methods: The patients were included in two study groups: a control group
(n532), and a group with metabolic syndrome and high rates of CVD events
(n535). LDL was isolated from the serum samples, by selectively precipitating
it at their isoelectric point (pH 5.12). The LDL susceptibility to in vitro induced
lipid peroxidation was evaluated following its incubation with a FeSO4/ascorbic
acid prooxidant system. The lipid peroxidation end–products were assayed by
thiobarbituric acid reactive substances (TBARS).
Results: Our results showed a significant increase in LDL susceptibility to
lipid peroxidation in metabolic syndrome patients, compared to the control group
(9.5162.73 v.s. 5.1163.03 mmoles MDA/dL serum, p50.001) and constitute an
evidence that the metabolic syndrome was associated with higher levels of
LDLox due to a higher fraction of LDLox and glucose.
Conclusion: The results fit well into the current concept of LDLox as a key
mechanism in the development of atherosclerosis. In vivo, LDLox has a wide
range of atherogenic properties. Examples of these atherogenic effects are
increased expression of adhesion molecules on endothelial cells, monocyte chemotaxis and up-regulation of inflammatory genes. In conclusion, the metabolic
syndrome, a risk factor for cardiovascular disease, is associated with higher levels of circulating LDLox that are associated with a greater disposition to atherothrombotic coronary disease.
EFFECTS OF METHYL-BETA-CYCLODEXTRIN ON CRYOSURVIVAL
OF C57BL/6 MOUSE SPERMATOZOA.
Ghasem Saki1,2 and Shabnam Movassaghi2
1
Department of Embryology and Cell Biology, Ahwaz Jondishapour of
Medical Science University, 2Department of Embryology and Cell Biology,
Apadna Clinical Reseach Center, Iran
MBCD and cholesterol-loaded-cyclodextrin (CLC) were examined for their
abilities to increase the cryosurvival of C57BL/6 mouse sperm, the main strain
of genetically engineered mice. The intactness of acrosome and motility of frozen/thawed spermatozoa were used to monitor cryosurvival.
In this experimental study, male mice were randomly divided into six groups:
control 1, experimental 1, experimental 2, control 2, experimental 3 and experimental 4. In experimental groups 1 and 2, spermatozoa were exposed to 0.75
and 1 mM MBCD and in experimental groups 3 and 4 were exposed to two different concentrations of CLC (1 & 2 mg/ml) over a period of 1 hour and were
subsequently cryopreserved. Spermatozoa in control 1 group were frozen without
any exposure to CLC or MBCD and in control 2 (vehicle), sperms were incubated with 4mM MBCD. The post-thaw sperms were evaluated for their motility
and acrosome status. The values of the intact acrosome and motility increased
significantly with concentration of CLC compared to controls and MBCD experimental groups (P\0.05). These results indicate that cryosurvival of C57BL/6
mouse spermatozoa is enhanced by exposure to MBCD loaded with cholesterol
(CLC) before freezing; MBCD alone can not efficiently protect sperm from
freeze-thaw damage as compared to CLC.
P-76
METABOLIC SYNDROME-INDUCING FACTOR FOR
CARDIOVASCULAR DEVELOPMENT
Gianina Ioana Constantin and Simona Opris
Biology of Aging, National Institute of Gerontology and Geriatrics,
Bucharest, Romania
P-77
P-75
THE EFFECT OF HUMAN RECOMBINANT LEUKEMIA INHIBITORY
FACTOR ON SPERM MOTILITY AND SURVIVAL
COMPARISON OF LMTRYP6 (EU251502) AND LMTRYP6
(LMJF15.1140) USING GENOMIC AND PROTEOMICS
SOFTWARE TOOLS
Ghasem Saki1 and Fakherolsadat Sajjadian2
1
Department of Embryology and Cell Biology, Faculty Of Medicine, Ahwaz
Joundishapour University of Medical Science-Physiology Research Center,
Iran, 2Department of Anatomy, Tabriz University of Medical Sciences, Iran
Gilda Eslami1, Rasoul Salehi2, Hossein Hejazi3, and Ali Khamesipour4
1
Department of Parasitology and Mycology, Yazd Shahid Sadoughi
University of Medical Sciences, Iran, 2Department of Genetics and Cell
Biology, Isfahan University of Medical Sciences, Iran, 3Department of
346
Parasitology and Mycology, Isfahan University of Medical Sciences, Iran,
4
Department of Microbiology, Tehran University of Medical Sciences, Iran
P-78
Leishmaniasis is a complex disease caused by about 20 different species of
Leishmania. Macrophage activation is hallmark of natural leishmaniasis control
in vertebrate hosts, and the parasites cope with the oxidative burden, especially
peroxides, by tryparedoxin pathway containing tryparedoxin peroxidase (TRYP).
In this study, LmTRYP6 gene (EU251502) was compared with the one in database (LmTRYP6, LmjF15.1140) using appropriate software tools for genomics
and proteomics analysis. One of the important differences between them is the
location of Cp residue, the first catalytic site in typical 2-Cys peroxiredoxines,
before the beginning of helix a2 in predicted LmTRYP6 protein (ABX26130)
and location of Cp residue in the first turn of helix a at LmTRYP6
(LmjF15.1140). This structure in LmTRYP6 protein (ABX26130) might access
Cp for CR in order to resolving and reduction as soon as possible. On the other
hand, a rationale for why only some peroxiredoxins are hyperoxidized to the sulfinic acid form comes from comparing the structure of S. typhimurium AhpC
(C46S mutant) with human PrxII. AhpC lacks an YF motif-containing, C-terminal helix which in PrxII interacts with a conserved GGLG-motif in close proximity to the peroxidatic cysteine. It is thought that these additional structural motifs
hinder or slow the ability of the resolving Cys to access the peroxidatic Cys. As
a result this kinetic ‘‘pause’’ leaves the Cys-SpOH intermediate increasingly
exposed to attack by another peroxide molecule resulting in hyperoxidation to
the sulfinic acid form. LmTryP6 (ABX26130) has GGLG-motif in the loop
between a3-b4 but like AhpC in prokaryotes lacks YF-motif. It might be an adaptation for viability and probably virulence of L. major with LmTRYP6
(ABX26130). Leishmanization with L. major is accompanied by rare complications. Introducing a Leishmania strain construct with tryparedoxin peroxidase
containing YF-motif in its C-terminal end is a useful candidate for Leishmanization since its less virulent. Presenting both of these motives in tryparedoxin peroxidase makes the parasite more sensitive to peroxides.
IMPORTANCE OF AVAILABILITY OF MOLECULAR GENETIC
TESTING IN FAMILIAL MEDITERRANEAN FEVER
Halil Ibrahim Atasoy1, Ayten Pamukcu-Uyan1, Eray Basman1,
and Selma Duzenli2
1
Department of Pediatrics, Abant Izzet Baysal University, Izzet Baysal
School of Medicine, Bolu, Turkey, 2Department of Medical Genetics, Abant
Izzet Baysal University, Izzet Baysal School of Medicine, Bolu, Turkey
Familial Mediterranean Fever (FMF) is a genetically determined disease
characterized with recurrences. Serositis and synovitis attacks are the most important features of the disease. The feared complication is the resultant amyloidosis of kidneys and the future need for dialysis. The disease is generally
accepted as population specific since increased rates are observed in Jewish, Armenian, Arab and Turkish people. FMF arthritis can be confused at times with
other rheumatologic and infectious diseases such as rheumatic fever arthritis, juvenile idiopathic arthritis, and infectious arthritides. Diagnosis could be clinical
supported by biochemical and genetic tests. Dysfunctional or absent pyrin tests
are not available to most laboratories today. Availability of confirmational
genetic tests is also an important challenge in diagnosis. Confirmation is required
in suspected cases not to order patients drugs that can not work properly and are
expensive. Confirmation of diagnosis is also important on a family basis since
members of the family might have the disease but are not aware of it. These
family members would develop renal amyloidosis and, if not treated with colchicine, would require dialysis, a therapy associated with high financial burden for
a country. Here we report a thirteen year old adolescent patient with FMF previously diagnosed as juvenile idiopathic arthritis and followed up with nonsteroidal
antiinflamatory drugs (NSAID) inappropriately for more than 7 years. She was
tested for the most common twelve mutations of FMF. E148Q mutation was
found on one side of the MEFV locus by the FMF hybridization and in-house
PCR-RFLP methods. Her young brother also had recurrent abdominal pain and
was homozygote for E148Q.The mother was symptomatic and test showed the
same unilateral mutation. Her mother, father, younger sister, older sister all had
the unilateral mutation for E148Q. We prescribed colchicine for the patient and
her brother and referred the mother for further evaluation. This FMF family was
a good example demonstrating the importance of availability of genetic testing
for confirmation of FMF cases. The index case was identified first then the other
family cases were diagnosed and treated appropriately. Availability of genetic
testing led us to the correct diagnosis which prevented inappropriate treatment
and side effects of inappropriate drugs. It also avoids to insurance agents
unnecessary payment for additional drugs and future probable dialysis costs.
P-79
TGF-bs ACTIVATE CELL DEATH IN MCF-7 CELLS AFTER
TREATMENT WITH GEMCITABINE OR 5-FU THROUGH THE
SYNERGISTIC COLLABORATION OF SMAD SIGNALING
PATHWAYS
H. Seda Vatansever1, Sevinc Inan1, Elgin Turkoz-Uluer1, Isil Aydemir1,
Nurcan Umur2, and M. Kemal Ozbilgin1
1
Department of Histology and Embryology, Faculty of Medicine, Celal
Bayar University, Manisa, Turkey, 2Department of Biochemistry, Vocational
School of Health Services, Celal Bayar University, Manisa, Turkey
Objective: Transforming growth factor-beta (TGF-beta) is a ubiquitous cytokine that affects various biological processes, such as regulation of cell proliferation, immune response, growth, differentiation, angiogenesis, and apoptosis of various cell types. TGF-beta is a potent growth inhibitor of most types of cells; therefore, perturbation of TGF-beta signaling is believed to result in progression of
various tumors. A wide variety of effects of TGF-beta are mediated by physical
interaction of signal transducer SMAD proteins with various transcription factors.
Intense investigations have revealed that SMAD proteins constitute the basic components of the core intracellular signaling cascade and that SMADs function by
carrying signals from the cell surface directly to the nucleus. In our study, we
investigated the effects of both Gemcitabine and 5-FU treatment on MCF-7 breast
cancer cell lines through TGF-beta-SMAD proteins signaling pathways.
Methods: The MCF-7 cells were cultured in vitro condition and they were
treated with Gemcitabine or 5-FU for 24 h. The cytotoxicity of cells was analyzed by measuring LDH levels with ELISA. The immunolabelling of TGF-bl,
TGF-b2, TGF-b3, SMAD 1/2/3, SMAD 6 and SMAD 7 were investigated using
indirect immuno-peroxidase staining.
Results: After cytotoxicity analyses, the number of dead cells were more in
5-FU treated cells when compared with Gemcitabine treated cells. The immunolabelling of TGF-bl, TGF-b2, TGF-b3, SMAD 1/2/3, SMAD 6 and SMAD 7
were detected in both control and treated groups. While the strong staining of
TGF-b3 and moderate staining of TGF-bl, TGF-b2, SMAD 1/2/3, SMAD 6 and
SMAD 7 was detected in control group, this staining was less in both Gemcitabine and 5-FU treated cells.
Conclusions: The MCF-7 cells were affected byGemcitabine or 5-FU treatment which triggered cell death and also decreased TGF-bs staining. Therefore,
the TGF-b and SMAD pathways, which are important for tumor cell proliferation, may be blocked after the treatments.
347
Familial Mediterranean fever (FMF, MIM# 249100) is an autosomal recessive disease with fever, peritonitis, arthritis, pleuritis and neuropathic amyloidosis. The disease is common in the Mediterranean Region among North African
and Iraqi Jews, Turks, Armenians, Middle Eastern Arabs and the other ethnic
groups living in this region. In more than 95% of patients with FMF, peritoneal
involvement mimicking acute abdomen exists and sometimes these patients
undergo unnecessary surgical intervention. From the anamnesis of 159 patients
referred to our department with suspect of FMF, FMF was detected in 25
(15.7%) patients of which 16 (10.1%) underwent appendectomy and 9 (5.7%)
underwent different abdomen surgery. As the mutation frequency in the studied
cases was evaluated, in 50 alleles of 25 patients, the most frequent mutations
were M694V (24%) and E148Q (14%). By systematic mutation analysis of suspect FMF patients, unnecessary surgical intervention will decrease significantly.
P-82
p53 CODON 72 POLYMORPHISM ASSOCIATED WITH HEPATITIS B
P-80
BETA-GLOBIN GENE MUTATIONS AND MICRO-HAPLOTYPE
POLYMORPHISMS OF BETA-THALASSEMIA PATIENTS IN
TRAKYA POPULATION
Hakan Gurkan1, Burhan Turgut2, Hilmi Tozkir3, Gokay Bozkurt4,
and Emre Tekgunduz2
1
Department of Medical Biology, Istanbul University Medical Faculty,
Istanbul, Turkey, 2Internal Medicine, Trakya University, Medical Faculty,
Edirne, Turkey, 3Department of Medical Biology, Trakya University,
Medical Faculty, Edirne, Turkey, 4Department of Medical Genetics, Adnan
Menderes University, Medical Faculty, Aydin, Turkey
Objectives: b-Thalassemia is an autosomal recessive inherited disease,
results from mutations of the b-globin gene. The high frequencies of b-Thalassemia in Turkish population have a major impact on public health. Molecular studies of b-Thalassemia in Turkey recorded the presence of 37 different alleles
associated with the disease. The frequency of b-Thalassemia gene is approximatelly 2.1% but in some regions such as Trakya, Mugla and Antalya the frequency
increase to 10%. The objective of the study is to explore the b-globin gene
mutations and micro-haplotype polymorphisms of b-Thalassemia patients in Trakya population with the automatized DNA sequence analysis method.
Methods: A total of 12 (7 female and 5 male) unrelated patients with b-thalassemia and 2 (1 female and 1 male) unrelated, healthy volunteers were
included in the study. Abnormal levels of hemoglobin were seen in all patients.
All controls and patients were Turkish and living in Trakya area. b-globin gene
regions multiplied with PCR and their nucleotids were determined with the
automatized DNA sequence analysis method.
Results: One of the patients was found to be IVS-2,1 (G-A) homozygous,
the other patients were found to be heterozygous for the IVS-I, 1 (G-A), IVS-2,1
(G-A), IVS-1, 110 (G-A) and codon 39 (C- T) mutations in b-globin gene
region. Furthermore the patients and healthy controls were found to be codon 2
nt.3 (C/T), IVS-II nt.16 (C/G), IVS-II nt.74 (G/T), IVS-II nt.81 (C/T) and IVS-II
nt.666 (T/C) single nucleotide polymorphisms in b- globin gene region.
Conclusion: In this study, the mutations that were found in b-globin gene in
Trakya population are compatible with the other related studies. The single nucleotide polymorphisms in b-globin gene region were defined as ‘‘framework’’
(FW) by Orkin et al. and Antonarakis et al. The haplotype for b-globin gene
mutations which were found in Trakya population in this study are associated
with FW 1 and FW 2. To our knowledge, this is the first report on b-globin
gene frameworks of b-Thalassemia patients in Turkish population using automatized DNA sequence.
P-81
THE FREQUENCY OF SURGERIES FOR THE REASON OF ACUTE
ABDOMEN AND MEVF MUTATIONS IN THE PATIENTS WITH FMF
Hale Samli
Department of Medical Genetics, Afyon Kocatepe University School of
Medicine, Afyon, Turkey
Halit Akbas1, Hilmi Isi1, Kendal Yalcin2, Selahattin Tekes1, Selda Simsek1,
and Turgay Budak1
1
Department of Medical Biology and Genetics, Dicle University, Faculty of
Medicine, Diyarbakir, Turkey, 2Department of Gastroenterology, Dicle
University, Faculty of Medicine, Diyarbakir, Turkey
Chronic viral hepatitis is the main cause of chronic liver disease, cirrhosis
and hepatocellular carcinoma throughout the world. Codon 72 polymorphism of
the p53 gene has been implicated in cancer risk, and it has been suggested that it
may have an impact on the clinical outcome of the disease. Hepatitis B virus
(HBV) has mutagenic effects on somatic cells and may show these mutagenic
effects through its viral proteins, such as HBX, or through integrating into host
genome. HBX protein could transcriptionally inhibit the expression of p53 tumor
suppressor gene, which might be a possible molecular mechanism responsible for
the development of HBV-associated hepatocellular carcinoma. Our objective was
to evaluate the association between p53 codon 72 polymorphism and patient
with hepatitis B infections.
Genomic DNA was extracted from peripheral blood cells of 40 patients with
hepatitis B and 25 healthy controls. The p53 codon 72 polymorphism was analyzed by the PCR-restriction fragment length polymorphism.
Our results showed higher allele frequencies of p53 codon 72 pro/pro for the
patient group with hepatitis B, than the control group (22.5% versus 8%). Based
on the findings of this study, it is suggested that p53 codon72 prolin homozygosity may have an impact on the clinical outcome of hepatitis B.
P-83
COEXISTENCE OF FMF AND HIDS IN A CHILD
Resul Yilmaz, Taner Sezer, and Haluk Esmeray
Department of Pediatrics, School of Medicine, Gaziosmanpasa University,
Tokat, Turkey
Hereditary periodic fever syndromes are Mendelian inherited single gene diseases which are also known as hereditary autoinflammatory syndromes, are characterized by recurrent attacks of fever and inflammation. Familial Mediterranean
Fever and Hyperimmunoglobulinemia D syndrome are prototypes and are inherited autosomal recessively. The diagnosis is based on clinical course, family history and is confirmed with genetic mutation analysis.
A 5-year-old Turkish boy was admitted to our pediatric clinic with abdominal pain, fever and rash. These complaints have been occurred every 2 or 3
weeks, lasted 3 days since 2 years old. During fever attacks, he had abdominal
pain, malaise, arthralgia and cervical lymphadenopathy. He also developed maculopapular rash on extremities. His parents were not consanguineous and family
history for periodic fever was negative. On admission physical examination
showed growth retardation (body weight and height \3p), purpuric lesions on
lower extremities, pallor and cervical microlymphadenopathy. Laboratory studies
revealed leukocyte count 4900/mm3, hematocrit 33.3%, platelet count 385000/
mm3, fibrinogen 367 U/L, serum amyloid A 8 mg/dL, C reactive protein 28.8 (05 mg/L) and erythrocyte sedimentation rate 42 mm/hour. Urinalysis was normal
and test for occult fecal blood was positive. Antinuclear antibody and antineutrophil cytoplasmic antibody were negative. Investigations for vasculitis, porfiria
and autoimmunlymphoprolifreative syndrome were negative. The serology for
348
various viral and bacterial infectious agents was negative. Enlarged mesenteric
lymph nodes were shown in abdominal ultrasonography. Serum immunoglobulin
levels were as given: IgA 2.06 gr/L, IgG 14.1 gr/L, IgE 1280 IU/mL, IgD level
160 U/L (the repeated value one month later was 148 U/L). Mutation analysis of
MEFV gene for FMF and MVK gene for HIDS revealed that he was homozygous for M694V and V377I respectively. In our patient, severe skin lesions and
the early onset of the symptoms aroused the suspicion of HIDS. Therefore, we
first examined IgD levels. Elevated IgD level was reported in patients with other
periodic fever syndromes, including FMF and TRAPS but in that study MVK
gene mutation were not studied. In our case, we measured serum IgD levels in
two occasions with at least one month apart and than examined V377I mutation
analysis for MVK gene. Due to our knowledge, FMF and HIDS coexistence has
not been reported before.
P-84
THE DETERMINATION OF TELOMERASE MRNA EXPRESSION
USING REAL-TIME POLYMERASE CHAIN REACTION IN
PERIPHERAL BLOODS OF PATIENTS WITH GASTRIC CANCER
Hamdullah Turhan1, Ebubekir Bakan2, Abdulkadir Yildirim2,
Gurkan Ozturk3, and Kerim Cayir4
1
Department of Biochemistry, Mus Bulanik State Hospital, Turkey,
2
Department of Biochemistry, Ataturk University, Faculty of Medicine,
Erzurum, Turkey, 3Department of Surgery, Ataturk University, Faculty of
Medicine, Erzurum, Turkey, 4Department of Medical Oncology, Ataturk
University, Faculty of Medicine, Erzurum, Turkey
Introduction: Early diagnosis of gastric cancer is important to reduce the
mortality rate. Unfortunately, only a limited number of biomarkers are available
for detection of gastric cancer. Telomeres are important in human cancer, repetitive DNA sequences at the ends of chromosomes, protecting them against incomplete replication and nuclease degradation. It is believed that telomerase enzyme
activation plays a significant role in the cell immortalization and carcinogenesis.
It has been found that the human telomerase reverse transcriptase (hTERT)
mRNA expression is correlated with telomerase activity and is up-regulated in
most pre-cancerous lesions and human cancer. We aimed to evaluate quantitative
determination of hTERT mRNA expression in the diagnostic value of gastric
cancer.
Materials and Methods: Our study was carried out in 25 patients with gastric cancer (2 early, 10 invasive, and 13 metastatic gastric cancer), 5 patients
with chronic atrophic gastritis and 16 healthy volunteers as controls. The levels
of hTERT mRNA expression were determined using Quantitative Real-Time RTPCR based on TaqMan fluorescence methodology. Statistical analysis was performed using Kruskal Wallis analysis and Mann Whitney U test by SPSS for
Windows version 11.5.
Results: The mean levels of hTERT mRNA in healthy individuals and
patients with chronic atrophic gastritis, early gastric cancer, invasive gastric cancer, and metastatic gastric cancer were 7831.16 6 5558.69, 13048.36 6
6631.44, 62156.85 6 31568.65, 44407.70 6 17493.43 and 721399.95 6
583319.10 copies/100 ng total RNA, respectively. The levels of hTERT mRNA
expression in early gastric cancer, invasive gastric cancer, and metastatic gastric
cancer were significantly higher than in healthy individuals (p<0.05, p<0.01 and
p<0.05, respectively). Although the levels of hTERT mRNA expression in
chronic atrophic gastritis were higher than in healthy individuals, no statistically
significant difference between these two groups (p>0.05) was found.
Conclusions: Our results suggested that hTERT mRNA overexpression in
peripheral blood may be suitable as early detection marker of gastric cancer in
Turkish population. However, a further study with long-term follow-up in a
larger number of patients is required to confirm the clinical application of this
molecular marker.
P-85
EFFECT OF CARNOSINE OR TAURINE TREATMENT ON
OXIDATIVE STRESS IN ISOPRENALIN INDUCED MYOCARDIAL
INFARCTION IN RATS
Hande Parildar-Karpuzoglu1, A. Fatih Aydin1, Nadir Arican2,
Semra Dogru-Abbasoglu1, and Mujdat Uysal1
1
Department of Biochemistry, Istanbul University, Istanbul Medical Faculty,
Turkey, 2Forensic Medicine, Istanbul University, Istanbul Medical Faculty,
Turkey
Free radicals play an important role in apoptotic and necrotic changes in tissues. Oxidative stress also affects tissue injury in acute myocardial infarction
(AMI). Isoprenalin (IP) is a synthetic b-adrenergic agonist which causes irreversible myocardial injury. When IP was applied in high doses to experimental animals, alterations in heart tissue resembled to human myocardial infarction. It has
been implied that oxidative stress contributes to the development of IP-induced
AMI.
In this study, IP treatment (100 and 300 mg/kg, i.p.) was given to rats to produce myocardial injury and serum troponin T levels and the prooxidant-antioxidant balance were determined in the rat cardiac tissue. For this reason, malondialdehyde (MDA), protein carbonyl (PC) and glutathione (GSH) levels as well
as superoxide dismutase (SOD) and GSH peroxidase (GSH-Px) were measured
3, 6 and 24 hours after IP treatment. IP treatment (100 and 300 mg/kg, i.p.)
caused significant increases in troponin T levels at 3, 6 and 24 hours. Although
there were no increases in MDA and PC levels, significant decreases were
detected in GSH levels and SOD and GSH-Px activities at 24 hours following
100 mg/kg IP treatment.
Carnosine (b-alanyl-L-hystidine) is a dipeptide which has powerful antioxidant effects. Taurine (2-aminoethanesulfonic acid) is a non-protein amino acid
which is synthesized from cysteine and it also has antioxidant effects. In this
study, we aimed to investigate whether carnosine (250 mg/kg, i.p.) or taurine
(250 mg/kg, i.p.) treatment prevent tissue injury and oxidative stress in cardiac
tissue in IP-treated rats. There were no changes in serum troponin T levels and
cardiac MDA and PC levels and SOD activities, but increases in GSH levels and
GSH-Px activities in IP-treated rats together with carnosine or taurine as compared to IP-treated rats.
P-86
ANTI-PROLIFERATIVE, ANTI-APOPTOTIC AND ANTI-ANGIOGENIC
EFFECTS OF GOSSYPOL IN COMBINATION WITH ZOLEDRONIC
ACID IN HUMAN OVARIAN CANCER CELL LINE OVCAR3
Harika Atmaca1, Selim Uzunoglu1, Gurbuz Gorumlu2, Burcak Karaca2,
Bulent Karabulut2, Ulus Ali Sanli2, and Ruchan Uslu2
1
Department of Molecular Biology, Celal Bayar University, Manisa, Turkey,
2
Department of Medical Oncology, Ege University, Izmir, Turkey
Objective: Despite the development of new chemotherapeutic drugs in recent
decades, epithelial ovarian carcinoma remains the leading cause of death from
gynaecologic cancers. Thus, new therapeutic targets and agents are urgently
needed. Gossypol (GP) is a natural polyphenolic compound isolated from cottonseed and has antiproliferative activity on some human tumor cell lines. Zoledronic acid (ZA) is the most potent nitrogen containing bisphosphonate compound that inhibits proliferation and induces apoptosis of various cancer cell
lines, in vitro. We investigated the possible synergistic cytotoxic, apoptotic and
antiangiogenic effects of gossypol in combination with zoledronic acid in human
ovarian cancer cell line, OVCAR-3.
Methods: The human ovarian cancer cell line OVCAR-3 was treated with
increasing concentrations of gossypol and zoledronic acid alone and also in combination for 24-48-72 hours. Cell viability was determined by XTT Cell Proliferation Assay. Cell Death Detection Elisa Plus Kit (Roche) and Caspase-Glo 3/7
Assay (Promega) were used to detect apoptosis. Human Angiogenesis Antibody
Array 1 (Raybiotech) was used to determine the angiogenic cytokines according
to the manufacturer’s instructions.
Results: Combination of both agents (5 lM ZA115 lM GP) resulted in synergistic cytotoxicity, enhanced apoptosis and caspase 3 and 7 cleavage. Moreover, significant decrease in VEGF and EGF proteins were observed in the antibody array from supernatant of OVCAR-3 cells treated with both gossypol and
zoledronic acid, compared to any agent alone and also to positive control.
Conclusion: The combination of gossypol and zoledronic acid leads to
enhancement of the anti-proliferative and apoptotic activity at clinically achievable drug concentrations by decreasing the levels of pivotal angiogenic cytokines
VEGF and EGF in human ovarian cancer cell line. These preliminary findings
suggest that gossypol and zoledronic acid combination might be a new candidate
therapy for effective treatment of ovarian carcinoma.
P-87
SYNERGISTIC INHIBITION OF ANGIOGENIN BY COMBINED
TREATMENT WITH GOSSYPOL AND CISPLATIN IN A HUMAN
OVARIAN CANCER CELL LINE, OVCAR-3
Harika Atmaca1, Burcak Karaca2, Selim Uzunoglu1, Bulent Karabulut2,
Ulus Ali Sanli2, and Ruchan Uslu2
1
Department of Molecular Biology, Celal Bayar University, Manisa, Turkey,
2
Department of Medical Oncology, Ege University, Izmir, Turkey
Objective: Development of multidrug resistance toward chemotherapeutic
agents, particularly to cisplatin, is inevitable in the initial stage or further stages
of the ovarian cancer. Therefore, new therapeutic agents and their combinations
with conventional chemotherapeutic agents are needed. Cis-diammininedichloroplatinum (II) (Cisplatin) is a DNA-damaging agent that is widely used in ovarian
cancer chemotherapy. Gossypol is a polyphenolic compound extracted from cotton plant (Gossypium species) and has anti-proliferative activity on some human
tumor cell lines. In this study, the possible synergistic cytotoxic and apoptotic
effects of combination of gossypol and cisplatin were investigated in human
drug-resistant ovarian cancer cell line, OVCAR-3. Besides, the effect of this
combination on some angiogenic cytokines was also studied.
Methods: OVCAR-3 cells were treated with increasing concentrations of
gossypol and cisplatin alone and in combination. Cytotoxicity was determined by
XTT Cell Proliferation Kit (Roche). Drug synergy was assessed by using CalculSyn 2.0 software (Biosoft). Apoptosis was detected by Cell Death Detection
Elisa Plus Kit (Roche) and confirmed by Caspase-Glo 3/7 Assay. Apoptotic cells
were also shown by Live/Dead Cell Viability Assay Kit (Invitrogen). Angiogenic
cytokines were determined by using Human Angiogenesis Antibody Array 1
(Raybiotech).
Results: Combined treatment (5 lM Cisplatin110 lM Gossypol) was shown
to have strong synergistic cytotoxic and apoptotic effects in OVCAR-3. Also,
significant inhibition of angiogenin cytokine was observed in the antibody array
from supernatant of OVCAR-3 cells treated with cisplatin and gossypol, compared to any agent alone.
Conclusion: The synergistic cytotoxic, apoptotic and antiangiogenic effects
of cisplatin and gossypol combination in drug resistant ovarian cancer cell line
OVCAR-3 should be further evaluated as a new therapeutic approach for the
treatment of drug resistant ovarian cancer.
P-88
THE EFFECTS OF CAFFEINE ON THE ANTIOXIDANT CAPACITY
AND AOPP LEVELS OF RAT BRAIN TISSUE
Hatice Pasaoglu1, Canan Demirtas1, Ebru OfIuoglu2, Ozge Tugce Pasaoglu1,
and Aydin Pasaoglu3
1
Department of Medical Biochemistry, Gazi University, Turkey, 2Zonguldak
Vocational School Health Services, Zonguldak Karaelmas University,
Turkey, 3Department of Neurosurgery, Gazi University, Turkey
Objective: The aim of this study was to investigate the oxidant-antioxidant
effects of caffeine in rat brain tissue by measuring antioxidant capacity level and
advanced oxidation protein products (AOPP).
Methods: Thirty rats were included in the study. The rats were divided into
three groups; a control group and two caffeine-treated groups (group1 and group
2). Group 1 and 2 were given 30mg/kg and 100 mg/kg doses of caffeine, respectively, for 14 days.
Results: We found a significant increase in the antioxidant capacities of the
caffeine-treated groups compared to control group (p50.027). The antioxidant
capacity level of group 1 was higher compared to control group but the difference was not statistically significant (p50.28). Group 2 antioxidant capacity levels were found to be significantly increased compared to control group (p50.0l).
No considerable difference between caffeine-treated groups was detected
(p50.08). When brain tissue AOPP levels were compared, we found significant
difference between groups (p50.0l).
AOPP levels of group 1 was diminished compared to control group, but difference was not statistically significant; whereas the difference between control
group and group 2 was significant (p50.0l). The result found when group 1 and
group 2 were compared, was statistically significant (p50.03).
349
Conclusions: In our study, we found that caffeine, in the given doses, causes
an elevation at the antioxidant capacity and decreases protein oxidation in rat
brain tissue. Our results support the antioxidant effect of caffeine.
P-89
STUDY OF GLIOMA CELL SURVIVAL IN RESPONSE TO
TEMOZOLOMIDE TREATMENT
Helena Carvalheiro1, Anália Carmo1,2, Inês Crespo1,
Maria Celeste Lopes1,2,3
1
Department of Cellular Immunology and Oncobiology, Center for
Neuroscience and Cell Biology, University of Coimbra, Portugal, 2Center of
Investigation in Environment, Genetics and Oncobiology (CIMAGO),
Portugal, 3Pharmacy Faculty, University of Coimbra, Portugal
Glioblastoma (GBM) is the most malignant and the most frequent primary
brain tumor, comprising approximately 50% of the cerebral gliomas. Despite
recent advances in therapies the median survival time for GBM patients remains
approximately 12-14 months. Temozolomide (TMZ) is an alkylating agent that is
considered the gold standard of GBM treatment after a clinical randomized
study. The cytotoxicity of TMZ is thought to be due to the formation of O6methylguanine (O6-meG) in DNA which mispairs with thymine during the next
cell cycle. Whereas the mechanism of death induced by O6-meG has been elucidated in various experimental systems it remains poorly understood in gliomas.
Therefore, we used a human glioma cell line to determine the effect triggered by
TMZ in two cell survival pathways: the PI3K/Akt and Ras-Erk1/2. In this study,
U-118 glioma cells were incubated with different concentrations of TMZ (from 5
to 1000 mM) for different periods of time (24 and 48 hours). Cell viability was
assessed by the MTT assay and cell proliferation was determined using BrdUrd
incorporation. The phosphorylation status of Akt and Erk1/2 was assessed by
western blot. Cell survival was addressed by confocal microscopy using two
stains Hoechst and propidium iodide and by flow cytometry. The results from
the MTT assay indicate that the U-118 cells viability is significantly reduced in
the presence of TMZ meaning that this cell line is sensible to this alkylating
agent. Regarding the activity of the cell survival pathways, our results showed
that the U-118 cell line presents a basal phosphorylation of both Akt and Erk1/2.
In the presence of TMZ, phosphorylation status of Akt and Erk1/2 is reduced
which indicates that the activity of the kinases is inhibited. The results of cell
survival obtained by confocal microscopy and flow cytometry showed that in
TMZ-treated cells, there is a significant increase in the number of apoptotic cells.
Taking in consideration that Akt and Erk1/2 control cell survival, our results
may indicate that apoptosis induced by TMZ is correlated with these two signalling pathways. In conclusion, our in vitro studies indicate that in the U-118 cell
line, TMZ induces cell death by apoptosis which seems to be mediated by the inhibition of Akt and Erk1/2 activity.
P-90
ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS)
1276 AND 1349 IN THE ADIPONECTIN GENE WITH TYPE 2
DIABETES MELLITUS IN TURKISH POPULATION
Hilal Arikoglu1, Dudu Erkoc Kaya1, Hulya Ozdemir1, Mustafa Sait Gonen2,
Suleyman Ipekci2, Ahmet Arslan3, Melda Aksoy Hepdogru1,
and Ferhan Paydak1
1
Department of Medical Biology, Selcuk University, Konya, Turkey,
2
Department of Endocrinology, Selcuk University, Konya, Turkey,
3
Department of Medical Biology, Gaziantep University, Gaziantep, Turkey
Type 2 Diabetes Mellitus (T2DM) is implied in the progression of insulin resistance in the target tissues and impairment of insulin secretion in pancreatic bcells. Several genes are promising candidates for insulin resistance. Adiponectin,
an adipose tissue specific protein encoded by the adiponectin gene, modulates insulin sensitivity and plays an important role in regulating energy homeostasis. In
recent studies, common single nucleotide polymorphisms (SNPs) in adiponectin
gene were shown as associated with low plasma adiponectin level, insulin resistance and an increased risk of T2DM. In this study, we investigated intronic
SNPs1276 and 1349 in the adiponectin gene, evaluated their allele frequencies
350
in Turkish patients, and analyzed the contribution of these polymorphisms to the
development of T2DM.
In this study, unrelated and non-obese 68 patients (Body Mass Index,
BMI30 kg/m2) were selected from Endocrinology Department, Selcuk University, Meram Medical Faculty. 20 non-diabetic individuals with no family history
of diabetes, age and body mass index matched, were included as control subjects.
Genomic DNA was isolated from peripheral blood leukocytes. SNPs were
screened by Polymerase Chain Reaction (PCR) amplification with specific primers, followed by single strand conformational polymorphism (SSCP) analysis.
Biochemical parameters (glucose, insulin, HbA1C, C-peptid, cholesterol, HDL-C,
LDL-C, triglycerides) of each individual were measured. Homeostasis model
assessment for insulin resistance (HOMO-IR) for each case and control individuals were calculated with the formula; fasting plasma glucose (mmol/l) x fasting
serum insulin (lU/ml)/22.5. According to our data, distributions of the genotypes
were; GT/AA (SNP1276 and 1349, respectively) (64%), GG/AG (31%), TT/
AA (5%). We evaluated the separate and combined effect of the SNPs
1276(G?T) and 1349(A?G) in the adiponectin gene on the biochemical parameters, insulin resistance, glucose tolerance statistically. Our study is continuing in order to have a more detailed analysis by increasing the number of
patients and healthy controls.
Nephrectomy (one kidney) was applied performed in all groups. Group A (n: 8):
Control, Group B (n: 8): Diabetic control, Group C (n: 8): Control1Vit-E, Group
D (n: 8): Diabetic1Vit-E. Vit-E was injected 40 mg/kg/every other day intraperitoneally for 2 weeks. At the end of eight weeks, all rats were anesthetized by
ether inhalation and sacrificed. The urinary bladder was taken from all of them.
The analyses of apoptosis in rat bladders were performed in formalin-fixed and
paraffin-embedded tissue sections using apoptosis detection kit and with the
TUNEL (TdT-mediated dUTP nick end labelling) technique. The tissues were
then directly examined under a fluorescence microscope.
Results: There was no appreciable difference between the control group and
vit-E treated rats. The serum glucose levels were determined and found significantly higher in diabetic control than untreated and vitamin E supplemented
groups in time intervals from 24 h to 8th week. Apoptotic cells, observed in
bladder urothelial cells, significantly increased in diabetic rats compared to control and decreased after vit-E treatment (p<0.001).
Conclusion: Vit-E, known as an anti-oxidant, diminishes apoptosis in urothelial cells of urinary bladder in diabetic rats.
P-91
EFFECT OF VITAMIN E ON OXIDATIVE STRESS MEASURED AS
ERYTHROCYTE HEMOLYSIS IN STREPTOZOTOCIN-INDUCED
DIABETIC, NEPHRECTOMISED RATS
Mehmet Cengiz Ustuner1, Hilmi Ozden2, Sahin Kabay3, Gul Guven2,
Derya Ustuner4, Nedim Unal2, and Irfan Degirmenci1
1
Department of Medical Biology, Eskisehir Osmangazi University, Faculty
of Medicine, Turkey, 2Department of Anatomy, Eskisehir Osmangazi
University, Faculty of Medicine, Turkey, 3Department of Urology,
Dumlupinar University, Faculty of Medicine, Turkey, 4Department of Medical
Genetic, Eskisehir Osmangazi University, Faculty of Medicine, Turkey
A RARE CASE MOSAIC 16
Aysegul Turkyilmaz, Diclehan Oral, Hilmi Isi, and Mahmut Balkan
Department of Medical Biology and Genetic, Medical Faculty, University of
Dicle, Diyarbakir, Turkey
Trisomy 16 is the most frequent chromosome abnormality at conception,
associated with a high probability of fetal death. Trisomy 16 occurs when cells
have three copies of chromosome 16 instead of the usual two copies. Full trisomy 16, which occurs when all of the body’s cells contain an extra copy of chromosome 16, is not compatible with life. A similar but less severe condition
called mosaic trisomy 16 occurs when only some of the body’s cells have an
extra copy of chromosome 16. The signs and symptoms of mosaic trisomy 16
vary widely and can include slow growth before birth (intrauterine growth retardation), delayed development, and heart defects.
An eight days old patient was referred to the department of medical genetics,
because of ambiguous genitalia. The patient was the second child of a non-consanguineous married couple. The mother and father of the patient were 23 and
24 years old. The patient had growth retardation, facial dysmorphism, penoscrotal hypospadia, bifid scrotum and macrocephaly. The conventional cytogenetic
analysis on peripheral blood lymphocytes by using GTG banding technique at
550 band level showed 47,XY,116[42]/46,XY[6] (mosaic 16). In addition, this
chromosomal aberration was confirmed by FISH analysis using octochrome
probe. Six months later the patient died.
P-92
EFFECT OF VITAMIN E ON THE APOPTOSIS IN URINARY
BLADDER FROM RATS SUBJECTED TO
STREPTOZOTOCIN-INDUCED DIABETES AND NEPHRECTOMY
Hilmi Ozden1, Sahin Kabay2, Mehmet Cengiz Ustuner3, Gul Guven1,
Esra Gurlek Olgun4, Derya Ustuner5, Nedim Unal1, and Irfan Degirmenci3
1
Department of Anatomy, Eskisehir Osmangazi University, Faculty of
Medicine, Turkey, 2Department of Urology, Dumlupinar University, Faculty
of Medicine, Turkey, 3Department of Medical Biology, Eskisehir
Osmangazi University, Faculty of Medicine, Turkey, 4Department of
Pathology, Dumlupinar University, Faculty of Medicine, Turkey,
5
Department of Medical Genetic, Eskisehir Osmangazi University,
Faculty of Medicine, Turkey
Objective: Diabetic rats have increased levels of apoptosis when compared to
non-diabetes rats. Defects in the apoptotic signaling pathway result in many diseases
including autoimmune diseases by DNA damage. Diabetes is a kind of autoimmune
diseases. The aim of this study was to examine the effect of vitamin E (vit-E) on
apoptosis in the urinary bladder of STZ-induced diabetic, nephrectomised rats.
Methods: Male Wistar albino rats were made diabetic using a single intraperitoneal injection of 65 mg/kg STZ. Rats were divided into four groups.
P-93
Objective: Diabetes is an autoimmune disease and represents an ideal target
for studying the consequences of oxidative stress and its treatment, because oxidative stress has been repetitively shown to be a feature of many diseases related
with metabolic disorders. Vitamin E has antioxidant properties and has also been
shown to play a role in immune function. The aim of this study is to determine the
lipid peroxidation and the antioxidant enzymes in the erythrocyte hemolysates after vitamin E treatment in streptozocin induced diabetic, nephrectomised rats.
Methods: Thirty two Wistar albino male rats were divided into four groups:
control, streptozocin, vitamin E and streptozocin 1 vitamin E groups. The rats
were made diabetic using a single intraperitoneal injection of 65 mg/kg streptozocin, and afterwards nephrectomy (one kidney) was applied to all groups. Vitamin E was injected 40 mg/kg/every other day intraperitoneally for 2 weeks. At
the end of eight weeks, rats were anesthetized and blood from the left cardiac
ventricle was transferred to tubes containing EDTA as anticoagulant from which
the erythrocyte hemolysate was prepared. Erythrocyte hemolysate was used to
investigate malondialdehyde level, superoxide dismutase, glutathione peroxidase,
and catalase activities.
Results: Serum glucose levels were significantly higher in diabetic control
than untreated and vitamin E supplemented groups in time interval from 24 h to
8th week. The streptozocin treated rats malondialdehyde levels were significantly
increased compared to control group. In streptozocin 1 vitamin E group catalase,
superoxide dismutase and glutathione peroxidase activities remained at control
values.
Conclusion: These results showed that oxidative stress is involved in the
development of diabetic nephropathy and vitamin E treatment is protective on
erythrocyte hemolysate in nephrectomised, diabetic rats.
P-94
THE PROTECTIVE EFFECTS OF NITRIC OXIDE SYNTHETASE
INHIBITORS ON RATS WITH TRINITROBENZEN SULFONIC ACID
INDUCED COLITIS
Yilmaz Altuner1, Adnan Ayhanci1, Hilmi Ozden2, Kismet Civi3,
Derya Ustuner4, Mehmet Cengiz Ustuner5, and Hulyam Kurt5
1
Department of Biology, Eskisehir Osmangazi University, Arts and Science
Faculty, Turkey, 2Department of Anatomy, Eskisehir Osmangazi University,
Faculty of Medicine, Turkey, 3Department of Pathology, Inegol State Hospital, Turkey, 4Department of Medical Genetic, Eskisehir Osmangazi University, Faculty of Medicine, Turkey, 5Department of Medical Biology, Eskisehir Osmangazi University, Faculty of Medicine, Turkey
Objectives: Colitis is an inflammatory disease caused by microorganisms,
chemicals and genetic diseases and it can spread from colon to rectum. Experimentally, colitis was induced by trinitrobenzene sulfonic acid. The high level of
nitric oxide was determined in colitis. The aim of the study was to determine the
protective effect of L-nitro-l arginine, metallothionein 1 and 2 against excess nitric oxide at trinitrobenzene sulfonic acid treated rats.
Methods: In our study, 70 rats were divided into 7 groups: control, trinitrobenzene sulfonic acid (120 mg/kg), L-nitro-L arginine (35 mg/kg), 1 and 2 mg/kg
of metallothionein 1 and 2. After the administration of trinitrobenzene sulfonic
acid, dissections of the rats in all groups were made at the first, the second and the
third day under ether anesthesia and colons samples were obtained. The colon tissue slides were evaluated histopathologically with H&E staining and scored.
Results: Different results were obtained from the histological analyses of the
descending colons. The findings were macroscopic score, ulcer, loss of mucous
cell, crypt abscess, inflammatory cyst, mucosa atrophy, edema, inflammatory cell
infiltration, vascular dilatation and I-NOS which increased in descend colon at
trinitrobenzene sulfonic acid treated rats. All histological findings were decreased
in the trinitrobenzene sulfonic acid treated rats supplemented with L-nitro-L arginine, metallothionein 1 and 2.
Conclusions: In this study, the results suggest that L-nitro-L arginine and
metallothioneins were effective in protecting against the toxic effects of excess
nitric oxide in trinitrobenzene sulfonic acid treated rats.
P-95
NUCLEAR BUDDING IN CARBON TETRACHLORIDE AND
LYCOPENE IN VIVO
Hulyam Kurt1, Derya Ustuner2, Mehmet Cengiz Ustuner1,
and Hilmi Ozden3
1
Department of Medical Biology, Eskisehir Osmangazi University, Faculty
of Medicine, Turkey, 2Department of Medical Genetic, Eskisehir Osmangazi
University, Faculty of Medicine, Turkey, 3Department of Anatomy,
Eskisehir Osmangazi University, Faculty of Medicine, Turkey
Objective: Carotenoids are important biological compounds contained in fruits
and vegetables. Epidemiological studies have also demonstrated that the increased
consumption of lycopene rich foods such as tomatoes and tomato based products is
associated with decreased risk of cancer. Carbon tetrachloride treatments caused
classical damage in the rat bone marrow. The aim of this study was to observe the
cytotoxic effects of carbon tetrachloride and compare it with lycopene effect on nuclear buds using the micronucleus assay. Nuclear budding is unique mechanism of
micronucleus formation. It induces gene amplification. A strong correlation
between micronucleus formation and nuclear budding has been shown.
Materials and Methods: Thirty six, three month old Sprague-Dawley rats
were divided into three groups; liquid oil (0.2 mL/kg/day), carbon tetrachloride
(0.2 mL/kg/day) and lycopene (5 mg/kg/day) groups. At the end of twenty days,
bone marrow samples were collected into 1640 RPMI medium. The bone marrow cell were incubated for 72 h. Cytochalasin B was added at 44th hour. Nuclear budding was scored from at least 1000 binucleated cells.
Results: There were no appreciable differences in the number of nuclear
buddings between olive oil and lycopene treated groups. The nuclear budding
was significantly increased in carbon tetrachloride the treated group as compared
to the lycopene treated rat bone marrow cells.
Conclusion: Carbon tetrachloride has high cytotoxic and genotoxic effects
and nuclear budding, as a measure of DNA Damage, has been detected with
micronucleus technique.
P-96
MOLECULAR DETECTION OF EPSTEIN-BARR VIRUS IN CERVICAL
SQUAMOUS CELL CARCINOMA IN NORTHEAST OF IRAN
Hossein Ayatollahi1, Fatemeh Homaee2, Kiarash Ghazvini3,
Mohammad-Hadi Sadeghian1, Mohammad-Mehdi Akbarin1,
and Maliheh Hasanzadeh4, Mahmood Bagheri3
351
1
Department of Hematopathology and Blood Banking, Mashhad University
of Medical Sciences, Iran, 2Department of Oncology, Mashhad University
of Medical Sciences, Iran, 3Department of Microbiology, Mashhad
University of Medical Sciences, Iran, 4Department of Obstetrics and
Gynecology, Mashhad University of Medical Sciences, Iran
Background: Human papillomavirus (HPV) is the central etiologic factor for
cervical cancer. Epstein-Barr virus more considered as an oncogenic viral agent
in association with SCC that it may act as an HPV cofactor. Epidemiologic studies suggest that (EBV) infection correlate with the natural history of cervical
carcinoma however the level of this risk is unknown.
Materials and Methods: To study the association between exposure EBV
DNA and cause of cervical Squamous Cell Carcinoma (SCC), one hundred
twenty women with cervical squamous cancer were selected as case group. One
hundred twenty women, that had been hysterectomy for DUB but without organic lesion and SCC, during the past 10 years were selected as control group
and assessed by cytological and histological analysis. The two groups were
paired match by age. Viral DNA was detected by PCR technique in samples that
were prepared by chloroform extraction method from paraffin embedded tissue.
Results: There were only 4 specimens positive in the case group and one
specimen positive in control group for DNA Epstein-Barr virus (OR54.1)
CI595%.
Conclusion: EBV is not strongly associated with cervical SCC and EBV
infection does not seem to play a main role in cervical carcinogenesis in our
studied territory.
P-97
ASSOCIATIONS OF LPL S447X AND APO E GENTOYPES WITH
LOW-DENSITY LIPOPROTEIN SUBFRACTIONS IN TURKISH
PATIENTS WITH CORONARY ARTERY DISEASE
Hulya Yilmaz-Aydogan1, Selim Isbir2, Ozlem Kurnaz1, Uzay Gormus1, and
Turgay Isbir1
1
Medicine, Istanbul University, Capa, Istanbul, Turkey, 2Cardiovascular
Background: Serum lipoproteins are important determinants of coronary artery disease, CAD. Lipoprotein lipase (LPL) is involved in the transformation of
dietary lipids into sources of energy for peripheral tissues and plays an important
role in triglycerides metabolism, since it is crucial for the hydrolysis of triglycerides and chylomicrons. Several polymorphisms at the LPL locus have been
described that are associated with variations in LPL activity, serum lipid concentrations. The S447X polymorphism results in the premature truncation of LPL.
This polymorphism is associated with increased levels of LPL secretion Apolipoprotein E is a protein constituent of both triglyceride-rich lipoproteins (TRL) as
well as HDL, which plays an important role in liver uptake of TRL remnants.
Three apoE isoforms (E2, E3, E4) with Cys/Arg interchanges at positions 112
and 158 have been identified. Many studies, assessing the role of APOE genotypes on plasma lipids, have shown that the presence of the apoE4 is associated
with elevations in LDL-C, while the presence of E2 is associated with decreased
levels of LDL-C. This study investigates the associations of specific LPL-S447X
and ApoE allelic patterns with LDL size and subfraction profiles in patients with
CAD and healthy subjects.
Materials and Methods: We compared 41 cases with CAD and 23 controls
regarding the occurrence of the LPL Ser447X and ApoE polymorphisms. PCR
and RFLP techniques were utilized to perform genotyping and LDL size and
subfractions were assessed by a high-resolution, non-gradient polyacrylamide gel
electrophoresis technique.
Results: The lowest sdLDL level was observed at homozygous LPL-X447
genotype (6.0064.00) while highest sdLDL level was observed at LPLX447(1)/ApoE4(1) haplotype (14.336 20.55) in the patient group. When it was
together with ApoE4 allele, a protective effect of LPL-X447 allele on atherogenic LDL profile was not observed. Furthermore, the detrimental effect of LPLS447 on atherogenic LDL profile was increased when it was together with
ApoE4 allele.
Conclusions: Despite the effect of the LPL S447X and ApoE polymorphisms
on atherogenic lipoprotein phenotype, we did not observe any associations
between these genotypes and risk of CAD in the Turkish population. The X447
352
mutant allele of the LPL gene may protect from atherogenic LDL subfraction,
although this effect is small. We suggest that the S447X polymorphism of the
LPL gene may modify the risk of atherogenic sdLDL fraction in an APOE dependent fashion.
P-98
IN TOKAT REGION, FREQUENCY AND DISTRIBUTION OF
FREQUENTLY SEEN MUTATIONS IN MEFV GENE IN THE PERSONS
PREDIAGNOSED WITH FAMILIAL MEDITERRANEAN FEVER
Semsettin Sahin1, Huseyin Ozyurt1, Ali Akbas1, Oguzhan Saylan1,
Ismail Benli2, Leyla Aydogan3, Beytullah Yildirim4, and Resul Yilmaz5
1
Department of Biochemistry, Gaziosmanpasa University, Tokat, Turkey,
2
Department of Biology, Gaziosmanpasa University, Tokat, Turkey,
3
Department of Chemistry, Gaziosmanpasa University, Tokat, Turkey,
4
Department of Internal Medicine, Gaziosmanpasa University, Tokat,
Turkey, 5Department of Children Health And Diseases, Gaziosmanpasa
University, Tokat, Turkey
Familial Mediterranean Fever (FMF) is an autosomal recessive disease and
clinically characterized by periodic abdominal pain, fever, arthralgia/arthritis and
skin lesions. The prevalence of FMF is higher in Turks, Armenians, Arabs and
Sephardic Jews. A wide variety of mutations have been described in pyrin gene
which is known to be responsible for FMF.
In this study, in Tokat, 12 most frequent mutations in pyrin gene (E148Q,
P369S, F479L, M680I(G/C), M680I(G/A), 1692del, M694V, M694I, K695R,
V726A, A744S and R761H) have been screened in patients who were diagnosed
as FMF and results were compared with classical data and symptoms. Detection
of mutations in pyrin gene was carried out by using Vienna Lab FMF StripAssayTM which is a strip mutations screening kit. Strip mutation analyze is a
method that can determine a large number of mutations of genes associated with
some diseases such as Cardiovascular disease (CVD), FMF and Celiac. In this
study 929 patients who were admitted to our clinic with periodic abdominal
pain, fever and arthralgia/arthritis were included. 375 of these patients were children and 554 of them were adult. As a result, in 432 patients (46.5%) heterozygous/homozygous mutations have been detected. In 233 patients mutation
M694V (45 number homozigous), in 88 patients mutation M680I (G/C) (7
patient homozygous), in 90 patients mutation E148Q (1 patient homozygous), in
49 patients mutation V726A (1 patient homozygous), in 21 patients mutation
R761H (1 patient homozygous) has been observed. 94 patients had 2 mutations
and 2 patients had 3 mutations.
As a result, in Tokat region, M694V mutation has been detected in most of
the cases as reported from other studies in Turkish population. In 46.5% of all
patients, at least one mutation has been detected.
tested for HBeAg specific antibody response by indirect ELISA. Two mice with
high antibody titer for antigen were selected for fusion. Fusions were performed
by some modification of classical fusion procedure and positive clones were
selected by indirect ELISA.
Results and Conclusion: Lymphocytes from spleen and lymph nodes were
fused with F0 (ATCC CRL 1646) myeloma cells. After two fusions, it was found
that 57 hybrid cells had antibody activity among 1148 hybrid clones and only
13B10 was producing antigen specific monoclonal antibody. Isotype of 13B10
was found to be IgG2a class by using isotyping kit. Anti-HBeAg monoclonal
antibody was subcloned using limited dilution method; produced in large scale
by using tissue culture flasks; purified by ammonium sulfate precipitation and
Protein A column and then labeled with horse radish peroxidase (HRP) enzyme.
Labeled 13B10 monoclonal antibody will be used for the possible detection of
HBeAg in infected sera.
P-100
IS THERE ANY CORRELATION BETWEEN CYCLIN D1 G870A
POLYMORPHISM AND GASTRIC CANCER RISK IN
TURKISH PATIENTS
Ilhan Yaylim-Eraltan1, H. Arzu Ergen1, Soykan Arikan2, Bedia Agachan1,
Yemliha Yildiz1, Uzay Gormus1, and Turgay Isbir1
1
Istanbul Research and Education Hospital, Istanbul, Turkey
Background: Cyclin D1 (CCND1) is a regulatory protein involved in the
cell cycle of both normal and neoplastic cells. Polymorphism of this gene at
codon 242 in exon 4 (A870G) has an impact on the risk of several human cancers. The aim of this study was to investigate the relation between the CCND1
A870G gene polymorphism and the risk of gastric cancer in Turkish population.
Material and methods: The study population consisted of 34 patients with
gastric cancer and 95 cancer-free controls. CCND1 870A/G polymorphism was
genotyped by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay.
Results: CCND1 genotype distribution among our gastric cancer patients
was not significantly different from that among controls (p50.09). However G
allele were significantly higher in the gastric cancer patients than in the controls
when subjects with the AA genotype and this difference was statistically significant (p50.04).In other words, the risk of gastric cancer for subjects with the AA
genotype was lower that of subjects who have G allele. (OR:0.399 95% CI
0.151-1.055).
Conclusions: The CCND1870 GG genotype is associated with an increased
risk for gastric cancer in Turkish patients. Larger studies with multiple polymorphisms are needed to verify this finding and the function of this polymorphism
needs to be further investigated.
P-99
PRODUCTION AND CHARACTERIZATION OF MONOCLONAL
ANTIBODIES AGAINST rHBeAg
P-101
Ibrahim Hatipoglu1, Ibrahim Sogut1, Esin Akcael2, Ali Ihsan Manav2,
Harun Kocaaga2, Fatima Yucel2, and Aynur Basalp1
1
Immunology of Vaccine Laboratory, TUBITAK MAM Genetic Engineering
and Biotechnology Institute, Turkey, 2Hybridoma Laboratory, TUBITAK
MAM Genetic Engineering and Biotechnology Institute, Turkey
IS THERE ANY RELATIONSHIP BETWEEN NOS POLYMORPHISM
AND COLORECTAL CANCER
Objective: Hepatitis B Virus (HBV) is a small DNA virus causing acute and
chronic hepatitis, cirrhosis and hepatocellular carcinoma. HBV infection is still
considered as a significant health problem worldwide. HBeAg, as well as
HBsAg, is one of the markers indicating HBV infection. HBeAg is encoded by
core gene, appears normally during the stage of high infectivity and is an indicator of active viral replication. Thus, serological tests detecting the presence of
HBeAg and anti-HBe antibody have clinical significance. In this study, a monoclonal antibody specific for HBeAg was produced by hybridoma technology for
the further development of a HBe diagnostic kit.
Method: BALB/c mice were immunized three times with 10 lg recombinant
Hepatitis B Virus e Antigen (rHBeAg) at 3 week intervals, intraperitoneally.
Immunizations were performed in the presence of FCA and IFA. Mice were
Ilhan Yaylim-Eraltan1, Soykan Arikan2, Canan Cacina1, Uzay Gormus1,
Umit Zeybek1, Nilufer Yigit1, Bedia Agachan1, and Turgay Isbir1
1
Istanbul Research and Education Hospital, Istanbul, Turkey
Background: Nitric oxide (NO) is a molecule that plays a key role in many
physiologic and pathologic processes. It is produced in vivo from the amino acid
l-arginine by a family of nitric oxide synthases (NOS). Endothelial NOS (eNOS)
is a constitutively expressed isoform of NOS. The eNOS gene entails several
polymorphisms, of which certain were investigated in some cancer types.
Materials and Methods: Fifty-five patients with colorectal cancer and 86
healthy subjects were included in the study. Genotype analyses were performed for
eNOS exon 7, Glu298Asp (G --> T) polymorphisms by PCR-RFLP techniques.
Results: No significant differences in allele or genotype frequencies for individual polymorphisms were observed between patients with colorectal cancer
and controls. The NOS GT genotype was associated with the presence of mucinous component in colorectal cancer (p50.02). (OR, 2.5; 95% CI, 1.345-4.646;
p50.019) compared with other homozygote genotypes.
Conclusion: Therefore, our results suggest that Glu298Asp eNOS polymorphism plays a role as a genetic factor for the histologic type of colorectal cancer.
P-102
353
type was associated with NSCLC risk compared with other genotypes
(OR53.809; 95%CI 1.601-9.061; P50.006).
Conclusion: Our findings suggest that the TT genotype of the VDR TaqI
variant was significantly associated with an increased risk of NSCLC, while the
VDR FokI polymorphism was not significantly associated with it. However, further studies are necessary to confirm these findings.
P-104
BREAST CANCER AND CYCLIN D1 GENE POLYMORPHISM IN
TURKISH WOMEN
1
1
2
3
Ilhan Yaylim-Eraltan , H. Arzu Ergen , Soykan Arikan , Seden Kucucuk ,
Umit Zeybek1, Yemliha Yildiz1, Nilufer Yigit1, and Turgay Isbir1
1
Istanbul Research and Education Hospital, Istanbul, Turkey, 3Institute of
Oncology, Istanbul University, Istanbul, Turkey
Background: Cyclin D1 protein plays an important part in regulating the
progress of the cell during the G1 phase of the cell cycle. It has been suggested
that G870A polymorphism at the exon4/intron4 splicing region of the CCND1
gene may play a role in tumorigenesis and invasiveness.
Materials and Methods: We performed a case-control study to test the association between G870A polymorphisms in the CCND1 gene and breast cancer
risk and cancer progression. For this purpose, 38 patients with breast cancer and
64 healthy women controls were included in the study. The CCND1 G870A
polymorphisms in our study groups were genotyped by PCR-RFLP using peripheral blood samples.
Results: We found a significant difference, in the distribution of the GG, AG
and AA genotypes between the patient group and the control group (p50.021).
A decreased risk (OR 0.435, 95% CI 0.223-0.846) was found to be associated
with heterozygote AG individuals when compared with homozygote allele carriers in breast cancer. The cyclin D1 A870G genotype was associated with capsular invasion (p 5 0.02).
Conclusion: The risk of breast cancer development and prognosis may be
associated with genetic variation in the CCND1 genotypes which may be used as
an important biomarker for further studies.
P-103
ASSOCIATION OF VITAMIN D RECEPTOR GENE POLYMORPHISM
WITH NON-SMALL CELL LUNG CANCER IN TURKISH PATIENTS
Ilhan Yaylim-Eraltan1, Akif Turna2, Uzay Gormus1, Nilufer Yigit1,
Bedia Agachan1, Yemliha Yildiz1, Bahar Toptas1, and Turgay Isbir1
1
Thoracic Surgery, Yedikule Teaching Hospital for Chest Diseases and
Thoracic Surgery, Istanbul, Turkey
Background: The effects of 1,25-dihydroxyvitamin D3 are mediated by
binding to a specific intracellular vitamin D receptor (VDR), which has been
identified in a variety of tissues. Certain polymorphisms in the VDR gene have
been associated with various neoplasms. The purpose of this study was to determine whether polymorphism in the VDR gene might also influence non-small
cell lung cancer (NSCLC) risk in Turkey.
Materials and Methods: Polymerase chain reaction (PCR) and restriction
fragment length polymorphism (RFLP), and agarose gel electrophoresis techniques were used to detect VDR TaqI and FokI polymorphisms. Seventeen NSCLC
patients and 74 healthy controls were genotyped for the TaqI and FokI.
Results: The frequencies of the TT, Tt and tt genotypes were 58.8%, 41.2%,
0% in NSCLC cancer patients and 31.1%, 47.3%, 21.6% in healthy controls,
respectively. The genotype distribution for TaqI polymorphism was different
between NSCLC patients and controls (P50.035). VDR TaqI TT genotype was
significantly associated with an increased risk of NSCLC in our study groups
(OR51.893; 95%CI 1.122-3.192; P50.032). We observed the F allele in 68%
and 70%, and the f allele in 32% and 30% in NSCLC patients and the control
group, respectively. On combined genotype analysis, the TTFF combined geno-
REFERENCE VALUE OF SERUM CHITOTRIOSIDASE ACTIVITY
AND INCIDENCE OF CHITOTRIOSIDASE DEFICIENCY IN
TURKISH POPULATION
Ismail Kurt1, Dilek Abasli2, Adnan Hasimi1, and M. Kemal Erbil1
1
Department of Clinical Biochemistry, Gulhane Military Medical Academy,
Turkey, 2Department of Clinical Biochemistry, Etimesgut Soldier Hospital,
Turkey
Objective: Chitotriosidase (EC 3.1.4.12) is a member of mammalian chitinase family, a group of enzymes with the capability to hydrolyze chitin and
selectively secreted by activated macrophages and neutrophils. In certain pathological conditions, over-production of chitotriosidase by macrophages is for
instance induced by accumulation of glycosphingolipids, iron or glycogen in
lysosomes of macrophage. Chitotriosidase enzymatic activity is markedly elevated in serum patients suffering from lysosomal storage disorders (eg. Gaucher
disease) as well as other diseases in which macrophage activity predominate.
Therefore, activity pattern of the enzyme may be used as a surrogate marker in
order to diagnose and monitor the efficacy of therapeutic intervention of these
diseases. This enzyme is encoded by a gene located on chromosome 1q31-32.
Chitotriosidase deficiency is caused by a duplication of 24 bp in exon10. Homozygosity and heterozygosity prevalences of this mutation are reported at diverse
rates in different populations. In order to use chitotriosidase activity as a surrogate biomarker, we determined the reference value of serum chitotriosidase activity and the frequency of chitotriosidase deficiency in Turkish population.
Methods: The chitotriosidase activity in serum samples of 100 healthy subjects was measured by fluorimetric method by using 4MU-chitotriose substrate as
previously described by Guo et al (1995). We detected the 24-bp duplication in
exon 10 of the chitotriosidase genes from randomly chosen 90 blood samples.
Genomic DNA was extracted from peripheral blood leucocytes by alkaline lysis
method. The region of interest was amplified by standard PCR followed by electrophoretic analysis.
Results and Conclusion: The chitotriosidase activity in normal healthy subjects was 2-90 nmol/ml/h. The obtained results showed a heterozygosity frequency of the duplication of 24 base pairs in exon 10 as 36%, whereas corresponding value for homozygote chitotriosidase deficiency was 8%.
P-105
ANTIANGIOGENIC AND PROAPOPTOTIC EFFECTS OF NEWLY
SYNTHETIZED BENZOSUBERONES
Jan Mojzis1, Lenka Varinska1, Martina Pilatova1, Gabriela Mojzisova2,
and Ladislav Mirossay1
1
Department of Pharmacology, P.J. Safarik University, Slovakia,
2
Department of Experimental Medicine, P.J. Safarik University, Slovakia
Background: Chalcones, like flavonoids, are widely distributed in edible
plants. Chalcones and their heterocyclic analogues exert various biological activities including anti-inflammatory, analgesic and antipyretic and anti-mutagenic
effects. In the present study, we have investigated whether newly synthesized
chalcones possess an antitumor/antiangiogenic activity.
Material and methods: The present study was conducted to examine the
effects of selected chalcones on proliferation and cell death in the Jurkat and
HeLa cell line. Chalcone used: A-(E-2-(40 -methoxybenzylidene)-1-benzosuberone); B-(E-2-(40 -hydroxybenzylidene)-1-benzosuberone); C-(E-2-(40 -methylbenzylidene)-1-benzosu-berone) and D-(E-2-(20 ,40 -dimethoxybenzylidene)-1-benzosuberone). Furthermore, we have also tested the effects of the studied compounds
on the growth, endothelial cell migration (ECM), matrix metalloproteinase activity (MMP) and tube formation (CTF) by human umbilical vein endothelial cells
354
(HUVECs). The level of VEGF secreted by the cancer cells in the medium was
determined by ELISA kit.
Results: From compounds tested only chalcone A (E-2-(4‘-methoxybenzylidene)-1-benzosuberone) possess significant anti-proliferative effect. In Jurkat and
HeLa cells at concentration of 10-6 mol/L for 72h caused 87 and 45% reduction
in cell survival, resp. Furthermore, it caused initial G2/M arrest in both cell lines
followed by an increase in the proapoptotic sub-G0/G1 fraction. Apoptosis was
also confirmed by flow cytometry as well as by DNA fragmentation. In HUVECs
the cytotoxic effect of compound A was concentration-dependent and cell survival significantly decreased at c510-4-10-6 mol/L. Furthermore, it completely
inhibited CTF in non-toxic concentrations (10-7-10-8 mol/L). Moreover, in concentration 10-7 mol/L it blocks also ECM. Chalcone A also reduced MMP-9 activity in HUVECs in a concentration-dependent manner. Inhibitory effect on
MMP-2 activity was observed only at the highest concentration. VEGF secretion
was significantly reduced in cancer cells treated by chalcone A at concentrations
10-6 and 10-7 mol/L for 24 hours.
Summary: The present study demonstrates antiproliferative and antiangiogenic properties of selected chalcones. Calcone A turned out to be the most
effective agent of all.
This work was supported by the Slovak Research and Development Agency
under the contract No. APVV-0325-07, and by the Slovak Grant Agency for
Science (No. 1/4236/07).
P-106
CUMINUM CYMINUM REDUCES FEEDING, BODY FAT AND
IMPROVES INSULIN RESISTANCE IN WISTAR RATS SIMILAR TO
THE SIBUTIRAMINE
we hypothesized that NF-jB binding to the ADAMTS9 promoter is involved in
IL-1b stimulated chondrocytes.
Methods: Human chondrocytes were cultured in the presence of IL-1b with
or without specific inhibitors of NF-jB signaling pathways. ADAMTS9 mRNA
levels were quantitatively measured by real time RT- PCR. ADAMTS9 promoter
region was connected to a luciferase reporter vector and luciferase assay was
performed. Finally, we performed the chromatin immunoprecipitation (ChIP)
assay to examine the binding of NF-jB to the ADAMTS9 promoter.
Results: Real time PCR results showed that NF-jB pathway inhibitors attenuated cytokine-induced ADAMTS9 mRNA expression. IL-1b stimulated luciferase activity indicating that the ADAMTS9 promoter was activated by IL-1b.
ChIP assay demonstrated that p65 bound to the NF-jB elements located at
approximately 22 kbp in the ADAMTS9 promoter.
Conclusion: These results indicated that NF-jB may play an important role
in IL-1b-induced expression of ADAMTS9 in human chondrocytes. Therefore
we suggest novel therapeutical agent for the treatment of cartilage diseases.
P-108
VARIABLE NUMBER TANDEM REPEAT MARKER FOR CARRIER
DETECTION OF PKU IN CENTRAL IRAN
Kazem Parivar1, Seifati S. Morteza2, Koochmeshgi Jalal3, and
Hashemi Mehrdad1
1
Department of Biology, Islamic Azad University Science and Research
Branch, Iran, 2Department of Biology, Islamic Azad University Ashkezar
Branch, Iran, 3Department of Biology, Research Institute for Life, Iran
Objective: We studied the effects of the Cuminum Cyminum on feeding and
body weight in a rat model in compare to sibutiramine.
Design: Male Wistar rats were divided into three groups: group A was given
Cuminum Cyminum (400 lg/kg-1/day-1), group B was given sibutiramine (3mg/
kg-1/day-1) and group C deionized water (controls) for 25 days.
Results: Cuminum Cyminum and sibutramine decreased food intake throughout the treatment period in both groups (p<0.005) versus their control. Plasma
leptin concentrations was lower in sibutramine and Cuminum Cyminum treated
rats (1.6 6 0.15, 2.1 6 0.01) versus their untreated controls (5.1 6 0.27)
(P<0.005). Glucose was decreased in sibutramine (80.6 615) and Cuminum
Cyminum (80 65.1 ) treated rats versus their controls (115613.8) (P<0.005).
Using the homeostasis mode assessment (HOMA) as a measure of insulin resistance, both groups of rats were significantly less insulin resistant than controls
(P<0.005). The lipids levels were significantly decreased in Cuminum Cyminum
and sibutramine treated rats compare to their controls (P<0.005).
Discussion: Cuminum Cyminum, a plant drug, shows anti-obesity effect like
sibutiramine. Its effects appear to not be mediated by inhibition of ARCNPY
neurons.
Phenylketonuria (PKU) is the most common error in the metabolism of
amino acids and an important genetic disease, which- if untreated- results in irreversible brain damage and mental retardation. The incidence of PKU among the
Caucasians in general is 1 in 10000 live births and the highest prevalence for
PKU has been reported from Iran and its neighboring countries (around 1 in
4000). This is largely due to the high prevalence of consanguineous marriages in
the region. Due to the large number of PKU causing mutations in the phenylalanine hydroxylase gene, segregation analysis of the Variable Number Tandem
Repeat (VNTR) polymorphic marker associated with this gene is applied in carrier detection of PKU. Informative level of this marker in carrier detection of
PKU in a population depends on the number of its alleles and distribution pattern
of these alleles in the population. Considering the population heterogeneity in
Iran, we undertook an investigation of the allelic frequencies of this marker in
the province of Yazd (central Iran). 33 mutant and normal chromosomes from 9
unrelated PKU families were studied by PCR and gel electrophoresis. 5 VNTR
alleles were identified. All of them were present in normal chromosomes but
only 4 of them were present in mutant chromosomes. One of these 4 alleles
accounted for 78% of mutant chromosomes. Pattern of distribution of alleles in
normal chromosomes was markedly different from that in mutant chromosomes
(20%, 20%, 33%, 13.5%, 13.5%). Polymorphic Information Content (PIC) of this
marker was calculated as 63%, which indicates it as a suitable marker for carrier
detection of PKU in the population under study.
P-107
P-109
IDENTIFICATION OF NF-kB BINDING ELEMENTS IN HUMAN
ADAMTS9 PROMOTER
THE G352VFS MUTATION IN TUNISIAN PHENYLKETONURIA
POPULATION
Kadir Demircan1, Esra Gunduz2, Mehmet Zeynel Cilek1,
Omer Faruk Hatipoglu1, Kursat Oguz Yaykasli1, Mehmet Gunduz2,
Yoshifum Ninomiya1, and Satoshi Hirohata1
1
Department of Molecular Biology and Biochemistry, Graduate School of
Medicine Dentistry and Pharmaceutical Sciences, Okayama University,
Japan, 2Department of Oral Pathology and Medicine, Okayama University
Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Japan
Khemir Sameh1, Tebib Neji2, Siala Hajer3, Azzouz Hatem2, Cherif Wafa4,
Jemaa Riadh1, Khouja Naziha5, Messaoud Taieb3, Abdelhak Sonia4,
Ben Dridi Marie-Francoise2, and Kaabachi Naziha1
1
Laboratory of Biochemistry, Rabta Hospital, Tunisia, 2Department of
Pediatrics, Rabta Hospital, Tunisia, 3Laboratory of Biochemistry and
Children Hospital, Tunisia, 4Molecular Biology Pasteur Institute, Tunisia,
5
Department of Pediatrics Neurology Institute, Tunisia
Objective: NF-jB (nuclear factor-jB) plays a role for gene regulation in
inflammation. NF-jB forms dimers and binds to 9-11 bp unique sequences
known as NF-jB sites. ADAMTS9 is a cytokine-inducible gene, and it has binding sites for NF-jB in its promoter region. Since interleukin 1b (IL-1b) is known
to exert numerous effects on cartilage metabolism through the NF-jB pathway,
Background: Phenylalanine hydroxylase (PAH) deficiency is caused by
mutations in the PAH gene (12q22-q24), resulting in a primary deficiency of the
PAH enzyme activity. More than 500 PAH mutations have been identified, and
their distribution among different populations varies significantly. The G352Vfs
mutation was reported in different ethnic population: Algerians, Moroccans, Ital-
Javad Mohiti-Ardekani and Mohammad A. Akbarian
Department of Biochemistry, Yazd University of Medical Science, Iran,
355
ians, French-Canadians and Lebanons.Phenylketonuria was reported to as being
associated with some cases of autism. The objective of this study is to screen
Tunisian PKU patients for the presence of the G352Vfs mutation.
Patients and Methods: Twenty three PKU unrelated families (32 cases),
that originated from various geographic locations in Tunisia were investigated.
Diagnosis of PKU was based on plasma phenylalanine level. Patients were classified into phenotypes based on pre-treatment plasma phenylalanine level: classic
PKU ([phe] > 20 mg/dl), moderate PKU (10 mg/dl < [phe] < 20 mg/dl) and
mild hyperphenylalaninemia ([phe] <10 mg/dl). Ten patients had autistic behaviours. After informed consent was obtained, blood samples were collected and
DNA extracted by salting out. Exon 10 was amplified using polymerase chain
reaction (PCR) and screening for the mutation of interest performed by restriction enzyme assay followed by direct sequencing confirmation.
Results: Among the 21 unrelated PKU patients studied, the G352Vfs mutation was found in five alleles (two of which are homozygous for this mutation
and one heterozygous). The five homozygous cases (4 consanguineous females
and 1 male) had autism but the heterozygote male have not autistic behaviours.
Conclusion: This mutation has a relatively low frequency (5/42 mutant alleles,
11.9%) On the other hand, the IVS10nt546 mutation was found only in 5% of the
study population what testifies the heterogeneity of the tunisian PKU alleles.
Methods: 44 healthy dental students were included in this study. 24 of them
were classified to have dental caries (DMF-T55.6) according to the World
Health Organization (WHO) and 20 of them were caries-free (DMF-T50). DNA
of all the participants was extracted from peripheral blood cells by using High
PCR Template Preparation Kit (Roche, Germany) according to the manufacturer’s guide. MUC7 gene was amplified by using specific primers (50 GAAGGTCGAGAAAGGGA TCAT-30 sense and 50 - GTCTTGTGGAGCTGGGGAAT-30 antisense). The amplicons were sequenced with ABI Prism 3100
Genetic Analyzer by using DYNamic ET Terminator Cycle Sequencing Kit
(Amersham).
Results: We did not find any difference between groups. We detected one
common SNP that caused to change asparagine to lysine in codon 80 (N80K).
This alteration was found to be present in 29% and 40% of the subjects with and
without caries, respectively.
Conclusion: The SNP found in this study may be a specific polymorphism
affecting Turkish population. Further studies with a larger number of individuals
are necessary in order to clarify this finding.
P-110
ASSOCIATION BETWEEN N-ACETYLTRANSFERASE 2 GENE
POLYMORPHISMS AND RHEUMATOID ARTHRITIS
VDR TAQ I POLYMORPHISM IN ORAL SQUAMOUS CELL
CARCINOMA: A REPORT FROM TURKEY
Kivanc Bektas-Kayhan1, Meral Unur1, Ilhan Yaylim-Eraltan2,
H. Arzu Ergen2, Gunter Hafiz3, Ahmet Karadeniz4, and Turgay Isbir2
1
Istanbul University, Faculty of Dentistry, Department of Oral Surgery and
Medicine, Capa, Istanbul, Turkey, 2Istanbul University, Institute of
Experimental Medical Research, Department of Molecular Medicine, Capa,
Istanbul, Turkey, 3Istanbul University, Istanbul Faculty of Medicine,
Department of Otorhinolaryngology, Capa, Istanbul, Turkey, 4Istanbul
University, Institute of Oncology, Department of Radiation Oncology,
Capa, Istanbul, Turkey
Background: A connection between vitamin D deficiency and severe health
problems including various types of cancer has been demonstrated. The aim of
our study is to evaluate the relationship between VDR Taq I polymorphism of
vitamin D receptor gene polymorphism and oral squamous cell carcinomas
(OSCC) development.
Materials and Methods: The distribution of VDR Taq I polymorphism in
64 patients with OSCC was determined by polymerase chain reaction based
RFLP and compared with that of 99 healthy control subjects.
Results: There was a significant difference in the distribution of VDR Taq I
genotypes between OSCC patients and healthy controls. The allele frequencies T
and t in OSCC patients were 0.60:0.40 and those in normal individuals were
0.57: 0.43, respectively. Patients with the VDR tt genotype were found to be a
significantly lower risk for OSCC than those with another genotype (OR: 0.422;
95% CI 0.181–0.983) (p50.03).
Conclusion: Our results suggest that VDR Taq I polymorphism may be a
significant marker to asses susceptibility to OSCC in Turkey.
P-112
Hatice Yildirim1, Ali Bicer2, and Lulufer Tamer-Gumus1
1
Department of Biochemistry, Faculty of Medicine, Mersin University,
Turkey, 2Department of Physical Therapy and Rehabilitation, Faculty of
Medicine, Mersin University, Turkey
Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory disease that
causes articular and extraarticular symptoms and affects quality of life. It is possible that dietary and environmental factors and/or genetic polymorphisms in
xenobiotic metabolizing enzymes may contribute to the development of the disease. The N-acetyltransferase polymorphism 2 (NAT 2) is involved in the metabolism of many xenobiotics, as well as in susceptibility to some diseases such as
RA. The aim of the present study was to investigate whether the genetic polymorphism of the NAT2 play a role in susceptibility to rheumatoid arthritis.
The study population consisted of 62 patients with RA and 80 unrelated
healthy individuals. Blood was collected in EDTA-containing tubes and DNA
was extracted from leukocytes by high pure PCR template preparation kit.
NAT2*5A, NAT2*6A, NAT2*7A/B polymorphism of NAT2 were detected by
using a LightCycler- NAT2 mutation detection kit in real time PCR (Roche diagnostics, GmbH, Mannheim, Germany).
No association was observed between the NAT2 genotypes and RA. The frequencies of wild, heterozygous and mutant NAT2*5A genotypes were 38.7%,
50% and 11.3%; in cases and 41.3%, 55% and 3.7% in controls. The frequencies
of wild, heterozygous and mutant NAT2*6A genotypes were 61.3%, 30.6% and
8.1%; in cases and 57.5%, 40% and 2.5% in controls and the frequencies of wild
and heterozygous NAT2*7A/B genotypes were 69.4%, 30.6%; in cases and 61.2
%, 30.8% in controls. Further studies on larger groups are needed to determine
the prevalence of NAT2 polymorphisms in patients with RA.
P-113
P-111
INVESTIGATION OF SALIVARY MUC7 GENE ALTERATIONS IN
DENTAL STUDENTS WITH AND WITHOUT CARIES
Leyla Koc-Ozturk1, Aysen Yarat1, Korkut Ulucan1, Serap Akyuz2, and
Halit Furuncuoglu2
1
Basic Medical Sciences, Marmara University, Istanbul, Turkey, 2Clinical
Sciences, Marmara University, Istanbul, Turkey
Purpose: Human low-molecular-weight salivary mucin (MUC7), also known
as MG2, is a small, secreted glycoprotein that functions to help salivary defense
against oral bacteria by masking their surface adhesins and thereby inhibiting
colonization. Early studies indicate that MG2 takes place in the human salivary
non-immune defense system and interacts with oral micro-organisms, including
cariogenic bacteria. In this study, we aimed to identify the N- Terminal region of
MUC7 gene polymorphisms, if any, between individuals with and without caries.
SINGLE NUCLEOTIDE POLYMORPHISMS OF THE
FOLLICLE-STIMULATING HORMONE RECEPTOR GENE
IN FERTILE AND INFERTILE MALES
Mahmut Balkan1, Abdullah Gedik2, Selahaddin Tekes1, Selda Simsek1,
Aysegul Turkyilmaz1, Diclehan Oral1, and Turgay Budak1
1
Department of Medical Biology and Genetic, Dicle University, Diyarbakir,
Turkey, 2Department of Urology, Dicle University, Diyarbakir, Turkey
Single nucleotide polymorphisms (SNP) of the follicle-stimulating hormone
receptor (FSHR) gene influence follicle-stimulating hormone (FSH) concentrations
and the sensitivity of the FSHR to FSH in females. FSH in males plays a key role
in the maintenance of qualitatively and quantitatively normal spermatogenesis. It
controls gamete development through sertoli cells, via binding to its receptor. The
influence and importance of FSHR variants on Sertoli cell function is not completely understood and remains to be investigated. For this purpose, the possible
role of three FSHR SNP was evaluated in male infertility. SNP in exon 10 (codon
356
307 and 680) and in the core promoter region (at position -29) of the FSHR gene
were analyzed by polymerase chain reaction-restriction fragment length polymorphism technique in 50 men as a representative of the general population, 50 proven fathers, 50 normozoospermic controls, and 50 infertile patients classified
according to sperm parameters. Reproductive hormones were measured and compared in infertile males and normozoospermic men. LH and FSH were measured
by immunoradiometric assay and testosterone by radioimmunoassay.
2-fold. The increased ratio of nitrosylated sGC vs. total sGC suggests an endogenous regulation mechanism as was described in-vitro. However, the signaling
function of the pathway is unaffected as indicated in vasorelaxation experiments
and by increased VASP-phosphorylation in eNOS11.
P-114
ABSENCE OF CD38 COMPROMISES LEUKOCYTE RECRUITMENT
AND MACROPHAGE ACTIVATION DURING RESPONSES
TO MYCOBACTERIA
CASE REPORT: TETRASOMY 18p IN A MALE DYSMORPHIC CHILD
Mahmut Balkan1, Hatun Duran2, and Turgay Budak1
Department of Medical Biology and Genetic, Dicle University, Medical
Faculty, Turkey, 2Pediatric Surgery, Diyarbakir Children’s Hospital, Turkey
1
Tetrasomy 18p seems to display marked variability of phenotypic characteristics. However, combining certain common characteristics may point to a unique
picture of an individual with tetrasomy 18p. It is important to put on record such
rare cases of genetic conditions, as there are relatively small numbers of cases in
such category of chromosome anomalies providing opportunity to explore the
mechanisms of anomalies that may be generalized to other conditions. Here we
address these questions through our case, and we report tetrasomy 18p in a male
dysmorphic child. The case was 8-years-old male infant who was referred to our
laboratory for chromosomal analysis due to suspected Down syndrome. The clinical features of the case were summarized and shown to constitute a distinct and
recognizable syndrome. The phenotypic features were consistent when compared
to the standard clinical features of tetrasomy 18p. The cytogenetic analysis
revealed that the proband had 47,XY,1mar18. FISH analysis by using the whole
chromosome paints and subtelomeric probes confirmed that marker chromosome
was i(18p). The possible explanation for i(18p) could be somewhat the advanced
parental age at the time of conceiving the proband.
P-115
S-NITROSYLATION OF THE NITRIC OXIDE RECEPTOR SOLUBLE
GUANYLYL CYCLASE IN-VIVO
Marc Oppermann, Tatsiana Suvorava, and Georg Kojda
Institute for Pharmacology and Clinical Pharmacology Heinrich Heine
University, Duesseldorf, Germany
Purpose: Vascular soluble guanylyl cyclase (sGC) activity has been shown
to be regulated in-vitro by nitric oxide (NO) via a) down-regulation of the
expression and b) a post-translational S-nitrosylation mechanism. However, we
were previously able to show that sGC-activity was not affected in-vivo in nitrate-treated animals. In this study, we investigated the expression, activity, and
S-nitrosylation of sGC in mice with an endothelial-specific overexpression of
eNOS.
Methods and Results: eNOS11-mice with a 2.8-fold higher aortic eNOSexpression and a decreased systolic blood pressure were generated using the endothelium-specific Tie-2 promotor, transgene negative littermates served as controls (eNOSn). Western blots for both a1- and b1-subunits of sGC were performed in mouse lungs using polyclonal rabbit antibodies and showed similar
protein levels of sGCb1 (103.4610.3%) and sGC-b1 (109.7628.6%, P>0.05
each) in eNOS11 vs eNOSn. Phosphorylation of vasodilator-stimulated phosphoprotein (VASP) was increased by approximately 2-fold (P<0.05) as determined by Western blot with a P-ser239-VASP monoclonal antibody. Conversion
of [a-32P]-GTP to 32P-cGMP was used to measure sGC activity stimulated with
S-nitroso-N-acetyl-penicillamin (SNAP, 1 mmol/L). The activities of isolated
sGC were decreased in lungs of eNOS11 (8006250 vs. 15626250 pmol/mg/
min, n55, P<0.05). S-Nitrosylation of b1-sGC was semi-quantitatively evaluated
by labeling S-nitrosylated residues with biotin and performing Western blot after
separation on neutravidin-agarose (biotin switch assay). There was an increase of
nitrosylated/total sGC-b1 subunit of 158615.4% in eNOS11 (100% in eNOSn,
n54, P<0.05). Aortic vasorelaxation by acetylcholine and SNAP was analyzed
in organ bath experiments. Concentration-dependent vasodilation by endogenous
and exogenous NO was similar.
Conclusions: While expression of sGC does not seem to be affected by an
increased bioavailability of nitric oxide, activity of isolated sGC is decreased by
P-116
Marta Viegas da Silva1 and Teresa C. Martins1,2
1
Molecular Pathology Laboratory, Portuguese Institute for Oncology at
Coimbra, Portugal, 2Centre for Neurosciences and Cell Biology, University
Coimbra, Portugal
CD38 is a multifunctional ectoenzyme possessing signalling and enzymatic
properties. It is expressed in cells of several lineages, including B and T lymphocytes, and macrophages. CD38 was shown to play a role in the development of
T-cell dependent humoral immune responses against extracellular pathogens. Furthermore, CD38 also appears to be functionally important in macrophages, which
are the host cells of Mycobacterium avium. Our previous data showed that
CD38KO mice were more susceptible to mycobacterial infection than C57Bl.6
mice probably due to ineffective mycobacterial dissemination at early stages of
infection, compromised Th1 differentiation and polarization and inability to
physically restrain mycobacterial growth within target organs. We have now
investigated whether absence of CD38 altered macrophages’ ability to kill mycobacteria and to polarize Th1 immune responses. For that, we have established
bone marrow derived macrophages (BMDM) cultures from CD38KO and
C57Bl.6 mice and compared their response to mycobacterial infection. We have
found that CD38KO macrophages, similarly to those of the parental strain, were
able to ingest mycobacteria, form and acidify phagolysosomes, and produce similar amounts of reactive species and TNFa. However, when we performed co-cultures of BMDM and splenocytes, we found that production of TNFa, reactive
species and IFNgamma were decreased when CD38KO splenocytes were present
in culture. Our in vivo data showed that CD38KO mice, in contrast to C57Bl.6
mice, had a lower number of granulocytes and mononuclear cells at the site of
infection. Altogether, our data suggest that absence of CD38 does not alter macrophages’ intrinsic capacities but compromises the migration of responding cells
to the site of infection. Moreover, absence of CD38 on T cells leads to a
decreased production of IFNgamma which, in turn, compromises activation of
macrophages and the production of TNFa and of reactive species, increasing in
this way the susceptibility of CD38KO mice to mycobacterial infection.
This work was supported by FEDER and POTCI programs (POTCI/BCI/
44109/2002) and FCT (SFRH/BD/28570/2006).
P-117
THE PROTECTIVE EFFECT OF DEMETHOXYVIRIDINE ON
MICRONUCLEUS AND APOPTOSIS IN THE EARLY TYPE
HEPATOCARCINOGENESIS
Mehmet Cengiz Ustuner1, Derya Ustuner2, and Irfan Degirmenci1
Department of Medical Biology, Eskisehir Osmangazi Universty, Medical
Faculty, Turkey, 2Department of Medical Genetic, Eskisehir Osmangazi
Universty, Medical Faculty, Turkey
1
Objective: The attention was focused on the protective effect of demethoxyviridine on micronucleus and apoptosis in diethyl nitrosamine and 2-acetylaminoflourine administrated two month-old Sprague-Dawley rats. They cause the early
type hepatocarcinogenesis in MAP kinase pathway. The diethyl nitrosamine initiates preneoplastic liver lesions while 2-acetylaminoflourine promotes it in hepatocyte. The demethoxyviridine is fungal metabolite which inhibits PI-3 kinase.
Methods: Thirty two rats were divided into four groups of eight rats. The two of
them were designed for control administrations. The another groups included the
administration of the inducing agent (diethyl nitrosamine, 175 mg/kg) and the
promoting (2-acetylaminoflourine, 20 mg/kg) which were administrated intraperitoneally to the rats (0.1 ml in olive oil) through injection and gavages, respectively. The other one demethoxyviridine (1.5 mg/kg) was administrated with
inducer and promoter. Bone marrow samples were taken from rat femur after the
sacrification under ether anaesthesia at the 35th day. Apoptosis and micronucleus
were observed in bone marrow with cytokinesis blocked micronucleus method.
Results: The number of apoptotic cells and micronucleus increased in diethyl
nitrosamine and 2-acetylaminoflourine administrated rats while it was decreased
as compared to that in diethyl nitrosamine12-acetylaminoflourine1demethoxyviridine treated rats.
Conclusions: The demethoxyviridine inhibited PI-3 kinase in MAP kinase
pathway. This study revealed that demethoxyviridine prevents generation of
DNA damage in the early type hepatocarcinogenesis development.
P-118
NUCLEOPLASMIC BRIDGES IN CATECHIN AND CARBON
TETRACHLORIDE IN RAT BONE MARROW
Derya Ustuner1, Hulyam Kurt2, Mehmet Cengiz Ustuner2, and
Hilmi Ozden3
1
Department of Medical Genetic, Eskisehir Osmangazi University, Faculty
of Medicine, Turkey, 2Department of Biology, Eskisehir Osmangazi
University, Faculty of Medicine, Turkey, 3Department of Anatomy,
Eskisehir Osmangazi University, Faculty of Medicine, Turkey
Objective: Tea (Camellia sinensis) is one of the most popular beverages
worldwide. Among the various types of tea, green tea contains a relatively high
level of polyphenols which comprises flavanol monomers also referred to as catechin. It has chemo-preventive activity in animal models. The analysis of NPBs
(Nucleoplasmic Bridges) was recently validated as a biomarker of DNA damage.
The nucleoplasmic bridges were observed between daughter nuclei in cells. Carbon tetrachloride is a solvent used in petroleum industry, insecticides, furniture
industry, as cooling agent. Carbon tetrachloride treatments cause damage in rat
liver after 24 hour and a carcinogenic response.
Materials: Thirty six three-month old Sprague-Dawley rats were used in this
study. The animals were divided into three groups (each group was assigned
twelve rats) as carbon tetrachloride (0.2 ml/kg/day), catechin (5mg/kg/day) and
oil (0.2ml/kg/day) as control group. The bone marrow sample was taken into
1640 RPMI medium contained syringe from the rats. NPB was scored at least
1000 BN (Binucleated Cells) in rat bone marrow.
Results: Data showed that catechin reduced frequency of NPB against to carbon tetrachloride-treated (p<0.001). The NPBs were dramatically increased in
carbon tetrachloride treated rats.
Conclusion: The finding confirmed a decrease in NPBs and that catechin
affected the cytotoxic effect of carbon tetrachloride.
P-119
EFFECT OF FLUOXETINE IN PERIPHERAL BLOOD BY USING
CYTOKINESIS BLOCKED MICRONUCLEUS (CBMN) TECHNIQUE
Derya Ustuner1, Muhsin Ozdemir1, Mehmet Cengiz Ustuner2,
Beyhan Durak1, and Sevilhan Artan1
1
University, Faculty of Medicine, Turkey
Objective: Fluoxetine is an antidepressant medication. The aim of this study
is to determine the effects of different fluoxetine doses on peripheral blood by
using CBMN (Cytokinesis Block MicroNucleus) technique. Micronuclei provide
a measure of chromosome damage as classical metaphase chromosome analysis.
The cytokinesis-block technique using cytochalasin-B arrests division of cytoplasm or cytokinesis without inhibiting nuclear division and enables such cells to
be recognized by their binucleate appearance.
Methods: Peripheral blood samples were taken from 8 males and 8 females
aged between 25-35 years. The peripheral blood cells were incubated in 1640
RPMI medium. The cytochalasin B was added at 44th hour. The effects of different
dosages (10-5M, 10-6M, 10-7M), as well as incubation periods (24 hours, 48 hours)
of the fluoxetine were evaluated by the results of MI (Mitotic Index), numerical and
structural chromosomes irregularities and CBMN technique. Slices were stained
with conventional Giemsa staining and were scored at least 1000 binucleate cell.
Results: The effects of different dosages (10-5M, 10-6M, 10-7M), as well as
incubation periods (24 hours, 48 hours) with fluoxetine were evaluated by MI
357
(Mitotic Index), numerical and structural chromosomes irregularities and CBMN
technique.
Conclusions: In this study it was established that high doses of fluoxetine
had cytotoxic effects and caused mitotic delay. It was shown by Cytokinesisblock micronucleus assay that high doses of fluoxetine caused an increase of binnucleated (BN) cells with micronucleus due to chromosome aberrations.
P-120
NUCLEOPLASMIC BRIDGE AND NUCLEAR BUDDING IN
ADMINISTRATION OF FLUOXETINE IN VIVO
Derya Ustuner1, Mehmet Cengiz Ustuner2, and Muhsin Ozdemir1
1
University, Faculty of Medicine, Turkey
Objective: Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) for
oral administration. It was developed and marketed as an antidepressant. The
aim of this study is to determine the effects of high dose fluoxetine in bone marrow samples with nucleoplasmic bridge (NPB) and nuclear budding. Nucleoplasmic bridge (NPB) and nuclear budding are biomarkers of genomic instability
within the cytokinesis block micronucleus assay.
Methods: Twenty four male and 24 female two month-old Sprague Dawley
rats were used to study the effect of administration of fluoxetine. The fluoxetine
was injected intraperitoneally into rats with dosage of 5mg/kg, 7.5 mg/kg, 10
mg/kg, and 25 mg/kg for over a period of 3 weeks. Bone marrow samples were
prepared with CBMN (Cytokinesis Block Micro Nucleus) technique and slices
were stained with conventional Giemsa staining and examined under a light
microscope.
Results and Conclusions: Increased NPB (Nucleoplasmic Bridge) and nuclear
budding were observed in high dose fluoxetine treated bone marrow samples.
P-121
RECURRENT MISCARRIAGES IN A COUPLE WITH T(4,8)
AND INV (9)
Diclehan Oral, Sevgi Kalkanli-Tas, Mehmet Fidanboy, Aysegul Turkyilmaz,
and Turgay Budak
Departments of Medical Biology and Genetic, Medical Faculty,
Dicle University, Diyarbakir, Turkey
Nearly 60% of all first trimester spontaneous miscarriages are due to chromosomal abnormalities. The most frequent are numerical abnormalities (94%), followed by structural abnormalities (5%) and less frequently mosaicism (1%). We
report a 28 years-old couple in which an 4,8 translocation and 9 inversion has
been identified during the investigation for recurrent miscarriages. They were
phenotypically normal female and male. She had suffered four miscarriages in 7
years but no cytogenetic analyses had been performed in these aborts. In karyotyping by standard G-banding techniques of peripheral blood cultures showed
46,XY,t(4,8) and 46,XX,inv. The translocation was also confirmed by FISH studies. We conclude that cytogenetic analysis should be an integral part of etiological exploration in couples with recurrent abortions.
P-122
UTILIZATION OF ADAMTS1 AS A NEW TOOL FOR
DETECTING HYPOXIA
Mehmet Zeynel Cilek, Satoshi Hirohata, Omer Faruk Hatipoglu, Kadir
Demircan, Junko Inagaki, Tomoko Yonezawa, Toshikata Oohashi,
and Yoshifumi Ninomiya
Department of Molecular Biology and Biochemistry, Okayama University,
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
Objective: A disintegrin and metalloproteinase with thrombospondin motifs
(ADAMTS) is a newly discovered matrix metalloproteinase (MMP) with multiple domains, including metalloproteinase, disintegrin and spacer-region domains,
358
and domains containing thrombospondin (TSP) type I motifs. ADAMTS1 was
originally cloned from mouse cancer cell lines. Recently, we have reported that
ADAMTS1 was induced in the endothelium in myocardial infarction. Our data
indicated that ADAMTS1 may be a hypoxia-inducible gene expressed by endothelial cells. Interestingly, its expression is limited to a few hours’ hypoxia (i.e.,
acute ischemia). The aim of this study is to test the hypothesis that whether
ADAMTS1 promoter can be used for detecting acute hypoxia.
Methods: We cloned human ADAMTS1 promoter region. We performed luciferase assay driven by ADAMTS1 promoter in hypoxic cells. From the results
of luciferase assay, we identified the responsible promoter region of ADAMTS1
activated by hypoxia. We made green fluorescence protein (GFP) expressing construct under the control of the ADAMTS1 promoter. We then transfected GFP
construct into human umbilical vein endothelial cells (HUVEC), and examined
GFP production under hypoxia. For chemically mimicked hypoxia, cobalt chloride (CoCl2) was used. After 24 hours incubation, cells were fixed using 4%
PFA and observed under the fluorescence microscope.
Results: When GFP–transfected HUVEC were cultured in normoxic condition, GFP was not observed. In contrast, a considerable number of GFP-positive
HUVEC was observed when exposed to hypoxia or hypoxia-mimic.
Conclusions: The ADAMTS1 promoter may be used for detecting acute
hypoxia.
P-123
THE REGULATION OF DESMOSOMES IN
EPITHELIAL-MESENCHYMAL TRANSITION
Min-Che Chen
School of Biology, Chemistry and Health Science, Manchester Metropolitan
University, Chester Street, Manchester, M1 5GD, United Kingdom
Desmosomes are multimolecular complexes containing two transmembrane
glycoproteins, the desmosomal cadherins, desmocollins (Dsc) and desmogleins
(Dsg), two types of armadillo proteins, plakoglobin (PG) and plakophilins (PP);
and the plakin proteins, desmoplakins (DP), which link desmosomes to the intermediate filament cytoskeleton. Desmosomes represent major intercellular adhesive junctions at basolateral membranes of epithelial cells. Epithelial-mesenchymal transition (EMT) is an indispensable mechanism during embryogenesis,
tumour growth and epithelial degeneration. Epithelial cells must down-regulate
desmosomal adhesion to transform into mesenchymal cells. Hepatocyte growth
factor/scatter factor (HGF/SF) induces EMT in subconfluent Madin-Darby canine
kidney (MDCK) polarized epithelial cells. Time-lapse fluorescence microscopy
indicates that desmosomes are stable under normal conditions, whereas their stability is lost upon treatment with HGF/SF. Western blotting analysis of protein
expression demonstrates that Dsc2, Dsc3, Dsg3, PG, PP1, PP2, DP, E-cadherin
and b-catenin are down-regulated, but Dsg2 is not, after HGF/SF induced EMT.
Further investigation of the role of Dsg2 in EMT was conducted by generation
of stably expressing Dsg2 clones of MDCK cells. Unlike Dsc2-transfected
MDCK cells, Dsg2 transfected MDCK cells display a mesenchymal phenotype
in low cell density culture. Moreover, Dsg2 stably transfected clones exhibit
greater migration and invasion ability compared to non-transfected MDCK cells
or Dsc2-transfected cells. These novel findings have established a link between
desmosomal cadherins and EMT, contributing to a potential therapeutic target.
P-124
EPIGENETIC BIOMARKERS LINKED WITH METHYL GROUP
METABOLISM IN MAMMARY CANCER DIAGNOSIS
Natalia Cucu1, Gabriela Anton2, Otilia Zarnescu3, Anca Botezatu2,
Camelia Albu4, Mioara Matei5, Andrei Ovidiu-Tanase6, and Corina Posea7
1
Department of Genetics, University of Bucharest, Faculty of Biology,
Romania, 2Department of Molecular Biology, Institute of Virology ‘‘Stefan
Nicolau’’, Bucharest, Romania, 3Department of Developmental Biology,
University of Bucharest, Faculty of Biology, Romania, 4Department of
Bioanalysis, National Institute of Biological Sciences, Bucharest, Romania,
5
Department of Genetics, CF2 Clinic Hospital Bucharest, Romania,
6
Veterinary Faculty, University for Agronomical and Veterinary Medicine,
Bucharest, Romania, 7Department of Hemathology, University Emergency
Hospital Bucharest, Romania
Malignant transformation process may be determined by altered gene expression which defines the cancerous cell phenotype comprising tumor suppressor
genes silencing and oncogenes activation through aberrant DNA and histone
methylation dynamics. Methyl donor S-adenosylmethionine (SAM) and its product, S-adenosylhomocysteine (SAH), as well as MTHFR (methylenetetrahydrofolate) polymorphisms are also good indicators for the cell methylation reactions.
As mammary lesions are frequently spontaneous in dogs sharing the same environment with humans, we considered canines ideal models to study breast
cancerogenesis.
Tumour tissues were obtained from dogs and human patients during surgery.
Blood samples from patients and healthy human individuals were processed in 2
parts, designed for DNA/RNA and SAM/SAH extractions. A third part of tissues
was processed for fixation and immunolabelling. SAM/SAH ratio in tissues and
blood were estimated by HPLC. MSPCR and MS restriction were performed for
DNA hypermethylation in ER alpha, BRCA1 and RAR beta and p16 promotersand global DNA hypomethylated state. BRCA1/ER and H3 K9trime expression
in tumor tissues were estimated by IHC. Blood samples provided materials for
the analyses of methylation states in circulating DNA.
The results showed deregulation of cellular epigenetic process leading to
tumour morphogenesis. BRCA1, 2 promoter hypermethylation has been proved
by MSPCR, both in canine and human tumors; RT PCR/IHC confirmed the
methylation silencing effect upon these genes and, in humans, upon ERgene. The
decrease in nuclear H3K9 trimethylated or its perinuclear localization was associated with cell proliferation, probably with apoptosis or tumour progression stage
or with silencing processes. SAM/SAH ratio showed a pronounced decrease; typical for tumor tissues where global DNA hypomethylation occurs.
Circulating DNA showed similar methylation features linked with chromosomal instability and loss of cell cycle control, suggesting the need for further
investigation of such early crosstalks between the epigenetic marks in
cancerogenesis.
P-125
MOLECULAR BASIS OF HOMOCYSTINURIA: IDENTIFICATION OF
MUTATION IN CYSTATHIONINE BETA SYNTHASE (CBS) GENE
M.A. Kausar1 and M.D. Bashyam2
Department of Respiratory Allergy and Applied Immunology, VP Chest
Institute, Delhi, India, 2Department of Molecular Oncology, Centre for
DNA Fingerprinting and Diagnostics, Delhi, India
1
Introduction: Homocystine is a sulfur-containing amino acid, which is
known for congenitally acquired disease, homocystinuria. Homocystinuria is a
metabolic disorder due to cystathionine beta-synthase (CBS) deficiency, producing increased urinary homocystine and methionine. Major clinical manifestations
involve eyes, central nervous, skeletal and vascular systems. Mutation in cystathionine beta synthase gene has been identified as a molecular basis for the disease and is also responsible for its clinical heterogeneity. Hence, the present
study was performed to identify mutation(s) in CBS gene that are responsible for
homocystinuria in any individual.
Methods: Mutation analysis was carried out in CBS gene in the three
patients of having symptoms of homocystinuria, (a) patient 1 (5 year, female) 1,
(b) patient 2 (16year, male) and (c) patient 3 (30 year, male). Patient 1 had developmental delay and visual deformities. Patient 2 had operated congenital cataracts and deep vein thrombosis. In all the three patients, elevated levels of homocystine were found in urine. DNA was isolated from the blood sample of 3
patients. The exons 3, 4, 5, 6, 8, 10, 11, and 12 of CBS gene were amplified by
polymerase chain reaction (PCR) from the patient samples. Agarose gel electrophoresis was performed and amplified DNA was eluted out from the gel.
Sequencing was carried out to determine the sequence of amplified DNA. It was
then compared with the sequence of normal subject to determine the mutation(s).
Results and Discussion: The sequencing results indicated heterozygous
mutation found in 10th exon of patient 2 at position 1080 of CBS gene. No
mutation was observed in any other exon of patient 2. Further, there was no
mutation found in any exon(s) of patient 1 and patient 3. The heterozygous
mutation converted triplet code for alanine, GCC (normal) to GCT (patient). As
the mutation was observed in wobble base, it coded for the same amino acid,
even after mutation. So, there was no effect on the final protein (i.e. Cystathionine Beta Synthase).
Conclusion: Symptoms of homocystinuria and elevated level of homocystine
were not due to mutation in CBS gene in any of the three patients. They may be
due to several other disorders including methylcobalamin deficiency, vitamin
B12-responsive homocystinuria etc.
359
Results: Direct sequencing of exon 9 showed the presence of the same mutation at homozygous state. These results indicate that the fetus was affected by
LPI. The parents make a decision to interrupt the pregnancy.
Conclusion: This report reveals the utility of molecular diagnosis in clinical
practice and the importance of prenatal diagnosis by mutational analysis for the
families affected by LPI.
P-126
INVESTIGATION OF CRITICAL POLYMORPHISMS OF VITAMIN D
RECEPTOR (VDR) AND ESTROGEN RECEPTOR (ER) GENES IN
COLON CANCER
Mucteba Gunduz1, Yaman Tekant1, Ilhan Yaylim-Eraltan2, Turgay Isbir2,
and Ali Akyuz1
1
General Surgery, Istanbul University, Istanbul Faculty of Medicine,
Istanbul, Turkey, 2Department of Molecular Medicine, The Institute of
Experimental Medicine, Istanbul University, Capa, Istanbul, Turkey
It is well recognized that both genetic and environmental factors are involved
in the development of sporadic colon cancers. In sporadic cases, there may be
some genetic alterations that may increase the tendency to neoplastic changes
and this is a growing issue of research. Besides its effects on calcium metabolism, it is proposed that vitamin D regulates cell proliferation and differentiation.
Some authors say that estrogen may have similar effects on cell cycle. Both hormones act through their receptors and transformations in the genes of these
receptors may influence the function of these hormones. It has been reported that
particular changes in vitamin D and estrogen receptor genes may increase tendency to colon cancer. The aim of this study is to detect the association of single
nucleotide polymorphisms of vitamin D receptor (VDR) gene and estrogen receptor (ER) gene with colon cancer. Taq I and Bsm I polymorphisms of VDR
gene and PvuII polymorphism of ER gene were investigated at venous blood
samples. Genotype frequencies were found to be similar in both cases and controls. Alleles B and T were higher in the control group (p50.039 v2: 4.276; OR
0.312; 95% CI 0.100-0.973 and p50.039, v254.258, OR: 0.254, 95% CI 0.0641.000, respectively). The heterozygote genotype (Bb) was found to be higher in
the control group as well (p50.029, X254.795; OR: 2.783; 95% CI 1.0997.046). After combined genotype analysis, the genotypes TTBb were higher in
the control group at a statistically high significance (p50.001, v2: 11.854, OR:
0.122 95% CI: 0.032-0.460). Similarly, the genotype BbPb was higher in the
control group (p50.044, v2:4.073, OR: 0.359 95% CI: 0.131-0.984). In conclusion, allele B, allele T, the heterozygote genotype Bb, the genotype TTBb and
the genotype BbPp were found to lower the risk of colon cancer. Thus, BsmI
and TaqI polymorphisms of the VDR gene and PvuII polymorphism of the ER
gene may be one of the genetic factors that influence the development of colon
cancer.
P-128
THE COMMON 1471 DEL TTCT MUTATION OF SLC7A7
GENE FROM TUNISIAN PATIENTS WITH LYSINURIC
PROTEIN INTOLERANCE
N. Esseghir1, C. Bouchlaka Souissi2, N. Tebib3, S. Hadj Fredj4,
T. Messaoud4, Mf. Ben Dridi3, A. Ben Ammar Elgaaied2, and N. Kaabachi1
1
Laboratory of Biochemistry, Rabta University Central Hospital, Tunis;
2
Laboratory of Molecular Genetic, Immunology and Biotechnology, EL
Manar University, Tunis; 3Department of Pediatrics, Rabta University
Central Hospital, Tunis; 4Laboratory of Biochemistry and Molecular
Biology Children Hospital, Tunis, Tunisia
Objective: To identify the mutation of SLC7A7 gene from Tunisian patients
with Lysinuric Protein Intolerance (LPI) and to establish a basis of genetic counseling of their families.
Methods: Three affected children (2 girls and 1 boy) belonging to 2 unrelated consanguineous families, originated from a restrict area of the North of
Tunisia were diagnosed on the basis of clinical features and biochemical data.
Peripheral blood samples were collected from the affected (3), parents (6) and
unaffected sibs (6). DNA was extracted by standard procedure using a phenol
chloroform protocol. Mutation’s screening of all the SLC7A7 gene exons was
performed using the DHPLC assay (Transgenomic). The confirmation of the
mutation was carried out by direct sequencing (Applied Biosystem).
Results: Heteroduplex profiles were obtained by DHPLC within exon 9 in
the 3 probands. Direct sequencing of this exon showed the presence of the 1471
Del TTCT mutation at homozygous state in all patients. Among the unaffected
children, 3 were heterozygous.
Conclusions: In the present study, we have identified the 1471 Del TTCT
mutation in 3 patients with LPI. This mutation has previously been reported by
other authors including families originated from Tunisia. It seems to be a common mutation to a Tunisian population as it has been already identified. The
identification of this specific mutation provides a tool that can be applied in confirmatory diagnosis, genetic counseling, carrier prediction and prenatal diagnosis.
P-129
P-127
FIRST TUNISIAN EXPERIENCE OF PRENATAL DIAGNOSIS OF
LYSINURIC PROTEIN INTOLERANCE BY DIRECT
MUTATIONAL ANALYSIS
N. Esseghir1, C. Bouchlaka Souissi2, N. Tebib3, S. Hadj Fredj4,
T. Messaoud4, Mf. Ben Dridi3, A. Ben Ammar Elgaaied2, and N. Kaabachi1
1
Laboratory of Biochemistry, Rabta University Central Hospital, Tunis;
2
Laboratory of Molecular Genetic, Immunology and Biotechnology, EL
Manar University, Tunis; 3Department of Pediatrics, Rabta University
Central Hospital, Tunis; 4Laboratory of Biochemistry and Molecular
Biology, Children Hospital, Tunis, Tunisia.
Objective: To report the first Lysinuric Protein Intolerance ‘‘LPI’’ prenatal
diagnosis performed in Tunisia by direct molecular analysis.
Methods: A 30 years-old woman asked for a prenatal diagnosis because the
eldest child, a ten- years- old girl, is affected by LPI. This diagnosis was establish at twenty two months of age according to clinical and biochemical criteria.
The 1471 Del TTCT mutation was identified in exon 9 by direct sequencing. For
the prenatal diagnosis of the third pregnancy, amniotic fluid sample was carried
out at 15 weeks of gestation. DNA was extracted from peripheral blood samples
of both parents and from amniotic fluid sample, and direct sequencing of exon 9
was carried out.
CHARACTERIZATION OF SIGNALING PROPERTIES OF
THE ENDOTHELIN SYSTEM IN MOUSE MAMMARY
GLAND PHYSIOLOGY
Nadir Gul, Andreas Fisher, and Franz Theuring
Center of Cardiovascular Research, Institute of Pharmacology, Germany
Background: Endothelin 1 (ET-1) is a vasoactive peptide implicated in the
regulation of a variety of physiological and pathophysiological processes. In
addition to the activation of its G-protein coupled receptors, this peptide furthermore exerts its effect by modulating the activation of receptor tyrosine kinases
such as the EGFR, previously shown to be important both in mammary gland development and tumorigenesis. We therefore aimed to analyze the role of endothelin in the regulation of mammary gland development.
Methods: Transgenic mice expressing human endothelin in the mammary
gland were employed. Global function of the mammary gland was assessed by
recording weight gain of the pups and standard histology was used to analyze
morphology of the mammary gland. Expression of the major milk proteins
WAP, b-casein and a-lactalbumin as well as the EGFR ligands amphiregulin,
EGF, TGFa and HB-EGF was analyzed by quantitative RT-PCR.
Results: Lactational competence was impaired in ET-transgenic females as
reflected by reduced weight gain of offspring suckled by transgenic mothers as
compared to wild type pups. Histologically, mammary gland morphology showed
no abnormalities in transgenic animals and expression of the major milk proteins
360
was not altered. However, expression of amphiregulin was significantly up-regulated in ET-transgenic mammary glands whereas other EGFR ligands displayed
no abnormalities.
Conclusion: Our observations suggest that endothelin might have an important role in the regulation of lactation. We furthermore provide evidence that
amphiregulin, an EGFR ligand, previously shown to regulate mammary gland development, is induced by endothelin, providing a potential explanation for the
observed phenotype.
P-130
CRUCIAL ROLE OF PARENTAL MTHFR GENE
POLYMORPHISM INVOLVED IN HOMOCYSTEINE/METABOLISM
IN DOWN SYNDROME
Nadir Kocak1, Filiz Ozen2, Recep Kesli3, Ilhan Sezgin2,
and Ozturk Ozdemir2
1
Genetics Department, Konya Educational and Research Hospital, Turkey,
2
Department of Medical Genetics, Medical Faculty, Cumhuriyet University,
Sivas, Turkey, 3Molecular Diagnostic Unit, Konya Educational and
Research Hospital, Turkey
Introduction: Trisomy 21 is known as a complex phenomenon that occurs
due to the failure of abnormal chromosome segregation during meiosis. The current study was aimed at finding out the role of parental MTHFR and ACE gene
polymorphism as a risk for Down syndrome.
Material and Method: Down syndrome patients and their parents were analysed at molecular level, clinically and cytogenetically. Peripheral blood cells
were used for chromosome analysis, genomic DNA isolation and reverse hybridization of strip assay technique was used to screen the allelic polymorphism/
mutation in Down syndrome patients and their parents.
Results: Initial results of the current study showed the paternal polymorphisms of 677C-T and 1298A-C of MTHFR gene as risk factors for Down syndrome pregnancies.
P-131
PREVALENCE OF THE FAMILIAL MEDITERRANEAN FEVER
COMMON GENE MUTATIONS IN PATIENTS WITH SIMPLE
FEBRILE SEIZURES
Filiz Ozen1, Nadir Kocak2, Recep Kesli2
1
Department of Medical Genetics, Cumhuriyet University, Medical Faculty,
Sivas, Turkey, 2Department of Genetics, Konya Educational and Research
Hospital, Turkey
Introduction: Febrile seizures (FSs) are the commonest form of convulsions,
occurring in 2–5% of infants in Europe and North America, and 6–9% in Japan.
A simple FS is clinically defined as a brief generalized seizure that occurs only
once during a 24 h period in a febrile child who does not have an intracranial
infection or severe metabolic disturbance. A genetic predisposition to FSs is
known, based on family studies, twin studies, and complex segregation analysis.
Simple FSs may be more homogenous in their clinical manifestations, and show
better agreement with the multifactorial inheritance theory than the complex
type. Interleukin-1 (IL-1) is one of the pro-inflammatory cytokines that is postulated to be involved in the development of FSs. To determine whether or not
function-related polymorphisms of the IL-1 (IL1B) gene are associated with susceptibility to simple FSs, Interleukin-1 (IL-1) is one of the pro-inflammatory
cytokines that play important roles in host defense responses to infection and
inflammation both within and outside the central nervous system, and acts as an
endogenous pyrogen. Fever is regarded as the main trigger in the pathogenesis of
FSs. Otherwise, FMF patients are in a proinflammatory state in attack free periods, and during attacks, symptoms and signs of overt inflammation are observed.
IL-1 and pyrin play a major role in FMF disorder. These observations led us to
conclude that the FMF polymorphism might be a risk factor for FBs in human
population. Our aim in the present study was to investigate whether the most
common MEFV gene mutations has any role in the etiology of FBs.
Materials and Method: MEFV gene mutations are investigated with reverse–
hybridization method with three steps. DNA isolation from anticoagulated blood
(Invisorb Spin Blood Mini kit Invitek-Germany), in vitro amplification with thermal cycler (Bioer XP Cycler, Japan) and finally the amplification products are
selectively hybridized to the test strips (Vienna Lab, Austria) contains oligonucleotide probes with Auto-LiPA instrument (Tecan ProfiBlot T48, Austria). The
assay covers the following 12 MEFV mutations.
Results: Our results suggest that occurrence of MEFV Mutation Carriers in
patients with Simple Febrile seizures is a very frequent event (34.1%) compared
with the estimated gene frequency of Turkish population (15-20%).
P-132
DETERMINATION OF DISTRIBUTION OF COMMON MEFV GENE
MUTATIONS IN HEALTHY INDIVIDUALS WHO HAD BEEN
EXPOSED TO APPENDECTOMY IN CHILDHOOD AGE
Nadir Kocak1, Filiz Ozen2, M. Serif Arslan3, and Ozturk Ozdemir2
1
Molecular Diagnostic Unit, Konya Educational and Research Hospital,
Turkey, 2Department of Medical Genetics, Cumhuriyet University, Medical
Faculty, Sivas, Turkey, 3Department of Pediatric Surgery, Cumhuriyet
University, Medical Faculty, Sivas, Turkey
Introduction: Familial Mediterranean Fewer (FMF) resulting from mutation
in the gene MEFV, which is located in the short arm of chromosome 16, is one
of the most common causes of acute non-surgical abdominal pain in children
among the Mediterranean countries. Abdominal attacks are characterized by
severe abdominal pain with fever and abdominal tenderness in 95% of patients.
Abdominal pain with peritoneal signs is the first clinical presentation in 50% of
the patients. Laboratory findings include leukocytosis, increased level of acute
phase reactants. It may be difficult to differentiate FMF and other surgical acute
abdominal conditions during abdominal attack, especially for acute appendicitis
due to similar symptoms and laboratory findings. Approximately two third of
FMF patients undergo surgical intervention with the provisional diagnosis of appendicitis in any period of life and the appendix is found normal in most of the
cases.
Material and Method: Fifty-four healthy individuals with acute abdomen
history who underwent operation with the clinical diagnosis of acute appendicitis
were enrolled in this study. We analyzed 12 common MEFV gene mutations in
these individuals. MEFV gene mutations were investigated with reverse–hybridization method in three steps. DNA isolation from anticoagulated blood (Invisorb
Spin Blood Mini Kit Invitek-Germany), in vitro amplification with thermal cycler
(Bioer XP Cycler, Japan) and finally the amplification products are selectively
hybridized to the test strips (Vienna Lab, Austria) contains oligonucleotide
probes with Auto-LiPA instrument (Tecan ProfiBlot T48, Austria). The assay
covers the following 12 MEFV mutations.
Conclusion: Our results suggest that frequency of MEFV mutation carriers
in healthy individuals with appendectomy history is a frequent event compared
with the estimated gene frequency of Turkish population.
P-133
RESULTS OF 543 MUTATIONS DETERMINED ON FAMILIAL
MEDITERRANEAN FEVER (FMF) GENE (MEFV) OF EVENTS
CONSIDERED FMF
Recep Kesli1, Nadir Kocak2, and Bulent Atas3
1
Molecular Diagnostic Unit, Konya Educational and Research Hospital,
Turkey, 2Department of Genetics, Konya Educational and Research
Hospital, Turkey, 3Department of Genetics, Dr. Faruk Sukan Maternal and
Children’s Hospital, Turkey
Introduction: Familial Mediterranean Fever (FMF) is an autosomal-recessive
inherited inflammatory disorder which is characterized by recurrent, short, selflimiting bouts of fever, accompanied by pain in the abdomen, chest or joints,
and sometimes associated with erysipelas-like erythema. The most severe complication of the disease is amyloidosis which causing renal failure. In this study,
FMF gene (MEFV) mutations analyses were made of events considered FMF in
Konya Educational and Research Hospital Central Laboratory Molecular Diagnostic Unit.
Material and Method: MEFV gene mutations were investigated with reverse–
hybridization method with three steps. DNA isolation from anticoagulated blood
(Invisorb Spin Blood Mini Kit, Invitek-Germany), in vitro amplification with thermal cycler (Bioer XP Cycler, Japan) and finally the amplification products were
selectively hybridized to the test strips (Vienna Lab, Austria) contains oligonucleotide probes with Auto-LiPA instrument (Tecan ProfiBlot T48, Austria). The assay
covers the following 12 MEFV mutations: E148Q, P369S, F479L, M680I (G/C),
M680I (G/A), I692del, M694V, M694I, K695R, V726A, A744S, R761H.
Results: Total 543 mutations were determined in 829 persons. Fifty (6%)
persons were homozygous and 4 of those persons were double gene mutation.
111 (13%) of remaining 332 (40%) heterozygous persons were double gene
mutation. FMF gene mutation analysis results were like this; 197 (36%) mutations were M694V (147 heterozygous, 25 homozygous); 108 (20%) mutations
were M680I (G/C) (74 heterozygous, 17 homozygous); 86 (16%) mutations were
E148Q (84 heterozygous, 1 homozygous ); 66 (12%) mutations were V726A (66
heterozygous); 28 (5%) mutations were M680I(G/A) (8 homozygous, 12 heterozygous); 27 (5%) mutations were P369S (27 heterozygous); 11 (2%) mutations
were R761H (11 heterozygous); 8 (1%) mutations were M694I (4 heterozygous,
2 homozygous ); 11 (2%) mutations were A744S (11 heterozygous); 8 (1%)
mutations were K695R (7 heterozygous, 1 homozygous). F479L and I692del
mutations were not determined.
Conclusion: As a result of our study, documentation of FMF gene mutations
was determined in our region and the most frequent was M694V gene mutation.
P-134
ANGIOTENSIN CONVERTING ENZYME (ACE) GENE
POLYMORPHISM AND BUERGER’S DISEASE
Filiz Ozen1, Nadir Kocak2, Ozturk Ozdemir1, and Ilhan Sezgin1
1
Department of Medical Genetics, Cumhuriyet University, Medical Faculty,
Sivas, Turkey, 2Department of Medical Genetics, Konya Educational and
Research Hospital, Turkey
Introduction: The exact cause of Buerger’s disease is not known. Epidemiological studies have shown a strong correlation between cigarette smoking and
development and progression of cardiovascular disease such as Buerger’s disease.
The mechanism for the increased cardiovascular risk of cigarette smoke is not
well understood but it is presumed to be related to endothelial dysfunction. ACE
participates in the control of vascular resistance by generating AII and degrading
bradykinin. AII also acts as a vascular growth factor participating in angiogenesis, vascular remodeling, response to vascular wall injury, and atherogenesis. As
a regulator of AII production ACE may have an important role in atherosclerosis
and hypertension. In vitro, nicotine has been shown to be mitogenic for endothelial cells, smooth muscle cells and fibroblasts. It modulates the expression of various proteins such as bFGF, TGFß, TNF alpha, PAI-1, NOS and VEGF in endothelial cells. Nicotine has been reported to accelerate the growth of atherosclerotic plaques and tumors in experimental animals and it has been suggested that
VEGF has a role in these processes. Thus, synergistic interaction of nicotine and
VEGF in ACE induction may enforce detrimental growth processes in the vascular wall by increasing AII generation and bradykinin degradation.
Material and Method: Patients with Buerger’s disease were enrolled in this
study. We analysed ACE gene mutations of these patients. MEFV gene mutations were investigated with reverse–hybridization method with three steps.
Conclusion: Our results suggest that frequency of ACE gene mutation carriers in patients with Buerger’s disease is a frequent event compared with the
estimated gene frequency of Turkish population.
P-135
DNA HYPOMETHYLATING EFFECT OF A GERIATRIC PROTECTIVE
DRUG AND CORRELATIONS WITH EPIGENETIC BIOMARKERS
OF AGING
Natalia Cucu1, Gabriela Anton2, Anca Botezatu2, Ileana Turcu3,
Luiza Spiru3, and Liliana Burlibasa1
1
Romania, 2Department of Molecular Biology, Institute of Virology,
Bucharest, Romania, 3Research and Development, Ana Aslan International
Academy of Anti-Aging, Bucharest, Romania
361
Objective: Epigenetic biomarkers such as the global DNA hypo- and local
hypermethylation are used for an investigation of the genome stability linked
with the aging process. An experimental model is proposed to study the relation
between the DNA hypomethylation and certain potent geriatric protective agents
such as procaine in Gerovital drug, by in vitro (blood lymphocyte from untreated
individuals/cancer derived cell lines) and in vivo (blood lymphocyte from at least
30 years treated individuals) approaches.
Methods: In our study a cohort of six - elderly (55 plus years), adult (30-55
years), and young (18-30 years) groups, has been approached, as follows: one
young group with controlled sportive life, one young group with uncontrolled
lifestyle, two adult groups with (cancer) and without health problems, and two
elderly groups, with vs. without significant pathology. DNA local hypermethylation have been studied through methylation specific PCR method in ER alpha
promoter and general DNA methylation level through HPLC/methylation sensible
restriction methods.
Results: DNA methylation dynamics was similar both during aging and cancerogenesis process showing concomitant global DNA hypo- and local DNA hypermethylation, even targeting the envisaged genes. Specific epigenetic drugs,
such as Trichostatin A and 5Aza cyditidine, as well as Gerovital, a product containing procaine, were studied in order to demonstrate their putative hypomethylating power on global DNA and targeted genes promoters. In vivo studies
proved that persons treated with Gerovital during at least 30 years presented a
visible global DNA hypomethylation, however, a general healthy life extension.
Conclusions: One carbon (methyl group) metabolism is actually considered
a very important cell pathway linking the environment and the genome stability
with a dramatic impact on our health status. The study envisaged the importance
of the epigenetic biomarkers when new therapeutically or nutritionally personalized strategies have to be considered.
P-136
EPIGENETIC METHOD FOR THE EARLY DIAGNOSIS OF PRADER
WILLI SYNDROME
Natalia Cucu1, Gabriela Anton2, Anca Botezatu2, Maria Puiu3,
and Dorica Dan4
1
Romania, 2Department of Molecular Biology, Institute of Virology,
Bucharest, Romania, 3Department of Genetics University of Medicine and
Pharmacy, Timisoara, Romania, 4APWR, Zalau, Romania
Prader Willi (PWS) and Angelman (AS) syndromes are determined by two
identical genetic defects (large deletions, uniparental disomia and only rare balanced translocations - involving 15 chromosome), however they are clinical, metabolic and neurological different syndromes with 1 case for 15000 newborns.
Epigenetic modifications in the processes of imprinting may differentiate the parent germline which determined the defects and therefore may direct the early
proper interventions for either one of the two similar imprinting diseases. Our
epigenetic diagnosis involved the estimation of DNA methylation status at maternal and paternal alleles of SNRPN-SNURF locus from the critical chromosome.
Blood samples from patients and their relatives were monitored by the Romanian
PWS Association. DNA samples were obtained using Invitek blood extraction kit
and Methylation Specific PCR method has been performed for the estimation of
the DNA methylation at maternal and paternal alleles of SNRPN-SNURF locus
from critical chromosome 15. DNA covalent modification, together with certain
other epigenetically codified histone modifications contributes to gene expression
control in eukaryotes through normal developmental processes, an indirect relation between DNA methylation and gene expression providing an excellent biomarker of aberrant gene silencing in pathological states. FISH assay for
15q11:q13 region deletions provided identical results for both syndromes conducting us towards an epigenetic approach to differentiate the parental source of
this deletion. The MSPCR amplicons electrophoretic analyses indicated the methylation of the paternal allele, in the case of PWS, which was clinically diagnosed
based on developmental delay, cryptorchidism, hyperphagia obesity, short stature
and mild retardation.
Once the epigenetic diagnosis would be implemented, one major problem
remains that is referred to ‘‘transgenerational critical windows’ towards environment (such as diet and lifestyle) which may confer a specific feature of genome
instability in the critical 15 chromosome region, prone to be transmitted aberrantly to descendant, like ‘‘erasure’’ events during gametogenesis.
362
P-137
PLASMID DNA AND sIRNA TRANSFECTION OPTIMIZATIONS OF
HARD-TO-TRANSFECT HUMAN BONE MARROW MESENCHYMAL
STEM CELLS (hMSCs)
Nebiyyeh Kamaci and Sevim Isik
Department of Biology, Fatih University, Turkey
Objective: To get the highest efficiency at transfection of bone marrow
derived human Mesenchymal Stem Cells (hMSCs) with plasmid based siRNAs
and siRNA oligos by using several transfection reagents.
Methods: hMSCs were grown in Dulbecco’s modified Eagle’s medium
(DMEM, Gibco) with 15% hMSC qualified Fetal Bovine Serum (Gibco). Human
Embryonic Kidney 293 (HEK293) cells and Human Umbilical Vein Endothelial
Cells (HUVEC) were used as positive controls since they are easy cell lines to
make transfection. hMSCs, HEK293 and HUVEC cells were seeded into 96 well
plate one day prior to transfection. Just before the transfection, medium was
refreshed. Cells were transfected at a density of 70-80% confluency in the presence and absence of serum. Green Flourescent Protein (GFP) Plasmid DNA and
AlexaFlour labeled non target siRNAs were used to monitor transfection efficiencies of reagents. LipofectAMINE 2000 (Invitrogen) and FuGENE (Roche) were
evaluated for transfection efficiencies at different ratios of siRNA/reagent and
DNA/reagent. Medium was replaced 24 hours post-transfection with DMEM containing FBS. The cells were incubated at 37 8C for 24-72 hours prior to analysis
under fluorescent microscope.
Results: Both DNA and siRNA were successfully introduced into cell lines
such as HEK293 and HUVECs with either reagent. Transfection efficiency at
hMSCs was no more than 20% with FuGENE and 30% with LipofectAMINE2000. In the absence of serum, toxic effect of reagents was observed.
Also there was no increase in transfection efficiency at serum free transfections.
Conclusion: Primer cells especially hMSCs are very hard to transfect with
respect to other cell lines. More promising reagents in the transfection of primary
cells such as Lipofectamine RNAiMAX (Invitrogen) and PrimeFect siRNA
(Lonza) will be tested soon.
Table 1
Plasmid Transfection Efficiencies at hMSCs and HEK293 cells
Lipo 1 FBS
Lipo - FBS
FuG 1 FBS
FuG - FBS
hMSCs
HEK293
30%
10%
20%
5%
95%
90%
95%
90%
P-138
THE EFFECT OF PARAOXONASE 1 L/M55 AND Q/R 192 GENE
POLYMORPHISM ON PARAOXONASE 1 ACTIVITY AND LIPID
LEVELS IN TYPE 2 DIABETES MELLITUS
Muhittin Onderci1, Necip Ilhan2, Dilara Kaman2, and Yusuf Ozkan3
1
Department of Biochemistry, Veterinary Control and Research Institute,
Turkey, 2Department of Biochemistry, Faculty of Medicine, Firat
University, Turkey, 3Department of Endocrinology, Faculty of Medicine,
Firat University, Turkey
Background: Paraoxonase 1 (PON 1) is a HDL-associated enzyme with antioxidant function protecting LDL from oxidation. PON 1 has two amino acid
polymorphisms in coding region; one Leu-Met at position 55 (L/M55) and the
other Glu-Arg at position 192 (Q/R 192). L/M 55 and Q/R 192 polymorphisms
modulate paraoxonase activity and antioxidant function of the enzyme. In the
present study, distribution of PON 1 L/M 55 and Q/R 192 polymorphisms and
the effect of these polymorphisms on the activities of PON 1 and lipid profile in
102 non-diabetic healthy subjects (control group) and 200 patients with type II
diabetes mellitus (DM group) were examined.
Methods: PON 1 L/M 55 and Q/R 192 genotypes were determined by PCR,
RFLP and agarose gel electrophoresis techniques. Serum PON 1 activity was
determined using paraoxon and measured by spectrophotometer. Serum lipids
were determined by an autoanalyzer.
Results: Genotype distributions and allele frequencies for both polymorphisms
were not significantly different between controls and DM group (p>0.05). Serum
PON 1 paraoxonase activity was lower in DM group than in controls but not statistically significant (p>0.05). In both groups, the highest paraoxonase activities were
detected in LL and RR genotypes, intermediate activities in LM and QR genotypes,
and the lowest activities in MM and QQ genotypes. No significant difference in
HDL-cholesterol levels was detected between both groups but LDL-cholesterol,
total cholesterol and triglyceride levels were significantly higher in DM group
(p<0.001). Presence of R and M alleles in control group and presence of Q and M
alleles in DM group accompanied with higher atherogenic lipid profile.
Conclusions: In conclusion, data obtained from the present study indicates
that PON 1 L/M 55 and Q/R 192 polymorphisms cause significant alterations in
paraoxonase activity.
P-139
RELATIONSHIP OF CLASSICAL AND NON-CLASSICAL RISK
FACTORS WITH GENETIC POLYMORPHISM OF PON 1 RELEVANT
TO CORONARY ARTERY DISEASE
Dilara Kaman1, Necip Ilhan1, Kerem Metin1, Mehmet Akbulut2,
and Bilal Ustundag1
1
Department of Biochemistry, Faculty of Medicine, Firat University,
Turkey, 2Department of Cardiology, Faculty of Medicine,
Firat University, Turkey
Background: In addition to the well established cardiovascular risk factors,
evidence suggests a possible role of genetic and non-classical risk factors, such
as Lp(a), Oxide LDL, in the development and progression of atherothrombosis.
We aimed to determine the relationship of classical and non-classical cardiovascular risk factors with polymorphisms (PON55 L:M, PON191 Q:R) of paraoxonase gene potentially involved in cardiovascular risk in the Turkish population.
Increased concentrations of lipoprotein(a) [Lp(a)] have been considered a genetically determined risk factor for coronary artery and cerebrovascular disease. Oxidized low density lipoprotein (oxLDL) particles are known to initiate the development of coronary artery disease.
Methods: We have determined the prevalence of classical (lipid profile,
blood pressure, glycaemia, diabetes, smoking, body mass index and family history of coronary heart disease) and non-classical cardiovascular risk factors (lipoprotein(a), PON levels, Oxide LDL) in a population-based study. PON 1 L/M 55
and Q/R 192 genotypes were determined by PCR, RFLP and agarose gel electrophoresis techniques. We analyzed the relationship of these risk factors with PON
gene polymorphism potentially involved in cardiovascular risk.
Results: Genotype distributions for PON 1 L/M55 polymorphism were significantly different between controls and CAD (1) patient group (p<0.05), but
genotype distribution of PON 1 Q/R192 polymorphism, there was no significant
difference among groups (P>0.05). Serum PON 1 paraoxonase activity was
lower in CAD (1) patients than in controls. In both groups, the highest paraoxonase activities were detected in LL and RR genotypes, intermediate activities in
LM and QR genotypes, and the lowest activities in MM and QQ genotypes. The
highest levels of HDL and the lowest level of oxide –LDL was detected in MM
genotype in CAD (1) patients (p<0.05).
Conclusions: PON 1 L/M55 gene polymorphism, but not PON 1 Q/R192,
were associated with non-classical risk factors, specifically Lp(a), Oxide LDL
and PON levels in CAD(1) patients.
P-140
GLU298ASP POLYMORPHISM OF THE ENDOTHELIAL NITRIC
OXIDE SYNTHASE GENE AND PLASMA CONCENTRATIONS OF
ASYMMETRIC DIMETHYLARGININE IN TURKISH PATIENTS
Fahri Turan1, Necip Ilhan2, Dilara Kaman2, Kadir Ates2, and
Ayse Kafkasli3
1
Department of Biochemistry, Faculty of Medicine, Malatya University,
Turkey, 2Department of Biochemistry, Faculty of Medicine, Firat
University, Turkey, 3Department of Obstetrics and Gynecology,
Faculty of Medicine, Malatya University, Turkey
Objectives: Worldwide, pre-eclampsia is a leading cause of maternal death,
affecting 3% to 5% of all pregnancies. We analyzed the association of the
Glu298Asp polymorphism of the endothelial nitric oxide synthase (eNOS) gene
with pre-eclampsia and asymmetric dimethylarginine (ADMA) in 55 Turkish
patients with pre-eclampsia and in 54 healthy pregnant women.
Methods: Restriction fragment length polymorphism analysis of Glu298Asp
of the eNOS gene was performed by amplification of genomic DNA isolated
from whole blood followed by digestion with the restriction enzyme Frio. Preeclampsia was defined according to the Working Group (2000) criteria, as high
blood pressure (R140/90 mmHg after 20 weeks’ gestation) and proteinuria (R300
mg/24 h). Serum arginine, ADMA and SDMA levels were measured by HPLC.
Results: Genotypes were defined as GG, GT, and TT according to the presence of the G and T alleles. In this case-control study, we did not find any significant difference in either the genotypic distribution or allelic frequency of
Glu298Asp gene polymorphism between the pre-eclamptic patients and healthy
pregnant. Serum ADMA, arginine and SDMA levels were higher in patients with
pre-eclampsia compared with healty pregnant subjects (P<0.0001).
Conclusions: The results suggested the lack of association between the
Glu298Asp gene polymorphism and pre-eclampsia in the Turkish population.
Elevated ADMA and SDMA levels suggest that ADMA has a role in the pathogenesis of pre-eclampsia.
P-141
363
Genetics and Genomic Sciences Center, VKV American Hospital,
Istanbul, Turkey
Breast cancer is the most common cancer among women and the second
highest cause of cancer death. It remains a significant health problem and represents a significant worry for many women and their physician. During the
last years, intensive research has been focused on accurate risk estimation for
breast cancer development. Preventive strategies, including genetics susceptibility testing may be applied for the women at high risk for breast cancer development and hormone replacement therapy effects during menopause. In the
present study we examined the genes, that were reported as associated with
breast cancer risk and hormone replacement therapy-risks and benefits in the
literature, in Turkish female population; Transforming Growth Factor Beta Receptor Type 1 (TGFBR1) *9A[*6A, Progesterone Receptor (PGR) G[A Pos.
1331, Androgen receptor (AR) short[long alleles, Vitamin D Receptor (VDR)
IVS7 1283 G[A (b[B), Cytochrome P450 1A1 (CYP1A1) T[C Pos. 3801,
Cytochrome P450 1B1 (CYP1B1) Leu[val codon 432, Collagen Type Ia1
(COL1a1) Pos. 1546 G[T/S[s, Cytochrome P450 17 A1 (CYP17A1) T[C
Pos. -34, Estrogen Receptor-a (ESR1) IVS -401 T[C (p[P), Factor II/Prothrombin (F2) G[A Pos. 120210, Factor V Leiden (F5) Arg[Gln Codon 506,
Catechol-O-Methyltransferase (COMT) Val[Met codon 158, Cytochrome P450
1A1 (CYP1A1) Ile[Val codon 462, Cytochrome P450 1B1 (CYP1B1)
Asn[Ser Codon 453, Aromatase (CYP19A1) C[T Pos. 11558. We analyzed
32 female patients. We obtained the mutant allele frequencies for the SNP’s for
Turkish female population. Gene-environment or gene-gene interactions may
possibly give further insight into both the etiology and the prevention of breast
cancer and the hormone replacement therapy risks. Special population of
women, who carry SNP’s associated with breast cancer and also the hormone
replacement therapy-risks and benefits- requires unique management strategies
and deserves separate consideration.
PREIMPLANTATION GENETIC TESTING OUTCOMES FOR POOR
PROGNOSIS CRYPTIC TRANSLOCATION CARRIER COUPLES
Nesrin Ercelen1, Emel Tutar1, Meral Gultomruk1, Levent Erkan1,
and Bulent Urman2
1
Genetics and Genomic Sciences Center, VKV American Hospital, Turkey,
2
Women’s Health Center, VKV American Hospital, Turkey
Objective: To present the preimplantation genetic testing results of reciprocal and
robertsonian translocations that were analyzed in 22 ICSI/PGD cycles for 18 couples.
Materials and Method: Cytogenetic analysis was performed on cultured
lymphocytes derived from peripheral bloods of couples. Two telomeric probes
with two or one centromeric probes for the chromosomes involved in the translocation were analyzed on metaphase spreads of translocation carrier individual.
Each blastomere biopsied from third day embryo was analyzed with telomeric
and centromeric probes. They were also analyzed for common aneuploidies with
PGT probe [13, 18, 21, X, Y (Vysis, Inc.)]. Embryos available for transfer were
transferred on the fifth day of embryo development. Prenatal diagnosis was recommended for pregnant patients.
Results: Preimplantation genetic testing was performed for 22 translocation
carrier cycles. Average maternal age was 32.2 years. Average of 1.6 normal
embryos were transferred in 14 cycles. 133 embryos were analyzed and only
18% (24/133) were normal or balanced. 16.3% (14/86) of embryos were analyzed normal or balanced in reciprocal translocations, while 21.3% (10/47) of
embryos were analyzed normal or balanced in robertsonian translocations. Clinical pregnancy per embryo transfer (ET) was 20% (2/10) and 50% (2/4) in reciprocal and robertsonian translocations, respectively. 4 clinical pregnancies were
resulted. 3 babies were delivered, 1 twin pregnancy still going on.
Conclusion: Individuals that carry chromosomal translocations seem healthy,
however they can produce chromosomally unbalanced gametes and they are at an
increased 10-15% risk for chromosomally abnormal fetus, In conclusion, preimplantation genetic testing should be advised for poor prognosis translocation carrier couples
in order to reduce the number of pregnancy losses and to have a successful pregnancy.
P-142
SNP ANALYSIS FOR PREVENTIVE GENETIC DIAGNOSIS: BASED
ON BREAST CANCER RISK AND HORMONE REPLACEMENT
THERAPY-RISKS AND BENEFITS
Burcu Yazar, Orkan Ilbay, Havva Comert, and Nesrin Ercelen
P-143
A SURVEY OF PATIENTS WHO RECEIVED TESTING FOR
HEREDITARY THROMBOPHILIA PANEL DUE TO MISCARRIAGE:
A CLINICAL GENETICIST PERSPECTIVE
Nesrin Ercelen, Burcu Yazar, T. Serhan Bora, and Havva Comert
Genetics and Genomic Sciences Center, VKV American Hospital,
Istanbul, Turkey
This retrospective study aims to evaluate the outcome of patients who
received hereditary thrombophilia panel testing for recurrent miscarriage and
assisted reproduction failure (ARF). We sought to evaluate the clinical practice impact of thrombophilic panel to determine the association of prevalence
of genetic markers for thrombophilia with the risk of conception and adverse
pregnancy outcomes. The effectiveness of prophylactic interventions during
pregnancy was also evaluated. Hypercoagulability may cause obstetric complications. The practical applications of both diagnostic testing and genetic counselling for the major inherited thrombophilias, are discussed in this study. A
total of 34 patients were tested for hereditary thrombophilia in our department
and analyzed retrospectively in this study. 14 patients were referred for
assisted reproduction failure (ARF). 20 patients were referred for recurrent
miscarriage. Control group consisted of 40 age-matched women who received
thrombophilia testing for reasons other than miscarriage and without any
abnormal obstetric history. Thrombophilia panel included testing for presence
of Factor V Leiden (FVL), prothrombin and methylenetetrahydro-folate reductase (MTHFR) mutations. Thus there was no family history available about
cardiovascular diseases (CVD), venous thromboembolism (VTE) or any other
risk factors. None of the patients in both groups were referred for genetic
counselling other than thrombophilia testing. There was a great need for high
risk patients in the means of their risk for hereditary thrombophilia to consider
counselling for future prophylactic interventions in order to avoid related complications that might occur and testing family members at high risk. Obstetric
patients with a detected thrombophilic risk must be managed according to
expert opinion. Testing would clearly be helpful if it identified individuals
with increased risk who could than be considered for both prophylactic and
long term therapy.
364
P-144
Medical Biochemistry, Firat University, Elazig, Turkey, 3Department of
Dermatology, Firat University, Elazig, Turkey
5,10 METHYLENETETRAHYDRO-FOLATE REDUCTASE C677T GENE
MUTATION ASSOCIATED UNSUCCESSFUL ASSISTED
REPRODUCTION
Objective: Behcet’s disease (BD) is a chronic, inflammatory disease with
unknown pathogenesis. An imbalance of the oxidant and antioxidant systems is
important in the pathogenesis of BD. The goal of this prospective study was to
investigate whether there is any relationship among the oxidant/antioxidant system and nitric oxide (NO) and malondialdehyde (MDA) levels, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity in patients with BD and
its subtypes: active Behcet’s disease and inactive Behcet’s disease.
Materials and Methods: In our study, we use 42 patients with Behcet’s Disease and as control group, we selected 19 people who have not a serious infection and chronic disease. Of these patients, 31 (20 female, 11 male; mean age
33.5 [SD 9.5] years) presented with active BD, and 11 (6 female, 5 male; mean
age 38.7 [SD 15] years) presented with inactive BD. The control group consisted
of 19 (15 female, 4 male; mean age 34.3 [SD 8] years) age-matched, healthy
individuals. Blood specimens were collected in tubes with or without EDTA.
The present study aimed to examine the levels of plasma NO, erythrocyte and
plasma MDA concentration, which are the end products of oxidant stress, and
erythrocyte SOD, GSH-Px activity, which are enzymatic antioxidants, were
determined in BD patients and compared with that of healthy individuals.
Results: Generally, the patient with the active Behcet’s disease has significantly higher plasma NO and MDA, erythrocyte GSH-Px and MDA levels than
control group (P<0.001, P<0.005, P<0.002, P<0.001, respectively). But there is
no meaningful difference between inactive group and control groups. We
observed that, only the erythrocyte MDA level is significantly higher at inactive
group than control group (P50.036). But we could not observe a meaningful difference between patient with active and inactive Behcet’s disease (P>0.05).
Conclusions: These findings suggest that neutrophils from patients with the
active Behcet’s disease generate high levels of lipid peroxidation end product,
resulting in endothelial tissue damage. We conclude that measurement of such
oxidative stress factors as MDA and NO and an accompanying evaluation of the
antioxidant defense system can be significant in the diagnosis and treatment
follow-up of BD.
Berrin Ozturk1, Burcu Yazar2, Orkan Ilbay2, Havva Comert2,
and Nesrin Ercelen2
1
Department of Clinical Genetics, East Carolina University, Brody School
of Medicine, USA, 2Genetics and Genomic Sciences Center, VKV
American Hospital, Istanbul, Turkey
Thrombophilia has been associated with adverse pregnancy outcomes and
recurrent pregnancy loss. However, it may also contribute to assisted reproductive failure (ARF). To determine the association of specific inherited thrombophilias and ARF, the prevalence of factor V Leiden (FVL), prothrombin
G20210A and 5,10 Methylenetetrahydro-folate Reductase (MTHFR) C677T gene
mutations were investigated.
A consecutive series of 14 women with ARF was enrolled in the study
group. Control group was 40 women with at least one successful pregnancy and
no history of pregnancy loss. Mean age of the study group was 33.3 years
(range, 28-41) versus 33.8 (range, 24-45) in control group. Mean number of
ARF was 1.9 (range, 1-5) in the study group. At least one thrombophilic defect
was found in 85.7% of total study group women compared with 57.5% in controls (p\000.1, OR: 4.448, 95% CI: 2.2-8.8). Presence of MTHFR mutations
was associated with an extremely significant increased risk for ARF (71 versus
43%, p50.0001, OR: 3.245, 95 % CI: 1.8-5.8). The presence of FVL mutation
showed no significant increased risk for ARF (14 versus 13%, p51.0000, OR:
1.167, 95% CI: 0.1-6.8). In addition none of the patients in the study group had
prothrombin mutation (p51.0000, OR: 0.9080, 95% CI: 0.03-23.6). C677T
MTHFR mutation might be a risk factor for ARF especially in younger women.
Definitive conclusions require analysis of larger series to confirm the result.
P-145
HYPERCHOLESTEROLEMIA-INDUCED OXIDATIVE STRESS IN THE
BRAIN: THE ROLE OF VITAMIN E
Nurgul Aytan1, Tobias Jung2, Faruk Tamturk1 Tilman Grune2,
and Nesrin Kartal-Ozer1
1
Department of Biochemistry, Faculty of Medicine, Marmara University,
Istanbul, Turkey, 2Institute of Biological Chemistry and Nutrition,
University of Hohenheim, Stuttgart, Germany
In recent years, there has been increasing evidence that cholesterol plays a
role in the pathology of Alzheimer disease. Since hypercholesterolemia was
reported to increase the levels of reactive oxygen species and Alzheimer disease
has clearly involved an oxidative component, it is possible that hypercholesterolemia is via increased oxidant production facilitating the disease development of
the neurodegenerative disease. Therefore, we tested in an established model of
enhanced cholesterol feed in rabbits the effects of serum cholesterol increase on
oxidative stress parameters as well in serum as in the brain. In addition to that
we tested the effects of vitamin E on the cholesterol-induced oxidative stress.
Since Alzheimer disease is largely connected with increased protein oxidation
whereas cholesterol is rather connected with lipid peroxidation processes, we
tested both protein carbonyl levels and the formation of malondialdehyde, a
marker of lipid peroxidation. We could clearly demonstrate an increase in serum
malondialdehyde due to high cholesterol feeding, which is accompanied by an
increase in protein oxidation parameters in the brain, especially in the hippocampus. Therefore, we suggest that specific neuropathological changes occur during
the feeding of hypercholesterolemic diet.
P-146
ASSOCIATION OF OXIDANT AND ANTIOXIDANT BALANCE IN
PATIENTS WITH BEHCET’S DISEASE
Muammer Bahsi1, Nevin Ilhan2, and Demet Cicek3
1
Department of Biology, Firat University, Elazig, Turkey, 2Department of
P-147
ASSOCIATION OF ENOS GLU298ASP POLYMORPHISM AND
ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH END-STAGE
RENAL DISEASE
Kadir Ates, Nevin Ilhan, Necip Ilhan, Dilara Kaman, and Enver Sancakdar
Department of Medical Biochemistry, Firat University, Elazig, Turkey
Objective: Atherosclerosis is the most frequent cause of cardiovascular morbidity in patients with end stage renal disease (ESRD). In the vascular endothelium, nitric oxide (NO), is constitutively produced from L-arginine by the action
of an endothelial NO synthase (eNOS) thus eNOS Glu298Asp polymorphism
may cause atherosclerosis by effecting NO production in endothelial cells. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase.
The concentrations of both dimethylarginines are increased in patients with endstage renal disease, which may explain at least in part endothelial dysfunction
and cardiovascular complications in this patient population. We investigated, in
patients with ESRD, the relationship between plasma ADMA, plasma NO levels,
and eNOS gene polymorphism.
Materials and Methods: 200 ESRD (130 hemodialysis and 70 peritoneal dialysis) patients and 100 healthy controls were included in the study. Blood specimens were collected in tubes containing EDTA. Genomic DNA was isolated
with DNA extraction kit. Glu298Asp in exon 7 of the eNOS gene was determined by polymerase chain reaction, followed by restriction fragment length
polymorphism analysis, in ESRD patients and compared with that of healthy
individuals. Plasma levels of NO was measured by the method of Cortas et al.
Plasma levels of ADMA, SDMA and arginine were measured by high performance liquid chromatography.
Results: In hemodialysis and peritoneal dialysis patients, AA genotype frequency was 25 (21.0%) versus 13 (27.1%), GA heterozygote genotype frequency
was 48 (40.3%) versus 17 (35.4%), and GG homozygote genotype frequency
was 46 (38.7%) versus 18 (37.5%), respectively (P >0.05). No significant differences were found in genotype frequency of control and patient groups. In hemodialysis patients, significant increases were found in NO, SDMA and ADMA
levels compared with control group. Compared to healthy controls, lower plasma
365
nitrite concentrations and ADMA levels were found in patients with peritoneal
dialysis.
Conclusions: In this study, the eNOS Glu298Asp polymorphism was not associated with ESRD patients, but an increase in ADMA and NO concentration in
these patients were associated with endothelial dysfunction. In patients with ESRD,
renal function is inversely associated with plasma ADMA. Thus, decreasing
ADMA levels may be protective in ESRD patients accompanying atherosclerosis.
P-148
ENOS GLu298Asp POLYMORPHISM AND ATHEROSCLEROSIS IN
PATIENTS WITH END-STAGE RENAL DISEASE
Kadir Ates1, Nevin Ilhan1, Dilara Kaman1, Necip Ilhan1,
and Hanifi Yildirim2
1
Department of Medical Biochemistry, Firat University, Elazig, Turkey,
2
Department of Radiology, Firat University, Elazig, Turkey
Objective: Endothelial nitric oxide synthase (eNOS) is present mainly in the
vascular endothelium. Nitric oxide (NO) produced by eNOS acts as a vasodilator
and may also be able to regulate blood pressure via other mechanisms. It has
been reported that the eNOS Glu298Asp substitution is likely to have a functional effect on the eNOS protein. Asymmetric dimethylarginine (ADMA)
assumes a significant role in atherosclerosis by inhibiting the endothelial nitric
oxide synthase (eNOS). Intima-media thickness (IMT) of the common carotid artery, a surrogate for vascular diseases, is one of the most used imaging techniques for the assessment of atherosclerosis, and it is increasingly applied in
patients with ESRD. The present study aimed to elucidate the Glu298Asp polymorphism of eNOS in patients with ESRD and its role as a predisposing factor
for cardiovascular complications.
Materials and Methods: 200 ESRD (130 hemodialysis [HD] and 70 peritoneal dialysis [PD]) patients and 100 healthy controls were included in the study.
Blood specimens were collected in tubes containing EDTA. Genomic DNA was
isolated with DNA extraction kit. Glu298Asp in exon 7 of the eNOS gene was
determined by polymerase chain reaction, followed by restriction fragment length
polymorphism analysis, in ESRD patients and compared with that of healthy
individuals. Using B-mode ultrasonography, we measured intima-media thickness
in the carotid arteries in these patients and healthy controls.
Results: We examined the association between IMT and ADMA and found
IMT to be increased in a group of patients with respect to controls (1.1860.16
mm [HD], 1.1060.14 [PD] versus 0.946.10 mm, P<0.001). No correlation was
found between the duration of HD, PD and intima-media thickness. No significant differences were found in genotype frequency of control and patient groups.
To determine plasma ADMA are associated with known cardiovascular risk factors or carotid intima-media thickness (IMT) in patients with end-stage renal disease (ESRD).
Conclusion: In patients with ESRD, renal function is inversely associated
with plasma ADMA, which, in turn, is positively associated with carotid IMT.
Increased plasma ADMA may be a link between renal function and cardiovascular disease in patients with mild-to-moderate renal failure.
P-149
INVOLVEMENT OF INTEGRIN A5b1 IN NEURAL
TRANSDIFFERENTIATION OF HUMAN BONE MARROW DERIVED
MESENCHYMAL STEM CELLS (hMSCs)
Nihal Karakas, Busra Mammadov, and Sevim Isik
Department of Biology, Science and Engineering Institute, Fatih University,
Turkey
Objective: In this study, it is aimed to investigate the role of fibronectin receptor integrin a5b1 during neural differentiation of bone marrow derived
hMSCs followed by the analysis of in vitro neurogenesis.
Methods: Primary hMSCs isolated from bone marrow were obtained from
Hematology Department of Karadeniz Technical University, Medical School.
Cells were expanded in DMEM with 10% MSC qualified FBS on fibronectin
coated surfaces. At passage 3, hMSCs were exposed to efficiently modified in
vitro neural differentiation protocol involving enriched cytokine combinations
(bFGF, hEGF, BDNF,FGF-8 ) and chemical compounds (IBMX, dbcAMP) in
Figure 1. Integrin a5b1 Expression of Neuronal Induced hMSC. [Color figure
can be viewed in the online issue, which is available at www.interscience.wiley.
com.]
serum reduced Neurobasal Media (plus B27 supplement). They were cultured for
immunofluorescence staining and western blotting to evaluate the expression of
early and late neural markers (such as NF, NeuN, Nestin, bIII Tubulin), integrin
a5/b1 and as a control, actin filaments. Western Blot samples were collected at
distinct time points during 14 days of the neural differentiation process and also
immunostaining was performed at those stages. Protein bands were detected on
X-ray films and images were taken under fluorescent microscope.
Results: Neuronal induction of hMSCs presented stable typical cell morphology with progression in dentritic and side by side axonal structure formation.
Besides, both early (bIII Tubulin, Nestin) and late (NF, NeuN) neural expression
marker were detected in neuronal induced hMSCs. It is undeniable that highly
improved Integrin a5b1 expression levels were detected in neuronal induced
hMSCs when it is compared to control group.
Conclusion: Our data demonstrated that Integrin a5b1 is functionally important in neural differentiation of hMSCs as the cells mobilize the integrin a5/b1
activity which then leads to cell adhesion and motility on extracellular matrix
proteins in conjunction with the stimulation of signal pathways required for in
vitro neurogenesis.
P-150
CLINICAL SIGNIFICANCE OF VASCULAR ENDOTHELIAL GROWTH
FACTOR IN EGYPTIAN COLORECTAL CANCER
Nirvana Samy1 and Tarek Essam2
1
Department of Biochemistry, National Research Centre, Cairo, Egypt,
2
Department of Surgical Oncology, National Cancer Institute,
Cairo University, Cairo, Egypt
Tumor cells may express both proangiogenic and/or antiangiogenic factors.
Among angiogenic molecules, vascular endothelial growth factor (VEGF)
appears to have a central role in the angiogenic process. The objective of the
current study was to assess the prognostic significance of the preoperative VEGF
plasma level as an indicator of recurrent disease in patients with colorectal carcinoma who undergo surgery with curative intent, also to determine its relationship
with disease stage at presentation. A further objective was the study of protein
expression pattern of VEGF isoforms in colorectal tumors in comparison to the
corresponding adjacent normal tissues. Preoperative and postoperative VEGF
plasma levels were determined by enzyme-linked immunosorbent assay in 78
patients with colorectal cancer who were undergoing surgery. Fifty healthy individuals served to define normal VEGF plasma levels. The results showed that
preoperative VEGF, carbohydrate antigen 19.9 (CA 19-9) and carcinoembryonic
antigen (CEA) levels were significantly high in patients with colorectal carcinoma compared to control group. Moreover, VEGF plasma levels were significantly lowered in patients who underwent curative surgery, with a further downward trend until the 6 month postoperative (P \0.05). Multiple regression analysis demonstrated significant positive correlation (r) between preoperative VEGF
plasma levels and Dukes stages, TNM stages, T classes, CEA levels, and CA 199 (P\0.05). Combining of VEGF and CEA markers result in significant increase
366
in the specificity, positive predictive value (PPV) and accuracy (P\0.05) in comparison with CEA alone. Over-expression of VEGF in colorectal cancer tissue
was confirmed by western blot (WB); a main protein band was detected with
molecular weight of 23 kDa (VEGF165), which was expressed in both normal
and tumor tissues of colorectal, but the expression of VEGF165 was detected at
higher levels in tumor tissues and the intensities of the bands varied according to
plasma VEGF concentrations. This study revealed that preoperative plasma
VEGF concentrations might be a valuable parameter for tumor burden and to
predict the outcome of colorectal cancer curative surgery. VEGF165 expression,
considered as a predominant form of VEGF, secreted by a variety of normal and
transformed cells was correlated to the plasma levels of VEGF.
P-151
EFFECTS OF CYPIA1, GSTM1, GSTT1 AND GSTP1 GENE
POLYMORPHISMS ON COLON CANCER RISK IN
TURKISH POPULATION
Omer Faruk Kagnici1, Tulin Ozturk2, Mete Bora Tuzuner1, Suha Goksel3,
Sibel Erdamar3, Oguz Ozturk1, and Turgay Isbir1
1
Pathology, Bagcilar Education and Research Hospital, Turkey, 3Department
of Pathology, I.U. Cerrahpasa School of Medicine, Istanbul, Turkey
Cytochrome P450 1A1 (CYP1A1) plays an important role in both activation
and the phase I detoxification of carcinogens. Glutathione S-transferase M1
(GSTM1), Glutathione S-transferase T1 (GSTT1) and Glutathione S-transferase
P1 (GSTP1) enzymes are involved in the phase II detoxification of active metabolites of many potential carcinogens. CYP1A1 MspI, GSTM1 null, GSTT1 null
and GSTP1 Ile/Val polymorphisms have been reported to be associated with colon cancer risk. We used PCR, RFLP and gel electrophoresis techniques to detect
these polymorphisms on 117 colon cancer patients and 107controls. In our study,
we found statistically mean frequencies and significantly difference in the distribution of GSTT1 null genotypes on colon cancer patients and controls. The colon cancer risk statistically increased due to the GSTT1 null genotype (p50.001;
OR5 3.26, 95% CI 5 1.58-6.71). Also we found increased risk for colon cancer
in patients with GSTP1 IIe/Val mutant genotype (OR:1,416, 95% CI:0,7782,570). However we could not find the same relation for GSTM1 and CYP1A1
polymorphic variants. On the other hand, we compared these genetic frequencies
and patients pathological parameters. We found blood vessel invasion risk statistically increased due to the GSTT1 null genotype (p50.049; OR5 3.78, 95% CI
5 0.972-14.705). The null genotype of GSTT1was associated with increased risk
for regional lymph node metastasis (OR5 2.933, 95% CI 5 0.824-10.448).
In conclusion, we suggest that GSTT1 null polymorphism may be a risk factor for colon cancer.
P-152
INVESTIGATION OF GENE POLYMORPHISMS OF CYP1B1 AND
NAT2 DETOXIFICATION ENZYMES IN PATIENTS WITH BREAST
CANCER IN TURKISH POPULATION
Yasemin Kadriye Ozbek1, Tulin Ozturk2, Mete Bora Tuzuner1,
Zerrin Calay3, Sennur Ilvan3, Oguz Ozturk1, and Turgay Isbir1
1
Being a disease caused by both hormonal and environmental factors affecting
genetic structure; breast cancer risk has been established to be elevated due to
exposure to environmental chemicals such as heterocyclic amines and aromatic
amines and high levels of estradiol exposure. Epidemiological, experimental and
clinical data demonstrate that breast cancer is induced by long exposure of mammalian epithelium to estrogens. Estrogens are known to increase breast cancer
risk in two ways: the stimulation of cell proliferation via estrogen receptor-mediated signaling and the formation of catechol metabolites during its metabolism.
Cytochrome P450 1B1, has the characteristics of hydroxylating a huge variety of
substrates and catalyzing the hydroxylation of C17. Estradiol plays a dominant
role in breast cancer progression. The CYP 1B1 Codon 432 polymorphism has
been identified as an important susceptibility factor. N-acetyltransferase 2 functions for the metabolic detoxification of arylamines and heterocyclic amines and
is known to comprise a number of allelic variants with differing acetylation
capacities. In our study, we have investigated seven missense and four silent
polymorphisms of N-acetyltransferase 2 and CYP 1B1 codon 432polymorphism
(CYP lBl*3) by using PCR-RFLP analysis in order to classify subjects into
allelic groups. As a conclusion; distribution of NAT 2 enzymatic activity in
healthy groups is found to be correlated with that of the caucasians. Breast cancer patients have been shown to possess slow acetylator phenotypes 1.8 times
higher than healthy groups but no statistical difference has been validated.
(p[0.05, X253.24 OR51.82; 95% CI5 0.94-3.50). Moreover, NAT 2*5 allele
is much more statistically related with breast cancer patients rather than healthy
groups (p\0.05, X255.04 OR52.22; 95% CI51.10-4.50) with percentages
43.3% in patients and 15.8% in controls. Breast cancer patients are detected to
bear less CYP 1B1 L432V polymorphism mutations than healthy groups
(p\0.005, X253.911 OR5 2.58; 95% CI50.99-6.75). NAT 2 activity and CYP
181*3 variant together has no statistical difference.
P-153
THE RELATIONSHIP BETWEEN ESTROGEN METABOLISM GENES
CYP 17, CYP 19 AND BREAST CANCER
Mete Bora Tuzuner1, Tulin Ozturk2, Zerrin Calay3, Sennur Ilvan3,
Oguz Ozturk1, and Turgay Isbir1
1
Many gene polymorphisms of enzymes, ligands, and receptors potentially
related to carcinogenesis have been examined for breast cancer risk, but only
few have showed positive associations. It is widely accepted that estrogens are
involved in the development of breast cancer and that increased life time exposure to endogenous estrogen is known to increase risk. Epidemiologic studies
have identified several factors affecting this parameter, including early age at
menarche, nulliparity, late age at menopause, late age at first full term pregnancy, and postmenopausal obesity. Excess body fat is thought to increase postmenopausal breast cancer risk by increasing bioavailable estrogen as a result of
increase in extraovarian estrogen production as well as causing change in estrogen protein binding. Consequently, the enzymes involved in the biosynthesis and
metabolism of estrogens (CYP17, CYP19, CYP2D6, COMT, or CYP1A1) have
been main targets in attempts to identify genetic polymorphisms contributing to
breast cancer risk. Cytochrome P450 17 alfa (CYP17) and aromatase (CYP19)
play important roles in estrogen biosynthesis. Associations between breast cancer
risk and CYP17 50 untranslated region (UTR), CYP19 codon39 Trp/Arg polymorphisms have been reported. We used PCR, RFLP and gel electrophoresis
techniques to detect these polymorphisms on 55 breast cancer patients and
91controls. In our study, we found statistically mean frequencies and significantly difference in the distribution of CYP19 heterozygote mutant genotypes
(TC) on breast cancer patients (p\0.00l; X25 12.31; OR 5 7.30; 95% CI5
2.29-25.64). However we could not find same relation for CYP17 polymorphic
variants. But when we consider the CYP17 gene variants which carry A2 allele
with heterozygous variants of the CYP19 gene together, we found statistically
significant difference between groups. (p5 0.006; X25 7.01; OR 5 2.53; 95%
CI5 1.26-5.07) On the other hand, we analysed the relation between genetic frequencies and patients pathological features but no difference has been found. In
conclusion, we suggest that carrying A2 allele for CYP17 and TC genotype for
CYP19 together may be a risk factor for breast cancer.
P-154
EFFECT OF FIBRINOGEN ON BRADYKININ RECEPTOR TYPE-2
PROTEIN EXPRESSION
Oktay Kocgirli1, Sandra Valcaccia1, Murat Bas2, Vera Balz3, and
Georg Kojda1
1
Institute of Pharmacology, Heinrich-Heine University, Dusseldorf,
Germany, 2Otorhinolaryngology Clinic, Technical University, Munich,
Germany, 3Otorhinolaryngology Clinic, Heinrich-Heine-University,
Dusseldorf, Germany
Increasing evidence suggests a causative role of bradykinin receptor type 2
(BKR-2) activation in the development of hereditary and iatrogenic angioedema.
Recent data showed that fibrinogen plasma levels are increased during acute angiotensin converting enzyme inhibitor (ACEi)-induced angioedema which is associated with increased plasma bradykinin levels. We sought to determine the mechanism underlying the potentiation of bradykinin induced vasodilatation by fibrinogen
in pig coronary artery. We used various human head and neck squamous carcinoma
cell lines. The ability of these cells to express BKR-2 was tested in 25 different
lines by reverse transcriptase polymerase chain reaction and we chose a cell line
expressing BKR-2 (UDSCC-2) and another without BKR-2 expression (UDSCC-3)
to evaluate a Western blot protocol. For detection of BKR-2 we used one monoclonal and 4 different polyclonal antibodies. We used porcine aortic endothelial cells
(PAECs) and human umbilical vein endothelial cells (HUVECs). These cells were
incubated with fibrinogen and melagatran. Melagatran was used to inhibit conversion of fibrinogen to fibrin. Additional PAECs were incubated with 10 lM S-nitroso-N-acetylpenicillamine (SNAP) (NO-Donor) or Ca-Ionophore with NG-NitroL-Arginine-Methyl-Ester (L-NAME) (NOS inhibitor). The monoclonal antibody
showed only one specific signal at 42 kD and therefore was used for the additional
experiments. In PAECs, BKR-2 protein expression showed no change after 1 hours
of incubation with fibrinogen (144.7635.41%, n523, P50.2205), after 3 hours of
incubation (149,3631,00 %, n525, P50,1251) and after 6 of hours incubation(129.9625.56%, n527, P50.2526). In HUVECs BKR-2 protein expression
was not changed (108.6627.05, n56, p50.7635) after 1 h and significantly
increased to 196.8638.28 (n511, p50.03) after 3 h incubation). L-NAME showed
decreased BKR-2 protein expression in PAECs after 1 hour (54.98617.13%, n55,
P50.0583), 3 hours (44.20615.41%, n55, P50.0223) and after 6 hours of incubation(56.80 6 14.24 %, n55, P5 0.0386). SNAP has no effect on BKR-2 protein
expression. These data suggest that fibrinogen does not up-regulate the expression
of BKR-2 in PAECs. Inhibition of NOS decreases BKR-2. Exogenous NO does not
have any effect on the expression of BKR-2. Further experiments are necessary to
clarify the role of fibrinogen in ACE induced angioedema.
P-155
THE EVALUATION OF NINE MUTATIONS ASSOCIATED WITH CVD
IN TOKAT REGION
Semsettin Sahin1, Huseyin Ozyurt1, Omer Atis1, Leyla Aydogan2,
Ismail Benli3, and Koksal Ceyhan4
1
2
3
4
Department of Cardiology, Gaziosmanpasa University, Tokat, Turkey
Cardiovascular heart disease (CVD) is a multigenic disorder that various factors play role in its ethiopathogenesis. Factor V H1299R(R2), Factor XIII V34L,
B-Fibrinogen-455 G-A, PAI-1 4G-5G, GPIIIa L33P(HPA-1), MTHFR A1298C,
ACE I/D, ApoB R3500Q, APOE (E2,E3,E4) are important mutations/polymorphisms in the evaluation of CVD disease.
In this study, in Tokat, 9 mutations (Factor V H1299R(R2), Factor XIII
V34L, B-Fibrinogen-455 G-A, PAI-1 4G-5G, GPIIIa L33P(HPA-1), MTHFR
A1298C, ACE I/D, ApoB R3500Q, APOE (E2,E3,E4) ) have been screened in
patients who were accepted to our hospital with various ailments. Detection of
mutations was carried out by using ViennaLab CVD StripAssayTM kit. Strip
mutation analyze is a method that can determine a large number of mutations of
genes associated with some diseases such as CVD, FMF and Celiac. CVD test
panel was performed in 286 patients who were accepted to our hospital with various ailments. The results of this study is as follows: Factor V H1299R(R2)
mutation 43 heterozygous (15.03%), 2 homozygous (0.69%), Factor XIII V34L
mutation 61 heterozygous (21.32%), 8 homozygous (2.79%), b-Fibrinojen-455
G-A mutation 92 heterozygous (32.16%), 10 homozygous (3.49%), PAI-1 4G-5G
mutation 56 5G/5G (19.58%), 156 4G/5G (54.54%), 74 4G/4G (25.87%), GPIIIa
L33P(HPA-1) mutation 223 a/a (77.97%), 55 a/b (19.23%), 8 b/b (2.79%),
MTHFR A1298C mutation 131 heterozygous (45.80%), 36 homozygous
(12.58%), ACE I/D polymorphism 53 II (18.53%), 138 ID (48.25%), 95 DD
367
(33.21%), ApoB R3500Q mutation 4 heterozygous (1.39%), APOE (E2,E3,E4)
mutation 3 E2/E2 (1.04%), 40 E2/E3 (13.98%), 3 E2/E4 (1.04%), 207 E3/E3
(72.37%), 32 E3/E4 (11.18%), 1 E4/E4 (0.34%) . In conclusion, these mutations
have shown no difference in Turkey since our findings are near to mean values.
P-156
THE 30 - UNTRANSLATED REGION OF THE ADAMTS1 REGULATES
ITS EXPRESSION
Omer Faruk Hatipoglu1, Satoshi Hirohata1, Kursat Oguz Yaykasli1,
Mehmet Zeynel Cilek1, Kadir Demircan1, Ryoko Shinohata2,
Shozo Kusachi2, and Yoshifumi Ninomiya1
1
Department of Molecular Biology and Biochemistry, Okayama University,
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
Japan, 2Department of Medical Technology Okayama University, Graduate
School of Health Sciences, Japan
Objective: ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs1) is an inflammatory-induced gene. We have previously reported
ADAMTS1 was strongly but transiently expressed in the infarcted heart. In this
study, we investigated whether 30 -untranslated region (UTR) affects its mRNA
stability. The 30 -UTR ADAMTS1 mRNA contains multiple adenine and uridinerich elements, suggesting that 30 -UTR may regulate its stability. Furthermore, a
region containing multiple AUUUA motifs within the ADAMTS1-30 -UTR destabilized transfected EGFP mRNA expression. These data demonstrated that
ADAMTS1-30 -UTR may regulate the expression of ADAMTS1 mRNA.
Methods: The expression of ADAMTS1 mRNA was examined in Cos7
stimulated by TNFa and LPS. To examine the effect of 30 -UTR for the decay of
RNA, we amplified the 30 -UTR of human ADAMTS1 mRNA by PCR. Two constructs, PmaxLong and PmaxShort, were created, the Long consisting of
AUUUA motif absent in the Short. For the mRNA stability assay, cells were
transfected with each construct and after 24h of transfection, actinomycin-D was
added to the medium (100 lg/ml) and the mRNA was extracted as indicated.
Samples analyzed by quantitative real-time RT-PCR.
Results and Conclusion: ADAMTS1 expression is increased in TNFa-and
LPS stimulated cells. Induced ADAMTS1 mRNA level by TNFa was significantly decreased when actinomycin D was added. Induced GFP-ADAMTS1-30 UTR-long mRNA level was significantly decreased as compared with that of
GFP-ADAMTS1-30 -UTR-short mRNA under actinomycin D treatment. Our study
demonstrated that ADAMTS1-30 -UTR contains multiple AUUUA motifs may
regulate the expression of ADAMTS1 mRNA.
P-157
EFFECT OF TYPE I COLLAGEN COMPARED TO SPIRULINA IN
PREVENTING INTRAPERITONEAL ADHESIONS IN A RAT UTERINE
HORN MODEL
Onder Koc1, Bulent Duran1, Ata Topcuoglu1, Guler Bugdayci2,
and Fahri Yilmaz3
1
Department of Obstetrics and Gynecology, Abant Izzet Baysal University,
Faculty of Medicine, Bolu, Turkey, 2Department of Biochemistry, Abant
Izzet Baysal University, Faculty of Medicine, Bolu, Turkey, 3Department of
Pathology, Abant Izzet Baysal University, Faculty of Medicine, Bolu, Turkey
Objective: To compare the anti-adhesion potential of type I collagen and
spirulina on prevention of adhesion formation in rat uterine horn model.
Methods: This study was carried out in the Abant Izzet Baysal University
Medical Center in Bolu, Turkey, during the period of January 2008 to March
2008. We used thirty female Wistar albino rats. Before the operation, rats were
randomly divided into 3 groups, each consisting of 10 rats. We examined the
effects of intraperitoneal 10 mg/puff type I collagen 2 puff versus 1 mg/mL spirulina 1 mL to reduce the extent and severity of postoperative adhesions in a rat
uterine horn model: 1 mL of normal saline (NS) solution in NS group, 2 puff of
10 mg/puff type I collagen in collagen group, intraperitoneal 1 mg/mL spirulina 1
mL in spirulina group was instilled onto uterine horns of the rats. Adhesions were
scored according to their extent and severity. Glutathione (GSH) was assayed for
antioxidant condition. Malondialdehyde (MDA) levels were assayed as products
368
of lipid peroxidation. Reactive oxygen metabolite-induced inflammatory changes
were evaluated as myelopreoxidase (MPO) activity.
Results: The study was completed in all 30 rats assigned to 3 study groups.
Adhesion extent and severity scores were assessed in each of the groups (n510)
based on 20-horns.
The extent and severity scores of adhesions in the collagen group were significantly lower than those of control and spirulina groups. There was statistically significant difference between the extent and severity scores of adhesions between
collagen and spirulina groups. The extent and severity scores of adhesions in collagen groups were significantly lower than those of control groups. There was no
statistically significant difference between the extent and severity scores of adhesions between spirulina and control groups. Glutathione levels increased in collagen group significantly respect to the control group (p\0.05, p\0.05) and spirulina group (p\0.05, p\0.05). Malondialdehyde levels and myeloperoxidase activity decreased significantly in collagen group (p \0.05, p\0.05).
Conclusions: These findings suggest that collagen type I should be considered as an adjuvant in the prevention of postoperative intra-abdominal adhesions.
Spirulina does not have anti-adhesive properties. Future experimental and clinical
studies are required to find collagen optimal formulation and usage.
P-158
Bolu, Turkey, 3Department of Biochemistry, Ankara Numune Teaching and
Education Hospital, Ankara, Turkey
Objective: Visfatin/pre- Beta cell enhancing factor that is highly expressed in
visceral fat and whose blood levels are correlated with obesity. In this study, we
investigated whether plasma visfatin levels are altered in healthy obese women.
Methods: Cross-sectional comparison of plasma visfatin levels in 71 women:
46 with a body mass index (BMI) higher than 28 and a waist to hip ratio (WHR)
of 0.86 or higher, android obesity; 21 with a BMI higher than 28 and WHR
lower than 0.86 gynoid obesity; and 25 non-obese women with a BMI lower
than 25. Plasma visfatin level was measured by using enzyme-linked immunosorbent assay (ELISA) kit (Phoenix Pharmaceuticals; Belmond; CA, USA) in obese
and non-obese women. We also measured blood pressure, glucose, insulin, and
high sensitive C-reactive protein (hsCRP).
Results: Women with android obesity had higher levels of plasma visfatin
(29.45 6 1.77 vs 25.48 6 2.29 ng/ml) than non-obese women (p<0.0001).
Based on multiple regression analysis hsCRP levels and WHRs of 0.86 or higher
predicted the plasma visfatin. Plasma visfatin levels were higher in women with
android obesity than women with gynoid obesity (p<0.001).
Conclusion: These results indicate that obesity causes an increase in plasma
visfatin levels, both in people with android obesity and gynoid obesity. This
increase is also more prominent in android obesity because of increased visceral
adiposity.
EFFECT OF MONTELUKAST IN PREVENTING INTRAPERITONEAL
ADHESIONS IN A RAT UTERINE HORN MODEL
Onder Koc1, Ata Topcuoglu1, Bulent Duran1, Guler Bugdayci2,
Fahri Yilmaz3, and Melahat Donmez1
1
Department of Obstetrics and Gynecology, Abant Izzet Baysal University,
Faculty of Medicine, Bolu, Turkey, 2Department of Biochemistry, Abant
Izzet Baysal University, Faculty of Medicine, Bolu, Turkey, 3Department of
Pathology, Abant Izzet Baysal University, Faculty of Medicine,
Bolu, Turkey
Objective: To investigate the anti-adhesion potential of Montelukast solution
on prevention of adhesion formation in rat uterine horn model.
Methods: This study was carried out in the Abant Izzet Baysal University
Medical Center in Bolu, Turkey, during the period of September 2008 to October
2008. We used thirty female Wistar albino rats. Before operation, the rats were
randomly assigned to 3 groups each consisting of 10 rats. We examined the
effects of intraperitoneal 1 mL of Montelukast solution to reduce the extent and
severity of postoperative adhesions in a rat uterine horn model: no adjuvant therapy in sham operated group, 1 mL of normal saline (NS) solution in NS group,
1 mg/mL Montelukast solution in Montelukast group, was instilled onto uterine
horns of the rats. Adhesions were scored according to their extent and severity.
Glutathione (GSH) was assayed for antioxidant condition. Malondialdehyde
(MDA) levels were assayed as products of lipid peroxidation. Reactive oxygen
metabolite-induced inflammatory changes were evaluated by the myelopreoxidase
(MPO) activity.
Results: The study was completed in all 30 rats assigned to 3 study groups.
Adhesion extent and severity scores were assessed in each of the groups (n510)
based on 20-horns. The extent and severity scores of adhesions in Montelukast
group were significantly lower than those of control group. There was a statistically significant difference between the extent and severity scores of adhesions
between Montelukast and NS groups. Glutathione levels increased in montelucast
group significantly with respect to the control group (p<0.05). Malondialdehyde
levels and myeloperoxidase activity decreased significantly in Montelukast group
(p<0.05, p<0.05).
Conclusions: These findings suggest that Montelukast solution should be
considered as an adjuvant in the prevention of postoperative intra-abdominal
adhesions. Future experimental and clinical studies are required to find out the
optimal formulation and usage of Montelukast solution.
P-159
PLASMA VISFATIN LEVELS ON OBESE WOMEN
Guler Bugdayci1, Onder Koc2, and Dilara Uncu3
1
Department of Biochemistry, Abant Izzet Baysal University, Bolu, Turkey,
2
Department of Obstetric and Gynecology, Abant Izzet Baysal University,
P-160
SALIVARY ANTIOXIDANT CAPACITY DURING EXERCISE
IN ATHLETES
Guler Bugdayci1, Onder Koc2, Bekir Yuktasir3, Serife Ozen3,
Hasan Birol Yalcin3, and Gul Tiryaki-Sonmez4
1
Department of Biochemistry, Abant Izzet Baysal University, Bolu, Turkey,
2
Department of Obstetric and Gynecology, Abant Izzet Baysal University,
Bolu, Turkey, 3Department of Physical Education and Sports, Abant Izzet
Baysal University, Bolu, Turkey, 4Health Sciences, Lehman College,
New York, USA
Background: It is well known that aerobic exercise has health benefits for
humans. Long duration and exhaustive exercise, however, can overcome our
capacity for detoxifying reactive oxygen species and free radicals and cause
damage.
Aim: The aim of our study was to investigate the effect aerobic exercise on
salivary total antioxidant status (TAS) and salivary lipid hydroperoxides (LPO)
in athletes.
Methods: Seven subjects aged 18-21 yr participated in two, 4-h trials (exercise and control) in a random crossover design. Trials began at 09:00 in the
morning after an overnight fast. In the exercise trial, subjects ran for 105 min. at
50% of maximum oxygen uptake and 15 min. at 75% of maximum oxygen
uptake between 09:00 and 12:00. In the control trial, subjects rested quietly for
the same duration. The total antioxidant status (Randox, San Francisco, CA,
USA) and lipid hydroperoxide (Cayman Chemical Company, Ann Arbor, MI,
USA) concentration were determined by colorimetric commercial kits. Statistical
analysis of the data was performed by two-way repeated measures analysis of
variance during 4 hours of monitoring.
Results: The salivary TAS levels were higher in first hour exercise (1.39 6
0.12 mmol/L vs 1.60 6 0.14 mmol/L) and significantly higher second hour exercise (1.50 6 0.15 mmol/L vs 2.01 6 0.35 mmol/L), exercise trial and control
trial, respectively. The salivary LPO levels were lower in the first hour of exercise (0.460 6 0.038 nmol/L vs 0.394 6 0.059 nmol/L) and significantly lower in
the second hour of exercise (0.489 6 0.047 nmol/L vs 0.241 6 0.055 nmol/L),
exercise trial and control trial, respectively. The results obtained during the experimental period indicated the existence of a 4-hours rhythm of salivary TAS
and LPO levels between exercise and control group. Two-way repeated measures
analysis showed a significant effect of time (F514.34 p50.001; F512.92,
p50.000, respectively), effect of trial (F518.96, p50.001; F530.28, p50.000,
respectively) and effect of time and trial interaction (F5 4.99 p50.032; F5
30.28, p50.000, respectively) for salivary TAS and LPO levels.
Conclusion: The results suggested that aerobic exercise-induced increase in
total antioxidant status seems to inhibit lipid hydroperoxide generation, a marker
of oxidative stress in human saliva.
369
P-161
P-163
AN ANALYSIS OF DEATH CASES OF CHILDREN YOUNGER THAN
ONE YEAR BECAUSE OF HOUSE ACCIDENTS
NRAMP1 (SLC11A1): POSSIBLE CANDIDATE GENE FOR
SARCOIDOSIS IN TURKISH POPULATION
Riza Yilmaz1, Veli Özdemir1, Muhammed Can1, Ozge Oztop2
1
1st Specialty Committees, Ministry of Justice Council of Fore, Turkey,
2
Molecular Medicine, University of Istanbul, Institute for Experimental
Medicine, Istanbul, Turkey
P. Akcakaya1, B. Azeroglu1, I. Even1, O. Ates2, H. Turker3, G. Ongen4,
and A. Topal-Sarikaya1
1
Department of Molecular Biology and Genetics, Science Faculty, Istanbul
University, Istanbul, Turkey, 2Institute for Medical Biology, Medicine
Faculty, Gaziosmanpasa University, Tokat, Turkey, 3Chest Disease
Department, Sureyyapasa Chest Diseases and Thoracic Surgery Education
and Research Hospital, Istanbul, Turkey, 4Chest Disease Department,
Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey
The risk of injury and death because of accidents is very high in the childhood. The situations which are not dangerous for adults can be very risky for
children since they are physically small and weak and since they are inexperienced and lack coordination. In this study totally 7 cases have been analyzed by
Ministry of Justice Council of Medicine of Turkey which include children
younger than 1 year that are died because of accidents occurred at home. These
cases were analyzed in terms of crime scene investigation, internal and external
pathology at autopsy, pathology and toxicology. It is understood that in 3 of
these 7 cases the crime scene investigation was performed, in 4 cases medical
care was performed. Additionally, for all cases autopsy was performed. It is very
important to perform autopsy, crime scene investigation and to consider the medical documents and the judicial files if the child was medically cared, to be able
to determine the cause of death and the origin.
P-162
THE FYN KINASE INTERACTS WITH HEPATOCYTE GROWTH
FACTOR RECEPTOR c-MET UNDER HYPOXIA: IMPLICATIONS FOR
HGF-SF/cMET SIGNALLING IN HCC CELLS
Peyda Korhan, Gülay Bulut, Aslı Toylu, Esra Erdal, and Nese Atabey
Department of Medical Biology and Genetics, Dokuz Eylül University, The
Institute of Health Science, Turkey
Background and Aim: Hypoxia induces proliferation, angiogenesis, metastasis, chemoresistance, and radioresistance of hepatocellular carcinoma
(HCC) while it suppresses differentiation and apoptosis. Aberrant Hepatocyte
Growth Factor (HGF-SF)/c-Met signaling has role in many of these processes
especially including proliferation and metastasis. In addition hypoxic preconditioning increases cell motility and invasion via c-Met activation. Since intermittent hypoxia is a very common phenotype during hepato-carcinogenesis, it
is very exciting to determine the role of c-Met receptor in this model.
Methods: Phage display library was carried out to seek peptides, binding
specifically to multi docking sites (MDS) of c-Met receptor. Hypoxia treated
HCC cell line, Huh7, was used to established cDNA library. Fyn kinase has
been found to bind specifically to c-Met in-vitro. In order to show this interaction in vivo, immunoprecipitation was performed in both Huh7 and SNU449 cell
lines. Expression level in both phosphorylated and un-phosphorylated forms of cMet and Fyn were analyzed by western blotting after HGF treatment under hypoxia. c-Met inhibitor (SU11274) and Fyn kinase inhibitor (PP2) were used to
find out regulation mechanism of this interaction. Cell motility was assayed
under hypoxia to investigate possible biological role of c-Met and Fyn
interaction.
Results: Potential interaction of Fyn with c-Met shown by phage display
has been confirmed with IP in Huh7 cells. As a result, Fyn physically binds
with c-Met under hypoxia in HCC. Both phosphorylated and un-phosphorylated forms of c-Met and Fyn protein levels were increased in hypoxia and/or
HGF treatment. Increasing in the phosphorylated c-Met level shows receptor
activation while increasing in phosphorylated Fyn level in negative manner
proved inactivation of Fyn under the same conditions. Moreover, inhibition of
c-Met activity by SU11274 was induced negative regulation of Fyn kinase activity. Inhibition of Fyn kinase activity by PP2 decreased c-Met activity.
Finally, disruption of interaction between Fyn and c-met reduced motility and
invasion of HCC cells.
Conclusion: As a conclusion, Fyn and c-Met interaction has been described
for the first time in this study. This interaction offers new explanations about the
mechanisms for radio- and chemo-resistance characteristics of HCC and it could
be an important molecular target for prevention of invasive and metastatic phenotypes during hepato-carcinogenesis in hypoxia.
Objective: Sarcoidosis (SA) is an immune-mediated multisystemic disorder
of unknown etiology characterized by the accumulation of lymphocytes, mononuclear phagocytes and epithelioid cell granulomas in involved tissues. Through
differences in the prevalence among different populations and familial aggregation, genetic susceptibility to SA has been suggested. Given the possible role of
the Nramp1 gene in granulomatous disorders, the association with human tuberculosis and the involvement in macrophage function, we hypothesized that
human NRAMP1 is associated with susceptibility to sarcoidosis. Therefore we
investigated four NRAMP1 gene polymorphisms, including a single nucleotide
change in intron4, INT4; a nonconservative single-base substitution at codon543
(D543N), a TGTG deletion in the 3’untranslated region, 3’UTR; and a (GT)n
microsatellite in the 5’promoter of the gene, 5’(GT)n in 73 Turkish SA patients
and 150 healthy control subjects.
Methods: INT4, 3’UTR and D543N polymorphisms were analyzed by using
amplification refractory mutation system-polymerase chain reaction (ARMSPCR). The 50 (GT)n microsatellite analysis was performed as sequence analysis,
using a genetic analyzer.
Table 1
Relationship between NRAMP1 (natural resistance associated
macrophage protein 1) polymorphisms and sarcoidosis
NRAMP1 locus,
polymorphism
INT4
C/C
G/C
G/G
Allelic frequency
C
G
3’UTR
D/D
ND/D
ND/ND
Allelic frequency
D
ND
D543N
A/A
G/A
G/G
Allelic frequency
D
N
5’(GT)n*
Allele 2/2
Allele 2/3
Allele 3/3
Allelic frequency
Allele 2
Allele 3
SA patients
n573
Mean age5
43.79612.12
Gender 5
48 women
25 men
Control
subject
n5150
Mean
age 5 5864
Gender 5
60 women
90 men
p
9
26
38
2
33
115
44 (30%)
102 (70%)
37 (12%)
263 (78%)
0
4
69
0
6
144
0.73
4 (3%)
142 (97%)
6 (2%)
294 (98%)
0.42
0
4
69
0
6
144
0.73
4 (3%)
142 (97%)
6 (2%)
294 (98%)
0.42
3
14
8
3
50
97
20 (40%)
30 (60%)
56 (19%)
244 (81%)
95% CI
0.0001
3.07 (1.82–5.18)
0.000
0.0017
2.90 (1.45–5.72)
0.001
370
Results: The NRAMP1 polymorphism allele frequencies are summarized in
Table I.
Conclusion: A statistically significant difference in allele frequency distribution of 5’(GT)n and INT4 alleles was observed between patients and control subjects, suggesting that these polymorphisms are associated with susceptibility to
sarcoidosis in Turkish population. But no association between 3’UTR and
D543N polymorphisms and SA was observed, as previous studies in several populations showed. The D543N A allele is apparently in linkage disequilibrium
with the 3’ UTR del allele, thus these results were not independent of each
other.
Acknowledgement: This research was supported by The Scientific and Technological Research Council of Turkey.
P-164
INVESTIGATION OF ROSCOVITINE (CYC202)-INDUCED APOPTOSIS
RELATED WITH POLYAMINE METABOLISM IN MCF-7 HUMAN
BREAST CANCER CELL LINE
Elif Damla Arisan, Narcin Palavan-Unsal, and Pinar Obakan
Department of Molecular Biology and Genetics, Istanbul Kultur University,
Istanbul, Turkey
patients with total prostate-specific antigen values smaller than 11 ng/ml had normal expression of the alpha-methylacyl-CoA racemase gene. The results from
this study could have epidemiological and preventive importance for prostate
cancer as the main sources of branched chain fatty acids are dairy products and
beef the consumption of which has been associated with an increased risk for
prostate cancer. Comparison of alpha-methylacyl-CoA racemase RT-PCR with
known serum prostate-specific antigen levels shows that a combination of these
two parameters can increase the sensitivity for detection of progressive disease.
According to its consistency and magnitude of cancer cell-specific expression,
we propose alpha-methylacyl-CoA racemase as an important new biomarker for
the early detection of prostate cancer.
P-166
MOLECULAR DETECTION OF INTRON 22 INVERSION OF FACTOR
VIII GENE IN EGYPTIAN HEMOPHILIA A PATIENTS
Azza Ibrahim1, Samia Rizk1, Hoda AbdelGhany1, Mona Aziz1,
Heba Abou-Elew1, Rania Zayed1, Ola Ibrahim2, and Nadia Zaghlol3
1
Department of Clinical Pathology, Faculty of Medicine, Egypt,
2
Department of Child Health, National Research Center, Egypt,
3
Department of Pediatric, Faculty of Medicine, Egypt
Selective inhibitor of CDKs, roscovitine (CYC202) induces cell cycle arrest
at G1/S and G2/M checkpoints in mammalian cells. Roscovitine acts in different
ways, all of which converge towards cell cycle arrest and subsequent cell death,
thereby providing the observed anti-tumor effects. In addition, polyamines are
small aliphatic amines that play a major role in multi-cellular functions. Levels
of polyamines are under control of the rate limiting enzyme of polyamine biosynthesis; ornithine decarboxylase. It was previously shown that ornithine decarboxylase activity is higher in human breast cancer specimens than in normal or
benign breast tissue. Therefore, it might be concluded that polyamine biosynthesis promotes breast cancer development. In our previous studies we estimated
that increased polyamine level in different body parts is expected to be used as
future diagnostic tool for malignancies. The objective of this study is to be able
to reveal the potential biomarker utility of polyamines as diagnostic tools in
breast cancer prognosis. In this study, we investigate therapeutic potential of
roscovitine in MCF-7 breast cancer cells. In order to understand the roscovitineinduced apoptotic mechanism, pro-apoptotic and anti-apoptotic gene expression
profiles will be discussed. Additionally, polyamine content is going to be determined to monitor roscovitine-induced polyamine metabolism. Roscovitine
decreased cell viability in dose and time dependent manner. Apoptotic dose is
determined as 20 lM which decreases cell viability by 27 % within 24 h. Investigation of roscovitine-induced polyamine pathway related apoptotic responses
are in progress.
Objective: Hemophilia A (HA) is an X-linked bleeding disorder caused by a
wide variety of mutations in the factor VIII (FVIII) gene, leading to absent or
deficient FVIII. Direct mutation detection is essential for carrier detection and
prenatal diagnosis in the family members of Egyptian hemophilic patients, and
this if achieved, will decrease the incidence of the disease. In addition, it will
avoid the hazards complicating therapy, including disease transmission and the
development of inhibitors to FVIII substitutes.
Methods: We analyzed the FVIII gene of forty Egyptian patients with HA.
The analysis included the investigation of intron 22 restriction fragment length
polymorphism (RFLP) by PCR using Xbal restriction enzyme, screening of FVIII
gene for other molecular abnormalities as deletions and point mutations by multiplex PCR and screening FVIII for a point mutation in exon 25 by using BsrDI
restriction enzyme.
Results: Our research revealed the following molecular abnormalities in the
studied HA patients; FVIII gene RFLP in intron 22 by using Xbal restriction
enzyme: 30% Xbal (1), 67.5% Xbal (-ve), 2.5% intron 22 deletion, partial FVIII
gene deletion in 10% of cases and exon 25 point mutation in 7.5% of cases.
Conclusion: We concluded that the study of intron 22 polymorphism by
Xbal restriction enzyme may be used to select the HA patients who have a
severe disorder and are negative for the Xbal polymorphic marker, or who have
intron 22 deletion to be investigated for FVIII gene intron 22 inversion.
P-165
P-167
BIOMARKER FOR THE EARLY DETECTION OF PROSTATE CANCER
Raluca Dumache1, Maria Puiu2, Gabriela Anton3, and Natalia Cucu4
1
Department of Biochemistry, University of Medicine and Pharmacy,
Timisoara, Romania, 2Department of Medical Genetics, University of
Medicine and Pharmacy, Timisoara, Romania, 3Molecular Biology National
Institute of Virology, Bucharest, Romania, 4Epigenetics Laboratory
University of Bucharest, Faculty of Biology, Bucharest, Romania
ADJUVANT DENDRITIC CELL-BASED TUMOR VACCINATION FOR
PATIENTS WITH MALIGNANT GLIOMA
Ahmed Eissa1, Hala Metwally2, Ehsan ElGhoneimy3, Mohamed Elbeltagy1,
Rania Zayed2, Nevine Foud2, and Ahmed Ali1
1
Department of Neurosurgery, Faculty of Medicine, Egypt, 2Clinical
Pathology, Faculty of Medicine, Egypt, 3Clinical Oncology and Nuclear
Medicine, Faculty of Medicine, Egypt
Prostate cancer is the most commonly diagnosed cancer in men and is
strongly associated with aging. The enzyme encoded by the alpha-methylacylCoA racemase gene plays a critical role in the peroxisomal beta-oxidation of
branched fatty acid molecules. In our study, we want to show that alpha-methylacyl-CoA racemase has a higher sensitivity and specificity for the prostatic tissue
and can be used for the early detection of prostate cancer. We evaluated the
alpha-methylacyl-CoA racemase gene expression in 32 patients aged 55 to 80
with total prostate-specific antigen values in a range of 3 to 40 ng/ml. Blood
samples from this patients were tested at various stages of disease progression.
26 patients were diagnosed positive for prostate intraepithelial carcinoma and
they had prostate-specific antigen values greater than 11 ng/ml. In these 26
patients we found by using RT- PCR techniques on needle biopsies that they presented an overexpressed gene for alpha-methylacyl-CoA racemase. The other 6
Glioblastoma multiform is the most aggressive and common primary brain
tumor, with a median survival of 12-18 month without the possibility of spontaneous remission. Current treatment has not substantially changed the prognosis
for GMB patients. One of the most recent treatment options is immune therapy.
The present study included 20 patients; liquid culture was established for generation of dendritic cells from peripheral blood mononuclear cells, using GM-CSF
and IL-4, followed by pulsing of the generated dendritic cells by tumor lysate to
produce tumor vaccine. Patients were vaccinated five times four weeks apart.
Follow up of the patients was done using MRI and DEMSA scan. The median
disease free survival was significantly improved in the studied patients. We demonstrate that tumor pulsed dendritic cells can be used as a potential vaccine in
the immunotherapy of brain tumors which represents an important approach for
improvement of treatment outcome.
P-168
FREQUENCY AND GENOTYPING OF HUMAN PAPILLOMA VIRUS
IN WOMEN WITH HIGH RISK OF DEVELOPING CERVICAL
CANCER
Recep Kesli1, Celalettin Eroglu2, M. Ali Eryilmaz2, M. Guzel Kurtoglu3,
and Havva Dogan2
1
Central Laboratory Molecular Diagnostic Unit, Konya Educational and
Research Hospital, Konya, Turkey, 2Cancer Early Screening-Diagnosis and
Training Centre, Konya Educational and Research Hospital, Konya, Turkey,
3
Microbiology Laboratory, Konya Educational and Research Hospital,
Konya, Turkey
Introduction: Objective of this study was to determine frequency and genotyping of Human Papillomavirus (HPV) in women with high risk of developing
cervical cancer with molecular diagnostic methods.
Materials and Method: One hundred sixty eight healthy women with high
risk of developing cervical cancer and applied to Cancer Early Screening-Diagnosis and Training Centre during July 2007-January 2008 consisted of this study
group. Endocervical swab specimens were provided with Dacron swabs and transported to laboratory within liquid transport media. HPV existence and genotyping
were investigated with reverse–hybridization method in three steps; firstly DNA
isolation was performed with isolation kits and secondly in vitro amplification was
performed with thermal cycler and finally the amplification products were selectively hybridized to the test strips contains sequence specific oligonucleotide
probes with Auto-LiPA instrument. Comparison of the HPV positive and negative
groups according to age, age of beginning of sexual activity, smoking was made
statistically with Chi-Square test. The following genotypes were evaluated as to
have high risk for developing cervical cancer: 16, 18, 31, 33, 45, 51, 53, 58, 59,
66, 68. Genotype HPV 6, 11, 40, 54, 70 were evaluated as to be have low risk.
Results: Median age of study group was 31 years, between 20 and 54 years.
Of 168 women 151 were younger then 45 (90%), 17 were older than 45 (10%);
for 140 (83%), sexual activity begun at less than 20 years (83%), and for 28 at
more than 20 years (17 %); 42 were smoking (25%), 126 were non-smoking
(75%). The frequency rate of HPV infection was demonstrated to be 17 %
(n529). Distribution of positivity of HPV genotypes were found as follows: for
HPV 6, 7 % (n52), HPV 16, 38% (n511), HPV 18, 10% (n53), HPV 31, 33,
40, 54 and 58 all together 21% (n56), HPV 45, 10% (n53), HPV 52, 10%
(n53), HPV 68, 7% (n52), HPV 35, 39, 51, 70, and 66 for each other 3%
(n51). Of total HPV positive patients, 28 were found to have high risk for
developing cervical cancer (97%), only one patient was found to have low risk
(3%). Although there was no significant difference between HPV- positive and
negative women regarding age and age of beginning of sexual activity (p >
0.05), smoking was found to be significant (p50.02).
Conclusion: In conclusion, the frequency of HPV infection in high risk
women was determined in our region.
P-169
SCREENING OF CAUSATIVE AGENTS OF STD’S BY DNA-REVERSE
HYBRIDIZATION METHOD FROM MAN WITH CHRONIC
PROSTATITIS SYMPTOMS IN KONYA, TURKEY
Recep Kesli1, Kenan Korkmaz2, Akin Yolas2, and Nadir Kocak1
1
Central Laboratory, Molecular Diagnostic Unit, Konya Educational and
Research Hospital, Konya, Turkey, 2Urology Clinics, Konya Educational
and Research Hospital, Konya, Turkey
Introduction: The prostatitis syndrome is one of the most common problems
encountered in urologic practice. Neisseria gonorrhoeae, Treponema pallidum,
Human papilloma virus (HPV), Herpes simplex virus (HSV) and Chlamydia trachomatis are the most common bacterial, viral and chlamydial agents in STD’s
that manifest primarily as urethritis or prostatitis in males. The objective of this
study was to screen urology outpatient, males with symptoms suggestive of
chronic prostatitis for the presence of the most common causative agents of the
prostatitis. Molecular diagnostic methods have been found to be highly sensitive
and specific methods for detection of these agents.
Material and Methods: All the 27 patients were evaluated by the same urologist according to a protocol comprising medical and sexual history, physical
371
and genital examination and basic laboratory investigation include urinalisys,
standard urine culture and haemogram but not 2 or 4 glass test. Patients with
normal laboratory findings had a urethral swab for determination of the specific
DNA belonging to the six most common agents of STD’s (N. gonorhoeae, T.
pallidum, HPV, HSV type 1. Existence of DNA was investigated with reverse–
hybridization method with three steps; firstly DNA isolation was performed with
isolation kits and secondly in vitro amplification performed with thermal cycler
and finally the amplification products were selectively hybridized to the
test stribs contains sequence specific oligonucleotide probes with Auto-LiPA
instrument.
Results: Totally 4 (14.8%) positive results were found. C. trachomatis DNA
was detected in 1 (3.7%), N. gonorrhoeae DNA was detected in 2 (7.4%) and
HPV DNA was detected in 1 (3.7%) of the 27 patients, others were not detected.
In 23 of the 27 patients (85.1%), the urethral swab specimens were negative. Of
the patients with positive results, 2 of 4 were married and the others single, 3 of
4 had multiple sexual partners. All of them did not use condom in their sexual
relations and their age is below 30.
Conclusion: C. trachomatis and N. gonorrhoeae may be two of the causative
agents of chronic prostatitis, but not very common in our series and the literature.
Screening programmes might be used more commonly in men who have multiple
sexual partners and do not use condom during sexual activity; active screening of
men with chronic prostatitis symptoms aged < 30 years is highly recommended.
P-170
EFFECTS OF INSULIN-LIKE GROWTH FACTOR-1 ON
METASTATIC CELL BEHAVIORS OF HUMAN METASTATIC
BREAST CANCER CELLS
R. Mine Guzel and Mustafa B.A. Djamgoz
Department of Cell and Molecular Biology, Imperial College, London, UK
Objective: IGF-1 is known to play a major role in development/progression
of human malignancies, including breast cancer (BCa). The objectives of this
study were (1) to evaluate the effects of IGF-1 on the metastatic behaviors of
MDA-MB-231 human BCa cell line; and (2) to determine whether a functional
relationship existed between IGF-1 signaling and voltage-gated sodium channel
(VGSC) activity in the control of metastatic cell behaviors.
Methods: Using the strongly metastatic human BCa MDA-MB-231 cell line,
the influence of IGF-1 on metastatic behaviors was investigated. Expression of
IGF-1 receptor mRNAs was determined by PCR. The functional assays used
included proliferation, motility and single-cell adhesion. Since the tyrosine kinase
inhibitor AG1024 exhibits lower IC50 values for IGF-1 than insulin receptors, 5
lM AG1024 was used in standard growth medium to block endogenous IGF-1
signaling. The possible involvement of VGSC activity was tested using the specific blocker tetrodotoxin (TTX).
Results: MDA-MB-231 cells expressed IGF-1 receptor mRNA, as shown previously. At the working concentration, AG1024 (5 lM), like TTX, did not affect
proliferation of MDA-MB-231 cells after 48 hours of treatment. However, the
same concentration of AG1024 demonstrated a downwards trend in the motility of
MDA-MB-231 cells after 48 hours; there was no apparent effect on single-cell adhesion. Increasing the concentration of AG1024 to 10-20 lM decreased proliferation of MDA-MB-231 cells after 24 hours in a dose-dependent manner.
Conclusions: These results suggested that in the MDA-MB-231 cell model
of BCa, high-level endogenous IGF-1 signaling could enhance metastatic but not
proliferative activity, similar to the role of VGSCs. Thus, there is the following
possible triangular functional relationship: metastatic BCa $ VGSC $ IGF-1,
which merits further investigation.
P-171
INVESTIGATION OF VASOACTIVE ENDOTHELIAL GROWTH
FACTOR (VEGF) GENE POLYMORPHISM AND SERUM
CONCENTRATION IN ENDOMETRIOSIS
Rukset Attar1, Erkut Attar2, Ilhan Yaylim-Eraltan3,
Hulya Yilmaz-Aydogan3, Bedia Agachan3, and Turgay Isbir3
1
Department of Obstetrics and Gynecology, Yeditepe University Hospital,
Istanbul, Turkey, 2Department of Obstetrics and Gynecology, Division of
372
Reproductive Endocrinology and Infertility, Istanbul University, Istanbul
Medical School, Istanbul, Turkey, 3Department of Molecular Medicine,
Istanbul University, Institute for Experimental Medicine, Istanbul, Turkey
In this study, we aimed to investigate the roles of VEGF -460[T and
1405[C polymorphisms and serum VEGF levels on etiopathogenesis of endometriosis. The DNA materials of endometriosis cases and control group were purified
and polymorphism analysis was performed by PCR-RFLP method, serum VEGF
levels were measured by ELISA method. As a result of this investigation, there
was no difference in VEGF 405 gene polymorphism aspect. CC genotype was
increased in patient group compared to control group (X25 2.91, p50.08). CC genotype increased endometriosis risk 1.61 times (ODDs ratio51.612, %95 CI:
0.893-2.913). There was statistically insignificant increment of GC genotype in endometriosis patient group than in control group (X25 2.69, p5 0.101). GC genotype increasedendometriosis risk 1.395 times. VEGF 405 C allele was higher in
patient group than in control group, but this was statistically not significant
([X25Q.027, P50.870). VEGF 405 G allele frequency was higher in advanced
cases than in early ones, but this was also not statistically significant (p50.302).
P-172
ASSOCIATION OF IL-1b GENE POLYMORPHISM WITH
ENDOMETRIOSIS
Rukset Attar1, Bedia Agachan2, Ozlem Kucukhuseyin2, Bahar Toptas2,
H. Arzu Ergen2, Erkut Attar3, and Turgay Isbir1
1
Department of Obstetrics and Gynecology, Yeditepe University Hospital,
Istanbul, Turkey, 2Department of Molecular Medicine, The Institute of
Experimental Medicine, Istanbul University, Capa, Istanbul, Turkey,
3
Department of Obstetrics and Gynecology, Division of Reproductive
Endocrinology and Infertility, Istanbul University, Istanbul Medical School,
Istanbul, Turkey
Aim: Genetic-association studies have revealed associations between the development of endometriosis and certain genetic polymorphisms but the genes that
play a role in susceptibility to the development and progression of endometriosis
are still unknown. Cytokine-mediated immune and inflammatory responses have
been considered to play an important role in the pathogenesis of endometriosis.
The aim of this study was to investigate whether the interleukin-1 b gene polymorphism is responsible in part for the genetic susceptibility to endometriosis.
Methods: The IL-1b13953 genotype was determined in 40 patients with
endometriosis and 78 control women by polymerase chain reaction-restriction
fragment length polymorphism.
Results: The frequency of 13953 CC genotype of IL-1 b was lower in cases
(37.5%) compared with controls (39.7%). The frequency of 13953 CT genotype
of IL-1 b was lower in cases (48.7%) compared with controls (40.0%). The frequency of 13953 TT genotype of IL-1 b was higher in cases (22.5%) compared
with controls (11.5%). Although the frequency of 13953 TT genotype of IL-1 b
was two times higher in cases but no statistical difference was found in the distribution of genotypes of IL-1b13953 (P > 0.05).
Conclusions: Our study showed that the frequency of TT allele was increased
in the patients with endometriosis compared with the controls. However, the distribution of genotype and allele frequencies of IL-1b13953 were not statistically
different between the endometriosis patients and the controls. This may be due to
the small size of the groups. Therefore further studies have to be done.
P-173
INCREASE IN PLATELET REACTIVE OXYGEN SPECIES AND
SERUM LYSOPHOSPHATIDIC ACID AND LIPID PEROXIDES IN
HYPERCHOLESTEROLEMIC RATS
A. Yesim Gocmen1, Saadet Gumuslu1, and Durmus Burgucu2
1
Department of Biochemistry, Akdeniz University, Faculty of Medicine,
Antalya, Turkey, 2Department of Physiology, Akdeniz University, Faculty
of Medicine, Antalya, Turkey
Oxidative stress, with the generation of reactive oxygen species (ROS), is suspected to play a role in the pathophysiology of atherosclerosis, thrombosis and
acute coronary diseases. Among circulating cells, platelets produce ROS, such as
superoxide anion. This has an important role in atherosclerosis, thrombosis and
acute coronary diseases. In the present study, we measured ROS status in platelets
and the levels of serum total cholesterol (TC), low-density lipoprotein cholesterol
(LDL-C), high density lipoprotein cholesterol (HDL-C) thiobarbituric acid reactive
substances (TBARS), oxidized low density lipoprotein (oxLDL), lysophosphatidic
acid (LPA), hydrogen peroxide (H2O2) and paraoxonase-1 (PON1) activity in
hypercholesterolemic and control rats. We used 24 male Wistar rats (n512 in
each group) which have been divided into 2 groups as; control (C), and cholesterol
(Chol). Control rats were fed standard diet for 8 weeks, while Chol group was
given 5% cholesterol diet for 8 weeks. The TC and LDL-C levels were found to
be increased and HDL-C level and PON1 activity were decreased in Chol group
when compared to controls. Our results also showed that platelets obtained from
hypercholesterolemic rats contain higher ROS than do platelets from control rats,
indicating a state of oxidative stress. Serum TBARS and oxLDL levels were found
to be increased while PON1 activity is decreased in Chol group compared to controls. Compared to control group, LPA and H2O2 levels were increased in Chol
group. In hypercholesterolemic rats, platelet ROS status positively correlated with
serum TBARS, oxLDL, H2O2 and LPA levels and negatively correlated with serum PON1 activity. In conclusion, the study revealed that hypercholesterolemia is
accompanied with increased platelet ROS levels and increased levels of serum
lipid peroxides. This finding might suggest a role for platelet ROS status in favoring the accumulation of lipid peroxides in hypercholesterolemic rats.
P-174
METHYLATION OF SFRP2, P16, DAPK1, HIC1 AND MGMT GENES
AND RELATING K-Ras GENE (CODONS 12-13) MUTATIONS WITH
COLORECTAL CANCERS
Sacide Pehlivan1, Mehmet Artac2, Can Kilicarslan1, Hakan Bozcuk3,
Tugce Sever1, and Mustafa Pehlivan4
1
Department of Medical Biology, Gaziantep University, Turkey,
2
Department of Medical Oncology, Selcuk University, Turkey, 3Department
of Medical Oncology, Akdeniz University, Turkey, 4Department of
Hematology Gaziantep University, Turkey
The aim of this study is to detect whether there is any difference between
normal and tumour tissue DNA by investigating methylation of SFRP2, P16,
DAPK1, HIC1 and MGMT genes and amino acid mutations of codons 12and 13
In K-Ras gene in normal and tumour tissue DNAs of patients diagnosed with
colorectal cancer. Methylation of SFRP2, P16, DAPK1, HIC1, MGMT genes and
amino acid mutations of K-Ras codons 12 and 13 are investigated by PCR and
Reverse Hybridization techniques in DNA from tumour and normal tissues of 17
individuals diagnosed with colorectal cancer. In codon 12 of K-Ras gene, Asp
has been detected in 4 patients and both Asp and Ser mutations have been
detected in 1 patient whereas Asp mutation has been detected in 8 patients in
codon 13. In SFRP2 gene, whole promoter methylation (WPM) has been
detected in 1 normal tissue and 4 tumour tissues while partial promotor methylation (PPM) has been found in 2 normal and 5 tumour tissues. WPM has been
observed in 1 tumour tissue in P16 gene. In DAPKl gene, WPM has been
detected in 7 tumour and 5 normal tissues whereas PPM has been found in 2
normal tissues. In HIC1 gene, WPM has been detected in 3 tumours while PPM
has been detected in 2 tumours; however, no methylations have been found in
normal tissue. In MGMT gene, WPM has been detected in 5 tumour and 2 normal tissues whereas PPM has been found in 1 tumour. As a result of this study,
it has been demonstrated that methylation in genes is observed at a higher rate in
tumour tissues than in normal tissues and that more significant results can be
obtained provided that a large number of patients are studied.
P-175
ALLELE FREQUENCIES FOR 14 STRs LOCI IN WESTERN
ANATOLIA POPULATION AND THEIR FORENSIC EVALUATION
Aysun Baransel-Isir1, Abdulmuttalip Ozkorkmaz2, and Sacide Pehlivan3
1
Forensic Medicine, Gaziantep University, Gaziantep, Turkey, 2Department
of Biology, Ege University, Izmir, Turkey, 3Department of Medical Biology
and Genetic, Gaziantep University, Gaziantep, Turkey
Genetic analysis of 104 unrelated individuals from Turkish population was
analyzed by ABI PRISM 3100 Genetic Analyzer after amplification with multiplex PCR Amplification kit which contains D3S1358, D2S1358, D16S539,
D8S1179, D21S11, D18S51, THO1, D13S317, D7S820, CSF1PO, TPOX,
D5S818, FGA and amelogenin loci. DNA was extracted from peripheral leucocytes by salting-out method. Loci polymorphisms were evaluated for forensic
purposes. Polymorphic Information Content (PIC) frequencies for D3S1358,
D2S1358, D16S539, D8S1179, D21S11, D18S51, THO1, D13S317, D7S820,
CSF1PO, TPOX, D5S818, FGA and amelogenin loci were determined. In Turkish population; 83.7% in D3S1358 locus, 88.5% in amelogenin allele, 73.1% in
D16S539 locus, 95.2% in D2S1358 locus, 86.5% in D8S1179 locus, 89.4% in
D21S11 locus, 88.5% in D18S51 locus, 81.7% in D13S317 locus, 78.8% in
THO1 locus, 84.6% in FGA locus, 77.9% in D5S818 locus, 66.3% in TPOX
locus, 75% in CSF1PO locus and 86.5% in D7S820 were determined PIC value.
As a result, 14 STR loci with their high combined PIC values (0.9999) seem to
be a useful system in forensic studies.
373
chemistry laboratory. The patients were classified into three groups according to
allele status. These groups were compared with regard to clinical and demographic features.
Results: The most prominent clinical symptoms were abdominal pain
(94.9%), fever (89.7%), arthritis (33.3%), and pleuritis (30.8%). Seventeen different genotypes were identified. The mutations were homozygous in 25 (32.1%)
patients, compound heterozygous in 28 (35.9%) patients, and heterozygous in 22
(28.2%) patients. No mutation was detected in three (3.8%) patients. The most
frequent mutations were M694V (54.5%), M680I (16%), E148Q (9.6%), and
P369S (3.6%). The mean symptom severity score was highest in the homozygous
group, and high levels of CRP were also detected in this group.
Conclusions: In addition to clinical criteria, molecular studies for detecting
disease-causing mutations are needed to establish the diagnosis of FMF. FMF
patients who were homozygous for MEFV gene mutations had a higher symptom
severity score and higher incidence of appendectomy. The broad spectrum of
mutations may reflect intercultural interactions of ethnic groups in Anatolia.
Nationwide studies may help to determine the relationships among demographic,
clinical, and genetic features of FMF.
P-176
ANALYSIS OF CYTOKINE AND HLA DQ GENOTYPING IN TURKISH
PATIENTS WITH HAIM-MUNK SYNDROME: A FAMILY STUDY
P-178
Kamile Erciyas1, Sacide Pehlivan2, Serhat Inaloz3, Ali Fuat Erciyas4,
Can Kilincarslan2, and Tugce Sever2
1
Department of Periodontology, Gaziantep University, Gaziantep, Turkey,
2
Department of Medical Biology and Genetics, Gaziantep University,
Gaziantep, Turkey, 3Department of Dermatology, Gaziantep University,
Gaziantep, Turkey, 4Department of Orthodonthy, AFE,
Clinical of Orthodonthy, Turkey
PLASMA NITRIC OXIDE LEVELS AND NITRIC OXIDE SYNTHASE
ACTIVITY IN LUNG CANCER PATIENTS
Haim-Munk syndrome (HMS) is an extremely rare autosomal recessive disorder clinically characterized by palmoplantar hyperkeratosis, severe early-onset
periodontitis with severe alveolar bone destruction, onychogryphosis, pes planus,
arachnodactyly, and acro-osteolysis. Consanguinity seems a notable prerequisite.
We present five unusual cases of familial HMS, one of which only had severe
skin lesions and severe periodontal disease; the others had mild skin lesions and
relatively mild periodontal disease. The aim of this study is to detect whether
there is any difference among family members of HMS in DNA by investigating
cytokine and HLA-DQ genotyping. Genomic DNA was extracted from mononuclear cells obtained from EDTA-treated peripheral venous blood using the salting
out method techniques. Cytokine (IL-6, IL-10, IFN-c, TGF-ß1, TNFa) and HLADQ genotyping was performed by the polymerase chain reaction sequence-specific primer method. In conclusion, this study showed for the first time the strong
association between Cytokine Genotyping and HMS.
Sebnem Korkmaz1, Ebru Gurleyik1, Mehmet Taspinar2, Derya Oztuna3,
Cansel Atinkaya4, Ulku Yazici5, Aslihan Avci1, and Asuman Sunguroglu2
1
Ankara, Turkey, 2Department of Medical Biology Ankara University,
Faculty of Medicine, Ankara, 3Department of Biostatistics, Ankara
University, Faculty of Medicine, Ankara, Turkey, 4Department of Thoracic
Surgery, Kirikkale University, Faculty of Medicine, Kirikkale, Turkey,
5
Department of Thoracic Surgery, Ataturk Center for Chest Disease and
Thoracic Surgery, Turkey
Objective: Nitric oxide (NO) is a molecule that plays various roles in the
body tissues. NO plays important roles in vasodilatation, neurotransmission, platelet aggregation, cytokine stimulation, etc. NO also exerts dual functions as an
oxidant and antioxidant substance depending on its concentrations and environmental conditions. In this study, we aimed to examine NO levels, NO synthase
(NOS) activity and possible correlation between NO levels and NO synthase activity in the non-small cell lung cancer patients.
Materials and Methods: Our study was carried out on 24 cancer patients
and 5 non-cancer subjects as controls. Blood samples were collected and plasma
NO levels and NOS activity were determined by spectrophotometric methods.
Results: In this study, it was found that NOS activity was increased in the
plasma of lung cancer patients compared to that of controls (Figure 1). This
P-177
FAMILIAL MEDITERRANEAN FEVER GENE MUTATIONS IN
THE INNER SOUTHERN REGION OF TURKEY AND
GENOTYPE-PHENOTYPE CORRELATION IN CHILDREN
Resul Yilmaz1, Samet Ozer1, Unal Korkmaz2, Huseyin Ozyurt3,
and Semsettin Sahin3
1
Department of Pediatrics, School of Medicine, Gaziosmanpasa University,
Tokat, Turkey, 2Department of Biostatistics, School of Medicine,
Gaziosmanpasa University, Tokat, Turkey, 3Department of Biochemistry
School of Medicine Gaziosmanpasa Universty, Tokat, Turkey
Objective: Familial Mediterranean fever (FMF) is an autosomal recessive
disorder characterized by recurrent episodes of fever, polyserositis, and rash. It is
caused by a mutation in the MEFV gene, which encodes pyrin, a protein with an
important role in the regulation of inflammation. The aim of this study was to
determine the most common mutations and clinical features, and their
relationships.
Materials and Methods: The medical records of 78 patients were evaluated
retrospectively. All of the patients had been diagnosed with FMF according to
Tel Hashomer criteria between January 2005 and May 2008 in general pediatric
clinics of the School of Medicine at Gaziosmanpasa University. Twelve mutations were detected in the 78 patients, by PCR-ELISA, in our molecular bio-
Figure 1. NOS Activity. [Color figure can be viewed in the online issue, which
is available at www.interscience.wiley.com.]
374
increase of NOS activity is statistically significant (p<0.05). Both in the control
and patient groups, NO levels were found to be nearly the same. There was a
correlation between NO levels and NOS activity however it was not statistically
significant.
Conclusions: The actions of NO in the field of tumor biology are complicated because they appear to have both promoting and inhibiting effects in the
etiology of cancer. It has been suggested that NOS may also play a role in the
complex transformation from cellular dysplasia to lung cancer.
P-179
POLYMORPHISM OF P53 GENE EXON 4 IN PATIENTS WITH
CORONARY ARTERY DISEASE
Selahaddin Tekes1, Dilek Cakir2, Beran Yokus3, Kenan Iltumur4,
and Selda Simsek1
1
Department of Medical Biology, Dicle University, Diyarbakir, Turkey,
2
Department of Biochemistry, Canakkale University, Turkey, 3Department
of Biochemistry, Dicle University, Diyarbakir, Turkey, 4Department of
Cardiology, Dicle University, Diyarbakir, Turkey
Coronary artery disease (CAD) is a multifactorial disorder. Hypertension
(HT), smoking, hyperlipidemia, diabetes mellitus (DM) and family history are
important risk factors for CAD. These risk factors have been described by many
studies. p53 is a tumour suppressor protein involved in the control of cell growth
and has an established role in carcinogenesis. Polymorphism in the p53 gene at
exon 4 has been linked to the development of certain diseases including cancer.
A possible association between such polymorphism and the development of coronary artery disease (CAD) is being investigated, but no conclusive evidence has
been reached yet. We investigated the possible association of the p53 gene polymorphism in patients with coronary artery disease living in south-eastern of
Turkey.
Seventy patients who required coronary angiography due to symptoms
related to coronary artery disease (CAD) and thirty control subjects were
screened for p53 exon 4 polymorphism using PCR-RLFP method. After analysis,
p53 genotypes were found to be as 11 Arg/Arg, 34 Arg/Pro, and 19 Pro/Pro
genotypes.
The next step will be to evaluate these genotypes and allele frequency of
p53 exon 4 polymorphism with the risk of CAD under the light of previous
studies.
P-181
MEFV GENE ANALYSIS RESULTS OF PATIENTS WITH PROBABLE
FAMILIAL MEDITERRANEAN FEVER
Esra Tug1, Selma Duzenli1, Dilek Dogruer1, and MusaKazim Caglar2
Department of Medical Genetics, Medical Faculty, Abant Izzet Baysal
University, Bolu, Turkey, 2Department of Paediatrics, Medical Faculty,
Abant Izzet Baysal University, Bolu, Turkey
1
Objectives: Familial Mediterranean Fever (FMF) is an autosomal recessive
disease, predominantly affecting people of Mediterranean descent (Turks, Arabs,
Jews, and Armenians). The disease is caused by mutations located in the MEFV
gene, and characterised by attacks of serositis, fever and amyloidosis. We
searched for the most common 12 MEFV gene mutations (M694V, M694I,
E148Q, M680I (G/C), M680I (G/A), V726A, R761H, P369S, F479L, I692del,
K695R and A744S) in Bolu, a region of Turkey. People living in this region are
commonly former nomads and there is not much immigration from other places,
unlike other regions of Turkey. This is the first attempt to evaluate MEFV mutations in this region.
Methods: Mutation detection was undertaken after DNA isolation. The most
common 12 mutations on the exons 2 and 10 were determined by hybridisation
method and an in-house PCR-RFLP method with appropriate restriction enzymes
(HinfI, AluI, Mva1) used for confirmation of the 5 most common mutations
V726A, E148Q, M680I (G/C), M680I and M694V.
Results: 24 of 30 patients with FMF susceptibility were found to have
MEFV mutations. We found 16 M694V alleles, 11 E148Q alleles, 3 V726A alleles, 2 P369S alleles, 1 M680I (G/C) allele, 1 F479L allele and 1 R761H allele.
The F479L, R761H and P369S mutations that we found in this homogenous population are rare mutations in Turks. Of the 24 patients 12 were heterozygote, 8
were homozygote and 4 were compound heterozygote.
Conclusion: Our study is part of a pre-study to determine the clinical results
of MEFV gene mutations in this region. It will be valuable to compare the study
results with results with other regions of Turkey and other Mediterranean countries. Our study needs to be strengthened by increasing the patient number and
with undertaking surveillance studies, to match the results with the above mentioned population groups.
P-182
P-180
PRENATAL DIAGNOSTIC RESULTS OF 241 CASES WITH HIGH RISK
IN SCREENING TESTS
Selda Simsek, Aysegul Turkyilmaz, Halit Akbas, Selahattin Tekes,
Mahmut Balkan, and Turgay Budak
Department of Medical Biology and Genetic, Dicle University,
Diyarbakir, Turkey
The practice of prenatal diagnosis in chromosomal abnormalities has been
used for nearly half a century. When Steele and Breg showed that the chromosome constitution of a fetus could be determined by analysis of cultured cells
from the amniotic fluid in 1966, prenatal diagnosis had its beginning. Karyotype
analysis has been the standard method for prenatal cytogenetic diagnosis since
the 1970s. Nowadays, a number of different noninvasive tests have been developed. Such noninvasive testing includes maternal serum screening (sometimes
called double screen or triple screen) together with ultrarasonographic examination to screen for fetuses with Down syndrome, trisomy 18, neural tube defects
(NTDs) and other abnormalities. In this study, we investigated fetal chromosome
constitution of 241 cases with high risk in double screening test and triple
screening test, which were referred to Medical Biology and Genetic Department
Diagnostic Laboratory of Dicle University, in the period of January 2007-August
2008.Two Lymphocyte Cultures were prepared for each case. Karyotype analyses
were carried out totally on ten slides for each specimen. One of the ten slides
was stained directly with Giemsa staining method and others with Giemsa Banding Technique (GTG Banding). In case of necessity, molecular cytogenetic techniques were applied.
ROUTINE RESULTS OF FACTOR V LEIDEN, FACTOR
V H1299R, FACTOR II PROTHROMBIN (G20210A),
MTHFR A1298C AND MTHFR C677T POLYMORPHISMS
WITH RT-PCR
Selma Duzenli, Esra Tug, Hatip Aydin, and Dilek Dogruer
University, Turkey
Objectives: It is postulated that some polymorphisms, alone or in combination, increase the predisposition for coagulation defects. Patients from different
clinics with pathologies like this were tested for some gene polymorphisms such
as Factor V Leiden, Factor V H1299R, Factor II Prothrombin (G20210A), methylene tetra hydropholate reductase (MTHFR) A1298C and MTHFR C677T.
Allelic distribution of these polymorphisms was evaluated in this patient group
with predisposition to clotting defect.
Methods: After extraction of genomic DNA, appropriate Real Time-PCR protocols were undertaken to determine each of the above mentioned polymorphisms.
Results: Of the analyzed 54 subjects, the allele number of the susceptibility
polymorphisms in total was 52. 18% of the total alleles were susceptibility alleles. We found 20 patients owning only 1 of this group of alleles. 24 owned 2
alleles, 8 had 3 alleles and 1 patient had 4 alleles.
Conclusion: Our study is a pre-study, aiming to show the distribution of susceptibility alleles for coagulation defects in patients with predisposition to diseases affecting the coagulation pathway. Of course, it is necessary to increase
the patient number and investigate a control group to get reliable results. In addition it is indispensable to add other susceptibility polymorphisms to studies like
this and make detailed clinical investigations.
P-183
MOLECULAR AND CLINICAL WORK-UP IN AN EXTENSIVE
FAMILY WITH BECKER MUSCULAR DYSTROPHY
Esra Tug1, Selma Duzenli1, Hatip Aydin1, H. Ibrahim Atasoy2,
and Zeynep Ocak3
1
University, Turkey, 2Department of Paediatrics, Medical Faculty, Abant
Izzet Baysal University, Turkey, 3Molecular Genetics, Burc Diagnostics
Center for Genetics, Turkey
Objectives: Becker Muscular Dystrophy (BMD) often results from in-frame
mutations of the dystrophin gene that allows production of an altered but partially functional protein. A 9-year-old boy was referred for evaluation because of
elevated serum creatine kinase (CK) activity, during routine blood screening for
hypospadias surgery. Blood screening of the proband’s older brother (11-yearold) showed elevated CK activity, as well. Pedigree analysis showed that one
maternal uncle, one maternal grand uncle and two second degree male cousins
were also affected.
Methods: Molecular genetics analysis was undertaken for exons 3, 4, 8, 12,
17, 19, 32, 34, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 and 52 in the dystrophin
gene of the proband and his brother. Multiplex PCR was undertaken with the
extracted DNA, and the products were run on an agarose gel after digesting with
appropriate restriction enzymes.
Results: This molecular work-up showed the proband and his brother to be
carriers of a deletion implying exons 13-19 of the dystrophin gene. Family members showing elevated CK activity and were aged between 9-25 yrs, had no clinical symptoms at all. 40-years-old maternal uncle of the proband showed mild
muscle weakness and 60-years-old maternal grand uncle showed severe muscle
weakness.
Conclusion: Based on molecular findings, this family would be given a diagnosis of BMD. This diagnosis implies the development of clinical symptoms,
even though a family is clearly asymptomatic in the first decades. This report
underscores the caution which must be exercised when giving presymptomatic
diagnoses based on molecular studies.
P-184
LOSS OF HETEROZYGOSITY (LOH) OF THE CDH1 GENE IN
SPORADIC GASTRIC ADENOCARCINOMAS
Selma Duzenli1, Omer Faruk Bayrak2, and Ismail Yegin3
University, Bolu, Turkey, 2Department of Medical Genetics and
Bioengineering, Yeditepe University, Istanbul, Turkey, 3Department of
Medical Oncology, Medical Faculty, Ataturk University, Turkey
1
Objectives: E-cadherin is a member of the cadherin family of calcium dependent cell adhesion molecules which is detected in most kinds of tissues and
plays a major role in the process of intercellular adhesion between epithelial
cells. Loss of function is thought to contribute to progression in cancer by
increasing proliferation, invasion, and/or metastasis. It has been demonstrated
that loss of E-cadherin function is pivotal in both sporadic and hereditary forms
of diffuse gastric tumorigenesis. Gastric carcinomas are the most seen between
all gastrointestinal carcinomas. We sought to determine the prevalence of CDH1/
E-cadherin (E-cad) gene alterations in "sporadic" gastric carcinomas. We analyzed a series of 25 sporadic gastric adenocarcinomas for the presence of LOH
and determined this in a single case.
Methods: DNA was extracted from 25 tissue specimen from Turkish patients
diagnosed with gastric adenocarcinoma histopathologically, after obtaining
informed consent. Clinical information was obtained from medical records. Loss
of heterozygosity (LOH) analysis was performed using two restriction fragment
length polymorphism (RFLP) reactions, undertaking nested PCR.
Results: Of the 25 patient, two were 3 were diffuse and 22 were intestinal
type tumors. 18 were female and 7 were male. The median age was 59.667.1.
In our study only one case of the diffuse type specimen showed loss of heterozygosity of the wild-type allele. This is 4% LOH in the CDH1 gene, in our
study.
375
Conclusion: The most common genetic abnormalities in gastric cancer seem
to be loss of heterozygosity of tumor suppressor genes. This study shows that
LOH is mostly seen in diffuse type of gastric carcinoma. Of course, to fulfill the
classical two hit hypothesis of E-cadherin inactivation, more specimen are
needed and more other mechanisms, operating at transcriptional or at the posttranslational level have to be investigated.
P-185
NON-SYNDROMIC HEARING IMPAIRMENT: 35DELG MUTATION IN
SEVEN CONSANGUINEOUS FAMILIES FROM EASTERN TURKEY
Selma Duzenli1, Akin Seraraslan2, and Harun Ucuncu3
1
Department of Medical Genetics, Abant Izzet Baysal University, Medical
Faculty, Bolu, Turkey, 2Head, Neck and Otolaryngology Clinics,
Gumushane City Hospital, Turkey, 3Department of Head, Neck and
Otolaryngology, Ataturk University, Medical Faculty, Erzurum, Turkey
Objectives: In underdeveloped countries environmental factors such as infections, trauma, and ototoxic drugs are an important cause of hearing loss. However, due to a high percentage of consanguinity mating, heredity is another important cause of deafness. Congenital sensorineural hearing impairment affects
approximately 1/1000 child in western countries. About 60% of all cases of sensorineural deafness are estimated to be of genetic origin, mainly with autosomal
recessive inheritance. Of particular relevance among deafness genes is GJB2,
coding for connexin 26 (Cx26). The hearing impairment in these patients has
been described as severe or profound.
Material and Methods: The study was conducted in seven consanguineous
families with a total of 43 subjects of which 27 patients affected by congenital
non-syndromic sensorineural hearing impairment (severe-profound hearing loss).
Since there is no known study in patients from the eastern part of Turkey we
tried to evaluate the 35delG mutation frequency in this patient population. A
thorough clinical evaluation and audiometric assessment, together with a molecular analysis of the 35delG mutation, were performed. The 35delG mutation in
hearing impaired patients was detected by PCR, followed by a RE digest.
Patients homozygous for 35delG showed one band with 181 bp and heterozygous
patients displayed two bands (181 bp and 207 bp) after polyacrylamide gel
electrophoresis.
Results: All of the 27 patients affected by sensorineural hearing impairment
showed homozygote 35delG mutation and the other 16 family members were all
heterozygote for the mutation. Our results show that the mutation frequency is
higher than in developed countries. The reason can be found in the high consanguinity rate as well as in infections.
Conclusions: It is obvious that for more precise and accurate genetic counseling 35delG mutation diagnose should be a must in sensorineural hearing
impaired patients.
P-186
SEPTO-OPTIC DYSPLASIA (MORSIER’S SYNDROME) AND
DEVELOPMENTAL GENETICS: A CASE REPORT
Selma Duzenli1, Dilek Dogruer1, Serpil Yildiz2, Hatip Aydin1,
and Esra Tug1
1
Department of Medical Genetics, Abant Izzet Baysal University, Medical
Faculty, Bolu, Turkey, 2Department of Neurology, Abant Izzet Baysal
University, Medical Faculty, Bolu, Turkey
Objectives: This is a report of a 23 year old female adult with septo-optic
dysplasia (SOD) which is a rare, highly heterogeneous condition comprising a
variable phenotype of optic nerve hypoplasia condition. Additionally, midline
brain abnormalities and pituitary hypoplasia with consequent endocrine deficits
are usual findings. The majority of cases are sporadic. SOD incidence is reported
as 1/10,000, and several aetiologies have been suggested to account for the
pathogenesis of the condition.
Material and Methods: Our proband is the child of a healthy non-consanguineous couple. The couple lost their first two babies for unknown reasons. In
addition to the proband, the family has 3 healthy children. It is reported that the
proband had his first seizure with 5 months and progressed like this. The patient
376
has almost no speech but moderate mental development. The ocular investigations showed nistagmus and sight defect. Spastic hemiparesis was the major disability of the body. Dysmorphologic findings like synophris, high palate, open
mouth, crowded teeth with caries were additional clinical findings. Laboratory
showed no abnormal hormone levels. MRI coincided with septo-optic dysplasia,
septum pellucidum agenesis, cortical developmental anomaly.
Results: The identification of mutations in developmental genes including
HEX1, SOX2 and SOX3 in patients with SOD, and associated phenotypes suggests a genetic causation. The precise aetiology of SOD is most likely multifactorial involving contributions from environmental factors in addition to the developmental genes.
Conclusions: The mechanism by which SOD is transmitted is unknown. The
variations in the phenotypes of the disease give a special value to every single
SOD case. Because of different phenotypes and the heterogeneity of the disease
this case should be a contribution to the existing reports.
P-187
MOLECULAR AND CLINICAL FINDINGS IN AN
EXTENSIVE FAMILY WITH DUCHENNE/BECKER
MUSCULAR DYSTROPHY
Selma Duzenli1, Esra Tug1, Dilek Dogruer1, Sule Aydin-Turkoglu2,
Nebil Yildiz2, and Ebru Kaplan1
1
Department of Medical Genetics, Abant Izzet Baysal University, Izzet
Baysal School of Medicine, Bolu, Turkey, 2Department of Neurology,
Abant Izzet Baysal University, Medical Faculty, Bolu, Turkey
Objectives: Becker Muscular Dystrophy (BMD) is a type of dystrophinopathy related to Duchenne Muscular Dystrophy (DMD), but is a milder form due
to some functional dystrophin protein. A 30-year-old male with muscle weakness
was referred to the clinic for testing deletions in the DMD gene. The patient suffered of slowly progressive difficulties like progressive symmetrical muscle
weakness and atrophy, proximal greater than distal, with calf hypertrophy (only
weakness of quadriceps femoris), activity-induced cramping, flexion contractures
of the elbows for 10 years. His neck flexor muscle strength was still preserved.
Blood screening of the proband showed elevated creatine kinase (CK) blood concentrations. Needle-EMG showed myogenic activity. After evaluation of the family members, 6 individuals of three generations showed CK elevation and 2
showed muscle weakness. The inheritance shows an X- recessive pattern.
Methods: Analysis was done by multiplex PCR with specific dystrophin
gene primers and was run on agarose gel after digesting with appropriate restriction enzymes for exons Pm, Pb, 3, 4, 5, 6, 7, 8, 9, 12, 13, 17, 18, 19, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60 at position Xp21.2.
Results: Deletion was shown on exons 17-42 of the dystrophin gene. Family
members showing elevated CK activity were aged between 9-25 years and had
no clinical symptoms at all. The 60-year-old maternal uncle showed severe
muscle weakness.
Conclusion: Based on molecular and clinical findings, this family would be
given a diagnosis of BMD. This diagnosis implies the development of clinical
symptoms, even though this family is clearly asymptomatic for the first decades.
This report underscores the caution which must be undertaken when giving presymptomatic diagnoses based on molecular studies.
Acknowledgement: We thank gratefully Prof. Dr. Pervin DINCER from
Hacettepe University, Medical Faculty, Department of Medical Biology for
evaluation of the test.
P-188
APOLIPOPROTEIN E GENOTYPING IN STROKE PATIENTS
Selma Duzenli1, Ibrahim Pirim2, Akcahan Gepdiremen3, and Orhan Deniz4
1
Department of Medical Genetics, Abant Izzet Baysal University, Izzet
Baysal School of Medicine, Bolu, Turkey, 2Department of Medical Biology,
Ataturk University, Erzurum, Turkey, 3Department of Pharmacology, Abant
Izzet Baysal University, Bolu, Turkey, 4Department of Neurology, Ataturk
University, Erzurum, Turkey
Objective: Human apolipoprotein E (apo E) alleles are polymorphic and
have been associated with increased risk of coronary heart disease and postulated as a major genetic susceptibility locus for Alzheimer’s disease. Studies
undertaken in different populations have shown different association patterns
between apoE genotype and stroke. The aim of this study was to determine
the risk of apoE genotype in stroke patients living in the eastern part of
Turkey.
Methods: The apoE genotypes and allele frequencies of 229 individuals
from the same geographic area were determined by PCR and RFLP, of which
103 were patients with a documented history of stroke without other apparent dementia and 126 age-matched healthy subjects as a control group.
Results: Our results show statistically significant association of apoE epsilon
3/4 genotype, A reduced epsilon 3/4 genotype frequency (p<0.05) was found in
subjects with stroke and the epsilon E2/3 genotype frequency (p<0.05) was elevated in patients with previous stroke which suggests that epsilon 2/3 genotype
is a survival factor for stroke. There was no association between apoE epsilon 4
allele and stroke.
Conclusion: This is the first report to have examined the association of apoE
genetic polymorphism with stroke patients from eastern Turkey. Compared with
other studies, the APOE alleles had divergent effects in this population. Association between APOE alleles and stroke in this population may be altered due
to interaction with other genetic effects. In conclusion, stroke is associated
with APOE alleles and genotypes and it requires further study in different
populations.
P-189
WHEATGRASS JUICE REDUCES NITRIC OXIDE PRODUCTION IN
LPS INDUCED RAW 264.7 CELLS
Sena Aydos1, Tulin Ozkan2, Arzu Z. Karabay3, Asli Koc3, Aynur Karadag1,
Buket Altinok2, and Asuman Sunguroglu1
1
Ankara University, Faculty of Medicine, Department of Medical Biology,
Graduate School of Health Sciences, Turkey, 2Ankara University, Institute
of Biotechnology, Turkey, 3Ankara University, Faculty of Pharmacy,
Department of Biochemistry, Graduate School of Health Sciences, Turkey
Objective: Wheatgrass, the young grass of Triticum aestivum contains chlorophyll, amino acids, minerals, vitamins, and enzymes and claimed to have antioxidant properties. Because of its probable potential to improve the digestive system, prevent cancer, diabetes and heart disease, it has been used as a folk medicine. However there is limited evidence to support these claims. LPS induces
macrophages and activates iNOS to produce excess nitric oxide. In this study,
we investigated the effect of wheatgrass juice on LPS-induced NO production in
macrophages.
Material and methods: Cell Culture and stimulation: RAW 264.7 cells
were cultured in RPMI 1640 medium supplemented with 10% heat inactive
Fetal Calf Serum (FCS), 2mM L-Glutamine, antibiotics (1.000.000 U/ml penicillin and 1 g/mL streptomycin) at 378C in a humidified atmosphere of 5 %
CO2 in air. After counting with 0.4% trypan blue, cells (4 x104 ) were seeded
to 96-well micro plates and incubated in the absence or presence of LPS (1
lg/mL) for 20 hours. Different concentrations of wheatgrass juice (0.5%;
1.5%; 2.5%; 3.5%, 5%, 7.5%, 10% v/v) was added to cultures 1 hour before
stimulation with LPS. Nitrite measurements: Samples (100 ll) of culture
media were incubated with an equal amount of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride and 2.5% phosphoric
acid) at room temperature for 10 min in a 96-well micro plate before determining the absorbance at 548 nm. The absorbance at 548 nm was compared
with standards of NaNO2. Statistical analysis: Differences among groups were
evaluated by One Way Anova analysis. P values less than 0.05 were considered as statistically significant.
Results: LPS induced NO production was reduced by wheatgrass juice in a
dose dependent manner. We found significant reductions of nitrite levels in lypopolysaccharide LPS induced cells which were treated with wheatgrass juice at
concentrations of 1.5%; 2.5%; 3.5%, 5%.
Conclusion: Our investigation shows that wheatgrass juice has an inhibitory
effect on NO production in LPS induced macrophage cells. Within the support
of further experimental data, it may have the potential to be used as an alternative for disorders related with NO production.
P-190
POLYMORPHISMS IN TNF-ALPHA (-308), IL-6 (-174) AND IL-10
(-1082) GENES AND THE RISK FOR SPORADIC LATE-ONSET
ALZHEIMER’S DISEASE
Pervin Vural1, Sevgin Degirmencioglu1, Hande Parildar-Karpuzoglu1, Semra
Dogru-Abbasoglu1, Hasmet A. Hanagasi2, Berrin Karadag3, Hakan Gurvit2,
Murat Emre2, and Mujdat Uysal1
1
Turkey, 2Department of Neurology, Istanbul University, Istanbul Medical
Faculty, Turkey, 3Department of Internal Medicine, Division of Geriatrics
Istanbul University, Cerrahpasa School of Medicine, Turkey
There is increasing evidence suggesting that inflammation and cytokines
might be involved in the pathogenesis of Alzheimer’s disease (AD). Multiple
cytokines are produced in response to Ab and are over-expressed in activated
microglia surrounding plaques in AD. The balance between pro- and anti-inflammatory cytokines determines the magnitude of the inflammatory response. TNFalpha, IL-6 and IL-10 have opposing effects. While TNF-alpha and IL-6 are generally pro-inflammatory, IL-10 limits and terminates inflammatory reactions.
TNF-alpha can increase the production of amyloid (Ab). IL-6 is thought to lead
to accumulation of acute phase proteins in plaques and is associated with
increased amyloid precursor protein (APP) synthesis. IL-10 limits inflammation
by reducing the synthesis of pro-inflammatory TNF-alpha, IL-1, and IL-6, by
suppressing cytokine receptor expression and by inhibiting receptor activation in
the brain. Single nucleotide polymorphisms (SNPs) in the regulatory regions of
TNF-alpha, IL-6 and IL-10 have been suggested to influence the risk of AD with
conflicting results. To further investigate the possible association between cytokines and AD susceptibility, we analyzed genotype and allele distributions of
TNF-alpha (-308), IL-6 (-174) and IL-10 (-1082) in 101 sporadic AD patients
and 138 healthy controls. Individuals with AA genotype at TNF-alpha (-308) had
2.2-fold increased risk for developing AD compared to wild homozygous. Heterozygotes (AG) or combined genotype (AG1AA) for IL-10 (-1082) were associated with approximately two-fold increase in the risk of AD. Carriers of A alleles of both TNF-alpha (-308) and IL-10 (-1082) had 6.5 times higher risk for
AD in comparison with non-carriers. Similarly, concomitant presence of both
mutant TNF-alpha (-308) A and IL-6 (-174) C alleles raised three-fold the AD
risk, whereas there was no notable risk for AD afflicted by IL-6 (-174) polymorphism alone. Our results suggest that TNF-alpha and IL-10 promoter polymorphism might be a risk factor for AD. The combined effects of TNF-alpha (-308),
IL-6 (-174) and IL-10 (-1082) variant alleles may be more decisive to induce
functional differences and modify the risk for AD.
P-191
SINGLE NUCLEOTIDE POLYMORPHISMS OF DNA BASE-EXCISION
REPAIR GENES APE/REF-1 AND XRCC1 ARE NOT ASSOCIATED
WITH THE RISK FOR GRAVES’ DISEASE
Semra Dogru-Abbasoglu1, Sevda Tanrikulu1, Evin Ademoglu1,
Yesim Erbil2, and Mujdat Uysal1
1
Turkey, 2Department of General Surgery, Istanbul University, Istanbul
Medical Faculty, Turkey
Reactive oxygen species (ROS) have been related to the etiopathogenesis of
Graves’ disease (GD). ROS are particularly important genotoxic agents and generate many DNA lesions, such as oxidized DNA bases and apurinic/apyrimidinic
sites. Oxidative DNA damage have been found increased in peripheral leukocytes, urine and cultured human mononuclear cells of the GD patients. Base
excision repair (BER) is the major DNA repair pathway responsible for removing
oxidative DNA damage. Polymorphisms in DNA-repair genes have been associated with the increased risk of several disorders including various types of cancer
and could also be related to the etiology of GD. Therefore, we conducted a casecontrol study including 97 patients with GD and age- and sex-matched 170
healthy control subjects to examine the role of single nucleotide polymorphisms
(SNPs) of BER genes, APE/Ref-1 (Asp148Glu) and XRCC1 (Arg194Trp and
Arg399Gln) as a risk factor for GD. The SNPs were determined by quantitative
real-time PCR and melting curve analysis using LightCycler. The frequencies of
377
the APE/Ref-1 Asp148Glu and XRCC1-Arg194 and XRCC1-Arg399Gln variant
alleles in our control group were 0.36, 0.08, and 0.34, respectively. No significant association was observed between the variant alleles of APE/Ref-1
Asp148Glu (OR51.11, 95% CI5 0.77-1.60), XRCC1-Arg194Trp (OR51.0, 95%
CI50.52-1.90) and XRCC1-Arg399Gln (OR51.10, 95% CI50.76-1.59) and GD.
Our results suggest that the polymorphic variants of these BER genes are not
independent risk factors for GD.
P-192
MELATONIN INHIBITS UVB-INDUCED OXIDATIVE STRESS AND
REGULATES EXTRACELLULAR MATRIX PROTEINS IN HUMAN
DERMAL FIBROBLASTS IN VITRO
Semra Kocturk1, Mehtap Yuksel-Egrilmez2, Gulgun Oktay1, Halil Resmi1,
Sebnem Aktan3, Sebnem Ozkan3, and Gul Guner1
1
Dokuz Eylul University, School of Medicine, Department of Biochemistry,
Izmir, Turkey, 2Dokuz Eylul University, School of Medicine, Research
Laboratory, Izmir, Turkey, 3Dokuz Eylul University, School of Medicine,
Department of Dermatology, Izmir, Turkey
Objective: Human skin is exposed to solar UV radiation which results in
photoaging by activation of matrix metalloproteinases (MMPs). It was previously
reported that UVB causes photoaging in human dermal cells by leading to downstream signal transduction through phosphorylation of mitogen-activated protein
kinase (MAPK) pathways, such as c-Jun N-terminal kinase (JNK) resulting in
activation of c-Jun and c-Fos, subunits of AP-1 transcription factor. These pathways induce MMPs that degrade skin connective tissue. Generation of reactive
oxygen species by UVB is also critical in triggering MAPK pathways. Melatonin, the product of pineal gland, is the most effective free radical scavenger. We
examined whether melatonin ameliorates UV-B-induced responses in vitro.
Methods: Human Dermal Fibroblasts (HDFs) were isolated from punch
biopsies of healthy individuals. HDFs were pretreated with melatonin (10-6 M)
for 1 hour and exposed to UVB and then incubated for various time points for
every parameter. Epidermal growth factor receptor activation, MMP-1 and
MMP-3 activities, levels of nitrotyrosine and tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured by ELISA. Procollagen type I C-peptide (PIPC)
levels were determined by EIA. JNK activation was analyzed by Western blotting. c-Jun and c-Fos activities were measured by DNA binding activity assay.
Levels of malondialdehyde and oxidized/reduced glutathione were analyzed by
HPLC.
Results: Data showed that UVB significantly increased EGFR activation,
nitrotyrosine, MDA levels, oxidized/reduced glutathione ratio and MMP-1 and
MMP-3 activities at different time points (p<0.05). UVB also induced phosphorylation of JNK and c-Jun and c-Fos activities. Pretreatment with melatonin significantly decreased EGFR activation, nitrotyrosine levels and MMP-1 and
MMP-3 activities in HDFs (p<0.05). Melatonin inhibited UVB-induced JNK
activation and c-Jun and c-Fos activities. MDA levels and oxidized/reduced glutathione ratio were also reduced by melatonin (p<0.05). Pretreatment with melatonin increased UVB-induced protein levels of TIMP-1 and PIPC (p<0.05).
Conclusion: Our results show that melatonin has protective effect on oxidative damage induced by UVB irradiation. This antioxidant effect may also ameliorate tissue homeostasis by reducing activation of MMPs and increasing TIMP-1
and PIPC levels via inhibiting JNK and AP-1 activity.
This study was supported by TUBITAK (Project number-2708).
P-193
EVALUATION OF RELATIONSHIP BETWEEN BIOCHEMICAL RISK
FACTORS AND GENE POLYMORPHISM OF ANGIOTENSINE
CONVERTING ENZYM (ACE) IN CORONARY HEART DISEASE
Semsettin Sahin1, Huseyin Ozyurt1, Omer Atis1, Ismail Benli2,
Leyla Aydogan3, Koksal Ceyhan4, Orhan Onalan4, and Fatih Altunkas4
1
2
3
4
Department of Cardiology, Gaziosmanpasa University, Tokat, Turkey
378
Objective: The aim of this study is to investigate the role of ACE(I/D) gene
polymorphism and ACE serum levels by evaluating the relation of biochemical
parameters that are risk factors for heart disease with ACE(I/D) gene polymorphism, which has been shown to play a role in the development of coronary
heart disease.
Materials and Method: 250 subjects who have not a history of coronary
hearth disease and 750 patients with angiographically diagnosed coronary heart
disease were included in this study. Biochemical risk factors, ACE(I/D) gene
polymorphism and ACE serum levels were compared. ACE genotypes were
determined by Real-Time PCR (RT-PCR). ACE serum levels were determined
by ELISA method.
Results: When the patient group was compared with control group, ACE serum levels were found to be meaningfully high (P<0.001). In DD genotype ACE
levels were found to be the highest. In the other genotypes the difference was
also meaningful. A meaningful difference between control and coronary patient
groups was also determined regarding the levels of total cholesterol, triglyceride,
HDL cholesterol, LDL cholesterol (P<0.05).
Result: ACE(I/D) genotypes and ACE serum levels with biochemical risk factors seem to be an important factor in the development of coronary heart disease.
P-194
IN TOKAT REGION, FREQUENCY AND DISTRIBUTION OF MOST
COMMONLY SEEN HLA GENE MUTATIONS IN THE PERSONS
PREDIAGNOSED WITH CELIAC DISEASE
Semsettin Sahin1, Huseyin Ozyurt1, A. Fikret Ozugurlu1, Muzaffer Katar1,
Ismail Benli2, Leyla Aydogan3, Beytullah Yildirim4, and Resul Yilmaz5
1
2
3
4
Department of Internal Medicine, Gaziosmanpasa University, Tokat,
Turkey, 5Department of Children Health And Diseases, Gaziosmanpasa
University, Tokat, Turkey
Celiac disease is an autoimmune enteropathy created by the immune
response, in genetically susceptible patients, triggered by the consumption of gluten containing wheat, barley and rye made foods. Celiac disease is the only lifelong food allergy. Celiac disease is a multigene disease and has a close relationship with human leukocyte antigens (HLA). The cases can be asymptomatic and
there is a wide range of clinical presentations. Because of delay in diagnosis,
patients can die. Celiac disease can cause high morbidity and mortality before diagnosis but after diagnosis it is no more a disease. It becomes a lifestyle.
In this study, in Tokat region, in 192 patients who were admitted to our
clinic with diarrhea, abdominal pain and growth retardation, the most commonly
seen 3 mutations in HLA gene have been screened. The results were compared
with literature data. Mutations analyses of HLA gene were carried out with
GenID which is a stripped celiac disease mutation screening kit. Strip mutation
analysis is a method that can determine a large number of mutations on genes
associated with some diseases such as cardiovascular disease (CVD) and FMF.
In 153 patients (79.69%) out of 192 included, in this study, at least one mutation
has been detected. In 100 of patients mutation DQA1*0501, in 54 of patients
mutation DQB1*0201, in 53 of patients mutation DRB1*04 has been detected.
In 6 (3.13%) patients all three mutations have been detected. The number of
patients in whom no mutation has been detected was 39 (20.34%).
P-195
CLINICAL EVALUATION OF CIRCULATING CYTOKERATIN 18 AS
A BIOMARKER IN COLORECTAL CANCER THERAPY
MONITORIZATION
Serpil Tanriverdi-Akhisaroglu1, Janserey Batu2, Zekiye Altun1,
Burcak Karaca3, Bulent Karabulut3, and Semra Kocturk2
1
Institute of Oncology, Dokuz Eylul University, Turkey, 2Faculty of
Medicine, Department of Biochemistry, Dokuz Eylul University, Turkey,
3
Faculty of Medicine, Department of Medical Oncology,
Ege University, Turkey
Objective: Circulating full-length (M65) and caspase-cleaved (M30) cytokeratin 18 (CK18) are considered biomarkers of cell death measured using a combination of the M30 (apoptotic) and M65 (apoptotic 1 necrotic) ELISAs. The aim
of this study is to determine serum Cytokeratin 18 in colon cancer patients during their chemotherapy treatments and compare these outcomes with CEA and
clinical stage.
Method: Twenty colorectal cancer patients (9 metastatic, 11 adjuvant) who
were admitted for chemotherapy treatment at Dokuz Eylul University and Ege
University Medical Faculties were included in this study. Blood samples were
collected at day 1, 2 and 14 of the cycles 1, 3 and 6. M30 and M65 levels were
determined by ELISA (Peviva, AB-Sweeden). These results together with CEA
data were compared with clinical outcomes.
Results: After 6 cycles of chemotherapy metastatic patients were clinically
evaluated using RECIST Criteria (Response Evaluation Criteria in Solid
Tumours). Four of them were assigned as partial responders, four as patients
with progressive disease and one patient has stable disease. Three patients of partial responders had an increase (1.2-3-fold) in M30 and M65 levels and in one
patient the M30 level did not change while M65 decreased (2 fold). CEA levels
decreased 2-10-fold in two patients of the partial responders. While the increase
in M30 and M65 levels occurred from the first cycle on (max. at cycle 3), CEA
did not fall at that early time. Three patients with progressive disease had a 3fold decrease in M30 and M65 levels and in one patient this levels increased.
CEA was decreased in all of the four patients.
The patient with stable disease had a decrease in both M30/M65 and CEA
levels. M30 and M65 levels in progression-free patients increased step-by-step
from the first cycle on and made a 3-fold increase after the 6th cycle.
Conclusion: The M30 peak observed in the 3rd cycle in patients with partial
response and the absence of it in patients with progressive disease suggests that
M30 could be a more sensitive marker than CEA for early response assessment.
In stable disease CK18 results were concordant with CEA levels. Increase in the
6th cycle in M30 and M65 in progression-free patients suggests that this peak is
due to chemotherapy-induced tumor cell death. Expanding the findings in enlarging the patient population is likely to show that CK18 is useful as a biomarker
for monitoring treatment response in colorectal cancer.
P-196
IN VIVO FLUORESCENT IMAGING OF ALLOGENEIC AND
XENOGENEIC ISLET GRAFTS FOLLOWING TRANSPLANTATION
Sevim Kahraman1, Ercument Dirice1, Ahter D. Sanlioglu1,
Mustafa K. Balci2, Abdulkadir Omer3, and Salih Sanlioglu1
1
Genetics, Faculty of Medicine, Akdeniz University, Turkey, 2The Division
of Endocrinology and Metabolism, Faculty of Medicine, Akdeniz
University, Turkey, 3Section on Islet Transplantation and Cell Biology,
Joslin Diabetes Center, Harvard Medical School, USA
Background: As pancreatic islet transplantation became a promising treatment for patients with type 1 diabetes, islet graft rejection following transplantation represented a challenging issue. We are aware that graft survival could be
prolonged by gene transfer to the islets. The follow up and quantification of reporter gene expression in vivo is very important for monitoring therapeutic gene
expression in islet grafts. Therefore, we decided to determine the duration of
adenovirus-mediated reporter gene expression in allogeneic and xenogeneic islet
grafts using CCD (cooled charged-coupled device) camera.
Methods: Pancreatic islets were first isolated from Wistar rats and then
examined for purity and viability. Isolated rat islets were transduced with adenoviral vectors encoding enhanced green fluorescent protein (Ad5-EGFP) at 1000
multiplicities of infection (MOI). Thirty-six hours after infection, transduced
islets were implanted under the left kidney capsule of streptozotocine (STZ)induced diabetic recipients. Wistar rats and BALB/c mice were used for allogeneic and xenogeneic transplantation. After transplantation, the intensity and duration of reporter gene expression were followed over time using CCD camera.
For in vivo fluorescent imaging, islet recipients were anesthetized and placed in
the light box illuminated by blue light. Fluorescent images were taken by CCD
camera.
Results: We successfully detected fluorescent signals from implanted regions
of the recipients after transplantation. Fluorescence intensities of grafts were
gradually reduced with each subsequent week and no signal was detected five
weeks after transplantation. It was observed that in vivo fluorescent signals from
grafts can be easily detected through direct exposing of kidney to camera
because of signal obstruction caused by thick tissue layers.
Conclusions: Pancreatic islet grafts can be monitored in vivo with the use of
fluorescence system for allogeneic and xenogeneic islet transplantation. In vivo
fluorescent imaging is a useful and practical tool for following transgenic expression in islet grafts while the animal is still alive. This method certainly provides
information on the status of islet grafts and may allow the monitoring of treatment efficacy in terms of post transplantation graft survival.
P-197
DISTRUBUTIONS OF CD44 AND CD24 IMMUNO-REACTIVITY
IN HUMAN OVARY CANCERS
Muzaffer Sanci1, Nihat Ata1, Sevinc Inan2, Nilgun Dicle3,
and Cigdem Sipahi1
1
Department of Obstetrics and Gynecology, Aegean Social Security
Hospital, Izmir, Turkey, 2Department of Histology and Embryology, Celal
Bayar University, Faculty of Medicine, Manisa, Turkey, 3Department of
Pathology, Aegean Social Security Hospital, Izmir, Turkey
Cell surface adhesion receptors anchor cells to their surroundings, regulate
cell mobility, and provide cells with critical sensors of their microenvironment.
CD44 is a cell-surface glycoprotein postulated to play a role in a variety of cellular behaviors, including adhesion, migration, invasion and survival. Its production is increased at the tumor-stroma interface, including in human cancers. Also
CD24 is a mucin –like cell adhesion molecule and highly expressed on B cells,
thymocytes and tumour cells. It was observed that CD24 expression was
increased in epithelial ovarian, breast, lung, prostate and pancreas cancers. The
aim of this study was to examine the immunohistochemical distributions of
CD44 and CD24 immunoreactivity on different types of ovarian cancers. Tissue
blocks from 33 patients, who had ovarian pathology (malignant serous adenocarcinoma (14), endometrioid carcinoma (10), endometrioid 1 clear cell carcinoma
(1), clear cell carcinoma (2), malignant mucinous adenocarcinoma (2), granulosa
cell tumour (2), trantitional epithelial carcinoma (1) and borderline serous tumor
(1) of the ovary), were included in this study. All formalin-fixed, paraffin-embedded tissue sections were stained with hematoxylin-eosin or primary antibodies
against CD44 and CD24 using the avidin-biotin-peroxidase method. Semi-quantitative grading system was used to compare immunohistochemical staining intensities. Positive CD44 and CD24 immunoreactivities were observed in the epithelial and stromal parts of all the ovarian samples. Increased immunoreactivity of
CD44 and CD24 were observed in malignant and mitotic cells. Strong immunoreactivity of mitotic cells may suggest an increase in cell invasion or metastasis,
and CD44 might be suggested as a novel prognostic marker for human cancers
and used as a target for cancer therapy. CD24 may also be used as diagnostic or
prognostic marker for ovarian cancer.
379
Tissue blocks from 60 patients, who had leiomyomas (n:20), cellular leiomyomas (n520) and leiomyosarcomas (n520) were included in this study. All
formalin-fixed, paraffin-embedded tissue sections were stained with hematoxylineosin or primary antibodies against Estrogen Receptor (ER), Progesterone Receptor (PR) and Ki-67 using the avidin-biotin-peroxidase method. Semi- quantitative
grading system was used to compare immunohistochemical staining intensities.
While positive Estrogen and Progesterone Receptor immunoreactivities were
observed in the leiomyomas, these immunoreactivities were decreased in leiomyosarcomas. Increased immunoreactivity of Ki 67 was observed in malignant
and mitotic cells.
Our results suggest that, increased immunoreactivity of Ki-67 in uterine leiomyosarcomas might be an indicator of the biological aggressiveness of these rare
tumors.
P-199
APPLICATON OF FREE AND IMMOBILIZED EPOXIDE HYDROLASE
IN ENANTIOSELECTIVE HYDROLYSIS OF STYRENE OXIDE
Deniz Yildirim, Seyhan Tukel, and Guzide Yucebilgic
University of Cukurova, Faculty of Arts and Sciences, Department of
Chemistry, 01330 Adana, Turkey
Epoxides, containing highly polarized carbon-oxygen bonds react with critical biological targets such as DNA and proteins, leading to mutagenic, toxic and
carcinogenic effects. Detoxification of epoxides are done with Epoxide hydrolases (EHs, EC 3.3.2.3) by converting the epoxides to 1,2-diols which can be
converted to water soluble products and thus easily eliminated. EHs are ubiquitous in nature and found in various living cells such as mammals, plants, insects,
bacteria, yeast and fungi. Recently, microbial EHs being able to enantioselectively hydrolyze several types of epoxides have been used for the kinetic resolution of the racemic epoxides. In this study, EH from Aspergillus niger was immobilized onto celite by adsorption and its activity in terms of the kinetic resolution of racemic styrene oxide, was compared with that of the free EH.
Immobilization of EH offered several advantages including its reuse, ease in
applications, better control of reactions, ease in removal from the reaction medium and improved stability. EH activity was assayed by measuring the rate of
hydrolysis of racemic styrene oxide. The quantity of formed diol was determined
by using HPLC equipped with a refractive index detector and a C18 column.
Retention times of diol and styrene oxide were 8.2 and 22 min., respectively. Km
and Vmax values were determined as 0.186 mM and 6.12 U/mg prot.min for free
EH and 0.465 mM and 6.80 U/mg prot.min. for immobilized EH at predetermined optimum pH and temperature. For the enantioselective reactions, the enantiomeric excess of the remaining (S)-styrene oxide and (R)-styrene oxide and
(R)-1-phenyl 1,2-ethanediol were determined by using ORpak CDC-453 Chiral
Column at 275 nm. The retention times were 14.3, 18.6 and 11.8 min. for (S)styrene oxide, (R)-styrene oxide and (R)-1-phenyl 1,2-ethanediol in case of both
free and immobilized EHs. Immobilized EH retained 97.8% of the efficiency of
the free EH upon immobilization.
P-198
DISTRIBUTIONS OF ESTROGEN AND PROGESTERONE RECEPTORS
AND Ki-67 ON HUMAN LEIOMYOMAS, CELLULAR LEIOMYOMAS
AND LEIOMYOSARCOMAS
Muzaffer Sanci1, Cihan Dikis1, Sevinc Inan2, Elgin Turkoz-Uluer2, and
Nilgun Dicle3
1
Department of Obstetrics and Gynecology, Aegean Social Security
Hospital, Izmir, Turkey, 2Department of Histology and Embryology, Celal
Bayar University, Faculty of Medicine, Manisa, Turkey, 3Department of
Pathology, Aegean Social Security Hospital, Izmir, Turkey
A role for ovarian sex steroid hormone in uterine leiomyoma growth is likely
because leiomyomas grow during the reproductive years, increase in size during
pregnancy and regress after menopause. Progesterone and estradiol cooperatively
stimulate leiomyoma cell proliferation by up-regulating growth factors. The aim
of this study was to examine the immunohistochemical distributions of Estrogen
Receptor, Progesteron Receptor and Ki-67 immunoreactivities on different types
of leiomyomas, cellular leiomyomas and leiomyosarcomas.
P-200
QUANTITATIVE DETECTION OF MAJOR BCR-ABL GENE
TRANSCRIPTS BY COMPETITIVE RT-PCR IN CHRONIC MYELOID
LEUKEMIA (CML) PATIENTS
Ali Nazemi and Shahrashoub Sharifi
Department of Biology, Tonekabon Azad University Branch, Iran
Objectives: Chronic Myeloid Leukemia (CML) is a disease characterized by
neoplastic overproduction of myelocytes and neutrophils. CML is caused by a
chromosomal translocation, and affected patients have an abnormal Philadelphia
chromosome that arises following a translocation between long arms of chromosome 9 and 22 (q34; q11). This translocation results in fusion of Breakpoint
Cluster Region and Abelson Murine (BCR/ABL) genes. Therefore, detection of
cells carrying BCR/ABL fusion is extremely important in monitoring response to
treatment, remission and relapse in CML patients. Although, BCR/ABL fusion
can be detected by some molecular diagnostic techniques, amplification of the
BCR/ABL fusion transcript and RT-PCR are considered most sensitive methods.
380
Materials and Methods: In this study, we used the competitive RT-PCR technique to determine the number of bcr/abl transcripts. The internal control was
designed from a non-human DNA source with the same flanking sequences but a
smaller size than the target segment. After amplification and purification of internal
control DNA, PCR products were quantified based on copy number. In order to optimize the reaction condition, competitive RT-PCRs were carried out separately on
target and internal control DNAs each with specific copy numbers, and final products of all concentrations were run on agarose gel electrophoresis.
Results: The simultaneous reactions of target RNA with different copy numbers of internal control DNA showed that the copy numbers of target RNA can
be determined via comparison of dye density absorbance of DNA bands (target
and control DNA) on the gel.
Conclusions: This study shows that competitive RT-PCR is a partially efficient and cheaper method for quantitative determination of BCR/ABL fusion,
compared to other methods such as Real-time PCR.
P-201
INFLUENCE OF OXIDATIVE STRESS IN HEART FAILURE
UPON NITRIC OXIDE AND CYCLIC GUANOSINE
MONOPHOSPHATE LEVELS
Simona Opris, Gianina Constantin, Victoria Andrei, and Anton Valuch
Biology of Aging, National Institute Gerontology and Geriatrics, Bucharest,
Romania
Objective: Heart failure (HF) is associated with reduced antioxidant reserve
and increased oxidative stress. Reactive oxygen species can be formed in the
heart, among other, by uncoupling of nitric oxide synthase (NOS). The aim of
this study is to investigate the influence of oxidative stress in HF upon urinary
nitric oxide (NO), cyclic guanosine monphosphate (cGMP) and NOS levels.
Methods: Urinary samples were obtained from patients (45-75 years) distributed in 2 groups: control- C and heart failure- HF. NO levels were measured
spectrophotometrically at 540nm and cGMP at 405nm with ELISA-analyzer.
From the NO levels we determin the enzymatic activity of NOS.
Results: Our data showed for NO a significant decrease at HF vs. C
(0.5360.29 vs. 0.9560.69 lmol/ml; p<0.05), for NOS a significant decrease at
HF vs. C (1.6860.93 vs. 3.0762.27 lmol/ml/min; p<0.05) and also for cGMP a
significant decreased at HF vs. C (0.5660.24 vs. 1.560.64 nmol/ml; p<0.001).
Between NO and cGMP, linear regression equations indicated a significant positive correlation at HF group (p< 0.01, r 5 0.514).
Conclusions: Our results emphasized for all studied parameters (NO, cGMP,
NOS) a decrease in HF vs. control and also a strong correlation between NO and
cGMP, respectively NO and NOS. General aging and age-related alteration in
the cardiovascular system have been attributed to the long-term cumulative
effects of reactive oxygen species. During HF, it’s more likely to occurred an
impaired NO synthesis and not an oxidative inactivation, because we found low
levels of both NO and cGMP.
P-202
RESEARCH OF CYTOTOXICITY AND APOPTOSIS IN COMBINED
EFFECT OF GENISTEIN AND HYPERTHERMIA IN BREAST CANCER
CELLS IN GENE EXPRESSION AND PROTEIN LEVEL
Sinem Numanoglu, Cigir Biray-Avci, Yavuz Dodurga, Sunde Yilmaz,
Zeynep Ozlem Dogan, and Cumhur Gunduz
Department of Medical Biology, Ege University, Medical Faculty,
Izmir, Turkey
Epidemiological studies have shown that there is a negative correlation
between soy consumption and low incidence of breast cancer. Isoflavonoid genistein is a prime anti-cancer component of soy bean and its activities include estrogen antagonism, inhibition of protein tyrosine kinase phosphorylation and topoisomerases, apoptosis, induction of cell differentiation, disruption of free radicals,
carcinogenesis and inhibition of tumor progression. Hyperthermia is the exposure
of tissues or cells to higher than normal psychological temperature (41-448C).
The aim of our study was to induce oxidative stress by treating MCF-7 breast
cancer cells with hyperthermia and investigate the dose of genistein and its combined effect with hyperthermia in terms of cytotoxic, apoptotic and gene expression levels in MCF-7 cell line. In our study, the changes resulted from hyperther-
mia and IC50 dose of genistein were examined by Trypan Blue Dye Exclusion
Assay, XTT, DNA Ladder, Comet assay, TUNEL assays and evaluated in the
level of p53, MDM-2 and hTERT gene expressions and PPAR-c protein level. It
is the first study which analyzes the combined effect of genistein and hyperthermia on breast cancer cell lines.
The IC50 dose of genistein was found to be 175 lM in MCF-7 cell line. No
cytotoxicity was detected in WI-38 cells whereas hyperthermia caused 40% cytotoxicity in MCF-7 cell line. The combination of hyperthermia and genistein
showed an obvious cytotoxicity in the 3rd day of the experiment in both of the
cell lines. In MCF-7 cells, apoptosis was detected in the hyperthermia group in
the 1st day by DNA ladder and comet assay, in the 2nd day, apoptosis was
observed in hyperthermia group by comet assay and in the 3rd day apoptosis
was detected in hyperthermia1genistein group by TUNEL assay. For WI-38
cells, an obvious apoptotic effect was detected in the 1st day in hyperthermia
group and in the 3rd day in hyperthermia1genistein group. In MCF-7 cell line,
genistein repressed hTERT gene expression whereas p53 was increased by genistein and hyperthermia reduced MDM-2 gene expression. PPAR-c was reported to
be increased in genistein group in the second day.
Consequently, we are of the opinion that hyperthermia and genistein combined
treatment can be a successful additional protocol in the therapy of breast cancer.
P-203
HYDROSTATIC PRESSURE INDUCED APOPTOSIS IN PC12 CELLS IS
RELATED TO INAPPROPRIATE CELL-SUBSTRATE ADHESION
Soheil Sadri
Department of Biology, Faculty of Science, Razi University, Kermanshah,
Iran and Fertility and Infertility Research Center, Kermanshah University of
Medical Sciences, Razi University, Iran
Objective: Cells of the various organ systems in human body are subjected
to a range of mechanical forces to which they must respond. Hydrostatic pressure
is one fundamental mechanical force of the cell’s environment, being a determinant of both integrity and function of cells. Disorders of this relationship
between the cells and this mechanical force, such as when pressure varies
beyond physiological limits, can acts as stimulus of apoptosis and thus lead to
disease or pathological states. Mechanisms whereby hydrostatic pressure triggers
apoptosis in neuronal cells and cell’s strategies for dealing with this challenge
remain to be fully explained.
Methods: In this study we examined the effect of hydrostatic pressure on apoptosis induction, morphology and cell-substrate interactions of PC12 cells. Both
native and staurosporine differentiated PC12 cells were subjected to increased
hydrostatic pressure of 100 mmHg above atmospheric pressure for 2 h. Total
area of native cells and neurite length of differentiated cells were assessed as
morphometry. TUNEL staining was used for apoptosis detection, and then cell
adhesion and migration were investigated.
Results and Conclusions: Hydrostatic pressure induced apoptosis in both
native and differentiated PC12 cells and apoptosis index in differentiated cells
was more than native cells. In addition, hydrostatic pressure reduced cell area,
total neurite length, adhesion and migration ability of these cells. We suggest
that the induced apoptosis in these cells may be by anoikis, cell death induced
by a loss of attachment to the substrate.
P-204
G PROTEIN-COUPLED RECEPTOR GENE (P2RY5) POLYMORPHISM
IN BLADDER CANCER
Sunde Yilmaz1, Cigir Biray-Avci1, Yavuz Dodurga1, Burak Turna2, Zeynep
Ozlem Dogan1, Sinem Numanoglu1, Serkan Demiryoguran2, Oktay Nazli2,
and Cumhur Gunduz1
1
Department of Medical Biology, Ege University, Turkey, 2Department of
Urology, Ege University, Turkey
Introduction: Purinergic receptors comprise a family of transmembrane
receptors that are activated by extracellular nucleosides and nucleotides. Purinergic receptors may be subdivided to two families: a P2X family of ligand-gated
ion-channel receptors and a P2Y family of G protein-coupled purinoreceptors.
P2RY5, is a G protein-coupled-receptor family member, activated by adenosine
and uridine nucleotide. This gene aligns with 17th intron of the retinoblastoma
susceptibility gene in the reverse orientation. We investigated the importance of
3 polymorphisms in exon 7 of P2RY5 gene in bladder cancer (BC).
Material and Method: DNA was isolated from blood samples of 22 cases
with (BC) and 20 healthy cases. Primers were designed for rs1060585,
rs4151553 and rs17071686 polymorphisms in exon 7 of P2RY5 gene, PCR products were genotyped by digesting with MseI, HpyCH4V and BsrI restriction
enzymes.
Results: For rs1060585 polymorphism of P2RY5 gene, all cases were found
wild type. In the cases with BC, wild type was not detected in rs4151553. Heterozygous and the mutant frequency were found 0.818 and 0.182, respectively. In
control group, wild type, heterozygous and mutant frequencies were 0.300, 0.500
and 0.200, respectively. There was a significant difference for rs4151553
(p50.016) in BC cases when compared to control group. There was no significant difference between two groups for rs17071686.
Conclusion: Due to its significance, the missense mutation of the 137th
amino acid of P2RY5 protein which is relevant to tryptophan changes to cysteine
contributes to susceptibility of BC, it is necessary to investigate this polymorphism in a wide scale study especially concerning expression of that protein.
381
Table 1
C278Y and H63D mutation in individuals
Genotype
WT/WT
C282Y/WT
C282Y/C282
H63D/WT
H63D/H63D
C282Y/H63D
Total
TS[45%
TS\45%
74 (58.7%)
4 (3.2%)
0
36 (28.6%)
12 (9.5%)
0
126
23 (67.6%)
0
0
8 (23.5%)
2 (5.8%)
0
34
P-205
HYDROGEN PEROXIDE INHIBITS EXERCISE-INDUCED INCREASE
OF CIRCULATING STEM CELLS WITH ENDOTHELIAL
PROGENITOR CAPACITY
Figure 1. H63D mutation in our sample.
Tatsiana Suvorava1, Stephanie Kumpf1, Volker Adams2, and Georg Kojda1
1
Medical Department Institute of Pharmacology Heinrich Heune University,
Duesseldorf, Germany, 2Heart Center, Clinic for Internal
Medicine/Cardiology, Leipzig, Germany
Background: The number of circulating stem cells with endothelial progenitor capacity (EPCs) inversely correlates with the number of cardiovascular risk
factors and is reduced in coronary artery disease.
Objectives: We sought to investigate whether effects of vascular hydrogen
peroxide are involved.
Methods: C57Bl/6 mice, transgenic mice with endothelial-specific overexpression of catalase (cat11) or eNOS (eNOS11) and their transgene-negative
littermates (catn, eNOSn) and eNOS knockout mice (eNOS-/-) were investigated.
Mice were randomly assigned to sedentary groups and groups subjected to either
freely moving, forced physical activity or voluntary exercise. Selected groups of
cat11 and catn were orally treated with the catalase inhibitor aminotriazole.
EPCs were measured using anti-mouse CD3, Flk-1 and CD34, CD133 or Sca-1
antibodies.
Results: Three weeks of exercise training failed to increase circulating EPCs
defined as double positive for Flk-1 and CD34, CD133 or Sca-1 in freely moving
C57BL/6 mice and in mice subjected to forced physical activity or voluntary
exercise. Neither insertion of additional genes encoding for catalase (cat11) or
eNOS (eNOS11) nor eNOS knockout (eNOS-/-) changed EPC counts in resting
mice. In contrast, inhibition of catalase by treatment with aminotriazole strongly
reduced circulatory EPCs in sedentary catn and cat11 (p<0.05, n55-8). When
cat11 mice were subjected to forced or voluntary exercise training, the number
of circulating EPCs was strongly increased (n54-8, p<0.05), an effect completely inhibitable by aminotriazole.
Conclusion: These data suggest that hydrogen peroxide, a known component
of vascular oxidative stress, likely contributes to the impairment of important stem
cell-induced endogenous vascular repair mechanisms in cardiovascular disease.
P-206
SCREENING MUTATIONS C282Y AND H63D OF HFE GENE TO
DETERMINE THE RISK OF HEMOCHROMATOSIS AMONG
INDIVIDIUALS WITH HIGH TRANSFERRIN SATURATION LEVEL
Taylan Demirci1, Orhan Terzioglu1, Baysal Karaca2, and I. Ethem Tankurt3
1
Department of Medical Biology and Genetics, Health Science Institute of
Dokuz Eylul University, Izmir, Turkey, 2Department of Clinical
Biochemistry, Educational and Research Hospital, Izmir ,Turkey, 3Internal
Medicine, Kent Hospital, Izmir, Turkey
Figure 2. C282Y mutation in our sample.
Hereditary Hemochromatosis (HH) is a genetic disease, which is inherited in
an autosomal recessive manner and characterized by inappropriately high absorption of iron by the gastrointestinal mucosa, resulting in excess storage of iron,
particulary in the liver, skin, pancreas, heart, joints, and testes. The HH frequency is about 1/200 to 1/400 in caucasian population. The mutations C282Y
and H63D in the HFE gene is related about 90% to the HH disease. The serum
iron concentration and total iron binding capacity of 730 individuals were measured. The working groups consist of 126 individuals as suspected HH patients
having transferrin saturation higher than 45% and 34 individuals as control having transferrin saturation lower than 45%. DNA were extracted separately from
both groups and amplified by PCR to detect C282Y and H63D mutations in
exon 2 and exon 4 of HFE gene. The frequency in the suspected HH individuals
and control groups for heterozygote C282Y mutations (C282Y/Wild) was 3.2
and 0, homozygote H63D mutation was 9.5% and 5.9%, heterozygote H63D
mutation (H63D/Wild) was 28.6% and 23.5% respectively. According to these
results the frequency of C282Y was 1.6% and the frequency of H63D was 24%
in all allele of suspected HH individuals. In conclusion, C282Y and H63D mutations which are related to risk of HH diseasewere screened for the first time in
Turkey. Moreover the method of screening of C282Y and H63D mutations were
set up in the laboratory of Medical Biology and Genetics, School of Medicine of
Dokuz Eylul University according to international standards.
382
P-207
POLYMORPHISMS IN DEFA1, 2 AND 3 AND DEFA5 ARE
ASSOCIATED WITH INCREASED RISK FOR HPV INFECTION AND
DEVELOPMENT OF CERVICAL CANCER
Rosete Pais1, Vera Goncalves1, and Teresa C. Martins1,2
1
Coimbra, EPE, Portugal, 2Centre for Neurosciences and Cell Biology,
University Coimbra, Portugal
High-risk papilloma viruses (HPV) are the main etiological agents of cervical
cancer, being detected in over 99% of invasive cervical cancer cases. However,
only a minority of high-risk HPV-infected women do develop cervical cancer,
the majority being able to solve the infection. Viral persistence is believed to be
required for development of cervical cancer. These observations suggest that
HPV-mediated cervical cancer is intimately associated with the host ability to
mount an effective immune response. Defensins are important molecules of the
innate immune system, which have an important role in anti-viral immune
responses, namely against HPV infection. Genetic changes in defensins’ coding
genes may alter their efficacy in the control of HPV infection and may therefore
modulate the host’s susceptibility to cervical cancer development. In this work
we have investigated whether alterations in the genes that code for defensins
alpha 1, 2, 3 (DEFA 1, 2 and 3) and 5 (DEFA 5) could constitute risk factors for
the development of cervical cancer, or to HPV infection. For that we have analysed defensins’ genetic changes in women with a history of normal pap smears,
and women presenting ASCUS, low-grade lesions, high-grade lesions and invasive cervical carcinomas (samples from the Cervical Cancer Screening Program
of IPO Coimbra, EPE; kindly provided by the Cytopathology Laboratory of IPO
Coimbra, EPE), by PCR and DNA sequencing. HPV detection was performed by
PCR using GP5/6 primers. We have found genetic changes in DEFA1, 2 and 3
and DEFA5 that were associated with increased susceptibility to HPV infection
and could this way alter the risk for development of cervical cancer. We have
also observed that some of the genetic alterations found were significantly more
prevalent in invasive carcinoma samples and high-grade lesions than in lowgrade lesions and normal samples. Altogether our data suggest that defensins
polymorphisms may predispose to cervical cancer development, either by modulating the host’s susceptibility to HPV infection, and/or by affecting the host
ability to solve infection and HPV persistence.
The developed method allows monitoring of DNA cleavage activity of chemical (ROS, antitumor drugs) and biochemical (DNA hydrolyzing proteins) in cancer and other research.
P-209
INVESTIGATION OF PON1 192 AND PON1 55 POLYMORPHISMS IN
OVARIAN CANCER PATIENTS IN TURKISH POPULATION
Aytac Arpaci, Uzay Gormus, Burak Dalan, Sinan Berkman,
and Turgay Isbir
Department of Molecular Medicine, The Institute of Experimental Medicine,
Istanbul University, Capa, Istanbul, Turkey
Ovarian cancer is the leading cause of death among gynecological malignancies. Oxidative stress is potentially harmful to cells and reactive oxygen species
are known to be induced in the initiation and progression of cancer. Paraoxonase
(PON) is an antioxidant enzyme, which eliminates lipid peroxides. PON has two
common polymorphisms (M/L55 and A/B192) that influence PON concentration
and activity. We studied whether the M/L55 or A/B192 genotype relates with
PON activity and over cancer in 50 patients and 52 controls. Polymerase Chain
Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP), and
agarose gel electrophoresis techniques were used to determine these polymorphisms. PON activity was measured by spectrophotometric assay of p-nitrophenol production following addition of paraoxone. We found that smoker cases are
significantly higher in patients than controls (26.9% vs 7%; X2:7.81, p:0.005;
OR: 4.88 95% CI: 1.49-15.99). Frequencies of PON 192 AA, BB and AB genotypes among the patients were 0.76, 0.12, 0.12; among the control subjects, there
were 0.33, 0.11, 0.56, respectively. AA genotype frequencies is increased significantly in patients than control groups (X2: 19.242, p50.000; Odds : 2.80 %95
CI:1.653-4.757). Frequencies of PON 55 LL, MM, and LM genotypes among the
patients were 0.53, 0.10, 0.37; among the control subjects, there were 0.46, 0.04,
0.50, respectively. MM genotype frequencies are higher in patients than control
groups, but not statistically significant (p[0.05). Our findings showed that the
two polymorphisms were associated with age onset of ovarian cancer, which
increased in the order of the AB \ AA \ BB genotype in the PON1 192 polymorphism and LM \ LL \ MM genotype in the PON1 55 polymorphism. In
conclusion, our results suggest that the PON1 192 AA genotype may play an important role as a risk factor for ovarian cancer in Turkish population and A and
L alleles may be associated with early onset of disease.
P-208
P-210
SIMPLE ELECTROCHEMICAL DETECTION OF CHEMICAL AND
BIOCHEMICAL DNA CLEAVAGE AGENTS
Timur Abdullin1, Irina Nikitina1, Oksana Bondar1, and Mustafa Culha2
Faculty of Biology and Soil Science, Kazan State University, Kazan,
Tatarstan, Russian Federation, 2Department of Genetic and Bioengineering,
Yeditepe University, Istanbul, Turkey
CHANGES IN NITRIC OXIDE LEVELS AND ERYTHROCYTE
ARGINASE ACTIVATES IN PATIENTS WITH DOWN SYNDROME
The accurate detection of factors of different nature affecting the structure
and function of nucleic acids is an important biomedical problem. For this purpose, electrochemical methods can readily be used due to their high selectivity
in the analysis of biological components. Here we propose a simple method to
evaluate the effect of chemical and biochemical DNA cleavage agents with the
use of electrochemical sensors modified with carbon nanotubes. On the surface
of these sensors DNA produces strong and specific signal at the potential of
about 11.1 V vs. Ag/AgCl corresponded to the oxidation of guanine nucleotide.
We found that the ultrasonic fragmentation of high molecular weight DNA was
accompanied with an increase of the DNA signal due to an increase of the effective concentration of DNA adsorbed on carbon nanotubes. We studied the influence of hydroxyl radical generated in Fenton’s reaction (the concentration of
Fe21 was 20 lM) on the electrochemical behaviour of DNA. 10 min exposure to
the reagent was found to increase DNA oxidation signal almost 2-fold compared
to untreated DNA presumably due to hydroxyl radical attacks deoxyribose residue causing polynucleotide strand cleavage. The increase of DNA signal was
also observed after DNAse I digestion of DNA. The response depended linearly
on the enzyme activity and reaction time (r [ 0.98).
Ulku Ozbey1, Cengiz Arslan2, Ayse Seyran3, Mine Erisir3,
and Fulya Benzer4
1
Firat University, Faculty of Medicine, Department of Medical Biology and
Genetics, Elazig, Turkey, 2Firat University, Department of Physical
Education and Sports School, Elazig, Turkey, 3Firat University, Faculty of
Veterinary, Department of Biochemistry, Elazig, Turkey, 4Veterinary
Control and Research Institute, Elazig, Turkey
Objective: Down syndrome (DS) is a chromosome abnormality with specific
clinical symptoms and mental retardation caused by trisomy of chromosome 21.
There is evidence that oxygen free radicals play an important role in the pathophysiology of many neuropsychiatric disorders. Although it has not been investigated yet, several recent studies proposed that nitric oxide (NO) related to oxidative stress and arginase may have a pathophysiological role in mental retardation.
The purpose of this study was to determine plasma NO levels and erythrocyte
arginase activities in patients with DS and in healthy children.
Methods: Plasma NO levels and erythrocyte arginase activity were measured
in 20 patients with DS (median age: 15.667.5) and 20 age-matched healthy subjects (median age: 16.267.4).
Results: Significant differences were found between erythrocyte arginase activity of the patients with DS (35.2612.1 U/g.Hb) and those of healthy children
(43.5113.8 U/g.Hb). (p<0.05). Whereas, we have not found any statistical difference between persons with DS and control group for NO (p>0.005).
Conclusions: It is concluded that the level of erythrocyte arginase activity
could be considered as a useful marker for assessing the level of mental retardation children in DS.
P-211
THE VALUE OF INTERLEUKIN-12B (P40) GENE PROMOTER
POLYMORPHISM IN PATIENTS WITH SCHIZOPHRENIA IN A
REGION OF EAST TURKEY
Ulku Ozbey1, Esra Tug2, Murat Kara2, and Mustafa Namli3
1
Genetics, Elazig, Turkey, 2Abant Izzet Baysal University, Izzet Baysal
Medical School, Department of Medical Genetics, Bolu, Turkey, 3Ataturk
University, Faculty of Medicine, Department of Medical Genetics, Erzurum,
Turkey, 4Psychiatry Hospital, Department of Psychiatry, Elazig, Turkey
Objective: It has been hypothesized that the activation of the immune system may be involved in the neuropathological changes occurring in the central
nervous system of schizophrenic patients. Cytokines play a key role in the activation of the immune system. Moreover, they strongly influence the dopaminergic, noradrenergic and serotonergic neurotransmission. To the best of our knowledge, in schizophrenic patients, plasma levels of interleukin (IL)-12 were investigated only in one study, where deregulation of IL-12 was determined. However,
genotypical variations of the IL-12B (p40) gene have not been investigated for
schizophrenic patients yet. Therefore, in the present study, we aimed to examine
polymorphic variants of IL-12B (p40) gene promoter region in patients with
schizophrenia in a population of the Elazig Region of East Anatolia, Turkey.
Methods: One hundred Turkish patients diagnosed with schizophrenia based
on the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV), and
116 healthy control subjects participated in the present study. The genotype characteristics were determined by polymerase chain reaction-based RFLP method
using DNA extracted from peripheral blood.
Results: Significant differences in both the genotype and allele frequencies
were found between schizophrenia patients and control groups (P<0.01).
Conclusions: These findings may support the hypothesis that activation of
the inflammatory response system and in particular, of Th-1 cells, is involved in
the pathophysiology of schizophrenia. We think that this study is the first trial
associated with IL-12 cytokine at the molecular genetic level on immune mechanisms for neuropsychiatric disorders including schizophrenia. This perspective
and the role of cytokines in the pathogenesis of schizophrenia may constitute a
reasonable target for the present and future treatment strategies and prognosis.
P-212
RELATIONSHIP BETWEEN BLOOD ANTIOXIDANT ENZYME
LEVELS AND GENOTYPE DURING MIGRAINE ATTACK AND
INITIAL PERIODS IN MIGRAINE PATIENTS
Ulku Ozbey1, Seda Ozel2, M. Said Berilgen2, Ayse Seyran3,
and Mine Erisir3
1
Genetics, Elazig, Turkey, 2Firat University, Faculty of Medicine,
Department of Neurology, Elazig, Turkey, 3Firat University, Faculty of
Veterinary, Department of Biochemistry, Elazig, Turkey
Objective: Migraine is a complex, disabling disorder of the brain that manifests itself as attacks of often severe, throbbing head pain with sensory sensitivity
to light, sound and head movement. Although many researches have been conducted to research migraine etiopathogenesis, no exact conclusion could be
reached yet. In recent years, importance of oxidative stress on migraine pathogenesis like on many diseases is being researched. The aim of this study is to
research the roles of antioxidant enzymes such as Superoxide Dismutase (SOD),
Glutathione Peroxidase (GSH-Px), Catalase (CAT), antioxidant Glutathione
(GSH) and Malondialdehyte (MDA) of a lipid peroxidation product in migraine
pathogenesis. Moreover, the incidence of this polymorphism in Turkish population is being researched by looking at the gene polymorphism of the enzymes of
Mn-Mn-SOD, GSH-Px3 and CAT.
383
Methods: GSH-Px, CAT, SOD activities and plasma MDA and blood GSH
levels were measured in 40 migraine patients (30 females and 10 males) and 40
age-matched healthy subjects and also between the attack period and initial period
in migraine patients. The genotype characteristics were determined by polymerase
chain reaction-based RFLP method using DNA extracted from peripheral blood.
Results: We detected a statistically remarkable decrease in GSH-Px, CAT,
SOD and GSH levels between migraine patients and control group (for GSH,
CAT p<0.001, for GSH-Px. p50.017, for SOD p50.04). In the molecular part
of our study, we haven’t found any statistical difference between migraine
patients and control group for Mn-SOD and CAT genotypes, but found a remarkable difference for GSH-Px3 genotype. For all of the three genes, there were no
difference between the patients and control group in terms of allele frequency.
Conclusion: Oxidative stress and antioxidant enzymes play an important role
in migraine pathogenesis, and according to our data GSH-Px3 gene polymorphism cause an inclination to migraine for our regional community.
P-213
INVESTIGATION OF DIFFERENCES IN P53 GENE POLYMORPHISMS
BETWEEN SCHIZOPHRENIA AND LUNG CANCER PATIENTS IN
THE TURKISH POPULATION
Ulku Ozbey1, Huseyin Yuce1, Mustafa Namli2, and Tamer Elkiran3
Firat University, Faculty of Medicine, Medical Biology and Genetic
Department, Elazig, Turkey, 2Psychiatry Hospital, Psychiatry Department,
Elazig, Turkey, 3Firat University, Faculty of Medicine, Department of
Medical Oncology, Institute of Oncology, Elazig, Turkey
1
Objective: The reduced incidence of cancer observed in schizophrenia
patients may be related to differences in genetic background. It has been suggested that genetic predisposition towards schizophrenia is associated with
reduced vulnerability to lung cancer, and p53 gene is one of the candidate genes.
In our study, we aimed to investigate polymorphisms in the BstUI in exon 4 and
MspI in intron 6 restriction sites of the p53 gene in Turkish schizophrenia, lung
cancer patients and controls.
Methods: Allele and genotype incidence of these polymorphisms with their
haplotype combinations were studied in 100 Turkish lung cancer and schizophrenia patients and 100 controls without malignant and schizophrenia diseases. The
genotype characteristics were determined by PCR-based RFLP method using
DNA extracted from peripheral blood.
Results: For the BstUI and MspI polymorphism, significant differences were
found in the genotype and allele frequencies between schizophrenia and lung
cancer patients relative to control groups (p<0.01). The analysis based on haplotype frequencies showed the presence of BstUI-MspI 2-1 haplotype in cancer
patients (12%) in contrast to the absence of this haplotype in schizophrenia and
controls. Only in lung cancer patients we found both a significant decrease of A1
allele of the p53 codon 72 (OR 0.23, 95% CI 0.9-0.58) and A1/A1 homozygous
genotype (P<0.0001, OR 0.19).
Conclusion: The results of this study suggest a protective effect of A1 allele
against lung cancer and the p53 MspI polymorphism may modify the susceptibility to lung cancer as a single factor rather than in combination with BstUI polymorphism.
P-214
INTERLEUKIN-10 GENE PROMOTER POLYMORPHISM IN PATIENTS
WITH SCHIZOPHRENIA IN A REGION OF EAST TURKEY
Ulku Ozbey1, Esra Tug2, and Mustafa Namli3
1
Genetics, Elazig, Turkey, 2Abant Izzet Baysal University, Izzet Baysal
Medical School, Department of Medical Genetics, Bolu, Turkey, 3Psychiatry
Hospital, Psychiatry Department, Elazig, Turkey
Objective: Schizophrenia is one of the most severe psychiatric disorders,
with a worldwide incidence of 1%. Immunological abnormalities have been
found to be associated with schizophrenia for decades. Cytokines are key proteins involved in the immune system activation. Interleukin-10 (IL-10), an impor-
384
tant immunoregulatory cytokine, is located on chromosome 1q31-32, a region
previously reported to be linked to schizophrenia in genetic studies. In the present study it was aimed to examine the IL-10 gene promoter region’s polymorphic
variants in patients with schizophrenia in a population of the Elazig Region of
East Anatolia, Turkey.
Methods: Polymorphisms at position _1082, _819 and _592 in the IL-10 promoter region were determined in 171 Turkish patients who were diagnosed with
schizophrenia, based on the DSM-IV, and 168 healthy controls, by polymerase chain
reaction (PCR)-restriction fragment length polymorphism (RFLP). We analyzed
allele, genotype, and haplotype distributions using a case-control association study.
Results: Genotyping was performed by RFLP. Statistically significant differences were observed in both allelic and genotypic frequencies of the _592A/C
polymorphism (Allele, p50.034, OR51.26, 95% CI 1.02–1.56; Genotype,
p50.048), while the other two polymorphisms in distribution of the alleles and
genotypes in patients with schizophrenia were not significantly different from
those of controls (p>0.05). Our results show a significant increase of GTA
homozygotes (the high IL-10-producing haplotype) in schizophrenic patients
compared to control subjects (p50.0001).
Conclusion: These data suggest that the IL-10 gene promoter polymorphism
may be one of the susceptibility factors to develop schizophrenia in the Turkish
population, and apparently in all humans.
(GPX), catalase (CAT) and plasma malondialdehyde (MDA), b-carotene, retinol
levels in adolescents with DS.
Methods: Twenty adolescents with Down’s syndrome (mean age: 16.261.0
y) performed a 12-week training program, 3 days/week at a work intensity of
60–75% of peak heart rate (HRmax5194.5–[0.56 age]). Body composition was
measured by bioelectric impedance method. GPX, CAT, MDA, retinol, b-carotene levels were determined spectrophotometrically.
Results: Pre-post exercise GPX and CAT activity, MDA, retinol and b-carotene levels in adolescents with DS were detected as 74.3640.9U/g Hb and
38.5615.1 U/g Hb, 23.568.9 k/g Hb and 27.565.6 k/g Hb, 2.461.3 nmol/ml
and 3.860.9 nmol/ml, 16.4610.9 lg/dl and 11.563.8 lg/dl, 37.5.4610.4 lg/dl
and 16.764.3 lg/dl, respectively. When compared to baseline, MDA content
was increased. GPX and b-carotene were decreased (p<0.05). There was no difference during pre-post exercise in terms of CAT and retinol (p>0.05). According to the body mass index, it was observed that 31.8% of the studied individuals
presented overweight and 31% of them were obese before starting our experiment. The mean value of percentage of fat mass was reduced from 31.966.8 %
to 2964.8% at the end of study (p50.031).
Conclusion: Regular exercise did not significantly increase GPX activity and
b-carotene level and did not affect the CAT activity and retinol level observed
in patients with DS. Adolescents with DS were able to reduce their fat mass percentage significantly after a 12-week exercise program.
P-215
P-217
OXIDATIVE STRESS AND ALTERED LEVELS OF OXIDANTS AND
ANTIOXIDANTS IN ACUTE ISCHEMIC STROKE PATIENTS IN A
REGION OF EAST TURKEY
CHANGES IN PLASMA NITRIC OXIDE LEVELS DURING MIGRAINE
INITIAL AND ATTACK PERIODS IN MIGRAINE PATIENTS
Ulku Ozbey1, Ayse Seyran2, Mine Erisir2, Seda Ozel3,
and M. Said Berilgen3
1
Genetics, Elazig, Turkey, 2Firat University, Faculty of Veterinary,
Department of Biochemistry, Elazig, Turkey, 3Firat University, Faculty of
Medicine, Department of Neurology, Elazig, Turkey
Objective: Ischemic stroke is a leading cause of mortality and disability particularly in the elderly. Hypertension is the most important risk factor in strokes,
representing roughly 70% of all cases. Oxidative stress is believed to be one of the
mechanisms taking part in neuronal damage in stroke. In 28 patients with ischemic-stroke and 28 healthy individuals were investigated with regard to relationship
between ischemic-stroke and glutathione peroxidase (GSH-Px), catalase (CAT)
activities, malondialdehyde (MDA), glutathione, b-carotene, vitamin A levels.
Methods: GSH-Px, CAT activities and MDA, GSH, vitamin A, b-carotene
levels were measured in 28 ischemic-stroke patients (12 females and 16 males,
median age 64) and 28 age-matched healthy subjects (15 females and 33 males,
median age 60).
Results: In the patients with ischemic-stroke, GSH-Px activity and MDA,
GSH, vitamin A levels increased (p<0.001), whereas no differences were found
in CAT activity and b-carotene level (p>0.005).
Conclusions: These results suggest that peroxidation reactions are accelerated in patients with ischemic-stroke and the antioxidant systems are activated.
P-216
OXIDATIVE STRESS AND ALTERED LEVELS OF ANTIOXIDANTS
IN ADOLESCENTS WITH DOWN SYNDROME DURING
PRE-EXERCISE AND POST-EXERCISE
Cengiz Arslan1, Ulku Ozbey2, Mine Erisir3, Ayse Seyran3, and Yuksel Savucu1
1
Firat University, Department of Physical Education and Sports School,
Elazig, Turkey, 2Firat University, Faculty of Medicine, Department of
Medical Biology and Genetics, Elazig, Turkey, 3Firat University, Faculty
of Veterinary, Department of Biochemistry, Elazig, Turkey
Objectives: Individuals with Down’s syndrome (DS) have been described as
having high levels of oxidative stress. This study was designed to determine the
influence of exercise program on activity of erythrocyte glutathione peroxidase
Mine Erisir1, Ulku Ozbey2, Ayse Seyran1, Fulya Benzer3, and Cengiz Arslan4
1
Department of Biochemistry, Faculty of Veterinary, Turkey, 2Department
of Medical Biology and Genetics, Faculty of Medicine, Turkey, 3Veterinary
Control and Research Institute, Turkey, 4Department of Physical Education
and Sports School, Turkey
Objective: The most important primary headaches are migraine, tension-type
headache and cluster headache. Migraine has a prevalence of 10% in the general
population and its social costs are high. Although the precise mechanisms underlying the pathophysiology of migraine are still elusive, the last decades have witnessed some progress (e.g. involvement of serotonin, calcitonin gene-related peptide, nitric oxide). Nitric oxide (NO) is believed to play a key role in migraine
pathogenesis. In our study, we compared migraine patients with healthy controls
by measuring plasma nitrite levels in order to investigate the effects of nitric
oxide in migraine patients.
Methods: A total of 26 patients with migraine in headache free period (median age: 33.3 6 6.3) and 24 patients with migraine (median age: 31.4 6 4.9) in
attack period were participated in our study. Control group was consisted of 24
healthy subjects (median age: 30.16 4.1). Plasma NO levels were measured
spectrophotometrically.
Results: In control group, plasma nitrite levels were found 85.8 6 3.2 lmol/
L. In migraine patients plasma nitrite level was detected 95.9 6 21.2 lmol/L
during headache free period and 85.2 6 2.7 lmol/L during attacks. According to
our results, plasma nitrite levels were significantly higher than controls during
headache free period (p<0.05). By contrast, the levels of plasma nitrite significantly decreased in attacks periods compared to headache free period. No significant change in plasma nitrite levels in migraine attacks period compared to controls was observed (p>0.05).
Conclusion: Results obtained indicate that the plasma nitrite levels were not
altered significantly after an attack in the patients with migraine. These results
suggested that increased level of plasma NO during headache free period may
play an important role in migraine pathogenesis.
P-218
MELATONIN PROVIDES NEUROPROTECTION THROUGH
REDUCING OXIDATIVE STRESS AND HSP70 EXPRESSION
AFTER CHRONIC CEREBRAL HYPOPERFUSION IN
OVARIECTOMIZED RATS
V. Haktan Ozacmak1, Figen Barut2, Hale Sayan-Ozacmak1
1
Department of Physiology, Zonguldak Karaelmas University Medical
School, Turkey, 2Department of Pathalogy, Zonguldak Karaelmas University
Medical School, Turkey
Oxidative stress is believed to contribute to functional and histopathological
disturbances after chronic cerebral hypoperfusion in rats. Melatonin has protective effects against cerebral ischemia reperfusion injury. This effect has mainly
been attributed to its antioxidant properties. In the present study, we evaluate the
effects of melatonin on chronic cerebral hypoperfused rats and observe its influence on oxidative stress, superoxide dismutase (SOD) and reduced glutathione
(GSH) levels, and heat shock protein (HSP) 70 induction. Chronic cerebral hypoperfusion was induced by permanent bilateral common carotid artery occlusion
in ovariectomized female rats. We found extensive neuronal loss in the hippocampus at day 14 after chronic hypoperfusion. The ischemic changes were preceded by increased MDA concentration, decreased GSH and SOD, and increased
HSP 70 induction. Melatonin treatment restored the levels of MDA, SOD, GSH
and HSP 70 induction as compared to ischemic group. Histopathological analysis
confirmed the protective effect of melatonin against cerebral hypoperfusioninduced histological alterations.
Taken together, our results suggest that melatonin provides neuroprotective
effects in chronic cerebral hypoperfusion by attenuating oxidative stress and
stress protein expression in neurons, and may be helpful of the treatment of vascular dementia and cerebrovascular insufficiency.
P-219
EFFECT OF ENDOGENOUS NO AND PENTAERYTHRITOL
TETRANITRATE ON MYOCARDIAL AT-2 RECEPTOR
EXPRESSION IN-VIVO
Vu Thao-Vi Dao1, Tanya Suvorava1, Oktay Kocgirli1, Marc Oppermann1,
Vera Balz2, and Georg Kojda1
1
Department of Pharmacology, Heinrich Heine University,
Germany, 2Department of, Otorhinolaryngology, Heinrich Heine
University, Germany
We hypothesized that pentaerythritol tetranitrate (PETN) and endothelial
nitric oxide (NO) might impact on the expression of angiotensin (AT) type 1
(AT-1) and type 2 (AT-2) receptors.
We generated mice with an endothelial-specific overexpression of endothelial
NO-synthase (eNOS) using the Tie-2 promoter and backcrossed these mice to
the C57BL/6 background. Two of these lines were characterized by eNOS-western blot analyses and blood pressure measurements in comparison to transgenic
negative littermates. In addition, C57Bl/6 mice were fed with either 6 or 60 mg
PETN/kg body weight/day for 4 weeks.
Analysis of line 1 of transgenic eNOS mice (1-eNOS11) showed a
2.360.15 fold higher aortic expression of eNOS and a reduction of blood pressure to 109.662.0 mmHg (P\0.01, n54-6). Analysis of line 2 of transgenic
eNOS mice (2-eNOS11) showed a 3.360.3 fold higher aortic expression of
eNOS and a reduction of blood pressure to 105.063.0 mmHg (n56, p\0.01).
Treatment of 2-eNOS11 with the NOS-inhibitor L-nitroarginine (L-NAME) for
30 days completely inhibited the difference in blood pressure suggesting that the
reduction of blood pressure in transgenic mice was caused by an increased bioavailability of endogenous NO. In lungs and left ventricular myocardium of 2eNOS11 the expression of AT-1-receptors was similar to transgenic negative
littermates (P[0.05, n58). In striking contrast, the expression of AT-2 receptors
was increased by eNOS overexpression in a gene-dose-dependent manner in the
myocardium. In 1-eNOS11 and 2-eNOS11 this increase was significantly
higher vs. control (P\0.05 and P\0.01, respectively). In lung tissue the AT-2 receptor expression was significantly increased in both lines vs. control (P\0.05).
Preliminary experiments with PETN-fed mice showed a significant increase in
AT-2-receptor expression in myocardial tissue (P\0.05) whereas the expression
of the AT-1 receptors did not change (P[0.05) either in myocardial or in aortic
tissue.
These results show that endogenous NO and NO-Donors can up-regulate vascular AT-2 receptor in-vivo. This newly discovered regulation might contribute
to the vasoprotective effects of NO.
385
P-220
GENETIC SUSCEPTIBILITY FACTORS FOR BREAST CANCER IN
THE PORTUGUESE POPULATION: AIB1, PPARGC1A, CYP1A1
AND DSS1
Vera Goncalves1, Hugo J. Prazeres1, Manuela Lacerda2, and
Teresa C. Martins1,3
1
Coimbra, Portugal, 2Histopathology Laboratory, Portuguese Institute for
Oncology at Coimbra, Portugal, 3Centre for Neurosciences and Cell
Biology, University Coimbra, Portugal
Breast cancer is the most common cancer among women. It occurs mainly in
sporadic forms but some characteristics such as early onset, familial aggregation
and bilateral involvement, suggest a familial transmission. This form of breast
cancer may be associated with germ-line mutations in BRCA1 and BRCA2
genes. However, these mutations have incomplete penetrance, suggesting that
other genetic or environmental factors may be involved. We have investigated
genetic factors that may modify the risk for breast cancer development, namely
genes that encode enzymes involved in oestrogen metabolism (CYP17, CYP1A1
and COMT) and signalling (ESRa, AIB1, PPARGC1A/B), as oestrogens increase
breast cancer risk. We have also analyzed alterations in DSS1, which encodes a
protein that interacts with and stabilizes BRCA2 during DNA damage response.
We have studied 47 familial and 37 sporadic breast cancer cases, and 19 normal
controls. Genetic alterations were studied by PCR, SSCP and DNA sequencing.
Statistical analysis was performed using Chi-square test. No significant differences were found with regards to CYP17 5’UTR T[C, CYP1A1 Thr461Asn,
COMT Val158Met, ESRa Ser10Ser and Arg243Arg, and PPARGC1B Ala203Pro
polymorphisms. However, we have found a higher prevalence of the AIB1 26CAG repeat allele in familial and sporadic cases. In addition, PPARGC1A
Thr612Met was present in a higher frequency in familial cases when compared
to sporadic and normal samples. DSS1 Lys5Lys was found in a higher prevalence in breast cancer cases, especially in sporadic forms, being absent in normal
controls. CYP1A1 Ile462Val was over-represented in the control group. Our data
suggest that, in our population, CYP1A1 Ile462Val, PPARGC1A Thr612Met,
AIB1 26 CAG repeats and DSS1 Lys5Lys may affect the risk for breast cancer
development in sporadic and familial settings.
This work was supported by FEDER and POCTI programs and FCT
(POCTI/MGI/48912/2002).
P-221
PROTEIN CONTAMINATION IN AUTOMATIC DNA
EXTRACTION METHODS
Victoriano J. Leon1, Alberto J. Leon2, and Juan Luis Garcia3
1
Analisis Clinicos, Hospital Universitario de Salamanca, Spain,
2
Lab Pediatria e Inmunologia, Universidad de Valladolid, Spain,
3
Centro Investigacion del Cancer Universidad de Salamanca, Spain
The automatic extraction methods of DNA from blood samples for molecular
biology histocompatibility studies, have similar physical-chemical principles in
contrast with manual methods which vary greatly. There are two clearly defined
stages. First, the cells must be destroyed in order to inactivate the nucleases present in the cell and free and suspend the DNA chains that they contain. In order
to achieve this, the cells are suspended using appropriate chaotropic salts or
detergents. In the second stage, the DNA fragments present in the mixture are
obtained, using the anionic nature of DNA are fixed to an appropriate matrix,
contaminants are removed with washing steps and finally the nucleic acid is
eluted in a salt buffer. In our study we have attempted to compare the quality of
the nucleic acid obtained in different automatic methods with different matrices
and quantity of blood, focusing on possible protein contamination. The techniques set up in our laboratory and applied to nucleated blood cells are summarized below, including there principal characteristics:
1) Chaotropic salts solubization; DNA separation matrix glass fiber, 2) Chaotropic salts solubilisation; DNA separation silica bead matrix, 3) Chaotropic
salts solubilisation; DNA separation silica membrane matrix. In the three methods we employed nucleated cells of 50, 200 and 500 mcl of blood. The quality
of the DNA was controlled by measuring at 260/280 nm. Protein contamination
386
was detected by electrophoresis in agarose gel at 1%, stained with bright blue
Coomassie R-250.
The results obtained indicate a decrease of the DNA quality and an increment of the protein contamination as the quantity of sample was increased, also
the contamination of the samples of 50 mcl always was bigger than the one present with manual methods. The quality of nucleic acid obtained by automatic procedures was sufficient for SSP and SSOP Histocompatibility techniques.
10/- 1082 (20 AA/50AG/30 GG), IL-12/- 1188 (60 AA/35 AC/5 CC), IFN
gamma/utr 5644 (35 AA/40AT/25 TT), TNF alpha/- 238 (0 AA/2O AG/80 GG),
TNF alpha/- 308 (5 AA/30 AG/65 GG), TGF beta1/codon 10 (25 CC/50 CT/25
TT), TGFbeta1/codon 25 (0 CC/10CG90 GG).
P-224
P-222
CONTRIBUTION TO INTERLEUKIN GENE POLYMORPHISMS
STUDY IN REUMATHOID ARTHRITIS
STORAGE OF DNA ON FILTER PAPER: QUALITY TEST ON
SAMPLES KEEPING 6 YEARS
Victoriano J. Leon1, Alberto J. Leon2, and Maria Luisa Hernandez-Cerceño1
1
Servicio Analisis Clinicos, Hospital Universitario de Salamanca, Spain,
2
Lab Pediatria e Inmunologia, Universidad de Valladolid, Spain
Victoriano J. Leon1, Alberto J. Leon2, and Juan Luis Garcia3
1
Analisis Clinicos, Hospital Universitario de Salamanca, Spain,
2
Lab Pediatria e Inmunologia, Universidad de Valladolid, Spain,
3
Centro Investigacion del Cancer Universidad de Salamanca, Spain
The creation of a registry of samples of genomic material usually requires a
painstaking process to obtain the DNA and to store it at -858C. All this implies a
high cost in terms of staff, material, and storage, which limits the size and the
number of archives. The conservation of blood samples (or previously extracted
DNA) on filter paper allows a great number of samples to be held within a small
space without any maintenance, making it very easy to access the DNA of the
sample and carry out different genomic studies, especially those that use PCR
technique. Several samples of 25 mcl of blood, or DNA were previously
extracted on Whatman n8 3 paper and dried at room temperature or in a heater
at 458C. The filter paper of the sample was inserted into an envelope and labeled. Several of these envelopes were enclosed in a plastic bag with hermetic
closure and silica gel desiccant and kept up to six years.
For the analysis the spot on the filter paper is cut and placed in an Eppendorf
tube, 200 mcl of a 4M solution of guanidine isotiocianate is added, and it is then
heated at 558C for 30 minutes. The DNA released from the paper can be extracted
either with saline precipitation, phenol chloroform or cationic resins. The technique
we have chosen was the Kit Direct II of Dynal, due to its ease and rapidity; it is
based on a resin with a metallic core which permits the separation of the DNA-resin
complex with a magnetic concentrator, and the complex is undone at 658C for 5
minutes. After removing the magnet with the associated resin, DNA remains in a
liquid phase in a quantity of 1-4 mcg, ready to be employed. We have applied the
DOP-PCR technique, when the amount of DNA available in a blood spot was not
enough for our purposes such as a complete HLA genomic study.
We have stored our samples over filter paper for periods of over six years,
using the DNA restored for the genomic typing of HLA class I and II, and also
for coagulation factors, with satisfactory results in all cases.
P-223
INTERLEUKIN GENES POLYMORPHISMS IN ALZHEIMER
DISEASE PATIENTS
Victoriano J. Leon1, Jesus L. Cacho2, and Erica Polo-Hernandez3
1
2
Servicio Neurologia, Hospital Universitario de Salamanca, Spain,
3
Instituto Neurociencias, Castilla y Leon, Unoversidad de Salamanca, Spain
Alzheimer’s disease (AD) is a complex disorder with many genetic factors
implicated in disease onset and pathology. We have studied 36 AD patients,
according to the NINCDS-ADRDA criteria, and 18 subjects as controls from our
community in order to establish a genomic interleukine polymorphism control
group. The DNA was obtained from blood samples extracted with the kit DNA
Direct II from Dynal; for the genomic studies Cytokine genotiping Kit, Pel-Freez
was employed. The AD results are detailed in the table; there are not significant
differences between patients and control group.
IL-1alpha/- 889 (52.5 CC/40 CT/7.5 TT), IL-1beta/-511 (35 CC/50 CT/15
TT), IL-1beta/3962 (55 CC/35CT/10 TT),IL-1RA/mspa 11100 (0CC/50 CT/50
TT), IL1R/psti 1970 (45 CC/52.5 CT/7.5 TT), IL-2/- 330 (15 GG/50 GT/35 TT),
IL-2/166 (55 GG/35 GT/10 TT), IL-4/- 1098 (10 GG/22 GT/70 TT), IL-4/- 590
(75CC/25 CT/0 TT), IL-4/- 33 (75 CC/20 CT/5 TT), IL-4R alpha/1902 (75 AA/
15 AG/10 GG), IL-6/- 174 (15CC/45CG/40 GG), IL-6/nt 565 (10 AA/50 AG/40
GG), IL-10/- 592 (5 AA/45 AC/50 CC), IL-10/- 819 (55 CC/35 CT/10 TT) IL-
We have been studied 28 Reumathoid Artrithis patients, also 32 subjects control from our community in order to establish a genomic interleukine polymorphism control group. DNA was obtained from blood samples extracted with the
kit DNA Direct II from Dynal, the genomic studies were employed with Cytokine genotyping Kit, Pel-Freez.
The results are detailed in the table; there are not significant differences
between patients and control group.
IL-1alpha/- 889 (57.5 CC/40 CT/2.5 TT), IL-1beta/-511 (40 CC/50 CT/10
TT), IL-1beta/3962 (50 CC/32.5CT/7.5 TT),IL-1RA/mspa 11100 (0CC/50 CT/50
TT), IL1R/psti 1970 (45 CC/52.5 CT/7.5 TT), IL-2/- 330 (15 GG/50 GT/35 TT),
IL-2/166 (55 GG/35 GT/10 TT), IL-4/- 1098 (10 GG/22 GT/70 TT), IL-4/- 590
(75CC/25 CT/0 TT), IL-4/- 33 (75 CC/20 CT/5 TT), IL-4R alpha/1902 (75 AA/
15 AG/10 GG), IL-6/- 174 (15CC/45CG/40 GG), IL-6/nt 565 (10 AA/50 AG/40
GG), IL-10/- 592 (5 AA/45 AC/50 CC), IL-10/- 819 (55 CC/35 CT/10 TT) IL10/- 1082 (20 AA/50AG/30 GG), IL-12/- 1188 (65 AA/35 AC/0 CC), IFN
gamma/utr 5644 (35 AA/40AT/25 TT), TNF alpha/- 238 (0 AA/2O AG/80 GG),
TNF alpha/- 308 (5 AA/30 AG/65 GG), TGF beta1/codon 10 (20 CC/50 CT/30
TT), TGFbeta1/codon 25 (0 CC/15CG85 GG).
P-225
STUDY OF SOME HLA ALLELES IN ALZHEIMER PATIENTS
Victoriano J. Leon1, Jesus L. Cacho2, and Erica Polo-Gutierrez3
1
2
Servicio Neurologia Hospital Universitario de Salamanca, Spain,
3
Instituto de Neurociencias Castilla y Leon Universidad de Salamanca, Spain
Introduction: Our Spanish region, Castilla y Leon, presents a fixed population with more than 2 million of inhabitants which has not received emigrants in
the last centuries; rather population has emigrated from it. Thus, it is suitable for
a population study of diseases that appear in young people with Alzheimer disease, AD. We have chosen for our study the markers HLA A3, B8 and DR4 in
patients with AD in our region.
Material and Methods: We have studied a series of 98 diagnosed patients
with AD according to the criteria NINCDS-ADRDA and 129 healthy controls
(HC). The DNA was extracted using the technique of Salting-Out. The genes
HLA were studied with Kits HLA ‘‘Low Resolution’’ Dynal. It was also studied
a well-defined genetic marker of EA as it is the Apo E, kit of Innogenetics.
Results: The frequency of allele HLA A3 was 32% in AD (24% in HC), the
allele HLA B8 was 36% (23% in HC) the allele DR 4 was 58% (30% in the
HC). The Chi-square test show differences (P< 0.001). The results, comparable
to those obtained by other authors, it makes at least appear the alleles HLA A3,
B8 and DR4 like risk factor markers in AD as ApoE.
P-226
MICROSATELLITES IN BLOOD, SUPERFICIAL BLADDER
CARCINOMA AND URINE SEDIMENTS IN PATIENTS WITH
BLADDER CARCINOMA
Victoriano J. Leon1, Javier Garcia2, Manuel Urrutia2, and Juan Luis Garcia3
1
Servicio de Analisis Clinicos, Hospital Universitario de Salamanca, Spain,
2
Servicio de Urologia, Hospital Universitario de Salamanca, Spain, 3Centro
Investigacion del Cancer, Universidad de Salamanca, Spain
Introduction: Microsatellite analysis has shown promise as highly sensitive
tools for the study of differences between cells populations (primary and recurrent cancer) and susceptibility for some pathologies (certain immune-mediated as
diabetes and immune-neurological diseases).
Material and methods: We selected blood, tissue of superficial bladder carcinoma and exfoliated cells of urine samples, as a source of DNA, were collected
from 32 patients, and 18 controls. DNA was extracted with DNAzol. A whole
genomic amplification (DOP-PCR, Roche) of exfoliated cells was necessary to
standardize the DNA quantities of samples. We used a diagnostic set of polymorphism microsatellite markers, D4S243, D9S162, IFNA, D9S171, D9S747. PCR
was performed as indicated by manufacturer of Taq-polymerase (BioLine). Polymerase chain reaction products were separated by denaturing 10% acrylamideurea gels, photographed and the images were analyzed with the program GelQuant (AMPL Software).
Results: There were no differences when comparing the microsatellites of
urine and blood samples with the control group. Significant differences were found
among the same patient’s samples in at least three of the microsatellites studied.
P-227
should be included in the HLA genomic studies; we have analyzed HLA A3, B7,
DR2 and DR4 allele by PCR.
Material and Methods: 96 patients with different rheumatological diseases
(50 Rheumatoid Arthritis, RA; 46, Psoriatic Arthritis PA) and 124 healthy controls
were included in our study. DNA was extracted with the DNA Direct II kit
(Dynal), which uses resin balls with a magnetic core in order to isolate DNA. The
genes were analyzed by PCR using the following primers: HLA A3: For 50 AgC
gAC gCC gCg AgC CA 30 , Rev 50 CAC TCC Acg CAC gTg CCA 30 ; para HLA
B7: For 50 ggA gTA TTg ggA CCg gAA C 3‘, Rev 50 TAC Cag CgC gCT CCA
gCT 30 ; para DR2 : For 50 TCC TgT ggC AgC CTA AgA g 30 , Rev 50 CgC TgC
ACT gTg AAg CTC TC 30 y DR4: For 50 gTT TCT Tgg AgC Agg TTA AAC A
30 , Rev 50 CgC TgC ACT gTg AAG CTC TC 30 . PCR conditions were as follows:
958, 5 min, 1 cycle; 958 30 sec, 538 30 sec, 728 30sec 34 cycles, and 728 10 min.
PCR products were visualized by electrophoresis with agarose 2%.
Results: Allelic frequencies for HLA A3 was 21.3%. in RA, 19.2% PE
(13.6% en CG); in HLA B7 18.3% RA, 17.5% PA ( 12.5% in CG); DR 2 17.6%
in RA, 17.2 PA (15.5% in CG); DR4 32.4% RA, 28.3% PA ( 15.6% CG).
They were similar to those previously published, significant differences were
found between controls and RA or PA (The Chi-square test, p\ 0.001, between
patients and control group).
POLYMORPHISM SER326CYS OF GEN HOGG1 IN
ALZHEIMER DISEASE
P-229
Victoriano J. Leon1, Jesus L. Cacho2, Yamisel Chong2, and Silvia Gamazo2
1
2
Servicio de Neurologia Hospital Universitario de Salamanca, Spain
THE PHARMACOGENETIC APPROACHES IN POLYCYSTIC
OVARY SYNDROME
The accumulation of damage in the DNA of cerebral cells with time seems
to play an important role in the pathogenesis of Alzheimer disease, AD. DNA
damage accumulates especially in mtDNA with time and hOGG1 is an enzyme
responsible for the repair of DNA oxidative damage both in nuclei and mitochondria. The Ser326Cys polymorphism was shown to diminish the hOGG1 activity in vitro, and it was suggested that the 326Cys allele might pose a higher
risk of 8-OHdG formation in DNA. Recently, some authors have demonstrated
that the capacity to repair oxidative DNA damage was significantly decreased in
healthy human subjects bearing the hOGG1 Cys326Cys homozygous variant genotype, compared to wild type individuals. In the present study, we intend to
study the possible relation of this polymorphism with AD.
Material and Methods: 46 cases of AD, according to the criteria NINCDSADRDA, and 94 controls were employed in this study. Genomic DNA was
extracted from peripheral blood lymphocytes by means of the DNA direct II
(Dynal) kit. Genotyping for the hOGG1 Ser326Cys polymorphism was performed by PCR-RFLP technique, briefly, a 234 bp product was amplified using
the forward: 50 -CCC AAC CCC AGT GGA TTC TCA TTG C-30 and the
reverse: 50 -GGT GCC CCA TCT AGC CTT GCG GCC CTT-30 primers. PCR
conditions were 4 min at 948C and followed by 30 cycles of 30s at 948C, 30s at
608C, and 1 min and 30s at 728C, and a final elongation step of 5 min at 728C.
Overnight digestion at 378C with Fnu4HI resulted in 213- and 21-bp products
for the wild type allele (Ser326), and in 164-, 49- and 21-bp products for the
mutant allele (Cys326). Digestion products were visualized after electrophoresis
on a 2% agarose gel containing ethidium bromide.
Result: The distribution of polymorphism in AD was: 62.3% SS, 33% SC
4.7% CC, in the control group: 66.4 SS, 30.4 SC 3.2 CC. The Chi-square test,
p< 0.01. In this study, there were no polymorphism correlations between AD
and control group.
P-228
HLA GENES AS MARKERS IN SOME RHEUMATOLOGICAL
DISEASES
Victoriano J. Leon1, Alberto J. Leon2, Susana Gomez-Castro3,
and Carlos Montilla3
1
2
Lab Pediatria e Inmunologia Universidad de Valladolid, Spain, 3Servicio
de Reumatologia, Hospital Universitario de Salamanca, Spain
Introduction: Epidemiological studies of HLA alleles related with susceptibility or resistance to certain diseases present the problem of the high price of
classical genomic HLA studies. In order to select those patients of interest that
387
Muammer Karadeniz1, Vildan Bozok-Cetintas2, Asli Tetik2, Ayhan Zengi1,
Mehmet Erdogan1, Sevki Cetinkalp1, Zuhal Eroglu2, and Candeger Yilmaz1
1
Endocrinology and Metabolism Disease, Ege University, Izmir, Turkey,
2
Department of Medical Biology, Ege University, Izmir, Turkey
Objective: The polycystic ovary syndrome (PCOS) is a disorder characterized
by signs and symptoms of hyperandrogenism and oligo- or amenorrhea, and is frequently associated with insulin resistance. Interleukin-6 and Interleukin-10 are
major anti-inflammatory cytokines that have been associated with obesity and type
2 diabetes as insulin resistance syndromes and cardiovascular diseases. PPARgamma is expressed mainly in adipose tissue and plays a pivotal role in the directive of energy storage, adipocyte differentiation and insulin sensitivity. PPARgamma polymorphism has been associated with decreased transcriptional activity
and the presence of Ala isoform has been related to elevated insulin sensitivity. The
aim of this study is to evaluate in the long term treatment responses of the patients
with PCOS that have different genotypes of PPAR-gamma, IL-6 and IL 10 genes.
Methods: Eighty-one women with PCOS were included in our study. PCOS
was defined by the Rotterdam PCOS consensus criteria. At the beginning of the
study, all subjects underwent venous blood drawing for complete hormonal
assays, lipid profile, glucose, insulin and genetic analyses. Patients were randomized according to the therapy they received as follows: metformin (MET) 2g/day
group, Rosiglitasone (ROSI) 4 mg/day group and 35 lg ethinyl estradiol–2 mg
cyproterone acetate group (ECA). IL-6 (G-174C), IL-10 (C819T) and PPARgamma Pro12Ala polymorphisms were genotyped by RFLP method.
Results: HDL levels (p50.038) and triglyceride levels (p50.049) showed a
meaningful increase in all genotypes of the PPAR-gamma gene in ROSI treatment group when compared to the beginning. In this study, it was seen that
MET treatment decreased the cholesterol levels meaningfully in patients that
have both Pro12Ala and Pro12Pro of PPAR exon6 (p50.0001). MET treatment
has showed a meaningful drop of total cholesterol level on CG and GG genotypes of IL-10 gene (p50.0001).
Discussion: Therapy with insulin sensitizer and oral contraceptive altered the
lipid and glucose metabolism, but these effects of the drugs are independentfrom
IL-6 (G-174C), IL-10 (C819T) and PPAR- Pro12Ala genotypes.
P-230
INCIDENCE OF FIBROBLAST GROWTH FACTOR RECEPTOR 3
(FGFR3) A248C, S249C, G372C, T375C MUTATIONS IN
BLADDER CANCER
Yavuz Dodurga1, Canten Tataroglu2, Zehra Kesen3, and
N. Lale Satiroglu-Tufan4
388
1
Department of Pathology, Adnan Menderes University, Aydin, Turkey,
3
Department of Pathology, Denizli State Hospital, Turkey, 4Department of
Medical Biology, Pamukkale University, Denizli, Turkey
2
Purpose: Bladder cancer is the most frequent cancer derived from urinary
system and 90% of them are transitional cell carcinomas. Fibroblast growth factor receptors (FGFR) belong to the tyrosine kinase family and have important
roles in cellular differentiation, proliferation and embryonic development.
FGFR3 is localized on chromosome 4p16.3 and missense mutations of FGFR3
are associated with autosomal dominant human skeletal disorders and some
oncogenic effects. In this study, incidence of FGFR3 thanatophoric dysplasia
mutations located at exon 7 A248C, S249C, exon 10 G372C, T375C and its
correlation to clinico-pathological parameters has been analyzed in a bladder
carcinoma patient population.
Materials and Methods: Fifty six paraffin embedded specimens of transitional cell carcinoma of the urinary bladder were obtained from Denizli State
Hospital Pathology department and included in this study. After DNA isolation
from the paraffin sections, PCR amplification of DNA segments including exon
7 A248C, S249C and exon 10 G372C, T375C was performed for Restriction
Fragment Length Polymorphism (RFLP) and DNA sequencing analysis.
Results and Conclusions: FGFR3 thanatophoric dysplasia mutations located
at exon 7 A248C, S249C and exon 10 G372C, T375C were detected in 33 of the
patients (58.9%) (heterozygous mutant) and 23 of the patients 41.1(%) were
found to be normal. Among the 56 transitional cell carcinomas, missense point
mutations were detected in 7 of them (12.5%) at codon A248C, 28 of them
(50%) at codon S249C, and 2 of them (5.4%) at codon T375C and these results
are in agreement with the previous research reports in the literature. When the
results of the FGFR3 thanatophoric dysplasia mutations located at exon 7
A248C, S249C and exon 10 G372C, T375C were analyzed one by one or as a
group, despite the findings of previous research reports, it has been suggested
that these mutations are detected homogenously regardless of the tumor classification and tumor grade in our study. Hence, it has been suggested to include
more early stage and low grade tumor samples in this research project.
P-231
THE EFFECTS OF QUERCETIN-INDUCED CELL DEATH ON A
NOVEL GENE ‘‘UP-REGULATED GENE 4’’ (URG4) EXPRESSION VIA
TELOMERASE ACTIVITY IN LEUKEMIA CELLS
Yavuz Dodurga1, Cigir Biray-Avci1, N. Lale Satiroglu-Tufan2,
Sunde Yilmaz1, Z. Ozlem Dogan1, Sinem Numanoglu1, Guray Saydam3,
and Cumhur Gunduz1
1
2
Department of Medical Biology, Pamukkale University, Denizli, Turkey,
3
Department of Hematology, Ege University, Izmir, Turkey
Along with being frequently consumed compounds of human diet, flavonoids
need multiple mechanisms to be defined for their biological and pharmacological
features. Muscadine grapes include sufficient amount of quercetin. Quercetin is a
commonly used flavonoid. It affects cell cycle kinetics, proliferation and induction
of apoptosis. Telomerase is composed of a RNA component called hTR and a
reverse transcriptase component that catalyses synthesis reaction is hTERT.
Although most of normal cells do not have telomerase enzyme activities, most of
tumors have it. Up-regulated gene 4 (URG4) is a novel gene that may be associated with the onset of leukemogenesis and cell cycle regulation. The present study
examined for the first time the expression of URG4 in leukemia. We aimed to
investigate the antiproliferative and apoptotic effect of quercetin on human leukemia cells. This study was also designed to answer the following question: does the
URG4 gene expression effect telomerase activity via treatment of quercetin?
Cell viability, cytotoxicity and evaluation of apoptosis were performed by
using Trypan blue dye exclusion, XTT and Acridine Orange/Ethidium Bromide
dye technique assay. hTERT mRNA and URG4 expression is determined by
using Real-time Online RT-PCR. As the effect of quercetin in cell viability evaluated in leukemia cell lines; CCRF-CEM, HL-60 and K562, IC50 was determined
as 25 lM and a distinct decrease was determined in time and dose dependent
manner in cell viability. In HL-60 and K562 cell lines, dose dependent apoptosis
, was 70% and 65%, respectively. hTERT mRNA expression decreases of 12%
and 25% at IC50 dose were detected in HL-60 and K562 cell lines, respectively.
The averages of URG4 mRNA gene expression in control groups of HL-60,
K562 and CCRF-CEM cells were found 2.73, 4.48 and 2.76 respectively. In the
IC50 -dose treated group at 24 hour, 99% , 52.3% , 90% reduction was observed
in HL-60, K562 and CCRF-CEM cells respectively. As a result, it was found
that quercetin decreased the URG4 mRNA gene expression at the IC50 dose in
leukemia cell lines.
In conclusion, URG4 may play important roles in the development of leukemia, and might be a useful molecular marker for predicting the prognosis of leukemia together with hTERT mRNA expression during quercetin treatment.
P-232
AN IN-VITRO STUDY TO EVALUATE THE SIGNIFICANCE OF CK-18
ON DETERMINATION OF CELL DEATH MODES TOWARD
CHEMOTHERAPEUTICS IN HUMAN COLON CANCER CELL LINE
Zekiye Altun1, Serpil Tanriverdi-Akhisaroglu1, Janserey Batu2, Halil Ates3,
Hatice Giray4, and Semra Kocturk2
1
Institute of Oncology, Dokuz Eylul University, Turkey, 2Department of
Biochemistry, Dokuz Eylul University, Faculty of Medicine, Turkey,
3
Hematology Laboratory, Dokuz Eylul University, Faculty of Medicine,
Turkey, 4Public Health, Dokuz Eylul University, Faculty of Medicine,
Turkey
Objectives: Colorectal cancer is the third most common cancer type in the
world. However there is still no useful marker to monitor the response of
patients to chemotherapy drugs during the treatment. Cytokeratin-18 (CK18) is
cleaved by caspases specifically during apoptosis, and the molecular form of this
protein (caspase cleaved vs. non-cleaved) released from dying tumor cells is
therefore diagnostic as to the type of cell death (apoptosis vs. necrosis). CK18 is
a cell death marker that could be a candidate for the follow-up in the early phase
of therapy efficiency. This study is the first in respect of evaluating the efficiency
of CK-18 on the determination of cell death in human colon cancer cell line
(HCT-116).
Methods: HCT-116 cells were treated with FOLFOX and FOLFIRI combinations in regard to in vivo doses for 24, 48 and 72 hours. MTT and LDH analyses were used in cell cytotoxicity determination. The necrotic (M65) and apoptotic forms (M30) of CK-18 were analyzed with special ELISA kits. The cell line
treated with chemotherapy combinations were also stained with Annexin-V/PI
and percentage of apoptotic/necrotic cells were analyzed by Flow Cytometer
(FC). Whole ELISA results (M30/M65) were compared with FC results.
Results: The maximum percentage of cell death was found at 72 hours with
FOLFOX (91%) and FOLFIRI (55%) treatments. FOLFIRI combination caused
an increase apoptotic and necrotic forms of CK-18 (M30 and M65) levels with
time. The proportion of apoptotic and necrotic results of CK-18 were compatible
with FC results. However FOLFOX combination caused an increase in apoptotic
and necrotic forms of CK-18 (M30 and M65) levels with time but these results
were not compatible with FC results. When all drug treatments were considered,
the results show that drugs lead more to necrosis than apoptosis in colon cancer
cell line.
Conclusion: Accordance between ELISA and FC results suggested for the
first time that CK-18 (M30 and M65) could be a useful marker to monitor cell
death modes in colon cancer cells. Our in-vitro drug treatment results showed
also that FOLFOX and FOLFIRI combinations caused more necrosis than apoptosis in human colon cancer cells.
P-233
RESEARCH OF PARAOXONASE GENE POLYMORPHISMS IN ORAL
SQUAMOUS CELL CARCINOMAS
Zeynep Boy-Metin1, Meral Unur1, Bedia Agachan2, Bahar Toptas2,
Salih Aydin3, Gunter Hafiz3, and Turgay Isbir2
1
Department of Oral Surgery and Medicine Istanbul University, Faculty of
Dentistry, Turkey, 2Department of Molecular Medicine, Institute of
Experimental Medical Research, Istanbul University, Turkey, 3Department
of Otorhinolaryngology and Head and Neck Surgery, Istanbul University,
Istanbul Faculty of Medicine, Turkey
Objective: Oxidative stress and free radicals have been associated with the
increased risks of various cancers. The human body has a number of endogenous
free radical scavenging systems, including paraoxonase. Serum paraoxonase
eliminates carcinogenic lipid soluble radicals. Genetic polymorphisms that result
in altered or absent activity of this enzyme have been consistently shown to be
risk factors for the development of numerous malignancies. The purpose of this
study was to investigate the association between polymorphisms in the paraoxonase gene and the risk of oral squamous cell carcinoma.
Methods: The distribution of PON 55 and 192 polymorphisms in 52 oral
squamous cell carcinomas were determined by polymerase chain reaction-based
restriction fragment length polymorphism analysis and compared with 51 healthy
control subjects.
Results: The distribution of PON 55 among cases was LL: 60.0%; MM:
0.0%; LM: 40.0% and among controls was LL: 61.1%; MM: 3.7%; LM: 35.2%.
The distribution of PON 192 among cases was AA: 34.6%; BB: 3.8%; AB: 61.5%
and among controls was AA: 54.9%; BB: 0.0%; AB: 45.1%. There was no significant difference in distribution of PON 55 and 192 genotypes between the case and
control subjects (P>0.05). The frequency of PON 192 B allele among case subjects (45.1%) was significantly higher than among control subjects (65.4%)
(X2:4.28, P:0.038). Individuals with PON 192 B allele showed 2.3 fold increased
risk of oral squamous cell carcinoma (OR: 2.3 95% CI:1.039-5.088).
Conclusions: PON 192 B allele appears to be associated with an increased
risk for oral squamous cell carcinoma.
P-234
EFFECT OF ARTICHOKE EXTRACT ON PEROXIDATION STATUS
ON APO-B-CONTAINING LIPOPROTEINS AND SERUM LIPID
COMPOSITION IN RATS FED ON HIGH CHOLESTEROL DIET
Zeynep Kusku-Kiraz1, Guldal Mehmetcik2, Semra Dogru-Abbasoglu1,
and Mujdat Uysal1
1
Department of Biochemistry, University of Istanbul, Istanbul Medical
Faculty, Turkey, 2Art and Science Faculty, Near East University,
Lefkosa, Cyprus
Oxidative modifications of apo-B-containing lipoproteins (LDL1VLDL)
appear to play a role in atherogenesis. Artichoke is a plant that is widely grown
in Mediterranean countries and is rich in natural antioxidants. The aim of this
study was to investigate the effect of artichoke leaf extract treatment on peroxidation status of LDL1VLDL and serum lipid composition in rats fed on 4%
cholesterol and 1% cholic acid supplemented diet for 1 month. Artichoke leaf
extract (1.5 g/kg/day) was given by gavage during the last two weeks. Baseline
diene conjugate and copper-induced malondialdehyde levels were determined in
LDL1VLDL fraction. In addition, lipid composition, lipid peroxides and antioxidant activity were measured in serum of rats. Our results indicate that artichoke
leaf extract may be useful for the prevention of high cholesterol-induced prooxidant state in LDL1VLDL fraction and the reduction of increased serum cholesterol and triglyceride levels.
P-235
QUANTITATION OF CELL FREE DNA WITH FOUR PROBES IN
PROSTATE CANCER
Zeynep Ozlem Dogan1, Cigir Biray-Avcı1, Sunde Yilmaz1,
Sinem Numanoglu1, Serkan Demiryoguran2, Burak Turna2, Oktay Nazlı2,
and Cumhur Gunduz1
1
Department of Medical Biology, Ege University, Medical Faculty, Izmir,
Turkey, 2Department of Urology, Ege University, Izmir, Turkey
We aimed to determine the quantization of copy number of human telomerase reverse transcriptase (hTERT), Sex-determining Region Y (SRY), Cyclooxygenase 2 (COX–2) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
genes in cell-free DNA of prostate cancer diagnosed cases.
389
Quantification of cell-free DNA from 29 patients, diagnosed and pre-diagnosed
with prostate cancer, has been evaluated. Plasma is isolated from 8 ml peripheral
blood sampling. The isolation of cell-free DNA from 3 ml plasma was performed
by using DNA Isolation Kit according to the manufacturer’s instruction. The gene
regions SRY, hTERT, COX–2 and GAPDH genes were chosen for the quantitative
analysis of cell-free DNA. Quantitative analysis was actualized by using TaqMan
5’ exonuclease method with SRY, hTERT, COX–2 and GAPDH primers and
probes on Real-time quantitative PCR in LightCycler instrument. Amplified region
sizes for hTERT, SRY, COX–2 and GAPDH were 98, 137, 826 and 226 base pairs,
respectively. For all the gene regions, the curve of concentration /cycle number was
constituted using standards which had known concentrations. By using formula of
conversion, the determined copy numbers was converted to ng/ml.
The average age (6SD) was 61.0067.91 (42–79, median 60) years for 29
cases. According to the TNM classification, 7 cases were T2a, 11 cases were
T2b, 6 cases were T3a, 4 cases were T3b and 1 case was T4Mb. Gleason score
of 13 of 29 cases were 6/10, 12 cases were 7/10 and 4 cases were 8/10. The average PSA value was 12.42610.06. The average of cell-free DNA for hTERT
gene region was 413.6861.366 ng/ml (3.5-7,590 ng/ml, median was 106.43 ng/
ml) and for GAPDH gene region, it was 338.736926.23 ng/ml (0.21-4,785 ng/
ml, median was 16.86 ng/ml). For cell free DNA amount, the significant relation
between GAPDH-hTERT, GAPDH-SRY and hTERT-SRY were 84.7%, 87.9%,
98.6%, respectively (p\0.05). No significant correlation between cell free DNA
amount and tumor grade, PSA and Gleason score was found. The quantization of
COX-2 gene region could not be performed. Being 826 bp of amplified COX-2
gene region, we are of the opinion that DNA fragmentation was longer than
expected (100-600 bp) during apoptosis. In conclusion, a large amount of cellfree DNA was released into the circulation following cell apoptosis. We believe
that by evaluating the ‘‘cut off’’ values by using 4 different probes and large series, cell-free DNA method could elevate the diagnostic value of prostate cancer.
P-236
OVER-EXPRESSION OF TATA BINDING PROTEIN ASSOCIATED
FACTOR 7 INDUCES CELL DEATH BY MICROTUBULE
STABILIZATION SIMILAR TO THE SOLUBLE FORM OF HIV-TAT
Dilek Gokturk1, Melek Pehlivan1, Ilknur Gorken,2 Zafer Karaguler2,
and Zeynep Sercan1
1
Department of Medical Biology and Genetics, Dokuz Eylul University,
Faculty of Medicine, Izmir, Turkey, 2Department of Radiation Oncology,
Dokuz Eylul University Faculty of Medicine, Izmir, Turkey
Understanding the immunopathogenesis and biology of HIV infection is of
great importance to introduce new therapeutic strategies and prophylactic vaccines. Tat is one of the nine genes encoded in the HIV genome and its functions
can be grouped under: 1) The transcriptional transactivation of the provirus 2) Inhibition of the host genes mainly at the transcriptional level 3) Inducing apoptosis
in host cells. It has been proposed that TAF7 – a component of the general transcription factor TFIID - could be a functional analogue of HIV Tat. TAF7 has
been shown to support elongation and inhibit transcription by mechanisms very
similar to HIV Tat. Previous reports have suggested that TAF7 acts as a transcriptional checkpoint regulator. Interestingly, there are no studies reporting a role for
TAF7 in apoptosis. In order to investigate TAF7 involvement with programmed
cell death, TAF7 expression was inhibited by siRNA in cell lines induced with cradiation, MGBG and etoposide. A 4-6-fold reduction in apoptotic cell death was
observed in the TAF7 siRNA group when compared to controls. In contrast TAF7
induced apoptosis in cells when over-expressed, as measured by caspase assay and
annexin V staining. We show that TAF7 over-expression is correlated with stabilized microtubule polymerization; a very similar mechanism was reported to be
used by the soluble form of HIV-Tat in apoptotic induction. In addition, we show
that TAF7 co-immunoprecipitates with b-tubulin, one of the building blocks of
microtubules. This is the first study reporting a functional role for TAF7 in programmed cell death. We believe that identifying functional similarities between
TAF7 and HIV-Tat will aid HIV research tremendously and its role in programmed cell death will impact on cell biology in general.

- Wiley Online Library

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