The Interplay
of Biomolecules
HDBMB2014
HDBMB2014
http://hdbmb2014.imi.hr
ISBN 978-953-95551-5-1
HDBMB2014
The Interplay of Biomolecules
Congress of the Croatian Society of
Biochemistry and Molecular Biology
24-27 September 2014
ZADAR, CROATIA
The Interplay
of Biomolecules
HDBMB2014
Congress of the Croatian Society of
Biochemistry and Molecular Biology
24-27 September 2014
ZADAR, CROATIA
Book of
Abstracts
Book of Abstracts of the Congress of the Croatian
Society of Biochemistry and Molecular Biology
“The Interplay of Biomolecules”, HDBMB2014
Publisher:
Croatian Society of Biochemistry and Molecular Biology
Editors:
Maja Katalinić and Zrinka Kovarik
Edition:
XII+163 pages, 200 copies
Cover page and logo design:
Sagita, Opatija
Photographers:
Jerolim Vulić, Zadar and Aleksandar Bonačić, Zadar
A CIP catalogue record for this book is available
in the Online Catalogue of the National and
University Library in Zagreb as 884683
ISBN 978-953-95551-5-1
Dear Colleagues,
Welcome to the Congress of the Croatian Society of Biochemistry and
Molecular Biology - HDBMB2014 entitled „The Interplay of Biomolecules“.
We are pleased that our Congress is being held for the first time in Zadar, a
city of exceptional history and rich cultural heritage.
HDBMB2014 is organised under the auspices of the Croatian Parliament
- Education, Science and Culture Committee, the University of Zadar, the
University of Zagreb, the University of Rijeka, the University of Osijek, the
University of Split, the Zadar County, and the Town of Zadar. We use this
opportunity to express our gratitude to them all as well as to our sponsors,
whose support is invaluable to the overall success of the Congress.
The scientific programme comprises 7 plenary lectures, 17 invited lectures, 12 short oral presentations, and 68 posters and will undoubtedly
provide an excellent opportunity to exchange ideas and experiences with
colleagues, establish new acquaintances, and renew old ones. In addition
to the main programme, we take great joy in organising the mini-symposium in honour of the 50th Anniversary of the FEBS.
HDBMB2014 will aspire to provide its participants a rewarding scientific
and personal experience in Zadar, a city surrounded by historical ramparts
boasting archaeological and monumental riches from ancient and medieval times and the Renaissance, as well as contemporary architectural
achievements such as the first sea organ in the world.
Have a very pleasant stay in Zadar during HDBMB2014!
Zrinka Kovarik
Chair of the Scientific Committee
Tihomir Balog
Chair of the Organising Committee
ORGANISER
Croatian Society of Biochemistry and Molecular Biology
www.hdbmb.hr
AUSPICES
Croatian Parliament - Education, Science and Culture Committee
Zadar County
Town of Zadar
University of Osijek
University of Rijeka
University of Split
University of Zadar
University of Zagreb
SCIENTIFIC COMMITTEE
Zrinka Kovarik, Chair
Members: Tihomir Balog, Damjan Franjević, Hrvoje Fulgosi, Maja Herak
Bosnar, Vladimir Mrša, Sonja Levanat, Ivan Sabolić, Janoš Terzić
ORGANISING COMMITTEE
Tihomir Balog, Chair
Maja Katalinić, Secretary
Ljubica Glavaš-Obrovac, Treasurer
Members: Jerka Dumić, Damjan Franjević, Hrvoje Fulgosi, Maja Herak Bosnar,
Zrinka Kovarik, Marijana Matek Sarić, Jasmina Rokov Plavec, Jadranka Varljen
CONGRESS SECRETARIAT
Maja Katalinić
Croatian Society of Biochemistry and Molecular Biology
Ksaverska cesta 2
10000 Zagreb, Croatia
tel: +385 1 4682551
http://hdbmb2014.imi.hr
VENUE
Hotel Kolovare
Bože Peričića 14
23000 Zadar, Croatia
SUPPORTED BY
Ministry of Science, Education and Sports of the Republic of Croatia
The Foundation of the Croatian Academy of Sciences and Arts
FEBS - Federation of European Biochemical Societies
Institute for Medical Research and Occupational Health
Ruđer Bošković Institute
Josip Juraj Strossmayer University of Osijek, School of Medicine
University of Rijeka, School of Medicine
University of Zadar
University of Zagreb, Faculty of Pharmacy and Biochemistry
University of Zagreb, Faculty of Science, Division of Biology
Zadar County
SPONSORS
Eli Lilly (Suisse) S.A.
Gorea Plus d.o.o.
Kandit d.o.o.
KEFO d.o.o.
Koestlin d.d.
Labena d.o.o.
Maraska d.d.
Obrnuta faza j.d.o.o.
UMNA
EXHIBITORS
AlphaChrom d.o.o.
Biosistemi d.o.o.
CRUX d.o.o.
Diagnostica skalpeli d.o.o
Fidelta d.o.o.
HEBE d.o.o.
INEL – medicinska tehnika d.o.o.
Kemomed d.o.o.
LKB Vertriebs Ges.M.B.H.
MERCK d.o.o.
Sartorius Croatia-Libra elektronik d.o.o.
Spiridion Brusina Medal
Spiridion Brusina is one of the most distinguished persons among 19th century Croatian scientists, an outstanding zoologist and palaeontologist and
farther of Croatian marine biology. He was the first university professor at
the Department of Zoology of the newly-established University of Zagreb,
founder of the Croatian Natural History Society (1885) and initiator and
first editor of its journal Glasnik (presently named Periodicum biologorum),
and founder and largely the designer of the rich and extremely valuable
library and collection of today’s Croatian Natural History Museum. Aided
by his immense organizational capabilities, he made an insurmountable
contribution to the advancement of natural sciences in Croatia, as well as
to their promotion in the world. His professional work and research in the
areas of ornithology, ichthyology, malacology, and mammalogy brought
him recognition domestically, whereas his basic research into the fauna of
neogene molluscs of Croatia and south-eastern Europe made him internationally renowned.
Spiridion Brusina was born on 11 December 1845 in Zadar. After finishing the local Gymnasium, he studied natural sciences at the University of
Vienna. After three months of work at the Zadar Gymnasium, in 1868 he
began working in the National Museum in Zagreb (today’s Croatian Natural
History Museum), where he remained until retirement in 1900. Professor
Brusina can especially be credited for the advancement and development
of zoological and paleomalacological collections, which he designed and
shaped according to the strictest professional principles. He was among
the first to recognize the immense importance of compiling scientific
bibliographies. He spent his entire life attempting to gather and organise
the fauna of the Adriatic Sea, which he in the twilight of his life, following
countless trips to the Croatian coast, succeeded by publishing a fauna still
unmatched in its comprehensiveness.
He was a man of tremendous energy and broad views. He published his
works across the globe, and cooperated with the Smithsonian Institution
from Washington. He kept close contact with many of his peers, with whom
he exchanged literature, debated, sought help – particularly when it comes
to obtaining the scarce literature of the time. He was a true man of his time,
endowed with the spirit of intellectual freedom, positivism, and patriotism.
He embraced Darwinism very early in his life and when he was striving to
initiate the Croatian Natural History Society, even exchanged letters with
Charles Darwin.
In memory of its founder, the Croatian Natural History Society
established the Spiridion Brusina Medal, which is awarded annually to distinguished foreign scientists who promote and support Croatian science and scientists. The
recipients are supposed to deliver a memorial lecture
after receiving the Spiridion Brusina Medal. The Medal
is an art work in bronze showing the profile of Spiridion
Brusina, designed by sculptor Ratko Petrić. Fifteen scientists have thus far received the award.
The Croatian Natural History Society will award the Spiridion Brusina Medal
for 2014 to Enrico Schleiff. The memorial lecture delivered by Professor
Enrico Schleiff is included into the programme of the Congress of the
Croatian Society of Biochemistry and Molecular Biology HDBMB2014, Zadar
– hometown of Spiridion Brusina. Professor Schleiff has been nominated
for the award by the HDBMB and this nomination was supported by the
Croatian Biological Society and Croatian Genetic Society.
Apart from heading his own research group, which studies the processes of protein intake via plastid membranes and RNA biogenesis, Professor
Schleiff is also the vice-president of the Goethe University and the director
of the Buchmann Institute for Molecular Life Sciences. During his scientific
career, Prof Schleiff worked at some of the most renowned universities
in the world alongside leading researchers in the field of photosynthetic
organelle biogenesis. For his contributions to the advancement of science,
he has received various awards and scholarships.
Professor Enrico Schleiff is one of the best younger German scientists who
has thus far brought up and mentored many Croatian scientists, who then
returned to Croatia to continue their careers. Almost a third of his publications in prestigious international journals were authored in cooperation
with young researchers from Croatia. This has made him a true friend of
Croatian science. Professor Schleiff’s laboratory still welcomes our young
researchers, whereas his reputation in the world of science actively promotes the development of Croatian research. That is why Prof Enrico
Schleiff without question deserves the 2014 Spiridion Brusina Medal.
Zrinka Kovarik and Hrvoje Fulgosi
In memoriam
Ivana Weygand-Đurašević was born in 1952 in Osijek, Republic of Croatia. She was a full professor of Biochemistry and Molecular Biology at
the Chemistry Department at the Faculty of Science, University of Zagreb
and an internationally renowned scientist in the field of tRNA and aminoacyl-tRNA synthetases, crucial molecules for protein biosynthesis.
Professor Weygand-Đurašević received a B.Sc. degree in chemistry (1975),
M.Sc. degree in molecular biology (1978), and Ph.D. degree in chemistry
(1981) from the University of Zagreb. From 1975, she was employed at
the Chemistry Department of the Faculty of Science, University of Zagreb,
where she first worked as an assistant, then assistant professor (1988),
associate professor (1995), and finally full professor (2000). She was the
Head of the Laboratory of Biochemistry in the
period 1999–2013. She served many administrative functions at the Faculty of Science,
being director of doctoral studies in chemistry and vice dean for science.
Professor Weygand-Đurašević always emphasized that her professional career was guided
by two mentors: Professor Željko Kućan, a distinguished Croatian biochemist and molecular
biologist, who introduced her into the wonders
of molecular life sciences, and Professor Dieter Söll, an eminent US scholar from Yale
University, who encouraged her to pursue an
independent and fruitful scientific carrier. She
spent six years in Professor Söll’s laboratory,
three years as a postdoctoral fellow (19841987) and three years as a visiting scientist
(1990-1993). Upon her return to Zagreb, she
set up her own laboratory and worked long but enthusiastic hours to make
her laboratory successful and internationally recognized. She was Principal Investigator on four projects funded by Croatian government and a PI
or leader of the Croatian team on eight internationally funded projects (two
NIH/FIRCA projects, two ICGEB projects, two SCOPES projects, a UKF project,
and an FP7-REGPOT project). During her entire scientific career, she collaborated with renowned and internationally recognized scientists such as
the previously mentioned Professor Dieter Söll (Yale University), Professor
Mike Ibba (Ohio State University), Professor Nenad Ban (ETH Zurich, Switzerland), and Professor Omar Orellana (University of Chile).
Her primary scientific interests were focused on interactions of nucleic
acids and proteins, quality control mechanisms in protein biosynthesis,
and protein engineering. She authored more than 70 papers and four book
chapters. Her major scientific contribution are, among others, the discovery
and study of unusual non-canonical seryl-tRNA synthetase from methanogenic archaea (EMBO Journal, 2006) and a new class of enzymes amino
acid:[carrier protein] ligases (PNAS, 2010). She loved to teach and had great
impact on many generations of chemistry and molecular biology students
at undergraduate, graduate and postgraduate study programs. She was a
supportive mentor in numerous diploma, master and doctoral theses and
trained many individuals who went on to make their own contributions to
research in molecular life sciences. Many of her former students today act
as principal investigators and heads of departments all across the world.
In 2006, she received the highly prestigious Croatian National Science
Award. For her outstanding scientific and teaching career, she was elected
into the Croatian Academy of Sciences and Arts in 2012.
She was a member of the editorial boards of Journal of Biological Chemistry and Croatica Chemica Acta. She also served as a member of the Committee on Ethics in Science and Higher Education (2006 - 2010) and the
Scientific Field Committee for Natural Sciences - Chemistry (2009 - 2014).
Professor Weygand-Đurašević was a member of the Croatian Chemical
Society, Croatian Biological Society, and Croatian Biophysical Society. She
was always a very active member of the Croatian Society of Biochemistry
and Molecular Biology (HDBMB) and participated in many of the Society’s
activities. She was recently appointed member of HDBMB’s Court of Honour. Together with her young collaborators, she actively contributed to the
programs of many HDBMB congresses.
Professor Weygand-Đurašević passed away on April 7, 2014. Her passing is
undoubtedly a huge loss for the entire community of Croatian biochemists
and molecular biologists and beyond. She was an outstanding scientist, a
dedicated teacher, and a wonderful colleague. We are all grateful for knowing her.
Jasmina Rokov Plavec
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Information
3
Programme
5
List of Posters
13
The 50th Anniversary of the FEBS
23
Abstracts of Plenary Lectures
27
Abstracts of Lectures
37
Abstracts of Short Presentations
57
Abstracts of Posters
71
Author Index
141
List of Participants
149
Sponsors
159
Table of Contents
Table of Contents
1
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Information
Registration
Registration will take place at the registration desk in the Hotel Kolovare
th
lobby from 10:00 on Wednesday, September 24 .
Registration fee for participants includes: admission to lectures and
exhibition, Book of Abstracts, congress materials, admission to all social
events and refreshments during the congress.
Registration for accompanying persons includes welcome party, the congress
dinner and Zadar city tour.
The certificate of attendance will be provided at the registration desk.
Language
The official language of the congress is English. There will be no simultaneous
translation.
Lectures and oral presentations
Standard personal computer and LCD projector will be available. The
speakers are kindly asked to deliver their presentations (CD, DVD or USB
stick) in advance to our technician, at least one hour before session start. The
use of personal laptops is discouraged due to potential incompatibility and
timing issues.
Poster presentations
Posters should be mounted according to the schedule and to the List of
posters in the Book of Abstracts.
Social events
19:30
15:00
20:00
Welcome party (Hotel Kolovare)
Zadar city tour
Congress dinner (Hotel Kolovare)
Information
Wednesday, Sept 24
Friday, Sept 26
Friday, Sept 26
3
Programme
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Programme
Wednesday, September 24, 2014
16:00
Opening ceremony
Chairs: Zrinka Kovarik and Israel Pecht
16:45 PL1 FEBS National Lecture
Hermona Soreq (Jerusalem, Israel)
FROM MICE TO MEN: FINE TUNING OF CHOLINERGIC
SIGNALING BY microRNAs
17:30
th
The 50 Anniversary of the FEBS
Chairs: Zrinka Kovarik and Tihomir Balog
SYM-L1 Israel Pecht (Rehovot, Israel)
FIFTY YEARS OF FEBS-ADVANCING SCIENCE ACROSS
EUROPE
SYM-L2 Jerka Dumić (Zagreb, Croatia)
LET’S JOIN THE CELEBRATION OF THE 50th FEBS
ANNIVERSARY!
Chairs: Jerka Dumić and Tihomir Balog
18:15 PL2 Israel Pecht (Rehovot, Israel)
ELECTRON TRANSFER IN PROTEINS: INSIGHTS INTO
PRINCIPLES AND POSSIBLE APPLICATIONS
Welcome party
Programme
19:30
7
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Thursday, September 25, 2014
Chairs: Maja Herak Bosnar and Matias Sprinzl
9:00
PL3 Ivan Mijaković (Göteborg, Sweden)
BACTERIAL SIGNALING AND REGULATION BASED ON
PROTEIN PHOSPHORYLATION
9:45
L1
Ivana Novak Nakir (Split, Croatia)
STEREOCHEMICAL ORIGINS OF THE GENETIC CODE FOR
GLUTAMINE AND GLUTAMATE
10:10 L2
László Buday (Budapest, Hungary)
TKs SCAFFOLD PROTEINS IN TYROSINE KINASE
SIGNALLING
10:35
Posters, exhibition and refreshment
Programme
Chairs: Hermona Soreq and Vladimir Mrša
11:30 L3
Marija Heffer (Osijek, Croatia)
WHAT IS WRONG WITH SIALYLTRANSFERASES KNOCKOUTS?
11:55 L4
Silva Hećimović (Zagreb, Croatia)
THE
ROLE
OF
LYSOSOMAL
PATHWAY
IN
NEURODEGENERATIVE DISEASES
12:20 SP1 Barbara Viljetić (Osijek, Croatia)
PIWIL1 REGULATES NEOCORTICAL STEM CELL CYCLE,
MIGRATION AND DENDRITOGENESIS
12:35 SP2 Dubravka Hranilović (Zagreb, Croatia)
INCREASED BONE STRUCTURE IN ANIMALS WITH
PERINATALLY ALTERED SEROTONIN METABOLISM
12:50 SP3 Katarina Gros (Ljubljana, Slovenia)
NON-SYNAPTIC ROLES OF AGRIN IN THE EARLY STAGES
OF SKELETAL MUSCLE REGENERATION
13:05 Lunch Break (on your own)
8
Chairs: Ljubica Glavaš-Obrovac and Ivan Mijaković
14:30 PL4 Matias Sprinzl (Bayreuth, Germany)
ELECTRICALLY READABLE BIOCHIPS FOR RAPID RNA
ANALYSIS
15:15 L5
Igor Jurak (Rijeka, Croatia)
THE miRNA WORLD OF HERPES SIMPLEX VIRUS
15:40 SP4 Mirna Ćurković-Perica (Zagreb, Croatia)
DIVERSITY OF Cryphonectria hypovirus 1 IN CROATIA:
IMPACT ON ITS FUNGAL HOST
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
15:55
SP5
Ivana Ivančić Baće (Zagreb, Croatia)
ACTIVATION OF ANTIVIRAL DEFENSE AT LOW
TEMPERATURE OF INCUBATION IN Escherichia coli
Morana Dulić (Zagreb, Croatia)
A SINGLE SYNTHETIC SITE RESIDUE MODULATES
PARTITIONING OF PRE- AND POST-TRANSFER EDITING
PATHWAYS IN OVERALL EDITING BY ISOLEUCYL-tRNA
SYNTHETASE FROM Escherichia coli
Petar Ozretić (Zagreb, Croatia)
FUNCTIONAL ANALYSIS OF CIS-REGULATORY ELEMENTS
FROM 5' UNTRANSLATED REGION OF PTCH1B GENE
16:10
SP6
16:25
SP7
16:40
Posters, exhibition and refreshment
Programme
Chairs: Maja Katalinić i Dubravko Jelić
17:30 L6
Branka Salopek-Sondi (Zagreb, Croatia)
PHYTOHORMONE AUXIN: A MEDIATOR OF PLANT STRESS
RESPONSE?
17:55 L7
Dubravko Jelić (Zagreb, Croatia)
PORPHYRINS AS NEW ENDOGENOUS MACROCYCLIC
ANTI-INFLAMMATORY AGENTS FOR TARGETING
PROTEIN-PROTEIN INTERACTIONS
18:20 L8
Zoran Radić (La Jolla, CA, USA)
NOVEL (BIO)MOLECULES FOR NEW ORGANOPHOSPHATE
INTOXICATION TREATMENTS
18:45 SP8 Florian Nachon (Grenoble, France)
CRYSTAL STRUCTURE OF TACRINE-BASED UNCHARGED
ACETYLCHOLINESTERASE REACTIVATORS
9
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Friday, September 26, 2014
Chairs: Željko Kućan and Janoš Terzić
9:00
PL5
Sanja Sever (Charlestown, MA, USA)
DYNAMIN OLIGOMERIZATION REGULATES ACTIN AND
REPRESENTS A NOVEL THERAPEUTIC TARGET IN
CHRONIC KIDNEY DISEASES
9:45
L9
Igor Weber (Zagreb, Croatia)
SIGNALING OF Rac1 GTPases TO THE ACTIN
CYTOSKELETON
10:10 SP9
Teuta Opačak-Bernardi (Osijek, Croatia)
THERMALLY RESPONSIVE ELP-DNMAML PROTEIN AND
IT'S EFFECT ON U251 CELLS IN VITRO
10:25 L10
Dunja Leljak-Levanić (Zagreb, Croatia)
THE MANY FACES OF MATH-BTB PROTEINS IN PLANTS
10:50 Coffee break
Chairs: Sonja Levanat and László Buday
11:10 L11
Judit E. Pongracz (Pecs, Hungary)
THE ROLE OF CELL-CELL INTERACTIONS IN TISSUE
MICROENVIRONMENT IN SIGNALLING STUDIES OF
CARCINOGENESIS AND DRUG RESPONSE
11:35 L12
Aleksandra Fučić (Zagreb, Croatia)
XENOESTROGENS AS CARCINOGENS
12:00 SP10
Neda Slade (Zagreb, Croatia)
THE EFFECT OF ΔNp73α ON THE CELL CYCLE CONTROL
AFTER DNA DAMAGE IN NORMAL AND TUMOR
HUMAN CELLS
Programme
Chairs: Zrinka Kovarik and Hrvoje Fulgosi
12:15 PL6
SPIRIDION BRUSINA MEDAL: Memorial Lecture
Enrico Schleiff (Frankfurt, Germany)
NEW FRONTIERS IN PROTEIN TARGETING AND
TRANSLOCATION IN PLANTS
10
13:00
Lunch Break (on your own)
15:00
Zadar guided city-tour
20:00
Congress dinner (hotel Kolovare)
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Saturday, September 27, 2014
Chairs: Silva Hećimović and Damjan Franjević
9:00
PL7
William Martin (Düsseldorf, Germany)
TWO KINDS OF ENERGY AT THE ORIGIN OF LIFE
9:45
L13
Tomislav Domazet-Lošo (Zagreb, Croatia)
INVASIVE TUMORS HAVE DEEP ROOTS IN ANIMAL
PHYLOGENY
10:10 SP11 Ana Bielen (Zagreb, Croatia)
DEEP SCANNING FOR GDSL MOTIFS ACROSS ECOLOGICALLY
DIVERSE ACTINOBACTERIA
10:25 L14
Iva Tolić (Zagreb, Croatia)
A YEAST'S TRICK FOR STAYING YOUNG
10:50
Coffee break
Chairs: Jadranka Varljen and Ivan Sabolić
11:10 L15
Olga Vitavska (Osnabrück, Germany)
SLC45, A NOVEL FAMILY OF MAMMALIAN SUGAR
TRANSPORTERS
11:35 L16
Bojan Polić (Rijeka, Croatia)
NK CELLS LINK OBESITY-INDUCED ADIPOSE STRESS TO
INFLAMMATION AND INSULIN RESISTANCE
12:00 L17
Balázs Sarkadi (Budapest, Hungary)
ABC TRANSPORTERS IN NORMAL AND CANCER STEM CELLS
12:25 SP12 Ivan Mihaljević (Zagreb, Croatia)
THE ROLE OF ORGANIC CATION TRANSPORTERS (OCTS,
SLC22A) IN ZEBRAFISH (Danio rerio)
Closing ceremony
Programme
12:40
11
List of Posters
P1
ANTIBACTERIAL EFFECT OF SELECTED ESSENTIAL OILS AGAINST
LEGIONELLA PNEUMOPHILA
Ana Babić, Mladenka Malenica Staver, Ivana Gobin
P2
GENDER DIFFERENCE IN EXPRESSION OF ESTROGEN AND LEPTIN
RECEPTOR IN SPRAGUE DAWLEY RAT ADRENAL GLAND AFTER ACUTE
AND CHRONIC STRESS
Marta Balog, Senka Blažetić, Irena Labak, Barbara Viljetić, Robert
Blažeković, Rosemary Vuković, Marija Heffer
P3
CD26 DEFICIENCY ALTERS VIP AND NPY LEVELS IN MURINE CROHN-LIKE
COLITIS
Lara Batičić Pučar, Dijana Detel, Natalia Kučić, Sunčica Buljević,
Jadranka Varljen
P4
INFLUENCE OF LIGNANS SUPPLEMENTATION ON ANTIOXIDANT
ACTIVITY OF APPLE JUICE
Drago Bešlo, Dejan Agić, Bono Lučić, Dragan Amić
P5
BRONCHODILATING BETA2-AGONISTS AS HUMAN CHOLINESTERASE
INHIBITORS
Anita Bosak, Ivana Gazić Smilović, Anamarija Knežević, Vladimir
Vinković, Zrinka Kovarik
P6
EXPRESSION AND CELLULAR DISTRIBUTION OF PRIMARY-, SECONDARYAND TERTIARY-ACTIVE BASOLATERAL MEMBRANE TRANSPORTERS FOR
ORGANIC ANIONS IN HUMAN KIDNEYS
Davorka Breljak, Marija Ljubojević, Vedran Micek, Daniela Balen, Ivana
Vrhovac, Hrvoje Brzica, Dean Karaica, Ognjen Kraus, Nikola Radović,
Yohannes Hagos, Maja Henjakovic, Roberto Antolović, Naohiko Anzai,
Birgitta C. Burckhardt, Gerhard Burckhardt, Ivan Sabolić
P7
TISSUE EXPRESSION PROFILING OF THE MOUSE Sglt1 mRNA AND
PROTEIN WITH SEX-DEPENDENT Sglt1 RENAL EXPRESSION
Ivana Vrhovac, Davorka Breljak, Dean Karaica, Hermann Koepsell, Ivan
Sabolić
P8
ASSOCIATION OF KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS
(KSHV) WITH BLADDER CANCER IN CROATIAN PATIENTS
Martina Paradžik, Viljemka Bučević-Popović, Marijan Šitum, Crystal J.
Jaing, Marina Degoricija, Kevin S. McLoughlin, Said I. Ismail, Volga
Punda Polić, Janoš Terzić
List of Posters
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
15
List of Posters
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
16
P9
TFF3 KNOCK-OUT MICE HAVE ALTERED FATTY ACID, PROTEIN AND
GLUCOSE METABOLISM IN THE LIVER
Maro Bujak, Martina Mihalj, Ivana Tartaro Bujak, Srđan Vučinić, Anita
Horvatić, Maja Tolušić Levak, Katarina Mišković, Tomislav Kopačin,
Branka Mihaljević, Ines Drenjančević, Mirela Baus Lončar
P10
EVALUATION OF THE POTENCY OF A NEW SERIES OF PYRIDOXAL OXIME
DERIVATIVES IN THE REACTIVATION OF TABUN, PARAOXON AND VXPHOSPHYLATED
ACETYLCHOLINESTERASE
AND
BUTYRILCHOLINESTERASE
Valentina Bušić, Dajana Gašo-Sokač, Maja Katalinić
P11
PREBIOTIC ACTIVITY OF HONEYDEW HONEY ON PROBIOTIC BACTERIA
LACTOBACILLUS PLANTARUM
Goranka Crnković, Mladenka Malenica Staver, Ivana Gobin
P12
THE RELATIONSHIP BETWEEN THE AUTOPHAGY-LYSOSOMAL
PATHWAY, CHOLESTEROL HOMEOSTASIS AND PROCESSING OF THE
ALZHEIMER’S APP PROTEIN
Stjepko Čermak, Mirsada Čaušević, Silva Hećimović
P13
COMPOSITION OF FREE FATTY ACIDS IN THE PLACENTA OF PREGNANT
WOMEN WITH TYPE 1 DIABETES
Vito Starčević, Ivančica Delaš, Tonko Dražić, Josip Juras, Josip Đelmiš
P14
IMMUNOMODULATORY PROPERTIES OF DIPEPTIDYL PEPTIDASE IV
(DPP IV/CD26) IN AN EXPERIMENTAL MODEL OF ULCERATIVE COLITIS
Dijana Detel, Lara Batičić Pučar, Ester Pernjak Pugel, Sunčica Buljević,
Jadranka Varljen
P15
CHOLESTEROL-MEDIATED OXIDATIVE STRESS IN NIEMANN-PICK TYPE C
DISEASE INVOLVES A DEFECT IN THE ACTIVITY OF SUPEROXIDE
DISMUTASE
Kristina Dominko, Martina Malnar, Domagoj Đikić, Silva Hećimović
P16
EXPRESSION OF GALECTIN-3, AN ANTI-APOPTOTIC MOLECULE IS NOT
AFFECTED IN APOPTISIS PROVOKED WITH 17-DMAG, AN INHIBITOR OF
HSP90
Jerka Dumić, Sanja Dabelić, Tamara Zorbaz, Sandra Šupraha Goreta,
Jozsef Petrik, Ognjen Čulić, Karmela Barišić
P17
SERUM GALECTIN-3 IN OSTEOPOROSIS AND OSTEOARTHRITIS
Mateja Prunk, Jerka Dumić, Janja Marc
P18
FUNCTIONAL CHARACTERISATION OF PARALOGOUS SsbB IN
Streptomyces coelicolor
Želimira Filić, Tina Paradžik, Nives Ivić, Ana Bielen, Babu A. Manjasetty,
Paul Herron, Dagmara Jakimowicz, Marija Luić, Dušica Vujaklija
P19
ALGAL ENDOSYMBIONTS IN EUROPEAN HYDRA STRAINS REFLECT
MULTIPLE ORIGINS OF THE ZOOCHLORELLA SYMBIOSIS
Nives Rajević, Goran Kovačević, Mirjana Kalafatić, Sven Gould, William
Martin, Damjan Franjević
P20
BIOMONITORING OF GENOME INTEGRITY IN HUMAN POPULATION
WITH THYROID DISEASES: A PILOT STUDY
Marko Gerić, Renato Janušić, Božena Šarčević, Vera Garaj-Vrhovac
P21
SUPPRESSION OF HIF-1 PATHWAY ENHANCES IL-6 ACTION IN
CULTURED HUMAN MYOBLASTS
Katarina Gros, Katarina Miš, Urška Matkovič, Matej Podbregar, Tomaž
Marš, Zoran Grubič, Sergej Pirkmajer
P22
THE ASSOCIATION BETWEEN 5-HT1A SEROTONIN RECEPTOR GENE
POLYMORPHISM AND EXTRAPYRAMIDAL SIDE EFFECTS IN
HALOPERIDOL-TREATED PATIENTS WITH SCHIZOPHRENIA
Mirko Grubor, Dubravka Svob Strac, Maja Mustapic, Maja Zivkovic,
Alma Mihaljevic-Peles, Marina Sagud, Nela Pivac, Dorotea Muck-Seler
P23
NMEGP1 GENE/PROTEIN FROM Capsaspora owcarzaki - STRUCTURE,
FUNCTION AND EVOLUTION
Helena Ćetković, Maja Herak Bosnar, Drago Perina, Andreja Mikoč,
Robert Belužić, Innaki Ruiz-Trillo, Matija Harcet
P24
ENHANCED NK CELL-DEPENDENT SUREVEILLANCE OF MELANOMA IN
NKG2D-DEFICIENT MICE
Vedrana Jelenčić, Felix M. Wensveen, Maja Gulin, Bojan Polić
P25
NON-BIOSYNTHETIC HUMAN MILK OLIGOSACCHARIDES – NATURAL
COMPONENTS OR ARTEFACTS?
Marko Jovanović, Richard Tyldesley-Worster, Gottfried Pohlentz, Jasna
Peter-Katalinić
P26
INFLUENCE OF EXTRACTION METHODS ON ANTIPROLIFERATIVE
POTENTIAL OF CHAMOMILE FLOWER EXTRACTS
Marijana Jukić, Aleksandra Cvetanović, Katarina Mišković, Jaroslava
Švarc-Gajić, Ljubica Glavaš-Obrovac
P27
CULTURABILITY OF MULTIDRUG-RESISTANT AND DRUG-SENSITIVE
STRAINS OF ACINETOBACTER BAUMANNII ON DRY PLASTIC SURFACES
Denis Juraga, Marina Matešić, Diana Jurčić-Momčilović, Ivana Gobin
List of Posters
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
17
List of Posters
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
18
P28
CELL LOCALIZATION AND SEX-DEPENDENT EXPRESSION OF
CHLORIDE/FORMATE EXCHANGER CFEX (Slc26a6) IN RAT KIDNEYS
Dean Karaica, Davorka Breljak, Jovica Lončar, Marija Ljubojević, Carol
M. Herak-Kramberger, Vedran Micek, Ivana Vrhovac, Jana Ivković, Ivan
Mihaljević, Petra Marić, Tvrtko Smital, Birgitta Burckhardt, Gerhard
Burckhardt, Ivan Sabolić
P29
CAN ACETYLCHOLINESTERASE MUTATIONS HELP CREATE MORE
EFFICIENT REACTIVATORS FOR ORGANOPHOSPHORUS COMPOUNDS
POISONING TREATMENT?
Maja Katalinić, Goran Šinko, Florian Nachon, José Dias, Nikolina Maček
Hrvat, Zrinka Kovarik
P30
IN VIVO AND IN VITRO ANALYSIS OF PLANT SERYL-tRNA SYNTHETASE
INTERACTOME
Mario Kekez, Jasmina Rokov Plavec, Nataša Bauer, Genadij Razdorov,
Vesna Hodnik, Gregor Anderluh, Ivana Weygand-Đurašević
P31
GLYCANS ARE A NOVEL BIOMARKER OF CHRONOLOGICAL AND
BIOLOGICAL AGE
Toma Keser, Jasminka Krištić, Frano Vučković, Cristina Menni, Lucija
Klarić, Ivona Bečeheli, Maja Pučić-Baković, Mislav Novokmet, Massimo
Mangino, Kujtim Thaqi, Pavao Rudan, Natalija Novokmet, Jelena Šarac,
Saša Missoni, Ivana Kolčić, Ozren Polašek, Igor Rudan, Harry Campbell,
Caroline Hayward, Yurii Aulchenko, Ana Valdes, James F. Wilson, Olga
Gornik, Dragan Primorac, Vlatka Zoldoš, Tim Spector, Gordan Lauc
P32
OBESITY PHENOTYPE OF RATS WITH CONSTITUTIONAL HYPERACTIVITY
OF SEROTONIN TRANSPORTER
Maja Kesić, Darko Orešković, Lipa Čičin-Šain
P33
GLOBAL HISTONE ACETYLATION IN DIABETIC EMBRYOPATHY
Marina Korolija, Mirko Hadžija, Sandra Sobočanec, Marijana Popović
Hadžija
P34
ISOLATION AND CHARACTERIZATION OF LYSOSOMES IN NPC MODEL
CELLS
Marko Kosicek, Tanja Jovic, Silva Hecimovic
P35
MACRODOMAIN PROTEIN FROM BACTERIUM STREPTOMYCES
COELICOLOR
Jasna Lalić, Andreja Mikoč, Drago Perina, Igor Sabljić, Bruna Pleše,
Mirna Imešek, Helena Ćetković, Marija Luić, Roko Žaja, Ivan Ahel
P36
CROSS-TALK BETWEEN ESTROGEN RECEPTOR ALPHA AND Hh-Gli
SIGNALING PATHWAYS IN BREAST CANCER CELLS
Diana Trnski, Maja Sabol, Zvonimir Uzarevic, Petar Ozretic, Vesna
Musani, Sonja Levanat
P37
CHOLINE BINDING SITE MUTATIONS IMPROVE HI-6 ASSISTED
REACTIVATION OF THE VX-ACETYLCHOLINESTERASE CONJUGATE
Nikolina Maček Hrvat, Zoran Radić, Palmer Taylor, Zrinka Kovarik
P38
REVERSIBLE INHIBITION OF CHOLINESTERASES WITH AROMATIC NSUBSTITUTED 2-HYDROXYIMINOACETAMIDES
Nikola Maraković, Anamarija Knežević, Vladimir Vinković, Zrinka
Kovarik, Goran Šinko
P39
THE ASSOCIATION OF FACTOR V LEIDEN, PROTHROMBIN G20210A,
MTHFR C677T AND PAI-1 5G/4G POLYMORPHISMS WITH DEEP VEIN
THROMBOSIS AND PULMONARY EMBOLISM IN EASTERN CROATIA
Saška Marczi, Stana Tokić, Nevenka Krajina, Ljubica Glavaš-Obrovac
P40
PRE-EXPOSURE TO OLIVE OIL POLYPHENOLS EXTRACT STIMULATES
LIVER REGENERATION IN MICE VIA STRESS SENSITIVE GENES
Jelena Marinić, Dalibor Broznić, Gordana Čanadi Jurešić, Marin Tota,
Čedomila Milin
P41
FRACTIONATION AND CHARACTERIZATION OF MAJOR BOVINE AND
GOAT WHEY PROTEINS
Andrea Markovinović, Marko Jovanović, Darko Gumbarević, Jasminka
Giacometti
P42
THE EFFECT OF A PHOSPHOLIPASE C GAMMA INHIBITOR ON THE
PROLIFERATION AND PHENOTYPE OF DU145 PROSTATE CANCER CELLS
Angela Mastelić, Nikolina Režić-Mužinić, Anita Markotić, Vedrana
Čikeš-Čulić, Milena Vuica-Ross, Ashley Ross, David Barker, Jóhannes
Reynisson
P43
ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES OF THE EXTRACTS
FROM PLANT LEAVES AS A POTENTIAL RICH SOURCES OF BIOACTIVE
PHENOLICS
Sanja Milovanović, Marina Bubonja Šonje, Marko Jovanović, Maja
Abram, Diana Jurčić-Momčilović, Jasminka Giacometti
P44
IN VITRO AND IN VIVO ACTIVITY OF THREE NOVEL MONOMETHINE
CYANINE DERIVATIVES - MCDS
Katarina Mišković, Tatjana Belovari, Jasmina Rajc, Vatroslav Šerić,
Ranko Stojković, Ivo Piantanida, Mirela Baus Lončar, Ljubica GlavašObrovac
List of Posters
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
19
List of Posters
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
20
P45
SEARCHING FOR PROTEIN-LIPID INTERACTIONS: GANGLIOSIDE
PROFILING OF NEUROPLASTIN KNOCK-OUT MICE
Kristina Mlinac, Rodrigo Herrera-Molina, Angela Kolodziej, Marko
Rožman, Željka Vukelić, Karl-Heinz Smalla, Dirk Montag, Svjetlana
Kalanj Bognar
P46
CHROMATIN REMODELING PROCESS AT THE YEAST PHO5 PROMOTER
IS ESSENTIALLY DEPENDENT ON THE ACTIVITY OF RSC REMODELING
COMPLEX
Sanja Musladin, Dora Hlevnjak, Nils Krietenstein, Philipp Korber,
Slobodan Barbarić
P47
SURVIVAL OF F. TULARENSIS SUBSP. NOVICIDA IN DIFFERENT SPRING
WATER SAMPLES
Mateja Ozanic, Valentina Marecic, Danijela Lenac, Vanda Piskur, Marin
Glad, Majda Meden, Marin Bajek, Marina Santic
P48
RECOGNITION OF ALKALI-LABILE GANGLIOSIDES BY TWODIMENSIONAL
THIN-LAYER
CHROMATOGRAPHY
AND
IMMUNOHISTOCHEMICAL LOCALIZATION AFTER ALKALI TREATMENT
FROM THE BRAIN OF TWO POIKILOTHERMIC FISH SPECIES
Valentina Pavić, Elizabeta Has-Schön, Ivan Bogut, Marija Heffer
P49
THE STRESS REGULATED ASR PROTEIN CAN BE DETECTED IN IN VITRO
GROWN TISSUES OF THE CACTUS MAMMILLARIA GRACILIS
Petra Peharec Štefanić, Tea Rogić, Dudy Bar-Zvi, Biljana Balen
P50
POLY(ADP-RIBOSYL)ATION IN THE RED SEAWEED CHONDRUS CRISPUS
Drago Perina, Andreja Mikoč, Josip Ahel, Helena Ćetković, Roko Žaja,
Ivan Ahel
P51
IN VITRO EVALUATION OF POLY(L-LYSINE)-COATED MAGHEMITE
NANOPARTICLES: APPLICATION IN BRAIN RESEARCH
Igor Pongrac, Marina Dobrivojević, Michal Babič, Marija Lovrić, Lejla
Ferhatović Hamzić, Miroslav Šlouf, Srećko Gajović, Daniel Horák
P52
INVESTIGATIONS OF THE KEY BINDING INTERACTIONS OF NOVEL
IMIDAZOLE- AND BENZIMIDAZOLE-BASED OXIMES WITHIN THE ACTIVE
SITE OF BUTYRYLCHOLINESTERASE
Ines Primožič, Srđanka Tomić, Tomica Hrenar
P53
ARABIDOPSIS THALIANA SERYL-tRNA SYNTHETASE PARTICIPATES IN
CELLULAR STRESS RESPONSE
Jasmina Rokov Plavec, Mario Kekez, Nataša Bauer, Ela Šarić, Ivana
Weygand-Đurašević
P54
THE EFFECT OF 17Β-ESTRADIOL ON THE EXPRESSION OF DIPEPTIDYL
PEPTIDASE III AND HEME OXYGENASE 1 IN LIVER OF CBA/H MICE
Željka Mačak Šafranko, Sandra Sobočanec, Ana Šarić, Nina Jajčanin
Jozić, Tihomir Balog, Marija Abramić
P55
COMPARATIVE ANALYSIS OF SSB GENES FROM PHYTOPLASMA
GENOMES AND THEIR POTENTIAL ROLE IN GENOME INSTABILITY
Martina Šeruga Musić, Anamarija Slović, Marija Pinterić
P56
RECA730 SUPPRESSES UV SENSITIVE PHENOTYPE IN RECA LOADING
MUTANTS OF ESCHERICHIA COLI
Ana Šimatović, Ignacija Vlašić, Krunoslav Brčić-Kostić
P57
EVALUATION OF BUTYRYLCHOLINESTERASE STEREOSELECTIVITY IN
INTERACTION WITH ENANTIOMERS OF ETHOPROPAZINE
Goran Šinko, Nikola Maraković, Jure Stojan
P58
THE PILOT STUDY OF VIROIDS IN ASYMPTOMATIC SOLENOSTEMON
SCUTELLARIOIDES (L.) CODD PLANTS
Dijana Škorić, Silvija Černi, Karlo Jezernik
P59
THE TRANSPORT ABILITY OF PROTECTIVE BIOMOLECULE QUERCETIN
THROUGH ARABIDOPSIS THALIANA IS IMPROVED BY THE RARE-EARTH
ELEMENT EUROPIUM
Ivana Šola, Ivo Piantanida, Ivo Crnolatac, Gordana Rusak
P60
SUBSTRATE-INDUCED CONFORMATIONAL CHANGES OF THE
ADENYLATION DOMAIN FROM TYROCIDINE SYNTHETASE 1 PROBED BY
INTRINSIC TRP FLUORESCENCE
Matilda Šprung, Viljemka Bučević-Popović, Barbara Soldo, Stjepan
Orhanović, Maja Pavela-Vrančič
P61
IGG FC N-GLYCOSYLATION PROFILING BY NANO-LC-ESI-QTOF-MS OF
GLYCOPEPTIDES
Jerko Štambuk, Maja Bučić Baković, Maurice H. J. Selman, Manfred
Wuhrer, Gordan Lauc
P62
THE EFFECT OF PROTEOLYTIC PROCESSING ON THE LOCALIZATION AND
PHYSIOLOGICAL ACTIVITY OF THE Saccharomyces cerevisiae CELL WALL
PROTEIN Scw4p
Renata Teparić, Antonija Grbavac, Sandra Kunštek, Vladimir Mrša
P63
THE INFLUENCE OF HLA-B27 IN PREDISPOSITION TO
SPONDYLOARTHROPATIES AMONG EASTERN CROATIANS
Stana Tokić, Marija Glasnović, Mario Štefanić, Ljubica Glavaš-Obrovac,
Saška Marczi
List of Posters
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
21
List of Posters
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
22
P64
PROTECTIVE EFFECT OF POLYPHENOLS FROM CHOKEBERRY JUICE AND
POWDER (ARONIA MELANOCARPA) ON OXIDATIVE STRESS AND
HYPERCHOLESTEROLEMIA IN C57BL MICE
Mandica-Tamara Tolić, Lana Nikolić, Domagoj Đikić, Ines Panjkota
Krbavčić, Nada Vahčić, Irena Landeka
P65
COMPARATIVE PROTEOME ANALYSIS OF YEAST’S ORGENELLES
TREATED WITH LEAD OR IMIDACLOPRID
Ana Vida, Iva Justinić, Gordana Čanadi Jurešić, Čedomila Milin
P66
MITE-LIKE ELEMENTS WITH INTERNAL TANDEM REPEATS IN THE
PACIFIC OYSTER Crassostrea gigas (Thunberg 1796)
Tanja Vojvoda Zeljko, Robert Bakarić, Miroslav Plohl
P67
SELENIZED ONION BISCUITS IN ATTENUATING OXIDATIVE STRESS,
INDUCED BY HIGH FAT DIET, IN THE LIVER OF OVARIECTOMISED RATS
Rosemary Vuković, Senka Blažetić, Ana Vuković, Kristina Vuković,
Martina Varga, Marta Balog, Zora Krivošíková, Marija Heffer, Elizabeta
Has-Schön
P68
REVERSIBLE DISSOCIATION OF THE YEAST V-ATPASE ANALYZED UNDER
IN VIVO CONDITIONS
Katharina Tabke, Andrea Albertmelcher, Olga Vitavska, Markus Huss,
Hans-Peter Schmitz, Helmut Wieczorek
The 50th Anniversary
of the FEBS
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
SYM-L1
FIFTY YEARS OF FEBS-ADVANCING SCIENCE ACROSS EUROPE
Israel Pecht, FEBS Secretary General
The 50th Anniversary of the FEBS
rd
On 23 of March 1964, representatives of 18 national biochemical societies
from across Europe met in London and founded the Federation of European
Biochemical Societies (FEBS). The first objective has been establishing intraEuropean meetings, later becoming the annual FEBS Congresses. It soon became
apparent that there was a need to support biochemistry and biochemists, and
encourage collaboration and exchange of information and ideas among
scientists, in particular across the boundaries in a Europe bitterly divided by the
‘Iron Curtain’. FEBS incorporated courses and summer schools and, crucially,
scientific publishing into its activities. A prestigious Fellowships programme for
research and training began in 1978. Further initiatives, such as promoting the
role of women in science, supporting education in the molecular life sciences at
both undergraduate and postgraduate level, and establishing the Young
Scientists’ Forum alongside FEBS Congresses, started around the turn of the
millennium. FEBS was a pioneer in all these, and is one of a few organizations
that have continuously focused on support of generations of young scientists.
FEBS has responded to the dramatic political changes in Europe that took place
in the last decades by integrating and supporting the scientific communities of
Central and Eastern European countries through the times of great political
upheaval, which are, to some extent, still continuing. Further, dramatic
differences still exist in the conditions of biochemical communities compared
with central or western parts of the continent and FEBS offers several different
means of support, mainly for young members in these countries.
Most noteworthy is the mode FEBS has always employed in its activities: These
are all based on devoted voluntary work of dozens of members of FEBS
Committees and Working Groups. All these members, elected democratically by
FEBS Council, work selflessly for many days every year in fulfilling their
respective FEBS responsibilities. We operate in a family-like atmosphere,
assisted by minute number of paid staff. A re-evaluation of the Federation’s
expenditure and future financial plans has already been started. The aim is to
build up investments that will maintain FEBS activities for the future, with less
dependence on the income from our journals, not least in light of the on-going
changes in the publishing market. During the past 50 years we have seen growth
in FEBS activities and impact, recognized and appreciated worldwide. It is all due
to this group of dedicated people that will be the base for future success in
promoting molecular life sciences in Europe.
25
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
The 50th Anniversary of the FEBS
SYM-L2
LET’S JOIN THE CELEBRATION OF THE 50th FEBS ANNIVERSARY!
Jerka Dumić, HDBMB Vice-president
26
The Croatian Society of Biochemistry and Molecular Biology (HDBMB) became a
regular member of the Federation of European Biochemical Societies (FEBS) in
1992; yet, Croatian biochemists joined the FEBS family already at the FEBS
Congress in Vienna in 1965, just one year after the FEBS has been established,
through the Section for Biochemistry of the Union of the Chemical (and later
Biochemical) Societies of Yugoslavia. Thanks to the vision, enthusiasm, and
dedicated work of our distinguished Professors Pavao Mildner, Elsa Reiner,
Blanka Ries, Željko Kućan, Mirna Flögel, Ljubinka, Vitale and many others, the
Croatian Biochemical Society, established in 1976, kept close contact with FEBS
to this day.
In addition to the active participation in the programs of the FEBS Congresses,
many Croatian biochemists and molecular biologists were awarded with the
FEBS fellowships, which provided them, especially young scientists, with the
opportunity to acquire knowledge and experience in laboratories all across
Europe. Thanks to these visits, numerous fruitful collaborations were
established and many of them last for many years. Through the Young Scientists
Fellowship program many PhD students and young postdocs got a chance to
participate in the FEBS Young Scientists’ Forum, thus contributing to the
networking and promotion of molecular life sciences all around Europe.
Croatian biochemists and molecular biologists also organised several FEBS
courses and summer schools on different topics (e.g., Enzymology,
Glycobiology, Plant Biochemistry, Immunology, and Bioinformatics). The first
summer school was organised in 1971 in Zadar (FEBS Summer School on
Catalytic and Regulatory Proprieties of enzymes), while several years later, in
1979, the Special FEBS Meeting on Enzymes was held in Cavtat. Members of the
Croatian Biochemical Society also expressed their profound dedication and
appreciation of the FEBS ideas through contribution to the organisation of the
18th FEBS Congress, held in Ljubljana in 1987.
The strong and wide support of FEBS to the HDBMB was evident during the visit
of the FEBS Working group for Central and Eastern Europe (today Working
group for Integration) in 2006, organisation of the Education Workshop (FEBS
Education Committee) in 2010, and especially through the support to the
FEBS3+ Meeting organised in Opatija in 2012 together with Hungarian and
Slovenian biochemical societies.
We are confident that the future collaboration between FEBS and HDBMB will
be even wider and stronger; therefore, we proudly join the celebration of the
first 50 successful years of the FEBS!
Plenary Lecture
Abstracts
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Continuous communication between the nervous and the immune system is
essential both for maintaining homeostasis and for ensuring rapid and
efficient to stressful and infection insults. The emerging level of microRNA
(miRNA) regulation provides an exciting and challenging model for studying
this communication in anxiety and inflammation. MiRNA regulators of gene
expression are yet evolving miniature genes (100-fold smaller than regular
genes) that can efficiently control neuronal signaling pathways and may have
contributed to the evolution of higher brain functions by simultaneously
dimming down the expression of multiple target genes each (1). Specifically,
miRNA controllers of acetylcholine signaling, which we designate
“CholinomiRs” modulate both anxiety and inflammation reactions to external
insults (2). We observe a physiologically relevant bidirectional competition of
CholinomiRs on the interaction with their targets (3). We found rapid
increases
of
the
evolutionarily
conserved
neuro-modulator
acetylcholinesterase (AChE)-targeted CholinomiR-132 in acute stress,
intestinal inflammation and post-ischemic stroke, inversely to its drastic
reduction in the Alzheimer’s disease brain (4). In comparison, we find single
nucleotide polymorphisms interfering with the AChE-silencing capacities of the
primate-specific CholinomiR-608 to associate with elevated trait anxiety,
inflammation and diverse aging-related diseases in human volunteers (5).
Deepened understanding of the evolution and complexity of neuronal miRNAs
may highlight their role in the emergence of human brain functions while
enhancing the ability to intervene with diseases involving cholinergic signaling
impairments.
1. Barbash, Shifman and Soreq, Mol Biol Evol. 2014 31(5):1237-47.
2. Shaltiel et al., Brain Struct Funct. 2013 Jan;218(1):59-72.
3. Nadorp and Soreq, Front Mol Neurosci. 2014 7:9
4. Lau et al., EMBO Mol Med. 2013 5(10):1613-34.
5. Hanin et al., Hum Mol Genet. 2014 23(17):4569-80.
Plenary Lecture Abstracts
PL1 FEBS National Lecture
FROM MICE TO MEN: FINE TUNING OF CHOLINERGIC SIGNALING BY
microRNAs
Hermona Soreq
The Institute of Life Sciences and the Edmond and Lily Safra Center of Brain
Science, Jerusalem, Israel
[email protected]
29
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
PL2
ELECTRON TRANSFER IN PROTEINS: INSIGHTS INTO PRINCIPLES AND
POSSIBLE APPLICATIONS
Israel Pecht
The Weizmann Institute of Science, Rehovot, Israel
Plenary Lecture Abstracts
Electron transfer (ET) reactions mediated by or via proteins are central to all
the biological energy conversion processes, from photosynthesis to
respiration. Furthermore, a rather wide and diverse range of biochemical
processes is catalyzed by redox enzymes employing electron transfer
reactions. Rates of the ET reactions between redox centers in proteins are
tightly controlled and tuned for efficiency and also for avoiding formation of
deleterious products. Our research aims at understanding the parameters that
control these ET rates in order to gain better knowledge of the underlying
principles and for possibly applying the gained insights to potential
applications. In studies of model systems such as the bacterial copper protein
azurin, we are examining correlation between the intramolecular ET rates and
specific structural changes introduced by mutations (1). In parallel, we also
study the intramolecular ET rates of reaction steps that are part of catalytic
cycles of redox enzymes in order to examine their possible evolution-driven
optimization.
Along a distinct research line electron mediating metallo-proteins like azurin
or cytochrome-C are investigated as potential components of bio-electronic
systems. This is mainly pursued by characterizing the conductance properties
of such proteins in the solid state (2).
1. O. Farver and I. Pecht. Prog. Inorg. Chem., 55, 1-78 (2007).
2. I. Ron, I. Pecht, M. Sheves and D. Cahen. Acc. Chem. Res., 43, 945-53 (2010).
30
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Reversible protein phosphorylation is a ubiquitous means of signaling in all
living cells. One of the hallmarks of cellular signaling in Eukarya are the
complex phosphorylation cascades involving hundreds of kinases and
thousands of phosphorylated protein substrates. In bacterial cells far less
kinases and phosphorylated proteins have been reported, engendering a view
that bacterial employ phosphorylation only sporadically. In the past decade,
with the advent of high-resolution mass spectrometry-based
phosphoproteomics and other complementary techniques for global analyses,
this view has slowly started to change. This talk will focus on the Grampositive model organism Bacillus subtilis and illustrate the emergent network
of protein phosphorylation which is in its complexity reminiscent of eukaryal
kinase cascades. Our quantitative phosphoproteomics studies tracked the
dynamics of occupancy of hundreds of phosphorylation sites across different
stages of bacterial growth and connected individual sites with kinases and
phosphatases responsible for their phosphorylation state. This approach has
been complemented by two-hybrid-based interactomics, which enabled us to
confirm kinase-substrate interactions and discover new kinase activators and
cross-phosphorylation events among different families of kinases. Case studies
of bacterial kinases and their substrates point to key regulatory roles in the
stationary phase, including nutritional shifts, competence development and
sporulation. Bacterial kinases are promiscuous, i.e. each kinase
phosphorylates a number of different substrates. Our results suggest that in
some cases kinase specificity towards a given substrate can be conveyed via
alternative protein activators. The promiscuity of bacterial kinases towards
substrates is an intriguing feature from the evolutionary standpoint. We argue
that bacterial protein kinases evolve faster than average bacterial genes, and
hence maintain the capacity to quickly evolve new substrate specificities
under conditions of evolutionary pressure.
Plenary Lecture Abstracts
PL3
BACTERIAL SIGNALING AND REGULATION BASED ON PROTEIN
PHOSPHORYLATION
Ivan Mijakovic
SysBio, Chalmers University of Technology, Kemivägen 10, 41296 Göteborg,
Sweden
[email protected]
31
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
PL4
ELECTRICALLY READABLE BIOCHIPS FOR RAPID RNA ANALYSIS
Mathias Sprinzl
Laboratorium für Biochemie, Universität Bayreuth, Germany
Plenary Lecture Abstracts
Transduction of biochemical events arising from nucleic acid hybridisation to
electrically readable signals is the objective of the presented research. The aim
is to develop analytical devices (biochips) for use in clinical and food
diagnostics, biotechnology, environmental analysis and biothreat detection.
In our approach, nucleic acid hybridisation directs a thermostable esterase for
binding to gold electrodes where an electrochemically detectable paminophenol is enzymatically synthesized and detected as a redox reaction –
dependent current.
All reactions are performed directly on chips-presenting electrodes in a
volume of about ten microliters, or less. Technical platforms and optimized
biochemical procedures improving the sensitivity and specificity of RNA
detection will be discussed. The presentation will focus on applications for
detection of bacteria and microRNA (1, 2,).
1). Pöhlmann C, Wang Y, Humenik M, Heidenreich B, Gareis M, Sprinzl M.
Rapid, specific and sensitive electrochemical detection of foodborne bacteria.
Biosens Bioelectron. (2009) 24, 2766-2771
2). Pöhlmann C, Sprinzl M. Electrochemical detection of microRNAs via gap
hybridization assay. Anal Chem. (2010) 82, 4434-4440.
Supported by German Federal Ministry of Education and Research
32
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
PL5
DYNAMIN OLIGOMERIZATION REGULATES ACTIN AND REPRESENTS A NOVEL
THERAPEUTIC TARGET IN CHRONIC KIDNEY DISEASES
Sanja Sever
Harvard Medical School, Nephrology Division, Massachusetts General Hospital,
Charlestown, MA 02129, USA
[email protected]
Plenary Lecture Abstracts
Dysregulation of the actin cytoskeleton in podocytes represents a common
pathway in the pathogenesis of proteinuria, the hallmark of chronic kidney
disease. We previously showed that the GTPase dynamin maintains the actin
cytoskeleton in podocytes and that dynamin downregulation underlies
proteinuria. Using in vitro and in vivo approaches and with the aid of a small
molecule called Bis-T-23 that promotes dynamin assembly into oligomers, we
demonstrate that dynamin oligomerization directly regulates the actin
cytoskeleton in podocytes. Stimulation of dynamin’s oligomerization cycle
partially restores organization of actin cytoskeleton and podocyte structure
and function in diverse genetic backgrounds. Administration of Bis-T-23 in
different animal models of kidney disease in zebrafish and mice ameliorates
proteinuria, attenuates glomerular scarring, and increases survival through an
effect on actin dynamics. This study establishes dynamin’s oligomerization
cycle as an important in vivo regulator of actin and an attractive therapeutic
target in chronic kidney disease.
33
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Plenary Lecture Abstracts
PL6
SPIRIDION BRUSINA MEDAL: Memorial Lecture
NEW FRONTIERS IN PROTEIN TARGETING AND TRANSLOCATION IN PLANTS
Enrico Schleiff
Cluster of Excellence Frankfurt & Center for Membrane Proteomics, Buchmann
Institute of Molecular Life Sciences, Department of Biosciences, Molecular Cell
Biology, Goethe University, D-60438 Frankfurt am Main, Germany.
34
Protein translocation across and into membranes is central for each cell as up
to 50% of all proteins synthesized in the cytosol need to traverse at least one
membrane to reach their place of function. Proteins destined for mitochondria
and chloroplasts are synthesized in the cytosol as precursor proteins
containing the targeting information in their amino acid sequence. The current
models suggest that these proteins are delivered to the surface of the
organelle by guiding complexes in a post-translational fashion. It is widely
discussed that the transport of precursor proteins largely depends on intrinsic
amino acid based signals as well as on cytosolic proteins like Hsp70 and Hsp90.
Recent studies have questioned this model. On the one hand, not only amino
acid based signals are required for targeting and in some cases protein
synthesis and translocation are coupled. In addition, the chaperones do not
only target proteins but participate in the quality control of precursor protein
targeting. Both, Hsp70 and Hsp90 are involved in the Chip (E3 ligase)dependent in the regulation of cytosolic precursor protein abundance. Thus,
the impact of cellular transport on the organellar protein content is revisited
during the presentation. At the surface, a membrane located multi-subunit
translocation machineries recognize the precursor proteins and facilitates
their translocation. Most if not all components of this complex have been
identified. The precursor protein recognizing translocon at the outer envelope
of chloroplasts (TOC) is a role model for the understanding of fundamental
processes during translocation. It involves chaperone recognizing receptors,
-barrel protein
required for translocation. Thus, general as well as TOC specific informations
are obtained while investigating the action of this macromolecular complex.
Based on structural investigations, single molecule experiments and
physiological studies on plant mutants we are now able to draw conclusions
about functional elements within the receptor unit of the complex. Within the
presentation, the structural and functional view on the translocation process
and the regulation of GADs will be presented.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
PL7
TWO KINDS OF ENERGY AT THE ORIGIN OF LIFE
William Martin
Institute of Molecular Evolution at Heinrich-Heine-Universität Düsseldorf,
Germany
[email protected]
Plenary Lecture Abstracts
Life is a chemical reaction. The more we learn about the metabolism of
anaerobic autotrophs that use the acetyl-CoA pathway of CO2 fixation
(acetogens and methanogens), the more evident become their similarities to
chemical reactions at hydrothermal vents. And the closer we look at chemical
reactions at hydrothermal vents, the stronger their similarities with biological
energy conversions become. Harnessing energy in the form ion gradients
across membranes is as universal as the genetic code. This is probably because
life arose in an environment like an alkaline hydrothermal vent where natural,
geochemically ion gradients existed. A model for the geochemical origin of life
is summarized that accounts for the ubiquity of chemiosmotic coupling, and
+
+
that suggests an important role for Na /H transporters in early bioenergetic
evolution. Natural proton gradients acting at alkaline hydrothermal vents
could have fostered the emergence of protocells within vent pores. Protocell
membranes that were initially leaky would eventually become less permeable,
forcing cells dependent on natural H+ gradients to pump Na+ ions. This would
accounts for the Na+/H+ promiscuity of many ancient bioenergetic proteins,
especially ATPases, as well as the deep divergence between bacteria and
archaea. However, carbon chemistry did not start with the advent of
chemisomotic coupling. Reactive carbon compounds and substrate level
phosphorylation probably played an important role at the onset of
biochemical evolution. Chemiosmotic harnessing and substrate level
phosphorylations are the only two ways that harnesses energy today. This
suggests that in the search for environments where life arose, we should pay
particular attention to those where natural geochemical processes could
support both kinds of energy.
35
Lecture Abstracts
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
L1
THE ROLE OF AUTOPHAGY RECEPTORS IN SELECTIVE REMOVAL OF
MITOCHONDRIA
1
1
2
3
Mija Marinković , Janoš Terzić , Anne Hamacher-Brady , Nathan Brady , Ivan
1,4
1
Đikić and Ivana Novak Nakir
1
University of Split, School of Medicine, Split, Croatia; 2Division of Theoretical
Bioinformatics, 3Systems Biology of Cell Death Mechanisms, German Cancer
Research Center (DKFZ) and Bioquant, University of Heidelberg, Heidelberg,
Germany; 4Institute of Biochemistry II, Goethe University Medical School,
Frankfurt am Main, Germany
[email protected]
Lecture Abstracts
Recent discoveries of autophagic receptors that recognize specific cellular
cargo have opened a new chapter in autophagy field. Selective removal of
damaged organelles or protein aggregates is essential for the proper cellular
homeostasis and survival. We have recently identified and characterized
Nix/Bnip3L mitochondrial protein as mitophagy receptor that, in LIRdependent manner, interacts with LC3/GABARAP proteins and recruits
autophagy machinery to damaged mitochondria. Moreover, we have shown
that Nix mediates mitochondrial clearance during reticulocyte differentiation.
Additionally, Nix forms strong homodimers and we propose that Nix
dimerization is required for the recruitment of LC3/GABARAP to damaged
mitochondria and is a trigger for mitophagy. Recent discovery of Optn, an
autophagy receptor involved in recognition of intracellular bacteria, showed
that its autophagic function is regulated by phosphorylation of the LIR motif.
We are investigating the phosphorylation status of Nix and its homolog Bnip3
in regulation of mitophagy. Our preliminary results indicate that
phosphorylation of mitophagy receptors Nix and Bnip3 is involved in fine
tuning of mitophagy.
39
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
L2
TKs SCAFFOLD PROTEINS IN TYROSINE KINASE SIGNALLING
László Buday
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian
Academy of Sciences, Budapest, Hungary
[email protected]
Lecture Abstracts
Signalling pathways utilising tyrosine kinases play crucial roles in the
regulation of cell proliferation and movement. Impairment of these pathways
may lead to important public health diseases such as malignant cancer or
diabetes mellitus. Our laboratory focuses on scaffold proteins which are
implicated in tyrosine kinase signalling. We have showed that the scaffold
protein Tks4 plays an important role in the EGF signalling as, in response to
EGF, Tks4 is tyrosine phosphorylated and associated with the activated EGF
receptor. This association is not direct but requires the presence of Src
tyrosine kinase. Treatment of cells with LY294002, an inhibitor of PI 3-kinase,
or mutations of the PX domain reduces tyrosine phosphorylation and
membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W)
Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient
shows aberrant intracellular expression and reduced phosphoinositide
binding. Silencing of Tks4 is shown to markedly inhibit HeLa cell migration in a
Boyden chamber assay in response to EGF or serum. In addition, we have
generated a Tks4 knock-out mouse which shows very dramatic morphological
changes compared to wild type mice. Our preliminary experiments suggest
that Tks4 may have a crucial role in the differentiation processes as
mesenchymal stem cells isolated from the KO mice have significantly reduced
potential to differentiate to adipose and bone tissues than that of stem cells
isolated from wild type mice.
40
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Syalylated glycoconjugates are more abundant in brain than in any other
mammalian tissue. In comparison with liver, protein-bound expression of sialic
acid is equivalent in both tissues whereas lipid-bound expression rises 15
times in brain, particularly in favour of ganglioside structures. Despite a huge
variety of up-to-now identified gangliosides, merely four structures, GM1,
GD1a, GD1b and GT1c, comprise 97 % of all human brain gangliosides. It was
proposed that a complete lack of these structures, like in B4Galt1-null mice,
would cause serious consequences, but initial reports found no abnormalities.
Further reports on one-year-old mice found significant axonal degeneration
and demyelination due to impaired myelin-axon cell-to-cell communication,
which was not compensated with a comparable synthesis of simple
gangliosides GM3 and GD3. Nonetheless, the interest in finding a specific
potential role for each of the four major brain glycolipids decreased.
Sialyltransferases are specific for glycolipid synthesis or act on both glycolipids
and glycoproteins during stepwise synthesis of the glycan chain in Golgi
apparatus. Enzymes responsible for syalilation of internal galactose on
tetrasaccharide ganglio-core are well described. In both cases knocking out of
the responsible gene (St3gal5 or St8sia1) is accompanied with the
compensatory synthesis of 0-series or a-series gangliosides, respectively. We
studied mice defective in terminal syalilation of ganglio-core; St3gal1, St3gal2,
St3gal3, St3gal4 and St3gal2/3 double-null mice. Disruption of either of these
genes did not change the total brain ganglioside quantity, but caused changes
in their relative ratio. While St3gal2-null mice express a half of the normal
amount of GD1a and GT1b, St3gal2/3 is 95% depleted of the same structures.
Despite the deficient synthesis of gangliosides St3gal2-null mice are robust,
whereas St3gal3 are somewhat smaller and St3gal2/3 are significantly smaller
and neurologically impaired in comparison with wild types. Further analysis
revealed a shift in the ratio of subgroups of interneurons that are critical for
cortical plasticity and have been implicated in the aetiology of complex
neuropsychiatric diseases like schizophrenia, autism, anxiety disorder and
seizures.
Lecture Abstracts
L3
WHAT IS WRONG WITH SIALYLTRANSFERASES KNOCK-OUTS?
Marija Heffer1 and Senka Blažetić2
1
2
University of Osijek, Faculty of Medicine, Department of Biology, Osijek,
Croatia
[email protected]
41
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Lecture Abstracts
L4
THE ROLE OF LYSOSOMAL PATHWAY IN NEURODEGENERATIVE DISEASES
Silva Hećimović
Division of Molecular Medicine, Ruder Boskovic Institute, Zagreb, Croatia
[email protected]
42
Lysosomes are specific cellular organelles that are normally involved in
degradation processes of proteins and other damaged organelles. Dysfunction
of lysosomes may lead to abnormal protein accumulation which may cause
cell degeneration and death. Such protein accumulation also occurs in the
brain cells and triggers development of the devastating neurodegenerative
diseases in humans, such as Alzheimer’s disease (AD) and Parkinson’s disease
(PD). Although AD and PD are the most common neurodegenerative diseases,
there are still no adequate therapies that would prevent or cure AD and PD. It
is intriguing that several human rare inherited disorders caused by dysfunction
of the lysosomal proteins, such as Niemann-Pick type C disease (NPC) and
Gauchers disease (GD), show neurodegeneration and accumulation of proteins
characteristic for AD and PD, respectively. Although these findings provide a
further evidence for the link between lysosomal dysfunction, protein
accumulation and neurodegenerative processes, molecular details of this
relationship are largely unknown. Using a lysosomal disorder NPC as a model,
our goal is to investigate a role of lysosomal impairment on accumulation of
amyloid-β peptide (Aβ) and α-synuclein (α-syn), the two characteristic
features in the pathogenesis of AD and PD, respectively. In our work we have
used CHO-NPC cells, primary mouse cerebral neurons and NPC mouse brains.
We have previously shown that an increase of Aβ in NPC cells is at least partly
due to sequestration of APP and BACE1, the two key proteins in the
pathogenesis of AD, in early/recycling endosomes leading to enhanced
processing of APP by β-secretase and Aβ formation. Our results of
immunostaining of several markers of the endolysosomal pathway revealed
enlarged endosomes and lysosomes in NPC cells, suggesting an impairment of
this pathway in NPC disease. Moreover, we have isolated lysosomes using
superparamagnetic nanoparticles and further characterized them.
Pharmacological treatments to stimulate/inhibit autophagy-lysosomal
pathway demonstrated a partial lysosomal dysfunction in NPC disease models.
We will further test whether enhancement of the lysosomal function can
rescue accumulation of Aβ/α-syn in NPC cells. Our results may provide
evidence that lysosomal dysfunction is a common mechanism that leads to
protein accumulation and neurodegeneration.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
L5
THE miRNA WORLD OF HERPES SIMPLEX VIRUS
Igor Jurak
Department of Biotechnology, University of Rijeka, R. Matejcic 2, 51000 Rijeka,
Croatia
[email protected]
Lecture Abstracts
Herpes simplex virus 1 (HSV-1) is important human pathogen widely known as
the causative agent of cold sores. Although infections with HSV-1 are usually
self-limiting, they can result in severe morbidity and life-threatening diseases.
HSV-1, similar to other herpesviruses, encodes numerous miRNAs, some of
which are conserved in closely related virus HSV-2, and are differentially
expressed in different phases of the virus replication cycle. miRNAs, small noncoding RNAs with a central role in posttranscriptional repression of gene
expression, have considerable potential for regulation of many biological
processes including virus replication. We have recently shown that HSV-1
miRNAs can target important viral genes to restrain their expression and to
prevent lethal disease, so the host survives with a latent infection. On the
other hand, some HSV miRNAs have been found to target host genes involved
in intrinsic antiviral defense to allow efficient virus replication. Moreover,
expression of host miRNAs dramatically changes during HSV-1 infection, which
can have a dramatic outcome for the virus replication and establishment of
latency. Thus, although investigating roles of miRNA in virus infection has been
very challenging there are several breakthroughs that indicate their roles and
importance in infection.
43
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
L6
PHYTOHORMONE AUXIN: A MEDIATOR OF PLANT STRESS RESPONSE
Branka Salopek-Sondi
Department for Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54,
10 000 Zagreb, Croatia
[email protected]
Lecture Abstracts
In the last decades the effect of environmental changes on agricultural
production challenges many scientists in the field of plant biology, agronomy,
ecology, and biotechnology to investigate and understand mechanisms
underlying plant stress responses and tolerance. Phytohormones are signalling
molecules crucial for the ability of plants to adapt to unfavourable
environmental changes by mediating wide range adaptive responses, such as
growth, development, nutrient allocation, and source/sink transitions. In
recent years emerging evidence suggests that phytohormone auxin (indole-3acetic acid, IAA) acts as a common player in the majority of hormonal
interactions in stress conditions, mostly interacting with the stress hormones:
salicylic acid (SA), abscisic acid (ABA), and jasmonic acid (JA).
Auxin levels and the regulation of auxin homeostasis as a mechanism of plant
stress response have been investigated in cabbage (Brassica rapa L. ssp.
pekinensis) upon drought and increased salinity. It was shown that stress
conditions caused perturbation of IAA levels with a tendency of increasing the
IAA conjugate level in comparison to free IAA. Correlations of IAA and stress
hormones were discussed. Gene expression experiments showed changes in
transcript level of enzymes involved in auxin homeostasis upon stress
conditions. Specifically, auxin amidohydrolases, enzymes involved in auxin
homeostasis have been investigated at biochemical and structural level. The
results implicated an important role of auxin and interactions with other stress
hormones in the plant stress response.
44
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Natural porphyrin complexes are essential for human life. However, besides
interesting optical, electronical and co-ordinating properties, and their
importance as phototherapeutic agents, finding successful application in
oncology, cardiology and dermatology, synthetic porphyrins and their
complexes have still very limited utility in drug discovery. A series of
porphyrins, tetrapyrrole natural organic compounds, are evaluated here as
endogenous anti-inflammatory agents. They directly inhibit the activity of Fyn,
a non-receptor Src-family tyrosine kinase, triggering anti-inflammatory events
associated with down-regulation of T-cell receptor signal transduction, leading
to inhibition of tumor necrosis factor alpha (TNF-α) production.
TNF-α is one of the major pro-inflammatory cytokines, associated with
diseases such as diabetes, tumorigenesis, rheumatoid arthritis, and
inflammatory bowel disease. To date, only protein-based TNF-α inhibitors
(anti-TNF-α antibodies) are available on the market. There is, therefore, a
need for small anti-TNF-α molecule as alternative to expensive and
complicated protein agents. Ideally, a small molecule TNF-α inhibitor should
possess immunomodulatory properties (decrease of the devastating effects of
inflammatory responses), but at the same time should not disturb cellprotecting mechanisms. Orally available small molecules for the treatment of
TNF-α associated diseases may thus be considered as answers to an “unmet
medical need”.
Porphyrins, as a chemical class, inhibited Fyn kinase activity in a noncompetitive, linear-mixed fashion. In cell-based in vitro experiments on
polymorphonuclear cells, porphyrins inhibited TNF-α cytokine production, Tcell proliferation, and the generation of free radicals in the oxidative burst, in a
concentration-related manner. In vivo, lipopolysaccharide-induced TNF-α
production in mice was inhibited by several of the porphyrins. These findings
may be very important for the overall understanding of the role(s) of
porphyrins in inflammation, by targeting protein-protein interactions, and
their possible application as new anti-inflammatory agents of endogenous
origin.
Lecture Abstracts
L7
PORPHYRINS AS NEW ENDOGENOUS MACROCYCLIC ANTI-INFLAMMATORY
AGENTS FOR TARGETING PROTEIN-PROTEIN INTERACTIONS
Dubravko Jelić
Fidelta Ltd., HR-10001 Zagreb, Croatia
[email protected]
45
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
L8
NOVEL (BIO)MOLECULES FOR NEW ORGANOPHOSPHATE INTOXICATION
TREATMENTS
1
2
3
3
Zoran Radić , Rakesh K. Sit , Suzana Žunec , Nikolina Maček Hrvat , Zrinka
3
2
1
Kovarik , Valery V. Fokin and Palmer Taylor
1
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of
California at San Diego, La Jolla, CA 92093, USA 2Skaggs Institute for Chemical
3
Biology, The Scripps Research Institute, La Jolla, CA 92037, USA Institute for
Medical Research and Occupational Health, HR-10001 Zagreb, Croatia
[email protected]
Lecture Abstracts
Persistent occurrence of potentially fatal human intoxications by pesticide and
nerve agent organophosphates (OPs) is at present insufficiently counteracted
by combined intramuscularly (i.m.) or intravenously (i.v.) administered
therapy
of
atropine
with
nucleophilic
pyridinium
aldoxime
acetylcholinesterase (AChE) reactivators. Therapeutic efficacy of pralidoxime
(2PAM), obidoxime, and HI-6, the most commonly used reactivator aldoximes,
is low in cases of massive OP pesticide or nerve agent exposure due to
constant AChE reinhibition by the excess circulating OP. Those oximes do not
penetrate Blood Brain Barrier, because of their charge, and can not reactivate
OP-inhibited brain AChE. Finally, their bioavailability is generally low, and
clearance is rapid, restricting useful administration modes to i.m. or i.v,
inconvenient for rapid treatment of larger number of exposed individuals and
populations.
Owing to discovery and structure-activity refinements of several novel
biologically active molecules, we were able, in recent years, to develop several
alternative and complementary therapeutic approaches to treatment of OP
intoxication. Our uncharged acetamide oxime RS194B shows low intrinsic
toxicity, BBB penetration, and high bioavailability and efficacy upon oral
administration. Our imidazole aldoxime RS2-33A efficiently reactivates
intrinsic, tissue butyrylcholinesterase inhibited by OPs promoting in situ
hydrolytic breakdown and removal of offending OPs in the exposed tissue.
Finally, combined administration of our aging resistant AChE mutant with
suitable novel oxime reactivator provides a useful means of hydrolytic clearing
of fast aging nerve agent Soman from the exposed circulation.
46
This research was supported by the CounterACT Program, National Institutes
of Health Office of the Director (NIH OD), and the National Institute of
Neurological Disorders and Stroke (NINDS), Grant Numbers U01 NS058046,
R21NS072086 and R21NS084904.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
L9
POLARITY IN CELL MIGRATION GOVERNED BY RAC1 GTPASES
Igor Weber1, Vedrana Filić1, Maja Marinović1, Jan Faix2
1
Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, HR10000 Zagreb, Croatia
2
Institute for Biophysical Chemistry, Hannover Medical School, Carl-NeubergStr. 1, D-30623 Hannover, Germany
[email protected]
Filić, Vedrana; Marinović, Maja; Faix, Jan; Weber, Igor (2012). A dual role for
Rac1 GTPases in the regulation of cell motility. Journal of Cell Science 125,
387-398.
Filić, Vedrana; Marinović, Maja; Faix, Jan; Weber, Igor (2014). The IQGAPrelated protein DGAP1 mediates signaling to the actin cytoskeleton as an
effector and a sequestrator of Rac1 GTPases. Cellular and Molecular Life
Sciences 71, 2775-2785.
Weber, Igor; Faix, Jan (2013). A dual role model for active Rac1 in cell
migration. Small GTPases 4, 110-115.
Lecture Abstracts
We have recently unraveled a mechanism wherein spatiotemporal dynamics
of Rac1 activity during migration of Dictyostelium cells is apparently regulated
by antagonizing interactions of Rac1-GTP with two distinct effectors (Filić et
al., 2012). By monitoring specific fluorescent probes, we found that activated
Rac1 is simultaneously present at the leading edge, where it participates in the
Scar/WAVE-mediated actin polymerization, and at the trailing edge, where it
induces formation of the DGAP1/cortexillin actin-bundling complex. In
addition to their opposed localizations, the two populations of activated Rac1
also display opposite kinetics of recruitment to the plasma membrane upon
stimulation by chemoattractants. Competition between DGAP1/cortexillin and
SCAR/Wave for the common activator, Rac1-GTP, might provide the basis for
the oscillatory re-polarization typically seen in randomly migrating
Dictyostelium cells (Filić et al., 2014). I will discuss the consequences of the
dual role exerted by Rac1 in the regulation of polarity in cell migration, and
propose that similar signaling mechanisms may be of general importance in
regulating spatiotemporal dynamics of the actin cytoskeleton by small
GTPases (Weber and Faix, 2013).
47
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Lecture Abstracts
L10
THE MANY FACES OF MATH-BTB PROTEINS IN PLANTS
Dunja Leljak-Levanić, Nataša Bauer, Martina Juranić
Department of Molecular Biology, Faculty of Science, University Zagreb,
Horvatovac 102a, Zagreb
[email protected]
48
The MATH-BTB protein family is common to both animals and plants. It is a
phenomenon that Arabidopsis and human genomes encode only few
members (six and two, respectively) of the MATH-BTB protein family but in
some plant and animal organisms, the same protein family has expanded
more than 10-fold. The first functionally characterized MATH-BTB protein is
Mel26 from Caenorhabditis elegans which is a key regulator required for the
first asymmetric zygote division. Three different plant MATH-BTB genes, from
Zea mays, Triticum aestivum and Arabidopsis thaliana were selected due to
their exclusive/strong expression during plant reproduction and functionally
characterized in our work. A gametophyte/zygote specific ZmMAB1 gene from
maize is expressed in both the female and male germline and its silencing
leads to defects in spindle organization and chromosome segregation during
gametophyte development. The zygotic induced gene TaMAB2 encodes a
protein that asymmetrically co-localises with microtubuli around the nuclear
envelope, what suggests its role in organizing the assembly and proper
position of microtubular spindles during asymmetric cell divisions of zygote.
Moreover, zygote deposited TaMAB2 is always inherited to the large basal cell
after first asymmetric zygotic division. The asymmetrically inheritance indicate
that the protein might be involved not only into establishment of asymmetry
but also into the cell specification in two-celled embryo.
ZmMAB1 and TaMAB2 as well as other yeast, animal, and plant BTB-domain
containing proteins interact with Cullin 3-based E3 ligases and are involved in
ubiquitin -dependent degradation pathway. In addition to Cullin 3-related
functions, Arabidopsis MATH-BTB protein AtBPM1, localizes predominantly in
nucleolus of plant cells indicating a Cullin3 independent function. We
identified, by mass spectrometry and other protein interaction analysis that
AtBPM1 interacts with proteins involved in RNA-directed DNA methylation
which represent a novel, yet undiscovered function of MATH-BTB protein in
regulation of DNA methylation.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
L11
THE ROLE OF CELL-CELL INTERACTIONS IN TISSUE MICROENVIRONMENT IN
SIGNALLING STUDIES OF CARCINOGENESIS AND DRUG RESPONSE
Judit E. Pongracz
Department of Pharmaceutical Biotechnology, Medical School, University of
Pecs, Pecs, Hungary
[email protected]
Lecture Abstracts
In recent years, comprehensive sequencing studies have revealed about 140
genes that when altered by mutations can promote tumorigenesis. The cancer
promoting or so called driver genes can be classified into 12 signaling
pathways that regulate cell fate, cell survival, and genome maintenance.
Despite intensive research and novel insights into signal regulation, a better
understanding of these pathways has remained one of the most pressing
needs in basic cancer research. Especially so, as clinical targeting of a specific
signalling pathway could offer the much needed curative approach in tumor
therapy.
To take cellular interactions into consideration during signalling studies, only
three dimensional (3D) cell culture based bioassays enable understanding of
inter and intracellular signalling pathways, identification of drug targets and
testing of drugs efficacy. The 3D microtissues are more predictive than
conventional cell-based models.
Our studies focus on cellular interactions and resulting inter-modifications of
signalling pathways of Wnt, Notch, TGFbeta and FGF that allows deeper
understanding of cancer initiation and treatment.
49
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Lecture Abstracts
L12
XENOESTROGENS AS CARCINOGENS
Aleksandra Fučić
Institute for Medical Research and Occupational Health, Ksaverska c 2, Zagreb,
Croatia
[email protected]
50
Estrogen is an endocrine, paracrine, and neuromodulating molecule that,
acting via several receptors, orchestrates growth, development, and
homeostasis in mammals. During the last decade, there has been growing
evidence on the significant impact of estrogen and estrogen receptor level
disturbances in the pathology and etiology of different types of cancers.
Consequently, there has also been a dramatic change in the therapeutic
approaches to cancer, which are becoming gender-tailored, as well as in
investigating cancer etiology. Synthetic xenoestrogens and phytoestrogens
usually have a lower coupling ability for estrogen receptors than animal
estrogens, but due to their strong biological effects at very low doses, there is
great concern about their effects in the process of carcinogenesis caused by
environmental factors. Environmentally caused cancerogenesis is the result of
a complex interplay between many factors, including genetics, lifestyle, diet,
endogenous hormone status, gender, age, and environmental factors. Levels
of susceptibility to xenoestrogens seem to be increased during prepubertal
and pubertal periods and change in postmenopausal women and men.
Xenoestrogens act on genome and non-genome levels. Both mechanisms are
especially critical if they occur during development and sexual maturation. On
genome level, xenoestrogens change methylation levels and can cause DNA
damage due to an increased release of free oxygen radicals. The
overstimulation of hormone receptors caused by xenoestrogens may change
production of estrogen levels and estrogen receptor levels. Constant increases
in testicular cancer incidence, shift of breast cancer towards younger women,
and sterility all reflect the impact of xenoestrogens present in food and air.
Understanding complex interactions between xenoestrogens and other
physical and chemical environmental agents demands new long-term
multiparameter approaches to investigation designs that have to involve
multidisciplinary teams. Results of such studies will have a critical role in the
preparation of future environmental health legislation as well as in oncology,
as they may suggest new diagnostic parameters and have an impact on
therapy. Thus, in the near future, xenoestrogens will no longer be defined as
endocrine disruptors but rather as endocrine carcinogens.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
L13
INVASIVE TUMORS HAVE DEEP ROOTS IN ANIMAL PHYLOGENY
Tomislav Domazet-Lošo1,2, Alexander Klimovich3 and Thomas C. G. Bosch3
1
Laboratory of Evolutionary Genetics, Division of Molecular Biology, Ruder
2
Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia; Catholic
3
University of Croatia, Ilica 242, HR-10000 Zagreb, Croatia Zoological Institute,
Christian-Albrechts-University Kiel, Am Botanischen Garten 1-9, D-24118 Kiel,
Germany
[email protected]
Lecture Abstracts
The molecular nature of malignant tumors is well studied in vertebrates, while
their evolutionary origin remains unknown. In particular, there is no evidence
for naturally occurring malignant tumors in pre-bilaterian animals, such as
sponges and cnidarians. This is somewhat surprising given that recent
computational studies have predicted that all metazoans are prone to develop
tumors. Here we provide first evidence for naturally occurring tumors in two
species of Hydra. Histological, cellular and molecular data reveal that these
tumors are transplantable and caused by differentiation arrest of female
gametes. Growth of tumor cells is independent from the cellular environment.
Tumor bearing polyps have significantly reduced fitness. In addition, Hydra
tumors show a greatly altered transcriptome that mimics expression shifts in
vertebrate cancers. Therefore, this study shows, that invasive tumors have
deep roots in animal phylogeny, and that early branching animals may be
informative in revealing the fundamental mechanisms of tumorigenesis.
51
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
L14
A YEAST'S TRICK FOR STAYING YOUNG
Miguel Coelho1 and Iva Tolić1,2
1
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr.
108, 01307 Dresden, Germany
2
Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, 10000
Zagreb, Croatia
[email protected], [email protected]
Lecture Abstracts
During their life, cells accumulate damage, which is inherited by the daughter
cells when the mother cell divides. The amount of inherited damage
determines how long the daughter cell will live and how fast it will age. Yet,
the mechanisms underlying damage segregation at cell division have remained
largely elusive. By combining live-cell imaging and genetics with a
mathematical model, we show that fission yeast cells divide aggregated
proteins, a form of damage, equally between the two daughter cells, but only
as long as the amount of damage is low and harmless. This equal partitioning
of damage makes fission yeast cells immune to aging. However, when the cells
are stressed and the damage accumulates to higher levels, the aggregated
proteins fuse into a single clump, which is then inherited by one daughter cell,
while the other cell is born clean. The cell that inherits a large amount of
aggregated proteins undergoes aging and eventually dies, while its sister cell is
rejuvenated. Our work shows that in response to increased damage,
symmetrically dividing cells increase the asymmetry of damage segregation,
thereby promoting the formation of damage-free cells. We have discovered
fusion of protein aggregates as a new strategy that cells use to asymmetrically
segregate damage throughout divisions. This form of damage control may be a
universal survival strategy for various cell types, including stem cells, germ
cells and cancer cells.
52
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
L15
SLC45, A NOVEL FAMILY OF MAMMALIAN SUGAR TRANSPORTERS
Rabea Bartölke1, Jürgen Heinisch2, Helmut Wieczorek1 and Olga Vitavska1
1
2
Division of Animal Physiology and Division of Genetics, University of
Osnabrück, Germany
[email protected]
Lecture Abstracts
Mammalian sugar transporters are dominated by the facilitative glucose
transporter family SLC2 and by the sodium dependent glucose transporter
family SLC5, all of which transport only monosaccharides. Here we report the
first mammalian sugar transporter family, namely the solute carrier family 45
(SLC45), that transports the disaccharide sucrose.
The members of SLC45 have been implicated with the regulation of glucose
homeostasis in the brain (SLC45A1), with skin and hair pigmentation
(SLC45A2), and with prostate cancer and myelination (SLC45A3). However,
apart from A1, a proton-associated glucose transporter, the function of these
proteins is still largely unknown although sequence similarities to plant
sucrose transporters mark them as a putative sucrose transporter family.
Heterologous expression of the three members SLC45A2, SLC45A3 and
SLC45A4 in Saccharomyces cerevisiae confirmed that they are indeed sucrose
transporters. 14C-sucrose uptake measurements revealed intermediate
transport affinities with Km values around 5 mM. Transport activities were best
under slightly acidic conditions and were inhibited by the protonophore CCCP,
demonstrating an H+-coupled transport mechanism. Na+, on the other hand,
had no effect on sucrose transport. Competitive inhibition assays indicated a
possible transport also of glucose and fructose. Real time PCR of mouse
tissues confirmed mRNA expression of SLC45A2 in eyes and skin and of
SLC45A3 primarily in the prostate, but also in other tissues, whereas SLC45A4
showed predominantly a ubiquitous expression. Altogether the results provide
new insights into the physiological significance of SLC45 family members and
challenge existing concepts of mammalian sugar transport, as they i) transport
a disaccharide and ii) perform secondary active transport in a proton
dependent manner.
53
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Lecture Abstracts
L16
NK CELLS LINK OBESITY-INDUCED ADIPOSE STRESS TO INFLAMMATION AND
INSULIN RESISTANCE
1
1
2
Felix M. Wensveen , Vedrana Jelenčić , Tamara Turk Wensveen , Sonja
1
1
3
4
Valentić , Marko Šestan , Sebastian Theurich , Davor Mendrila , Davor
Štimac2, F. Thomas Wunderlich3, Jens C. Brüning3, Ofer Mandelboim5, Bojan
Polić1
1
Department of Histology and Embryology, Faculty of Medicine, University of
Rijeka, Rijeka, Croatia; 2Department of Internal Medicine, University Hospital
3
Rijeka, Rijeka, Croatia; Institute for Genetics / Max Planck Institute for
Neurological Research Cologne, Cologne, Germany; 4Department of Surgery,
5
University Hospital Rijeka, Rijeka, Croatia; The Lautenberg Center for General
and Tumor Immunology, The Hebrew University Hadassah Medical School,
Jerusalem, Israel
[email protected]
54
Obesity is an increasingly common health issue that predisposes people to
metabolic disorders such as insulin resistance (IR), which can progress to
diabetes mellitus type 2 (DM2). An important underlying cause of obesityinduced IR is chronic systemic inflammation derived from accumulating proinflammatory macrophages in visceral adipose tissue (VAT). Currently, it is
unknown which signal initiates adipose tissue macrophage (ATM) activation in
VAT. We find that a unique subset of VAT-resident NK cells provides a crucial
link between obesity-induced adipose stress and ATM activation in VAT.
Ligands for the NK- activating receptor NKp46 are expressed in human and
mouse VAT. Feeding with high-fat diet causes up regulation of NKp46-ligands,
leading to localized activation and cellular increase of NK cells. IFNγ produced
by these cells drives early pro-inflammatory macrophage differentiation and
promotes obesity-induced insulin resistance. Lack of NK cells, NKp46 or IFNγ
prevents macrophage activation in VAT and greatly ameliorates glucose
tolerance and insulin sensitivity. Therapeutic blocking of NKp46-signaling
forestalls ATM activation. Our study identifies NK cells as key regulators of
macrophage polarization and insulin resistance in response to obesity induced
adipose stress. The NK-ATM axis therefore provides an attractive new target
for early treatment of patients with metabolic syndrome to prevent
progression to DM2.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
ABC multidrug transporters, including ABCB1 (MDR1, Pgp), ABCC1 (MRP1), and
ABCG2 (BCRP, MXR) play a key role in defending human cells and tissues
against harmful xenobiotics and endobiotics. Human progenitor and stem cells
preferentially express the ABCG2 multidrug transporter, capable of extruding
both hydrophobic and partially hydrophilic toxic compounds. The side
population (SP) phenotype, first recognized in hematopoietic stem cells, is
based on the ABCG2-dependent export of the Hoechst compound 33342, thus
resulting in a less efficiently stained sub-population of the cells after
incubation with the Hoechst dye. The SP population related to the abundance
of the ABCG2 protein in the cell membrane has been widely observed both in
normal and cancer stem cells. In our recent studies we have analysed the
expression and function of the ABCG2 transporter in pluripotent human
embryonic and induced pluripotent stem cells, and found an important
protective effect of this protein against drugs and stress conditions. We also
observed a down-regulation of ABCG2 expression in early stages of stem cell
differentiation, while selected differentiated tissues were found to express
again relatively high levels of ABCG2. Since the expression of the ABCG2
multidrug transporter may partially explain the drug resistance observed in
the potential cancer stem cells, we have generated various human cancer cell
lines overexpressing ABCG2. We found that these cells may show non-specific
resistance against specifically targeted signal transduction inhibitors, applied
in recently developing targeted cancer therapies. This resistance is based on
the wide-range drug extrusion capability of the ABCG2 protein. Targeted
anticancer molecules may be both substrates and inhibitors of the ABCG2
multidrug transporter, in the latter case also affecting the cellular response to
unrelated chemotherapeutic agents. Therefore, we suggest that a prescreening of targeted anticancer agents for their ABCG2 interactions may
significantly improve the clinical applicability of these new compounds.
Supported by OTKA 83533 and KTIA_AIK_12-1-2012-0025, Hungary.
Lecture Abstracts
L17
ABC TRANSPORTERS IN NORMAL AND CANCER STEM CELLS
Balázs Sarkadi1,2, Zsuzsa Erdei1, Csilla Hegedüs2, Ágota Apáti1
1
Research Centre for Natural Sciences, Hungarian Academy of Sciences, and
2
Department of Biophysics and Radiation Biology, Semmelweis University,
Budapest, Hungary
[email protected]
55
Short Presentation
Abstracts
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
SP1
PIWIL1 REGULATES NEOCORTICAL STEM CELL CYCLE, MIGRATION AND
DENDRITOGENESIS
1
1
1
Barbara Viljetic , Marina Dutra-Clarke , Althea Stillman , Mathew L.
1
1
1
2
Kraushar , Hema M. Arikala , Sagara H.R. Wijeratne , Kevin Chen , Mladen1
Roko Rasin
1
Department of Neuroscience and Cell Biology, Rutgers University, Robert
2
Wood Johnson Medical School, Piscataway, NJ 08854, USA; Department of
Genetics and BioMaPS Institute for Quantitative Biology, Rutgers University,
School of Art and Sciences, Piscataway, NJ 08854, USA
[email protected]
Short Presentation Abstracts
Distinct projection neurons are born sequentially from radial glia to migrate
in an "inside-out" fashion and ultimately form the six-layered neocortex.
Disrupted neocortical cell cycle, migration and dendritogenesis were
associated with a range of neurological and psychiatric disorders. Therefore,
a better understanding of the molecular mechanisms determining
neocortical cell cycle, migration and dendritogenesis is paramount. Here, we
show that PIWI-like 1 (PIWIL1; also known as Miwi) protein of the argonaute
protein family is required for proper neocorticogenesis in mice. In particular,
we found that Piwil1 depletion resulted in increased number of mitotic
figures at embryonic day 17 (E17), increased number of PAX6+ radial glia at
postnatal day 0 (P0) and disrupted placement of later born SATB2+ neurons
within deep layers at E17 and P7. In addition, in Piwil1 knock-outs, we found
disrupted neocortical circuitry represented by thinning of corpus callosum
and altered dendritogenesis. Finally, microarray analysis coupled to
bioinformatics revealed subset of genes associated with cell cycle, cell
adhesion, transcription and migration to be regulated by PIWIL1 in
developing neocortices. Thus, we found a novel role of Piwil1 in
neocorticogenesis and determined potential targets through which PIWIL1
acts.
59
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Short Presentation Abstracts
SP2
INCREASED BONE STRUCTURE IN ANIMALS WITH PERINATALLY ALTERED
SEROTONIN METABOLISM
1
2
1
2
Igor Erjavec , Sofia Blazevic , Slobodan Vukicevic , Dubravka Hranilovic
1
Laboratory for Mineralized Tissues, Center for Translational and Clinical
Research, University of Zagreb School of Medicine, Zagreb, Croatia;
2
Department of Animal Physiology, Division of Biology, University of Zagreb
Faculty of Science, Zagreb, Croatia
[email protected]
60
In mammals, serotonin (5HT) is present both in the brain and peripheral
tissues. In the brain, serotonin acts as a key regulator of serotonergic
outgrowth and synaptogenesis during development and later it assumes the
function of a neurotransmitter, while peripheral serotonin is involved in the
regulation of gastrointestinal and cardiovascular functions and platelet
aggregation. Additionally, serotonin is considered to be a negative regulator
of bone remodeling in which osteoblasts and osteoclasts maintain a fine
balance between bone formation and resorption. There is a growing
evidence for the involvement of central and peripheral 5HT in the regulation
of bone tissue, but the interplay between the two compartments in
maintaining bone mass still remains to be elucidated. In order to study the
effects of the two compartments, we examined bone structure in rats
perinatally treated with tranylcypromine (TCP), an irreversible inhibitor of
monoamine oxidase. Rats were treated with 2mg/kg TCP or saline from
gestational day 12 until postnatal day 21. In adult animals, 5HT concentration
was significantly increased in the peripheral compartment and significantly
decreased in the central compartment, in comparison to the saline treated
rats. Femurs were collected on postnatal day 70 and cortical and trabecular
bone parameters were examined by SkyScan 1076 micro CT device. In
comparison to the saline treated rats, TCP-treated rats displayed significantly
increased trabecular bone volume, trabecular number and connectivity
density, along with decreased cortical volume and thickness. Smaller, more
compact bones with increased trabecular structure in animals with
decreased brain 5HT concentrations may suggest a more prominent role of
the central 5HT compartment in bone maintenance.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Agrin is a heparan-sulphate proteoglycan that plays a major role in the
formation and maintenance of the neuromuscular junction. Accelerated
agrin degradation has recently been linked to degeneration of the
neuromuscular junction and development of sarcopenia. Ageing-related
sarcopenia is characterized by progressive loss of skeletal muscle mass and
strength. Thus, agrin-based pharmacological approaches represent a
promising strategy to alleviate muscle wasting in the elderly. However, data
suggest that agrin might also play a role as a negative regulator of muscle
regeneration, which would tend to accelerate ageing-related regenerative
decline and skeletal muscle loss. Whether and how agrin suppresses
regenerative muscle formation remains unclear.
Here we explored whether agrin modulates the early stages of muscle
regeneration. Using cultured human myoblasts we found that proliferation of
myoblasts from young donors remained unaltered upon acute or chronic
exposure to neural agrin. Conversely, acute and chronic treatment with
neural agrin enhanced proliferation of myoblasts from old donors. Myoblasts
expressed the canonical agrin receptor Lrp4/MuSK and inhibition of
downstream kinases ERK1/2 and Abl abrogated agrin-stimulated myoblast
proliferation. However, agrin exposure did not stimulate phosphorylation of
ERK1/2 and Abl, suggesting Lrp4/MuSK was not activated in agrin-treated
myoblasts. Consistent with this view, agrin-z0, a non-neural agrin isoform
that does not activate Lrp4/MuSK, increased myoblast proliferation as
efficiently as the neural agrin. Finally, myoblast fusion and maturation of the
excitation-contraction coupling in myotubes remained unaltered in the
presence of agrin, indicating increased proliferation was not linked to
impaired myogenic differentiation.
Taken together, our results demonstrate that agrin does not suppress
myoblast proliferation and myotube formation. Furthermore, we show that
agrin stimulates proliferation of myoblasts from old donors. Thus, agrin may
counteract ageing-related regenerative decline in skeletal muscle.
Collectively, our findings support the notion that agrin-based
pharmacological approaches may lead to novel treatments for sarcopenia.
Short Presentation Abstracts
SP3
NON-SYNAPTIC ROLES OF AGRIN IN THE EARLY STAGES OF SKELETAL
MUSCLE REGENERATION
Katarina Gros¹*, Sergej Pirkmajer¹*, Urška Matkovič¹, Giulia Parato², Katarina
,
Miš¹, Matej Podbregar¹ ³, Zoran Grubic¹, Tomaž Marš¹, Paola Lorenzon²
¹
University of Ljubljana, Faculty of Medicine, Institute of Pathophysiology,
Slovenia; ²University of Trieste, Department of Life Sciences, Italy; ³University
Medical Centre Ljubljana, Slovenia
*These authors have contributed equally to this work.
[email protected]
61
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Short Presentation Abstracts
SP4
DIVERSITY OF CRYPHONECTRIA HYPOVIRUS 1 IN CROATIA: IMPACT ON ITS
FUNGAL HOST
1
2
3
4
1
Ljiljana Krstin , Marin Ježić , Daniel Rigling , Igor Poljak , Zorana Katanić ,
2
Mirna Ćurković-Perica
1
University of J.J. Strossmayer in Osijek, Department of Biology, 31000 Osijek,
Croatia; 2University of Zagreb, Faculty of Science, Department of Biology,
3
10000 Zagreb, Croatia; WSL Swiss Federal Research Institute, CH-8903
Birmensdorf, Switzerland; 4University of Zagreb, Faculty of Forestry,
Department of Forest Genetics, Dendrology and Botany, 10000 Zagreb,
Croatia
[email protected]
62
Cryphonectria parasitica is the causal agent of chestnut blight, a severe
disease responsible for the decline of chestnut trees. However, this
aggressive fungus can be infected with naturally occurring Cryphonectria
hypovirus 1 (CHV-1). Hypovirus CHV-1 is unencapsidated dsRNA virus, one of
four species that belong to family Hypoviridae. CHV-1 reduces fungal
virulence and sporulation, consequently causing healing of cankers and
recovery of infected trees. This phenomenon is called hypovirulence and it
provides the basis for biological control of chestnut blight. The hypovirus can
be transmitted horizontally from infected to non-infected C. parasitica
strains via hyphal anastomosis and vertically into asexual conidia, but not
into sexual ascospores. CHV-1 isolates from Croatia were characterized by
sequencing part of the genomic ORF-A and the effect of virus isolates on the
fungal host was assessed by measuring growth of CHV-1-infected C.
parasitica strains on chestnut stems. Although all virus isolates from Croatia
belonged to the Italian subtype of CHV-1, their effect on fungal host was
significantly different. The results of other scientific groups imply that CHV-1
isolates that belong to Italian subtype have weaker effect on its fungal host
compared to French subtype isolates. However, our results reveal that one
CHV-1 isolate sampled on island Cres had strong effect on fungal growth,
similar to the strong French isolate EP713-CHV-1.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
CRISPR (clustered regularly interspaced short palindromic repeats) and its
associated Cas proteins is a recently discovered defence mechanism against
invading genetic elements in many bacteria and archaea. CRISPR loci
comprise arrays of sequence repeats separated by spacers that are
homologous to sequences of some phage and plasmids. Escherichia coli have
a type IE CRISPR/Cas system that targets invader DNA with CRISPR RNA
(crRNA) bound within a "Cascade" nucleoprotein complex. Cas3 helicasenuclease is thought to then degrade invader DNA. The most efficient
protection of cells depends on 5’-AWG-3’ PAM (protospacer adjacent motifs)
sequences that are immediately next to a protospacer on the target DNA
(e.g. phage) genome. Mutation or variations in PAM result in lower binding
affinity of Cascade to the target DNA and consequently absence of resistance
to phage infection. Co-expression of Cascade and Cas3 proteins with crRNA is
effective at protecting E. coli cells from propagation of bacteriophage into
lytic plaques, but the transcriptional repressor H-NS has been shown to
repress Cascade and crRNA expression from their chromosomal loci. Factors
that control transcription of E. coli cas3 gene have not been identified.
Previous studies showed that cells lacking H-NS have elevated levels of
Cascade and crRNA transcripts and are resistant to infection by phage vir if
they contain appropriate anti-lambda spacer. Surprisingly, resistance was
strongly dependent on post-infection temperature of incubation: at 30°C E.
coli Δhns cells containing anti-lambda spacer were fully resistant to phage
attack but were sensitive if incubated at 37°C.
In this work we wanted to understand the mechanism responsible for
temperature dependent resistance of E. coli cells to the phage attack. Our
genetic analysis showed that efficient resistance to the phage attack at 37 C
depended on the correct PAM sequence and expression of the cas3 gene.
Surprisingly, using qPCR we provide evidence that the expression of cas3
gene is induced in Δhns cells at both temperatures. This indicates that the
temperature of incubation might regulate the expression of cas3 gene at the
post-transcription level.
Short Presentation Abstracts
SP5
ACTIVATION OF ANTIVIRAL DEFENSE AT LOW TEMPERATURE OF
INCUBATION IN ESCHERICHIA COLI
1
2
1
Kristina Majsec , Edward L. Bolt , Ivana Ivančić Baće
1
Faculty of Science, University of Zagreb, Department of Molecular Biology,
Horvatovac 102a, 10000 Zagreb, Croatia; 2Edward L. Bolt School of
Biomedical Sciences, Queen's Medical Centre, Medical School, University of
Nottingham, Nottingham NG7 2UH, United Kingdom
[email protected]
63
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
SP6
A SINGLE SYNTHETIC SITE RESIDUE MODULATES PARTITIONING OF PREAND POST-TRANSFER EDITING PATHWAYS IN OVERALL EDITING BY
ISOLEUCYL-tRNA SYNTHETASE FROM Escherichia coli
1
2,3
1
Morana Dulić , John J. Perona , Ita Gruić-Sovulj
1
Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a,
10000 Zagreb, Croatia; 2Department of Chemistry, Portland State University, P.O. Box
751, Portland OR 97207; 3Department of Biochemistry & Molecular Biology, Oregon
Health & Sciences University, 3181 SW Sam Jackson Park Road, Portland OR 97239
Short Presentation Abstracts
[email protected]
64
Isoleucyl-tRNA synthetase (IleRS) covalently attaches isoleucine to its
cognate tRNAIle in a two-step reaction. First, isoleucine is activated by ATP
yielding isoleucyl-adenylate, followed by the transfer of isoleucyl moiety to
tRNA. IleRS also activates structurally similar noncognate valine and transfers
Ile
it to tRNA . To maintain accuracy of translation, IleRS, as many other
aminoacyl-tRNA synthetases, evolved a network of hydrolytic proofreading
activities. Rapid deacylation of misacylated tRNA or post-transfer editing
occurs in a separate editing domain dedicated to this activity alone. In
contrast, hydrolysis of aminoacyl-adenylate or pre-transfer editing is in the
most cases only a weak tRNA-independent activity. Rarely, as in IleRS, it is
stimulated by the presence of tRNA. We have recently shown that both
tRNA-independent and tRNA-dependent pre-transfer editing activities are in
IleRS localized within the confines of the synthetic site. Furthermore, kinetic
partitioning of valyl-adenylate between hydrolysis and the aminoacyl
transfer reactions is determined by the ratio of their corresponding rates. In
this work, we introduce an improved kinetic approach to distinguish editing
reaction from aminoacylation, since both reactions contribute to AMP
formation in the common editing assay. This approach revealed that tRNA
dependent pre-transfer editing is indeed a significant pathway contributing
up to 30 % to overall Escherichia coli IleRS editing. Using this methodology in
a quantitive analysis of the synthetic site mutants, we found that the
conserved Tyr59 is important for both synthetic and editing reactions. This
indicates that IleRS catalyzes competing aa-tRNA synthesis and Val-AMP
hydrolysis in the overlapping subsites with the synthetic site, providing a
rationale for hindered evolution of pre-transfer editing as the major
proofreading step. Substitutions of Tyr59 with Thr diminished tRNAdependent pre-transfer editing providing the first determinant of the
synthetic site based pre-transfer editing model. We also observed that IleRS
consumes significantly less ATP in proofreading than homologous leucyl- and
valyl-tRNA synthetases although it maintains the similar rate of misacylation
with these enzymes. As IleRS experiences more error-prone environment in
E. coli, relatively unique operational mechanism in the synthetic and editing
pathways in IleRS seems to be driven by cellular energetic requirements.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
SP7
FUNCTIONAL ANALYSIS OF CIS-REGULATORY ELEMENTS FROM 5'
UNTRANSLATED REGION OF PTCH1B GENE
1
2
1
1
1
Petar Ozretić , Alessandra Bisio , Vesna Musani , Diana Trnski , Maja Sabol ,
1
2
Sonja Levanat , Alberto Inga
1
[email protected]
PTCH1 tumor suppressor gene encodes for a transmembrane receptor with a
negative regulatory role in Hedgehog-Gli signaling pathway. PTCH1 germline
mutations cause Gorlin syndrome which is characterized by developmental
abnormalities and tumor susceptibility. Most of malformations are caused by
PTCH1 haploinsufficiency, indicative of fine-tuning needed to properly
regulate pathway activity. After identifying 5 to 8 CGG repeats close to
translation initiation site, our aim was to examine how 5’ untranslated region
(5’UTR) regulates the expression of PTCH1 transcript 1b.All potential PTCH1b
5’UTR cis-regulatory elements were studied by various in silico tools and
gene reporter assays. We tested the influence of 5’UTR length and CGGrepeat polymorphism by cloning either 188- or 300bp-long 5’UTR, each
harboring 5 to 8 CGG repeats, in pGL3-P plasmid upstream of firefly
luciferase gene (Fluc). Site-directed mutagenesis (SDM) was used to test the
significance of predicted upstream open reading frames (uORF). Bicistronic
pRuF vectors were constructed by cloning PTCH1b 5’UTRs between Renilla
and firefly luciferase genes, since the last 76bp in PTCH1b 5’UTR were
predicted as an internal ribosome entry site (IRES). Dual luciferase assays and
qPCR were performed in MCF-7, HCT 116 and HEK 293T cells. Reporter
assays showed that shorter 5’UTR significantly increased reporter activity,
with a subtle reduction with higher repeat number. Longer 5’UTR led to
much reduced reporter activity, without a difference among repeats. Fluc
mRNA levels showed that both 5’UTRs significantly increased transcription.
SDM proved hypothesis that 2 potential uORFs, present only in 300bp-long
5’UTR, might account for this severe reduction in Fluc activity. Both 5’UTRs
significantly increased Fluc activity of pRuF vectors (proved by PCR this is not
due to alternative splicing), with no difference among repeats, while Fluc
activity was significantly reduced after removing predicted IRES motif.
Firefly/Renilla luciferase mRNA ratios were the same as for empty vector,
indicating that observed higher Fluc activity should be due to a posttranscriptional regulation, i.e., cap-independent translation of Fluc mRNA.
All our results point to exceptionally complex and so far unexplored role of
5’UTR in the regulation of PTCH1b expression, whilst the existence of an IRES
would enable PTC1 protein to be synthesized under conditions when general
level of protein synthesis is reduced, such as in hypoxia.
Short Presentation Abstracts
Laboratory for Hereditary Cancer, Division of Molecular Medicine, Ruđer Bošković
Institute, Zagreb, Croatia; 2Laboratory of Transcriptional Networks, Centre for
Integrative Biology (CIBIO), University of Trento, Mattarello (Trento), Italy
65
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Short Presentation Abstracts
SP8
CRYSTAL
STRUCTURE
OF
TACRINE-BASED
UNCHARGED
ACETYLCHOLINESTERASE REACTIVATORS
1
2
2
Gianluca Santoni , Julien de Sousa , Maria Kliachyna , Jacques-Philippe
1
3
3
2
Colletier , Ludovic Jean , Pierre-Yves Renard , Rachid Baati , Florian
1,4
1
Nachon , Martin Weik
1
Commissariat à l'Energie Atomique, Institut de Biologie Structurale, F-38054
Grenoble, France, CNRS, UMR5075, F-38027 Grenoble, France, Université
Joseph Fourier, F-38000, Grenoble, France; 2Université de Strasbourg, Faculté
de Pharmacie, CNRS/ UMR 7199 BP 24, 74 route du rhin 67401 Illkirch,
France; 3Normandie University, COBRA, UMR 6014 and FR 3038, University of
Rouen, INSA of Rouen, CNRS, 1 rue Tesniere 76821 Mont-Saint-Aignan,
4
Cedex; Département de Toxicologie, Institut de Recherche Biomédicale des
Armées BP73, 91993 Brétigny/s/Orge, France.
[email protected]
66
Acetylcholinesterase (AChE), a key enzyme of the central nervous system, is
the main target of organophosphorus chemical warfare. A family of
molecules bearing an oxime group has been developed to restore AChE
function by displacing the inhibitory phosphyl group of nerve agents from the
catalytic serine. But these reactivators have a narrow spectrum of efficiency
and have a permanent charge, which prevents them to cross the blood brain
barrier and reactivate central AChE. The development of broad-spectrum
central reactivators has been a hot topic in the recent years. We take
advantage from the knowledge of the structure and function of AChE to
design new reactivators based on a tacrine moiety linked to a non-charged
oxime group. The crystal structure of non-phosphylated AChE in complex
with one of these reactivators reveals two binding sites responsible for the
submicromolar affinity of this molecule, deep into the active site gorge and
at the gorge entrance or peripheral site of the enzyme. Modifying the tacrine
moiety by adding a chlorine substituent prevents binding into the gorge as
shown by the crystal structure of the complex, and lead to a 10-fold decrease
in affinity, but only 2-fold decrease in VX-reactivation efficiency. The crystal
structure of the chlorinated derivative bound to the peripheral site of tabuninhibited AChE is also described.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Mastermind-like (MAML) family of proteins has 3 members in mammalian
organisms and has gotten its name because of the similarities between them
and the Drosophilla Mastermind protein both in structure and in function.
MAML proteins function as transcriptional co-activators in Notch signaling
and several other pathways. MAML is essential for the assembly of
transcription activation complex of Notch target genes. MAML proteins are
structurally simple and can be the base for binding more complex proteins
and getting them in close contact necessary for proper function. dnMAML1
mutant is a truncation of MAML1 protein consisting of 62 amino acids (1374) from the N-terminal basic domain of MAML1. Since N-terminus is the
most conserved part of all MAML proteins it can inhibit all four receptors and
MAML proteins. Depending on the part of MAML involved dnMAML is
presumed to be able to mimic MAML in Notch independent functions as
well. That is true for p53 where binding is accomplished between N-terminal
part of MAML and the DNA binding domain of p53. MAML family of coactivators makes an excellent candidate for targeting since they modulate a
wide number of signaling pathways. N-terminal fragment of MAML1 protein
further named dnMAML was cloned into a vector carrying ELP and SynB1 cell
penetrating peptide. SynB1-ELP-dnMAML inhibition potential was tested in
U251 glioblastoma derived cell line and confirmed by appropriate controls.
Precise mechanism of inhibition was explored by testing levels of apoptosis
induction and cell cycle distribution. SynB1-ELP-dnMAML show cytoplasmic
accumulation in the cells. Protein uptake in U251 cells doubles when heat is
applied. Cells were treated with growing concentrations of SynB1-ELPdnMAML for 1h at different temperatures in two 72h cycles. Same was done
with SynB1-ELP to show that the vehicle protein itself is not cytotoxic.
Treatment with dnMAML inhibits cell growth significantly up to 60%. The
effect can partially be explained by increased apoptosis (up to 20% in heated
samples). There is no significant cell cycle arrest. dnMAML also negatively
effects expression levels of key regulatory protein MAPK, pAKT and p53,
which is mutated in the tested cell line.
Further investigation is necessary but SynB1-ELP-dnMAML holds great
potential for targeted molecular therapy.
Short Presentation Abstracts
SP9
THERMALLY RESPONSIVE ELP-DNMAML PROTEIN AND IT'S EFFECT ON U251
CELLS IN VITRO
1,2
2
2
Teuta Opačak-Bernardi , Jung Su Ryu , Drazen Raucher
1
Faculty of Medicine, J.J. Strossmayer University of Osijek, Josipa Huttlera 4,
31000 Osijek, Croatia; 2University of Mississippi Medical Center, 2500 North
State Street, Jackson, MS 39216
[email protected]
67
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Short Presentation Abstracts
SP10
THE EFFECT OF ΔNp73α ON THE CELL CYCLE CONTROL AFTER DNA DAMAGE
IN NORMAL AND TUMOR HUMAN CELLS
1
2
1
1
Anđela Horvat , Vjekoslav Dulić , Arijana Zorić , Neda Slade
1
Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia;
2
Institute of Molecular Genetics of Montpellier, CNRS UMR5535, Montpellier,
France
[email protected]
68
ΔNp73α represents a potentially oncogenic isoform of p73 protein. Its
overexpression has been detected in various human tumors often correlating
with increased chemoresistance and worse prognosis. Exploring ΔNp73α
tumorigenic properties upon its overexpression revealed cell-type and
condition-dependent roles. Our aim is to investigate the effects of ΔNp73α
overexpression on cycle progression of normal and tumor cells and
expression of different cell cycle regulators after DNA damage.
Normal human fibroblasts (NHF) and U2OS human osteosarcoma cell line
were infected with retrovirus carrying ΔNp73α gene. Cells were treated with
different doses of γ-irradiation (5 and 10 Gy) or topoisomerase II inhibitor
ICRF-193, and harvested at different time points. The expression of proteins
involved in cell cycle regulation and DNA damage response (p21, p27, p53,
pRB, cA, cB1, Chk1, Chk2) was determined by western blot. Distribution of
cells in different cell cycle phases was determined using flow cytometer by
measuring DNA content after propidium iodide staining.
U2OS cells overexpressing ΔNp73α showed higher percentage of cells with
4N DNA content upon γ-irradiation compared to control cells, especially for
longer time periods post-treatment (48 and 72h), and also increased number
of cells with >4N DNA content (polyploidy). Significantly higher percentage of
polyploid U2OS ΔNp73α overexpressing cells was also found after 48h of
ICRF-193 treatment compared to control cells. The effect of ΔNp73α
overexpression on cell-cycle distribution of NHF was weaker, possibly due to
lower ectopic ΔNp73α levels compared to U2OS cells. Protein expression
analysis of both cell types revealed various differences between control and
cells overexpressing ΔNp73α upon both γ-irradiation and ICRF-193
treatment.
ΔNp73α isoform was shown to influence cell response to different genotoxic
treatments in U2OS, and to a lesser extent in NHF cells. Effects obtained in
response to ICRF-193 and γ-irradiation suggest its potential role in G2/M
DNA damage checkpoint, representing interesting basis for more detailed
analysis of signalling pathways involved.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Enzymes of GDSL family exhibit high potential for pharmaceutical and
biotechnological applications, due to their broad substrate specificities and
diverse catalytic activities like lipase, phospholipase, esterase and
thioesterase. They are characterized by five distinct protein motifs (or
Blocks), among which Block I, III and V exhibit higher conservation and carry
important catalytic residues. The number of proteins assigned to the GDSL
family has increased rapidly over the last few years and continues to expand.
Preliminary analysis of Uniprot database showed that Actinobacteria could
be a rich source of GDSL hydrolases. Due to low overall sequence similarity
within members of the GDSL family, we used motif scanning as a tool for
identification of actinobacterial GDSL sequences. We scanned 77
actinobacterial proteomes and found 484 GDSL enzymes in total (up to 26
per proteome). CLANS clustering and phylogenetic analysis provided new
insights into evolution of GDSL family in Actinobacteria. Newly identified
protein sequences formed eight well defined groups, each possessing
undescribed variations in GDSL motifs that possibly reflect novel enzyme
properties of biotechnological interest. Further, our results pointed out that
horizontal gene transfer had a significant impact in evolution of
actinobacterial GDSL genes. These bacteria exhibit a variety of lifestyles and
acquiring multiple GDSL genes by horizontal transfer could significantly help
them in their saprophytic lifestyle in soil and in adaptation to novel
ecological niches.
Short Presentation Abstracts
SP11
DEEP SCANNING FOR GDSL MOTIFS ACROSS ECOLOGICALLY DIVERSE
ACTINOBACTERIA
1,2
3
4
1
Ana Bielen , Branka Bruvo-Mađarić , Ivan Vujaklija , Tina Paradžik , Siniša
4
3
5
1
Biđin , Željka Pezer Sakač , Pavle Goldstein , Dušica Vujaklija
1
Laboratory for Molecular Genetics, Ruđer Bošković Institute; 2Laboratory for
Biology and Microbial Genetics, Faculty of Food Technology and
3
Biotechnology; Laboratory of Evolutionary Genetics, Ruđer Bošković
4
Institute; Department of Electronics, Microelectronics, Computer and
5
Intelligent Systems, Faculty of Electrical Engineering; Department of
Mathematics, Faculty of Science, University of Zagreb, Zagreb, Croatia.
[email protected]
69
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Short Presentation Abstracts
SP12
THE ROLE OF ORGANIC CATION TRANSPORTERS (OCTS, SLC22A) IN
ZEBRAFISH (Danio rerio)
Ivan Mihaljević, Roko Žaja, Marta Popović, Jovica Lončar, Tvrtko Smital
Division for Marine and Environmental Research, Rudjer Boskovic Institute,
Zagreb, Croatia
[email protected]
70
Polyspecific organic cation transporters (OCTs) of the SLC22 family play
important roles in distribution and elimination of cationic drugs and toxins in
mammals. Our study is the first attempt directed towards the identification,
molecular characterization and understanding of physiological role of fish
Octs. Using phylogenetic analysis we identified two zebrafish Oct coorthologs, Oct1 and Oct2, on chromosomes 20 and 17, respectively, which
are syntenic with human SLC22 cluster on chromosome 6. Quantitative realtime PCR (qPCR) revealed high expression of Oct2 in testes and moderate in
kidney. Oct1, which is closer to mammalian OCT orthologs according to
phylogenetic analysis, showed very high expression in kidney and high
expression in liver and testes of male zebrafish. In order to determine the
substrate specificity of zebrafish OCT1, we transiently expressed the
transporter in HEK293 cell line. We found that OCT1 acts as a high affinity
transporter for several fluorescent cations: 4-(4-Dimethylaminostyryl)-Nmethylpyridinium (ASP+; Km=11 μM), ethidium bromide (Km=2 μM),
amiloride (Km=206 μM), berberine (Km=1.8 μM), 4',6-diamidino-2phenylindole (DAPI; Km=10 μM) and rhodamine123 (Km=0.06 μM). Using the
ASP+ as fluorescent probe we then tested 50 compounds known to interact
with human OCTs. Among the tested physiological substrates, high affinity
was found for hormones androstenedione (Ki=1.6 μM) and progesterone
(Ki=2.8 μM), while moderate affinity was found for β-estradiol (Ki=29 μM)
and testosterone (Ki=16 μM). Among the tested xenobiotics, we found
strong interaction with organotines, especially tri-n-butyltin chloride (Ki=3.9
μM). Taken together, our results imply that: (1) zebrafish OCT1 has a pivotal
physiological role in uptake of hormones in testes and their clearance from
blood through kidney and liver, and (2) OCT1 may have an important
defensive role in fish metabolic response to organotines as globally
ubiquitous and harmful marine contaminants. Further research will be
focused on determination of substrate/inhibitor interaction of tested
compounds, based on Michaelis-Menten kinetics, and comparative analysis
of human OCT1-3 through biochemical characterization and homology
modelling of both human and zebrafish transporters, which will shed new
light on transport mechanism of zebrafish Oct1.
Poster Abstracts
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Legionella pneumophila, the causative agent of Legionnaires' disease, is
ubiquitously found in aquatic environments. With increasing awareness of
legionellosis associtaed with spa and bathtubs, regular clean - up and
disinfection becomes essential. In addition to superheating and chlorine
disinfection, antibacterial agents extracted from a variety of plants have
been increasingly evaluated. The aim of this study was to determine the in
vitro anti - Legionella activity of 7 different plant essential oils from Croatia.
Using a broth dilution method, the antibacterial activity of 7 different plant
essential oils (Immortelle spring and autum oil – Helichrysum arenarium,
Sage oil - Salvia officinalis, Laurel oil –Laurus nobilis and Juniper oil –
Juniperus communis, Thuja oil - Thuja occidentalis, Chamomile oil Matricaria chamommilla, Lavandin oil - Lavandula hybrida ) against different
L. pneumophila were tested. The minimum inhibitory concentrations (MIC)
were determined by visual inspection as the lack of visual turbidity and
minimum bactericidal concentration (MBC) as well as no growth after
culturing on BCYE agar. Additionally, the time– kill studies were performed
with 106 cfu/mL of L. pneumophila and different plant essential oil at 1xMIC
value. The results showed that L. pneumophila is sensitive to all tested
essential oils, but the best antibacterial activity was demonstrated with
Immortelle summer essential oil, with minimal inhibitory concentration of
0,1 µg/mL and a bactericidal activity at 0,2 µg/mL. There was a significant
drop in bacterial burden within 48 h with Immortelle summer and autum
essential oil, Juniper essential oil, Laural essential oil and Lavandula hybrid
essential oil.
Our results suggest that Immortelle summer essential, as well as Immortelle
autumn, Juniper and Laurel oil possess strong anti - Legionella activities, and
have a great potential to be used as a disinfectants in control of legionellosis
associated with spa facilities.
Poster Abstracts
P1
ANTIBACTERIAL EFFECT OF SELECTED ESSENTIAL OILS AGAINST Legionella
pneumophila
1
2
1
Ana Babić , Mladenka Malenica Staver , Ivana Gobin
1
Department of Microbiology and Parasitology, School of Medicine,
University of Rijeka, Croatia; 2Department of Biotechnology, University of
Rijeka, Croatia
[email protected]
73
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P2
GENDER DIFFERENCE IN EXPRESSION OF ESTROGEN AND LEPTIN RECEPTOR
IN SPRAGUE DAWLEY RAT ADRENAL GLAND AFTER ACUTE AND CHRONIC
STRESS
1*
2
2
1
Marta Balog , Senka Blažetić , Irena Labak , Barbara Viljetić , Robert
3
2
1
Blažeković , Rosemary Vuković , Marija Heffer
1
University of Osijek, Faculty of Medicine, Osijek, Croatia; 2University of
3
Osijek, Department of Biology, Osijek, Croatia; Department of Cardiac and
Transplantation Surgery, University Hospital Dubrava, Zagreb, Croatia
[email protected]
Poster Abstracts
AIM: To analyze clinically relevant biochemical markers for development of
metabolic syndrome and differences in expression of estrogen receptor ß
(ER-ß) and leptin receptor (Ob-R) in adrenal gland of Sprague Dawley male,
non-ovariectomized female (NON-OVX) and ovariectomized female (OVX)
rats under acute and chronic stress.
METHODS: Study included 72 four-months-old Sprague-Dawley rats, 24
males and 48 females. Animals were divided into male, NON-OVX and OVX
group. Each animal group of was further divided into – acute stress, chronic
stress and control group. Weight, glucose tolerance test (GTT) and plasma
concentration of glucose, cholesterol and urates were measured. Adrenal
glands were collected at the age of 26 weeks and immunohistochemical
staining was performed using Erβ and Ob-R antibodies.
RESULTS: Concentration of plasma glucose, cholesterol and urates decreased
after acute and chronic stress. Acute and chronic stress significantly changed
GTT profile in males and NON-OVX, but not in OVX group. ERβ expression in
zona glomerulosa was significantly higher in OVX than NON-OVX control (p =
0.009) and also in OVX chronic stress group than NON-OVX chronic stress
group (p = 0.004). ERβ expression in medulla was also significantly higher in
OVX than NON-OVX group (p = 0.03). Ob-R expression in zona reticularis is
significantly higher in NON-OVX than OVX after chronic stress (p = 0.03).
CONCLUSION: Chronic stress is connected with the loss of weight in both
genders and decrease in plasma glucose, cholesterol and urates after acute
and chronic stress. Intense nuclear staining obtained with Erβ and Ob-R
implies a possibility of long-term changes in transcription profile in adrenal
glands.
74
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Inflammatory bowel disease (IBD) is a group of chronic inflammatory
conditions that affect different parts of the gastrointestinal tract. The
etiology of IBD is unclear, but increasing scientific evidence confirms a
bidirectional connection between central and enteric nervous system, known
as gut-brain axis. In this context, peptidases exert a key role in maintaining
the homeostasis in the gut by regulating the biological activity of their
bioactive substrates at local, circulating and central level. Dipeptidylpeptidase IV (DPP IV/CD26) is a pleiotropic membrane glycoprotein found
also in soluble form in different biological fluids, associated with important
processes in the organism, both in physiological and pathological conditions.
Neuropeptide Y (NPY) and Vasoactive intestinal peptide (VIP) are substrates
of DPP IV/CD26, which involvement in chronic inflammatory processes,
including IBD, has been indicated. Our hypothesis was that DPP IV/CD26
plays an important neuroimmunomodulatory role in IBD pathogenesis by
influencing circulating and tissue levels of NPY and VIP in a chemicallyinduced model of colitis in mice. In order to evaluate the impact of DPP
IV/CD26 on NPY and VIP levels among the gut-brain axis, a
trinitrobenzenesulfonic acid (TNBS-induced; Crohn-like) model of colitis has
been induced in CD26 deficient (CD26-/-) and wild type (C57BL/6) mice. NPY
and VIP concentrations and protein expressions have been determined at
both systemic and local levels. Results of our study showed that VIP
concentrations and protein expressions in serum, colon and brain of both
mice strains reach their maximum values in the acute phase of colitis, but the
increment was more pronounced in CD26-/- mice (p<0.05). The increase of
serum NPY concentration was more emphasized in CD26-/- mice, while NPY
colon concentration and protein expression was higher in C57BL/6 mice.
Furthermore, colon inflammation induced an increase in brain NPY
concentration in the acute phase in C57BL/6 mice and, adversely, a decrease
in CD26-/- mice. Our results indicate the importance of the gut-brain axis in
the pathogenesis of IBD, as well as an important impact of DPP IV/CD26 on
VIP and NPY levels during experimental colitis.
Poster Abstracts
P3
CD26 DEFICIENCY ALTERS VIP AND NPY LEVELS IN MURINE CROHN-LIKE
COLITIS
1
1
2
1
Lara Batičić Pučar , Dijana Detel , Natalia Kučić , Sunčica Buljević , Jadranka
1
Varljen
1
Department of Chemistry and Biochemistry, 2Department of Physiology and
Immunology, School of Medicine, University of Rijeka, Braće Branchetta 20,
51000 Rijeka, Croatia
[email protected]
75
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P4
INFLUENCE OF LIGNANS SUPPLEMENTATION ON ANTIOXIDANT ACTIVITY
OF APPLE JUICE
1
1
2
1
Drago Bešlo , Dejan Agić , Bono Lučić , Dragan Amić
1
Josip Juraj Strossmayer University of Osijek, Faculty of Agriculture in Osijek,
Kralja Petra Svačića 1d, 31000 Osijek, Croatia; 2NMR Center, Rudjer Bošković
Institute, P.O. Box 180, 10002, Zagreb, Croatia
[email protected]
Poster Abstracts
For many years sinthetic antioxidants have been used effectively to retard
autoxidative deterioration of food and food products but some of them have
been restricted recently because they are reported to be carcinogenic and
toxic. Lignans are plant-derived phenolic products formed by the coupling of
two phenylpropanoid (C6-C3) units and most of them containing hydroxyl
groups that was suggested to exhibit antioxidant activity. Therefore, we
aimed to investigate the influence of plant lignans supplementation on
antioxidant activity of apple juice during storage. In this study apple juice was
treated with three different lignans: secoisolariciresinol (SECO), matairesinol
(MAT) and isolariciresinol (ILARI) at concentrations 1 and 10 μg mL -1, while
the control was apple juice without the addition of lignans. Antioxidant
activity of the apple juice were measured after one day, and after 30, 60, 90,
120 and 150 days of storage using the 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging assay and results were expressed as ascorbic acid
equivalence antioxidant capacity (AEAC). The results show that the
supplementation of lignans increased antioxidant activity of apple juice,
particularly at higher concentrations. During the storage, antioxidant
activities of apple juice samples treated with lignans decreased, but for each
sample they are higher than in controls. Apple juice treated with 10 μg mL-1
ILARI had the highest antioxidant activity during storage among the lignans
that were studied. Based on the in vitro assay, plant lignans ILARI, SECO and
MAT may have potential application in food and health industry as natural
antioxidants.
76
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Beta2-agonists are one of three types of prescription bronchodilating drugs.
They possess the ability to relieve bronchoconstriction. Besides dilating
bronchial passages, this class of compounds also causes vasodilation in
muscles and liver, relaxation of the uterine muscle, and insulin release. Also,
recent studies suggest that chronic treatment with certain bronchodilating
beta2-agonists relieves neuropathic pain. The beneficial effect of
bronchodilators albuterol and adrenaline in congenital myasthenic syndrome
and congenital endplate acetylcholinesterase deficiency has already been
proven. Terbutaline, isoproterenol and metaproterenol have been
demonstrated to be reversible cholinesterase inhibitors. Most
bronchodilators are derivatives of catecholamines, resorcinol or saligenin.
We focused our research on resorcinol (fenoterol), catecholamine
(isoetharine and adrenaline), and saligenine (albuterol) derivatives as
inhibitors of human acetylcholinesterase (AChE; EC. 3.1.1.7) and of the usual,
atypical and fluoride-resistant butyrylcholinesterase (BChE; EC 3.1.1.8). As a
measure of inhibition potency, we determined the dissociation constants of
the enzyme-inhibitor complex (Ki). All of the tested compounds reversibly
inhibited cholinesterases and the Ki constants ranged from 0.02 mM to 6.8
mM, which is typical for low-potency inhibitors. Generally, the affinity (1/Ki)
of AChE toward the tested compounds was lower than the affinity of the
tested BChE variants. Moreover, the highest selectivity of binding was
revealed in the case of albuterol, where the affinity of usual BChE was about
75 times higher compared to AChE. Usual BChE and AChE showed the lowest
affinity for adrenaline. Usual BChE showed the highest affinity for fenoterol
and the lowest for adrenaline. An analysis of systematic structural
differences between compounds revealed that the inhibition potency of the
studied compounds could not be related to the disposition of two hydroxyl
groups on the benzene ring, but to the size of N-substituents on the aliphatic
part of a compound. Our results suggested that, when using beta2-agonists
as bronchodilators, one should always keep in mind that they also possess a
week inhibition potency for cholinesterases, which leads to a reduction in
the level of hydrolytic activity of these enzymes.
Poster Abstracts
P5
BRONCHODILATING BETA2-AGONISTS AS HUMAN CHOLINESTERASE
INHIBITORS
1
2
2
2
Anita Bosak , Ivana Gazić Smilović , Anamarija Knežević , Vladimir Vinković ,
1
Zrinka Kovarik
1
Institute for Medical Research and Occupational Health, Zagreb, Croatia;
2
Ruđer Bošković Institute, Zagreb, Croatia
[email protected]
77
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P6
EXPRESSION AND CELLULAR DISTRIBUTION OF PRIMARY-, SECONDARYAND TERTIARY-ACTIVE BASOLATERAL MEMBRANE TRANSPORTERS FOR
ORGANIC ANIONS IN HUMAN KIDNEYS
Poster Abstracts
Davorka Breljak1, Marija Ljubojević1, Vedran Micek1, Daniela Balen1, Ivana Vrhovac1,
Hrvoje Brzica1, Dean Karaica1, Ognjen Kraus2, Nikola Radović3, Yohannes Hagos4, Maja
Henjakovic4, Roberto Antolović5, Naohiko Anzai6, Birgitta C. Burckhardt4, Gerhard
Burckhardt4, Ivan Sabolić1
1
Molecular Toxicology, Institute for Medical Research and Occupational Health,
Zagreb, Croatia; 2Urology, University Hospital Sisters of Mercy and 3Clinical Hospital
Dubrava, Zagreb, Croatia; 4Institute of Systemic Physiology and Pathophysiology,
Center of Physiology and Pathophysiology, University Medical Center Göttingen,
Germany; 5Biotechnology, University of Rijeka, Croatia; 6Pharmacology and
Toxicology, Kyorin University School of Medicine, Tokyo, Japan; [email protected]
78
In human kidneys, endogenous and exogenous organic anions (OA) are removed by
organic anion transporters (OAT) that belong to the SLC family of transporters and
reside in the basolateral membrane (BLM) of epithelial cells along the nephron,
predominantly in proximal tubules. The active contributors to this process are the
primary-active sodium-potassium adenosine triphosphatase (Na/K-ATPase),
secondary-active sodium-dicarboxylate cotransporter 3 (NaDC3/SLC13A3) and
tertiary-active OA exchangers OAT1/SLC22A6, OAT2/SLC22A7, and OAT3/SLC22A8.
Several previous studies showed that the expression of renal OAT1-3 was sexdependent in rodents (rats and mice), but similar investigations in human kidneys
have not been reported. Accordingly, here we studied the expression and distribution
of various BLM transporters that contribute to OA secretion along the human
nephron by immunofluorescence cytochemistry (IC) in kidney cryosections and by
Western blotting (WB) of total cell membranes (TCM) isolated from the kidneys of
men (M; n = 7) and women (W; n = 9). Virtually healthy part of kidney tissue was
obtained following surgical removal of the carcinoma-diseased organs. Specificity of
the used polyclonal antibodies (Ab) for human NaDC3 and OAT1-3 was confirmed in
the transporters-transfected HEK293 cells by IC and WB, whereas the proper
transfection was validated in uptake experiments with radiolabeled succinate
(NaDC3), p-aminohippurate (OAT1), cGMP (OAT2) and estrone-3-sulfate (OAT3). In IC
studies, in human kidneys we confirmed colocalization of Na/K-ATPase, NaDC3 and
OAT1-3 in the BLM of proximal tubules, whereas Na/K-ATPase and NaDC3 were also
colocalized in the BLM of collecting ducts. In WB of TCM, the protein bands of 85,
80, 75 and 110 kDa were detected for OAT1, OAT2, OAT3 and Na/K-ATPase,
respectively, whereas two protein bands of 70 and 90 kDa were detected for
NaDC3. The density of protein bands in WB studies, and the staining intensity of
various transporters in IC studies were similar in M and W kidneys. Therefore, various
primary-, secondary- and tertiary-active transporters that contribute to OA secretion
along the human nephron exhibit fair colocalization in the proximal tubules, but in
more distal nephron segments, the co-expression is restricted to Na/K-ATPase and
NaDC3. Sex differences in the expression of tested transporters were not observed,
thus indicating that the sex-dependent expression of some renal transporters is
species-related.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
One of the diseases in humans, with most growing incidence, is diabetes mellitus
type 2, which is characterized by hyperglycemia and various successive
complications. Novel antidiabetic drugs have been proposed that would inhibit
the activity of SGLT1 (SLC5A1 in humans)/Sglt1 (Slc5a1 in rodents) in the brushborder membrane (BBM) of small intestinal enterocytes and renal proximal
tubules (PT), and would impair absorption and reabsorption, respectively, of
glucose in these organs. The impact of such drugs on other organs is unknown,
since the expression of SGLT1 in other organs/tissues has been poorly studied, or
is contradictory. Since mice are frequently used in preclinical studies, knowledge
of the Sglt1 distribution in various mouse organs/tissues as possible targets for
such drugs would be of great importance. In order to determine the Sglt1 mRNA
and protein expression by qPCR, Western blotting (WB) and
immunocytochemistry (IC), we compared these parameters in various
organs/tissues of wild-type (WT) and Sglt1 knockout (KO) mice. By qPCR: a)
highest expression of Sglt1 mRNA was observed in the small intestine, b) high
expression was measured in seminal vesicles, kidneys, salivary glands (parotid,
submandibular and sublingual), and prostate, c) medium expression was
observed in eyes, tongue, pancreas, lungs, and liver, and d) low expression was
found in skeletal muscle, testes, heart, fat, epididymis, cerebellum, cerebrum,
and spleen. Cell localization of the Sglt1 protein was studied by WB and IC in the
Sglt1 mRNA positive tissues using the antibody whose specificity was confirmed
in Sglt1 KO mice. By IC, in the kidneys of WT mice, Sglt1 protein was localized to
the BBM of PT and luminal membrane of TALH, exhibiting segmental (S2>S3),
zonal (cortex>outer stripe), and sex (males (M)>females (F)) differences. In the
small intestine, liver and pancreas, it was localized to the BBM of enterocytes,
bile ducts and pancreatic ducts, respectively. By WB, a single Sglt1-related
protein band of 75 kDa was detected in the BBM of kidneys and small intestine.
The Sglt1-related staining and protein bands were not observed in the respective
organs of Sglt1 KO mice. Contrary to the M-dominant Sglt1 protein expression,
the renal Sglt1 mRNA expression was F-dominant. Our data show that Sglt1 is
expressed in various organs/tissues, and thus indicate that the potential
inhibitors of SGLT1 activity may target various organs/tissues with unpredictable
consequences.
Poster Abstracts
P7
TISSUE EXPRESSION PROFILING OF THE MOUSE Sglt1 mRNA AND PROTEIN
WITH SEX-DEPENDENT Sglt1 RENAL EXPRESSION
1
1
1
2
Ivana Vrhovac , Davorka Breljak , Dean Karaica , Hermann Koepsell , Ivan
1
Sabolić
1
Molecular Toxicology Unit, Institute for Medical Research and Occupational
Health, Zagreb, Croatia; 2Anatomy & Cell Biology, University of Würzburg,
Würzburg, Germany; [email protected]; [email protected]
79
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24-27 September 2014, Zadar, Croatia
P8
ASSOCIATION OF KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS (KSHV)
WITH BLADDER CANCER IN CROATIAN PATIENTS
1
2
3
Martina Paradžik , Viljemka Bučević-Popović , Marijan Šitum , Crystal J.
4
1
4
5
Jaing , Marina Degoricija , Kevin S. McLoughlin , Said I. Ismail , Volga Punda
Polić1, Janoš Terzić1
1
School of Medicine, University of Split, Croatia; 2Department of Chemistry,
3
Faculty of Science, University of Split, Croatia; Department of Urology,
4
Clinical Hospital Split, Croatia; Global Security, Lawrence Livermore National
5
Laboratory, Livermore, USA; Molecular Biology Research Lab, Department of
Biochemistry, Faculty of Medicine, University of Jordan, Jordan
[email protected]
Poster Abstracts
As the seventh most common human malignancy, bladder cancer represents
a global health problem. In addition to well-recognized risk factors such as
smoking and exposure to chemicals, various infectious agents have been
implicated as cofactors in the pathogenesis of urothelial malignancies. The
aim of the present study was to assess the possible association of viral
infection and bladder cancer in Croatian patients. Biopsy specimens were
collected from a total of 55 patients diagnosed with different stages of
bladder cancer. Initial screening of DNA extracts for the presence of viruses
on Lawrence Livermore Microbial Detection Array revealed Kaposi’s
sarcoma-associated herpesvirus (KSHV) in each of three randomly chosen
biopsy specimens. The prevalence of infection with KSHV among study
population was then examined by KSHV-specific polymerase chain reaction
(PCR) and immunoblotting. By nested PCR, KSHV DNA was detected in 55 %
of patients. KSHV, also known as human herpesvirus 8, is an infectious agent
known to cause cancer. Its oncogenic potential is primarily recognized from
its role in Kaposi's sarcoma, but it has also been involved in pathogenesis of
two lymphoproliferative disorders. A high prevalence of KSHV infection in
our study indicates that KSHV may play a role in tumorigenesis of bladder
cancer and warrants further studies.
80
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P9
TFF3 KNOCK-OUT MICE HAVE ALTERED FATTY ACID, PROTEIN AND
GLUCOSE METABOLISM IN THE LIVER
Maro Bujak1, Martina Mihalj2, Ivana Tartaro Bujak3, Srđan Vučinić4, Anita
Horvatić1, Maja Tolušić Levak5, Katarina Mišković6, Tomislav Kopačin7, Branka
Mihaljević3, Ines Drenjančević2, Mirela Baus Lončar1
1
3
Trefoil family factor 3 (TFF3) is a small polypeptide first described in the intestine,
characterized with unique structure of trefoil motif, a 40-amino acid domain that
contains three conserved disulfide bonds. TFFs have a well established role in the
process of restitution after mucosal injury and the maintenance of epithelial
barrier integrity, moreover their role in other systems started to emerge. TFF3
gene was identified as one of the genes involved in liver steatosis. The aim of this
study was to determine the rate of fatty acids in the liver of Tff3 knock-out mice,
hepatic proteomic profile as well as the serum glucose and lipid content.
TFF3 knock-out (129/Sv) and wild type (129/Sv) mice (n=20 per group) were
used. Care and use of the animals and the experimental protocol were reviewed
and approved by the local Ethics Committee. Mice were anesthetized at the age
of 5 weeks and the blood was collected by cardiac puncture. Following mice were
sacrificed by cervical dislocation and the liver tissues collected and snap frozen in
liquid nitrogen or fixed in 10% formalin and processed for regular histological
analysis (HE, PAS). Total lipids were extracted from tissue homogenates
according to Bligh and Dyer. Lipids were analyzed by gas chromatography (Varian
450-GC). Serum lipids and glucose were analysed in routine laboratory using
Architect c8000 (Abbott Diagnostic). Proteom profiles of different groups were
analysed by 2D-PAGE and differentially displayed proteins were identified by
MALDI TOF/TOF MS.
TFF3 knock-out mice compared to WT mice have significantly higher amount of
polyunsaturated fatty acids- arachidonic acid (AA, C20:4n-6) (9.51±0.63% vs.
6.36±0.89%; p<0.05) and docosahexaenoic acid (DHA, C22:6n-3) (10.63±1.77%
vs. 6.67±0.48%; p<0.05). Total serum levels of the cholesterol, triglycerides, HDL
or LDL cholesterol did not differ among the groups. Postprandial glucose levels in
TFF3 knock-out mice were higher than in controls but did not reach statistical
significance (p=0.072). There were no differences in the total body weight or the
liver mass. Microscopic analysis of the liver sections revealed normal tissue
architecture (HE) and glycogen reserves (PAS). Proteomic analysis revealed
several proteins involved in lipid, carbohydrate, citric acid cycle and protein
metabolism. TFF3 deficient mice have altered caloric metabolism and TFF3
protein could be a potential target to develop therapeutic strategies against
metabolic disorders.
Poster Abstracts
Laboratory for System Biomedicine, Laboratory for Radiation Chemistry and Dosimetry, Ruđer
2
5
Bošković Institute, Zagreb, Croatia; Department of Physiology and Immunology, Department of
6
Histology and Embryology, Department of Medical Chemistry and Biochemistry, Faculty of
4
Medicine, University of Osijek, Osijek, Croatia; Croatian Academy of Sciences and Arts, Zagreb,
7
Croatia; “Laboratory for Medical Biochemistry Kopačin”, Health Centre “Drava”, Osijek, Croatia;
[email protected]
81
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P10
EVALUATION OF THE POTENCY OF A NEW SERIES OF PYRIDOXAL OXIME
DERIVATIVES IN THE REACTIVATION OF TABUN, PARAOXON AND VXPHOSPHYLATED ACETYLCHOLINESTERASE AND BUTYRILCHOLINESTERASE
1
1
2
Valentina Bušić , Dajana Gašo-Sokač , Maja Katalinić
1
Faculty of Food Technology, University J.J. Strossmayer, Kuhačeva 20, 31000
Osijek, Croatia; 2Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, POB 291, HR-10001 Zagreb, Croatia
[email protected]
Poster Abstracts
Quaternary salts of pyridoxal oximes were synthesized by the quaternization
of the pyridoxal oxime with substituted phenacyl bromides using microwave
heating. Microwave-assisted rapid synthesis was done in solvent (acetone)
and under solvent-free conditions. In this study we investigated these new
oximes interaction with human recombinant acetylcholinesterase (AChE, EC
3.1.1.7) and human butyrylcholinesterase (BChE, EC 3.1.1.8). We tested the
oxime´s reversible inhibition of enzymes and oxime-assisted reactivation of
phosphylated enzymes. All of the tested oximes reversibly inhibited AChE
and BChE with inhibition constants (Ki) in micromolar to milimolar range but
showing week enzyme selectivity. Furthermore, the oximes were tested in 1
mM concentration as reactivators of AChE and BChE inhibited by
organophosphorus compounds tabun, paraoxon and VX. Unfortunately, the
tested oximes were not efficient in reactivating either tabun-, paraoxon- or
VX-inhibited enzymes, especially compared to the oximes currently used in
medical treatment practice. However, two of the tested oximes showed a
potency to reactivate BChE inhibited by VX, and therefore could be
considered in the area of organophosphorus compounds bioscavenger
development and pre-treatment drugs.
82
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P11
PREBIOTIC ACTIVITY OF HONEYDEW HONEY ON PROBIOTIC BACTERIA
Lactobacillus plantarum
1
2
1
Goranka Crnković , Mladenka Malenica Staver , Ivana Gobin
1
Department of Microbiology and Parasitology, School of Medicine,
University of Rijeka, Croatia; 2Department of Biotechnology, University of
Rijeka, Croatia
[email protected]
Poster Abstracts
Numerous studies have shown that honey, besides antimicrobial properties
also possess prebiotic activity on probiotic bacteria, which as part of the
natural human microflora play an important role in preserving health and
fighting against diseases and various pathogens. One of the most quality,
although still relative unknown type of honey is honeydew honey. In this
work, prebiotic effects of honeydew on probiotic bacterium Lactobacillus
plantarum have been tested. Results have shown that honeydews have
prebiotic effect and enhance growth of probiotic bacteria Lactobacillus
plantarum at a concentration of 1% and 5%. Also, honeydew improve
fermentation abilities but do not enhance autoagregation of Lactobacillus
plantarum. Therefore, honeydew could be used as prebiotic in functional
food.
83
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P12
THE RELATIONSHIP BETWEEN THE AUTOPHAGY-LYSOSOMAL PATHWAY,
CHOLESTEROL HOMEOSTASIS AND PROCESSING OF THE ALZHEIMER’S APP
PROTEIN
Stjepko Čermak, Mirsada Čaušević, Silva Hećimović
Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia
[email protected]
84
Lysosomes are intracellular organelles with a crucial role in the clearance of
protein aggregates, damaged organelles, food particles and engulfed
microorganisms. Dysfunction of the autophagy-lysosome pathway has been
found in various neurodegenerative diseases, and is considered one of the
first pathological changes in Alzheimer’s disease. Conversely, majority of the
lysosomal storage diseases that are primarily caused by the dysfunction of
the lysosomal proteins show the most severe phenotype in the brain.
We are using a lysosomal storage disease, Niemann Pick type C (NPC), as a
model of Alzheimer’s disease. There are many similarities between the
diseases including perturbations in cholesterol homeostasis and in the
processing of the amyloid precursor protein (APP). Amyloid plaques,
aggregates of Aβ peptides and one of the hallmarks of Alzheimer’s disease,
are produced by β- (BACE1) and γ-secretase cleavage of the APP protein.
Previous work done in our group has demonstrated that in NPC disease APP
protein is preferentially cleaved through the amyloidogenic pathway. We
have also shown a significant increase in co-localization of APP and BACE1 in
late endosomes and lysosomes of the NPC cells. Cholesterol accumulation
was found to be vital for these changes and through the intracellular
depletion of cholesterol we were able to reverse them.
In this research we are using cellular and mice models of NPC disease. We
have found an increase in LC3II protein levels (autophagosomal marker) in
NPC. Starvation treatment further increased LC3II, as did blocking the
lysosome function using Leupeptin/NH4Cl treatment. These results point to a
conserved function of autophagy activation in NPC and to lysosome
dysfunction as the main culprit for increased LC3II levels. We have also used
different treatments designed to modulate autophagy-lysosome pathway
and followed their effect on Aβ levels. Cholesterol was monitored using the
Amplex Red cholesterol assay (Molecular Probes®) and by determination of
protein levels of the main brain cholesterol transporter ApoE, which is the
main genetic risk factor for sporadic Alzheimer’s disease and also for an
increased severity in the pathogenesis observed in Niemann Pick type C
patients. We believe our results further elucidate the role of autophagy and
lysosomal degradation in neurodegeneration and show a possible
therapeutic role in modulation of this system.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
INTRODUCTION
Diabetes-induced changes and poorly regulated glycemia in diabetic
pregnant women are reflected on placental tissue and metabolism, and
consequently induce a series of complications in both the embryo and later
fetus, and the pregnant woman. For normal growth and development of the
fetal central nervous system of particular importance is the availability and
supplementation of essential long-chain polyunsaturated fatty acids. The
purpose of this study was to determine how changes in carbohydrate
metabolism can affect the lipid content of the placenta in pregnant women
with type 1 diabetes mellitus (DM 1).
MATERIALS AND METHODS
Pregnant women (38 with DM 1, and 34 healthy) were recruited for the
study at the Reference Center for Diabetes in Pregnancy, Department of
Gynecology and Obstetrics, Clinical Hospital Center Zagreb. All pregnancies
were finished by caesarean section at the expected term of birth. Samples
were collected from placental tissue, homogenized and total lipids were
extracted by solvent mixtures. Free fatty acids were separated by thin layer
chromatography, trans-esterified to methyl esters and analyzed by gas
chromatography.
RESULTS
The amounts of free fatty acids were significantly reduced in placental tissue
of the pregnant women with DM1 than in the control group. In DM1 group
the ratio of lauric acid (C12:0) was significantly lower and the ratios of
miristoleic (14:1), palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2)
and docosahexaenoic (22:6) fatty acids were significantly higher than in the
control group.
CONCLUSION
Decreased amounts of free fatty acids in the placenta of pregnant women
with DM1 might indicate an increased lipolytic activity. Well regulated DM1
results with stable metabolism of carbohydrates and lipids, and with the
appropriate fatty acid composition. Especially encouraging for the
development and function of the fetal nervous system is the presence of
sufficient amounts of docosahexaenoic acid.
Poster Abstracts
P13
COMPOSITION OF FREE FATTY ACIDS IN THE PLACENTA OF PREGNANT
WOMEN WITH TYPE 1 DIABETES
1
2
2
1
1
Vito Starčević , Ivančica Delaš , Tonko Dražić , Josip Juras , Josip Đelmiš
1
Department of Gynecology and Obstetrics, Clinical Hospital Center Zagreb,
Croatia; 2Department of Chemistry and Biochemistry, School of Medicine,
University of Zagreb, Croatia
[email protected]
85
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P14
IMMUNOMODULATORY PROPERTIES OF DIPEPTIDYL PEPTIDASE IV (DPP
IV/CD26) IN AN EXPERIMENTAL MODEL OF ULCERATIVE COLITIS
1
1
2
1
Dijana Detel , Lara Batičić Pučar , Ester Pernjak Pugel , Sunčica Buljević ,
1
Jadranka Varljen
1
Department of Chemistry and Biochemistry, 2Department of Histology and
Embriology; School of Medicine, University of Rijeka, Braće Branchetta 20,
51000 Rijeka, Croatia
[email protected]
86
Disturbances in the balance between tolerance and an active immune
response to antigens are fundamental to the pathogenesis of inflammatory
bowel disease (IBD). Therefore, immune cells including dendritic cells (DC)
and macrophages, as well as T cells play a crucial role in the control of
intestinal inflammation and immune tolerance at both systemic and local
level. A role for dipeptidyl peptidase IV (DPP IV/CD26) in the pathogenesis of
IBD has been proposed owing to its involvement in immune regulations via
its expression on immune cells and ability to cleave biologically active
molecules. The aim of the study was to investigate the influence of DPP
IV/CD26 deficiency on infiltration of specific immune cells in colonic mucosa,
in a model of dextran sulfate sodium (DSS)-induced ulcerative colitis in wildtype (WT) and CD26-deficient mice. Colitis development and severity was
assessed by clinical and histological parameters while distribution and
expression of specific cell markers in colonic mucosa by
immunohistochemical staining. In the acute phase of colitis, loss of body
mass and disease activity in WT mice was more severe than in CD26-deficient
mice, in spite of similar histopathological changes at the local level. Although
acute inflammation induced a significant increase in the number of
macrophages and DC in both mouse strains, in CD26-deficient mice the
increase of macrophages was twice than in WT animals (18.0 4.5 versus
41.3 5.8), whereas the increase in DC was more pronounced in WT mice.
Moreover, in the acute phase of inflammation an increased activation of NFB p65 subunit in the colonic mucosa of CD26-deficient animals was
determined. Observed modulatory effect of CD26 deficiency on immune cells
and activation of NF- B p65 subunit at the site of inflammation expands
current understanding and provides important insights into the role of DPP
IV/CD26 in the acute phase of DSS colitis.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Niemann-Pick type C (NPC) disease is an inherited, neurodegenerative
lysosomal storage disorder caused by mutations in NPC1/2 genes. At the
cellular level, the most prominent feature of the NPC disease is lysosomal
sequestration of low density lipoprotein derived cholesterol. The
mechanisms underlying the progressive neurodegeneration in the NPC
disease are still not fully established. In addition to defects in intracellular
lipid transport and disturbed endo/lysosomal pathway, a number of studies
are consistent with increased oxidative stress being a contributing factor in
the pathophysiology of NPC disease. Moreover, abnormalities in
mitochondria, the main source of reactive oxygen species in the cell, have
also been reported in NPC. Interestingly, it was shown that mitochondrial
cholesterol content was higher in NPC1-deficient cells than in wild-type (wt)
cells.
To determine the effect of cholesterol accumulation on oxidative stress in
NPC, we analysed antioxidant defence systems in CHOwt and CHO NPC1-null
cells. We measured the concentration of total glutathione and activity of
antioxidant enzymes superoxide dismutase (SOD) and catalase. While the
concentrations of glutathione and catalase activity do not seem to be
affected, our results show a significantly higher activity of SOD in NPC1-null
cells. Under conditions of oxidative stress (treatment with hydrogen
peroxide), NPC1-null cells show lower SOD activity than wt cells indicating a
defect in the antioxidant defence. We confirmed that the observed
alterations in SOD activity in NPC1-null cells are partially due to disturbed
activity of mitochondrial SOD (SOD2). The expression levels of SOD1
(cytosolic) and SOD2 are not changed in NPC1-null cells vs. wt cells.
Additionally, we showed that the defect in SOD activity in NPC1-null cells is
dependent on cholesterol levels since cholesterol depletion in these cells
completely reversed SOD activity to the levels as in wt cells.
Our results indicate that mitochondria are especially susceptible to oxidative
stress caused by cholesterol accumulation in NPC. Our findings suggest that
oxidative stress might contribute to the NPC disease and that lipid storage
reducing therapies could protect against this pathological process.
Poster Abstracts
P15
CHOLESTEROL-MEDIATED OXIDATIVE STRESS IN NIEMANN-PICK TYPE C
DISEASE INVOLVES A DEFECT IN THE ACTIVITY OF SUPEROXIDE DISMUTASE
1,2
1
2
1
Kristina Dominko , Martina Malnar , Domagoj Đikić , Silva Hećimović
1
2
Rudjer Boskovic Institute, Zagreb, Croatia; Department of Animal
Physiology, Faculty of Science, University of Zagreb, Zagreb, Croatia
[email protected]
87
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P16
EXPRESSION OF GALECTIN-3, AN ANTI-APOPTOTIC MOLECULE IS NOT
AFFECTED IN APOPTISIS PROVOKED WITH 17-DMAG, AN INHIBITOR OF
HSP90
Jerka Dumić, Sanja Dabelić, Tamara Zorbaz, Sandra Šupraha Goreta, Jozsef
Petrik, Ognjen Čulić, Karmela Barišić
University of Zagreb, Faculty of Pharmacy and Biochemistry, Ante Kovačića 1,
HR-10000 Zagreb, Croatia
[email protected]
88
Heat shock protein of Mr 90 kDa (Hsp90) comprises 2 homologous (Hsp90α and
Hsp90β) stress-inducible molecular chaperons that are encoded by separate
genes. These highly conserved, homo-dimeric ATP-dependent proteins,
abundantly expressed in eukaryotic cells, account for the maturation and
functional stability of a plethora of polypeptides termed Hsp90 client proteins,
including many proteins involved in tumorigenesis (e.g. anti-apoptotic proteins,
transcription factors, signal-transduction proteins, Tyr-kinase receptors, etc.).
Inhibition of HSP90 has been shown to be a promising therapeutic approach with
clinical relevance for treatment of specific tumour types. Orally bio-available
derivative of ansamycin antibiotic geldanamycin, 17-DMAG [17(dimethylaminoethylamino)-17-demethoxygeldanamycin] through inhibition of
Hsp90 causes down-regulation of Hsp90 client proteins which lead to impaired
signalling of apoptosis. Galectin-3, a β-galactoside lectin is a multifunctional
protein that is ubiquitously expressed in both intracellular and extracellular
environments as well as on the surface of different types of cells of many tissues.
Intracellular galectin-3 was shown to be a strong anti-apoptotic molecule, and it
is well known for its roles being correlated with the development and malignancy
of cancers and cancer drug resistance.
It this study we explored the effects of 17-DMAG on the expression of galectin-3,
different heat shock protein family members (Hsp90α, Hsp90β, Hsp70, Hsp27)
and Hsp90 client proteins involved in cell cycle regulation (cdk1, p(Tyr)-cdk1 and
cdc2) in human acute monocytic leukaemia THP-1 cells. Cell we treated with 0.5,
2, or 3 μM 17-DMAG for 24, 48 and 72 hours. Cytotoxic and pro-apoptotic effects
of 17-DMAG estimated by ApoToxGlo assay, were time and concentration
dependent. 17-DMAG slightly induced the expression of both Hsp90α and
Hsp90β, tremendously up-regulated the expression of HSP70, but did not affect
the expression of Hsp27. In parallel, 17-DMAG did not affect expression of
intracellular expression of galectin-3, an anti-apoptotic molecule important for
cell survival. It seems that molecular pathways resulting in apoptosis provoked by
inhibition of Hsp90 with 17-DMAG bypass galectin-3. These results encourage for
further studies focused on elucidation of galectin-3 role in apoptosis, but also the
effects of 17-DMAG on molecular level.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Osteoporosis and osteoarthritis are disorders common in elderly people and
closely associated with disability and morbidity. Development and
progression of both diseases is caused by the imbalance of the
osteoclastogenesis, osteoblastogenesis and cartilage metabolism. Galectin-3,
a member of the β-galactoside–binding proteins family, was found to be
involved in all of these processes. Consequently the aim of our study was to
optimise the method for determination of galectin-3 in serum and to assess
if serum concentration of galectin-3 could be used as a biochemical marker in
osteoporosis and osteoarthritis diagnosis.
Using the optimised ELISA method the concentration of galectin-3 in sera of
131 Slovene participants, 106 women and 25 men was determined. Galectin3 concentration was significantly higher in women (p = 0.026, N = 131) while
association with body mass index (p = 0.119, N = 126) and age (p = 0.990, N =
131) was not statistically significant. In addition, we found no difference in
serum galectin-3 concentration between participants with osteoporosis,
osteoarthritis and healthy controls (p = 0.277, N = 131). Moreover the
concentration did not correlate with bone mineral density, 25-hydroxyvitamin D concentration or any of the studied biochemical markers related to
bone turnover (C-terminal cross-linking telopeptide of type I collagen,
receptor activator of nuclear factor kappa-B ligand, osteoprotegerin or
parathormone). However we did found an association with dickkopf 1
concentration in the group of all participants (p = 0.012, N = 47) and in the
subgroup of healthy participants (p = 0.023, N = 26), but the association was
not significant in the subgroup of osteoporotic participants (p = 0.222, N =
21).
Based on the obtained results we concluded that serum galectin-3
determination is not useful as a biochemical marker of osteoporosis or
osteoarthritis diagnosis. Statistically significant association between galectin3 concentration and dickkopf-1 concentration points to a possible role of
galectin-3 in wnt/β-catenin signalling pathway that could be further
evaluated in additional studies.
Poster Abstracts
P17
SERUM GALECTIN-3 IN OSTEOPOROSIS AND OSTEOARTHRITIS
Mateja Prunk1, Jerka Dumić2, Janja Marc1
1
2
University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenija; University
of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
[email protected]
89
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P18
FUNCTIONAL CHARACTERISATION OF PARALOGOUS SsbB IN Streptomyces
coelicolor
1
1
2
1,3
4
Želimira Filić , Tina Paradžik , Nives Ivić , Ana Bielen , Babu A. Manjasetty ,
5
6
2
1
Paul Herron , Dagmara Jakimowicz , Marija Luić , Dušica Vujaklija
1
Division of Molecular Biology, 2Division of Physical Chemistry, Rudjer Boskovic
Institute, Croatia; 3Department of Biochemical Engineering, Faculty of Food
Technology and Biotechnology, Croatia; 4European Molecular Biology Laboratory,
5
Grenoble Outstation and Unit of Virus Host-Cell Interactions, France; Strathclyde
Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, UK;,
6
Department of Molecular Microbiology, University of Wroclaw, Poland
Poster Abstracts
[email protected]
90
Single stranded DNA binding proteins (SSBs) are essential for DNA metabolism in
all organisms and viruses. These proteins protect single stranded DNA (ssDNA)
intermediates and disrupt unproductive secondary structures. Our bioinformatics
analysis of sequenced bacterial genomes revealed the presence of paralogoues
SSBs in many bacteria thus indicating highly dynamic evolution of SSB proteins in
Eubacteria. However the role of duplicated SSB proteins is poorly studied. S.
coelicolor is the model representative of Streptomyces bacteria, the largest genus
of Actinobacteria. Streptomyces species exhibit a complex lifecycle involving
mycelial growth, spore formation and ability to produce a plethora of secondary
metabolites including antibiotics and many other useful compounds. Since S.
coelicolor has two paralogus genes encoding SSBs (ssbA and ssbB) we have
selected this complex bacterium to study biological role(s) of paralogous SSB
proteins. SSB proteins from most prokaryotic species function as tetramers that
bind to ssDNA through structurally conserved N-terminal folding motifs (OB
folds), while the C-terminal domain is responsible for protein interactions. We
will show here interesting variations of 3D structure of two SSBs. Next molecular
analyses showed that promoter regions of ssb genes differ significantly. In
concert with this, expressions of ssb genes varied throughout development
indicating that SSB proteins acquired different cellular functions. Expression of
ssbA is constant and high during life cycle, decreasing towards late stationary
phase, while the expression of ssbB is low at all-time. In minimal medium the
ssbB is significantly upregulated. Gene disruptions strongly indicated that SsbA is
essential for survival while SsbB is important during the sporulation process. To
get a better insight into the role of SsbB and the complex mechanism during
chromosome segregation in reproductive phase of growth we have constructed
double and triple mutant strains carrying mutations in ssbB gene and genes
reported previously to be important for the chromosomal segregation (for
example parB or smc). By fluorescence microscopy we examined the effect of
these mutations on chromosome segregation and spore formation. The results of
these experiments showed severe defects in nucleoid segregations during
sporulation as well as unequal distribution of DNA in spore compartments.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P19
ALGAL ENDOSYMBIONTS IN EUROPEAN HYDRA STRAINS REFLECT MULTIPLE
ORIGINS OF THE ZOOCHLORELLA SYMBIOSIS
1
1
1
2
Nives Rajević , Goran Kovačević , Mirjana Kalafatić , Sven Gould , William
2
1
Martin , Damjan Franjević
1
Department of Biology, Division of Zoology, Faculty of Science, University of
Zagreb, HR-10000 Zagreb, Croatia; 2Instutute of Molecular Evolution,
Henrich-Heine University, 40225 Düsseldorf, Germany
[email protected]
Poster Abstracts
Symbiotic associations are of broad significance in evolution and biodiversity.
Green Hydra is a classic example of endosymbiosis. In its gastrodermal
myoepithelial cells it harbours endosymbiotic unicellular green alga, most
commonly from the genus Chlorella. Hydra is a single freshwater polyp that
inhabits shallow lakes and calm, slow-moving waters. It belongs to the
hydrozoan clade Aplanulata within the deep-branching eumetazoan phylum
Cnidaria. It provides useful model system for comparative research in
development and evolution, both for investigations of early branching
metazoans and for the study of plant-animal symbioses. Recent phylogenetic
analyses that included most globally identified Hydra species demonstrated
they can be divided into four groups. Here we investigate the phylogeny of
algal endosymbionts from green Hydra strains, collected from six different
geographical sites. All strains were endosymbiotic algae isolated from green
Hydra; four from different localities in Croatia, one from Israel and one from
Germany. We used nuclear (18S rDNA, the ITS region) and chloroplast
markers (16S, rbcL) for maximum likelihood phylogenetic analyses of 547
different sequences spanning chlorophyte diversity. We focussed on the
question of whether native symbionts of Croatian H. viridissima strains
descend from two or more symbiotic events or whether symbiosis with
Chlorella occurred only once in the distant past followed by subsequent
cospeciation and the secondary origin of free-living algal strains from
escaped endosymbionts. Resulting phylogenetic trees based on ITS region,
rbcL gene, 16S rRNA gene and 18S rDNA gene do not support a monophyletic
origin of endosymbiotic algal strains isolated from Croatian green Hydra
hosts. It thus appears that Hydra – algal endosymbioses have been
established multiple times during the evolution of these strains.
91
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P20
BIOMONITORING OF GENOME INTEGRITY IN HUMAN POPULATION WITH
THYROID DISEASES: A PILOT STUDY
1
2
2
1
Marko Gerić , Renato Janušić , Božena Šarčević , Vera Garaj-Vrhovac
1
Institute for Medical Research and Occupational Health, Zagreb, Croatia;
2
Clinical Hospital for Tumours, Zagreb, Croatia
[email protected]
Poster Abstracts
Presented results are part of three-phase-study that aims to improve cancer
prevention. In the phase one of the study, the genome integrity is
biomonitored using micronucleus and comet tests in patients with thyroid
diseases. Thyroid cancer is one of the fastest growing types of cancer in the
world. Its molecular pathogenesis and mechanisms are closely related to
changes in the genome what makes it a good model for such study.
The study population consisted of 24 volunteer patients (18 female: 6 male,
average age 48.75 years) diagnosed with follicular adenoma (9), papillary
cancer (8), goitre (6) and thyroiditis (1). The analysis of DNA damage in
peripheral blood lymphocytes for this group resulted with average total
number of: micronuclei (MNi) 12.43±4.14, nucleoplasmic bridges (NPBs)
4.05±3.49, and nuclear buds (NBs) 6.43±4.36 per 1000 binuclear
lymphocytes. Comet assay parameters were analysed in 200 lymphocytes
and the mean values were for tail intensity (TI) 3.83±2.00 and for tail
moment (TM) 0.18±0.12. When compared to control population that
consisted of 24 healthy volunteers (18 female: 6 male, average age 48.46
years), significantly (p<0.05) lower average total number of MNi (5.55±3.10),
NPBs (0.55±0.69), NBs (2.80±1.54), TI (2.19±0.76) and TM (0.09±0.04) was
observed. At the same time cytokinesis-block proliferation index (CBPI)
2.031±0.098 vs 2.067±0.082 did not differ statistically between two groups.
The results of this pilot study suggest that patients with thyroid diseases
have more DNA damage. In the next phases of the study the DNA damage of
larger number of volunteer patients will be associated with mutant proteins
from diseased thyroid tissues and with the telomere length. Overall results
will provide great basis for detection of biomarkers that could be used for
better risk assessment of cancer diseases and therefore for improvement of
cancer prevention.
92
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P21
SUPPRESSION OF HIF-1 PATHWAY ENHANCES IL-6 ACTION IN CULTURED
HUMAN MYOBLASTS
1,*
1,*
1
1,2
Katarina Gros , Katarina Miš , Urška Matkovič , Matej Podbregar , Tomaž
1
1
1
Marš , Zoran Grubič , Sergej Pirkmajer
1
[email protected]
Interleukin-6 (IL-6), a prototypical muscle-derived cytokine, regulates various
aspects of skeletal muscle function, including energy metabolism and
myogenesis. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric
transcription factor, comprised of the oxygen-responsive HIF-1α subunit and
the constitutive HIF-1β subunit. Once activated, HIF-1 leads to plethora of
downstream events that help to maintain homeostasis under oxygendeprived conditions. Emerging data suggest that IL-6 and HIF-1 may
participate in an autocrine signalling loop, however the extent to which the
two factors are linked in skeletal muscle has not been explored. Here we
determined whether HIF-1 modulates IL-6 signalling in cultured human
myoblasts. Using siRNA we suppressed the expression of HIF-1α and/or HIF1β. In siRNA-transfected myoblasts up-regulation of HIF-1 target genes
(vascular endothelial growth factor-A, phosphoglycerate kinase-1 and the
natural antisense HIF-1α transcript aHIF) was markedly diminished upon
exposure to hypoxia or HIF-1α inducer CoCl2, indicating efficient suppression
of HIF-1 pathway. Conversely, gene silencing of HIF-1α or HIF-1β enhanced
Tyr705
IL-6-stimulated phosphorylation of STAT3
, a marker for the activation of
the canonical JAK/STAT pathway downstream of IL-6 receptor complex
gp130/IL-6R. Furthermore, concurrent suppression of HIF-1α and HIF-1β had
an additive effect on phosphorylation of STAT3, even as the expression of
total STAT3 remained unaltered. These results indicate that HIF-1 inhibits IL6-stimulated activation of the JAK/STAT pathway. To explore the underlying
mechanisms, we determined expression of IL-6 receptor subunits gp130 and
IL-6Rα. Expression of the signal-transducing subunit gp130 remained
unaltered in HIF-1α-depleted myoblasts. Conversely, the IL-6-binding subunit
IL-6Rα was up-regulated upon depletion of HIF-1α. Consistent with the
augmentation of STAT3 phosphorylation, expression of IL-6Rα was further
increased when HIF-1α and HIF-1β were concurrently suppressed. These
results demonstrate that suppression of HIF-1 pathway enhances IL-6stimulated activation of the JAK/STAT pathway in cultured human myoblasts.
Elevated expression of IL-6Rα may provide one possible underlying
mechanism for the enhanced lL-6 action in HIF-1-depleted myoblasts.
Collectively, our findings implicate a role for HIF-1 as a negative regulator of
IL-6 signalling in skeletal muscle.
Poster Abstracts
University of Ljubljana, Faculty of Medicine, Institute of Pathophysiology, Ljubljana,
Slovenia; 2University Medical Centre Ljubljana, Slovenia
*
These authors have contributed equally to this work.
93
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P22
THE ASSOCIATION BETWEEN 5-HT1A SEROTONIN RECEPTOR GENE
POLYMORPHISM AND EXTRAPYRAMIDAL SIDE EFFECTS IN HALOPERIDOLTREATED PATIENTS WITH SCHIZOPHRENIA
1
1
1
2
Mirko Grubor , Dubravka Svob Strac , Maja Mustapic , Maja Zivkovic , Alma
3
3
1
1
Mihaljevic-Peles , Marina Sagud , Nela Pivac , Dorotea Muck-Seler
1
Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia;
2
3
Clinic for Psychiatry Vrapce, Zagreb, Croatia; Clinic for Psychiatry University
Hospital Center Zagreb, Croatia
[email protected]
94
Schizophrenia is a serious chronic psychiatric disorder with etiology and
neurobiological basis still insufficiently known. Different studies suggest the
involvement of many environmental factors and genes in the development of
schizophrenia, as well as interactions between genes and the environment. The
disease is treated with so-called typical or first generation antipsychotics
(haloperidol, fluphenazine), effective in the treatment of positive symptoms, and
atypical or second generation antipsychotics (olanzapine, risperidone,
quetiapine), which can reduce both positive and negative symptoms of
schizophrenia. Despite various antipsychotic drugs, some schizophrenic patients
do not respond satisfactorily, while others develop side-effects that substantially
compromise the treatment, leading to discontinuation of therapy and frequent
relapse of the disease. The most common adverse effects of typical
antipsychotics are acute (dystonia, parkinsonism, akathisia) and chronic (tardive
dyskinesia) extrapyramidal motor side effects (EPS). As the role of serotonin
receptor genes in the development of antipsychotics side effects is not clear, the
aim of the study is to examine the association of serotonin type 1A receptor gene
(HTR1A) polymorphism with the development of acute EPS in schizophrenic
patients following haloperidol therapy. The study included about 200 patients
with schizophrenia diagnosed according to the DSM-IV criteria. The severity of
EPS in schizophrenic patients following 2 weeks haloperidol (15 mg/d)
monotherapy was evaluated using Simpson Angus Rating Scale for
Extrapyramidal Side Effects, Barnes Akathisia Rating Scale and Extrapyramidal
Symptom Rating Scale. The genomic DNA was extracted from the whole blood
and a polymorphism (rs6295) located in HTR1A was genotyped using TaqMan
Real-Time allelic discrimination. The results demonstrated significant differences
in the genotype and allele frequencies of HTR1A polymorphism between
schizophrenic patients with or without EPS, suggesting potential protective role
of HTR1A variant. The results imply that in addition to the dopaminergic system,
serotonergic mechanisms might be also involved in the development of EPS,
either by the effects on dopamine release or via serotonergic receptors as
molecular targets for antipsychotics. However, further studies are needed to
confirm these results and to elucidate biological mechanisms underlying present
findings.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Nme (Nucleoside diphosphate kinases NDPK, Nm23) is an evolutionary
conserved family of proteins present in all three domains of life. Their
canonical role is the maintenance of the NTP pool in the cell by the
phosphorylation of NDPs, although many other functions have been
described. In vertebrates, Nme proteins are divided in two distinct groups.
Group I has diversified after the appearance of vertebrates. Group II contains
members that mostly have more ancient origins, with homologues present in
unicellular eukaryotes. Ten Nme proteins have been identified in human:
Nme1-4 belong to Group I, and Nme5-9 to Group II. Human Nme10, aka
XRP2, has only a partial NDPK domain and does not belong to either group.
Human Nme1 is the first know and the most studied metastasis suppressor.
Decreased expression of Nme1 has been linked to metastatic phenotype of
several tumor types. Due to a large research effort, we have a good
knowledge of Nme1 properties in complex animals such as mammals. Nme
seems to be involved in many biological processes such as metastasis,
proliferation, development, differentiation, ciliary functions, vesicle
transport and apoptosis in vertebrates. Biochemical mechanisms of these
processes are still largely unknown. In our earlier study, we demonstrated
that the earliest-branching simple animals – sponges (Porifera) possess a
NmeGp1 homolog with similar biochemical properties as human Nme1/2.
The work on Nme1/2 in unicellular eukaryotes has been so far very limited.
No data are available on its properties in unicellular relatives of animals.
Herein, we present the results of our research on NmeGp1 of Capsaspora
owczarzaki, a member of Filasterea – a group of unicellular organisms closely
related to animals. We show that the NmeGp1Co has properties strikingly
similar to the sponge and human homologues. The protein did not change
significantly during the transition to multicellularity and it has all the
properties that make it a metastasis suppressor. This implies that metastasis
suppression in mammals may be related to the intricate Nme1 pathways and
interaction networks rather than a simple change of biochemical properties.
Poster Abstracts
P23
NMEGP1 GENE/PROTEIN FROM Capsaspora owcarzaki - STRUCTURE,
FUNCTION AND EVOLUTION
1
2
1
1
Helena Ćetković , Maja Herak Bosnar , Drago Perina , Andreja Mikoč , Robert
2
3
1,3
Belužić , Innaki Ruiz-Trillo , Matija Harcet
1
Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia;
2
Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia,
3
Institut de Biologia Evolutiva (UPF-CSIC), Barcelona, Spain
[email protected]
95
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P24
ENHANCED NK CELL-DEPENDENT SUREVEILLANCE OF MELANOMA IN
NKG2D-DEFICIENT MICE
Vedrana Jelenčić, Felix M. Wensveen, Maja Gulin, Bojan Polić
School of Medicine, University of Rijeka, Rijeka, Croatia
[email protected]
Poster Abstracts
NKG2D and NCR are both activating receptors expressed on all murine NK
cells early in NK cell development. For NKG2D receptor majority of its’
ligands are known, such as Rae 1 family, H60 and MULT, while for NCR
receptor most of the ligands are still unknown.
It has been published that NKG2D deficient NK cells show perturbation in size
of some NK cell subpopulations, impairments in NK cell development,
enhanced proliferation of NK cells and augmented sensitivity to apoptosis.
NKG2D deficient mice show an enhanced NK cell-mediated resistance to
MCMV infection. NKG2D deficient mice are also less responsive to tumor
targets expressing NKG2D ligands. However, ability of NKG2D deficient mice
to control tumors which don’t express NKG2D ligands is still unknown.
In our model we are using B16 melanoma cell line, which does not express
NKG2D ligands. We observed prolonged survival of NKG2D -/- mice in
comparison to wild type mice. Prolonged survival of NKG2D deficient mice is
result of NK cell activity since after NK cell depletion these differences were
lost. Although B16 cells don’t express NKG2D ligands they expresses
unknown NCR ligands, so to further analyse this observations we included in
our study also NCR deficient mice ( NCR gfp/gfp) and double knock-out mice
(NKG2D-/-NCR gfp/gfp). NKG2D deficient mice were the best in controling
gfp/gfp
methastasis development while the NCR
mice were the worst. Same
results were observed after MCMV infection at early time points (4th day post
infection).
Our findings indicate at possible interaction between these two receptors
and show us that NKG2D deficiency results in hyperactive NK cells which are
then better in controlling MCMV infection and tumor growth.
96
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P25
NON-BIOSYNTHETIC HUMAN MILK OLIGOSACCHARIDES – NATURAL
COMPONENTS OR ARTEFACTS?
1
2
3
Marko Jovanović , Richard Tyldesley-Worster , Gottfried Pohlentz , Jasna
1
Peter-Katalinić
1
Department of Biotechnology, University of Rijeka, Croatia; 2Waters
Corporation, Wilmslow, United Kingdom; 3Institute for Hygiene, University of
Muenster, Germany
[email protected]
1. Zivkovic, A.M.; German, J.B.; Lebrilla, C.B.; Mills, D.A. Human milk
glycobiome and its impact on the infant gastrointestinal microbiota. Proc.
Natl. Acad. Sci. USA 2011, 108, 4653–4658.
2. Newburg, D.S.; Ruiz-Palacios, G.M.; Morrow, A.L. Human milk glycans
protect infants against enteric pathogens. Annu. Rev. Nutr. 2005, 25, 37–58.
3. O’Hara, A.M.; Shanahan, F. The gut flora as a forgotten organ. EMBO Rep.
2006, 7, 688–693.
4. Jovanović M, Tyldesley-Worster R, Pohlentz G, Peter-Katalinić J., MALDI QTOF CID MS for diagnostic ion screening of human milk oligosaccharide
samples. Int J Mol Sci 2014, 15, 6527-6543
5. Newburg, D.S.; Ruiz-Palacios, G.M.; Morrow, A.L. Human milk glycans
protect infants against enteric pathogens. Annu. Rev. Nutr. 2005, 25, 37–58.
Poster Abstracts
Human milk oligosaccharides (HMO) represent an important class of
biomolecules which provide health benefits to infants. The most understood
function is their interaction with infant’s gut microflora, stimulating the
growth of probiotic bacteria (1,2) and providing immunological benefits to
the infant (3). Their structural characterization as an essential prerequisite to
understanding their function has been followed in a number of studies. We
have recently published a MALDI Q-TOF MS analysis of a chromatographic
HMO fraction (4) and found alongside the classical structures a number of
non-biosynthetic HMOs. Such HMO structures were not reported in the
literature previously, raising the question whether these structures are an
artefact of the work-up procedure, or if they are produced in vivo. Limited
enzymatic hydrolysis of HMO structures in vivo has been reported previously
(5), with focus on β-D-galactosidase and N-acetyl-β-hexosaminidase. In
contrast, our analyzes of HMO structures suggested other sources of
truncation were present, not reported previously. The data suggest that
these truncated structures may not be exclusively an artefact of
chromatographic procedure, but further studies directly comparing different
HMO purification procedures with appropriate controls are necessary to
clarify this feature.
97
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P26
INFLUENCE OF EXTRACTION METHODS ON ANTIPROLIFERATIVE POTENTIAL
OF CHAMOMILE FLOWER EXTRACTS
1
2
1
Marijana Jukić , Aleksandra Cvetanović , Katarina Mišković , Jaroslava Švarc2
1
Gajić , Ljubica Glavaš-Obrovac
1
Faculty of Medicine, J.J. Strossmayer University of Osijek, Huttlerova 4, HR31000 Osijek, Croatia; 2 Faculty of Technology, University of Novi Sad, Bulevar
cara Lazara 1, SR-21000 Novi Sad, Serbia
[email protected]
Poster Abstracts
Antiproliferative activity of seven chamomile (Matricaria chamomilla L.)
flower extracts (CFEs), prepared by different extraction processes, were
examined on four human tumour cell lines (HuT 78, K562, HeLa, and NCIH358), peripheral blood mononuclear cells (PBMC) and normal Madin Darby
canine kidney (MDCK I) cells as well. Cytotoxicity of CFEs (range of
concentration 0.0001 mg/mL to 0.5 mg/mL) was tested by MTT assay after
72 hrs of incubation. A plant flavanoid apigenin was used in a concentration
range of 0.01 mg/mL to 0.5 mg/mL as a standard compound. To extract
flavonoids from native and fermented chamomile flowers different
extraction methods, as is water under high pressure, Soxhlet extraction,
microwave-assisted extraction, and ultrasound-assisted extraction were
used. Composition of analyzed extracts was determined by LC/MS method. A
highest concentration of apigenin and chlorogenic acid compared to all
tested CFEs were found in CFE III (isolated by ultrasound-assisted extraction,
fermented) sample; and apigenin-7-O-glucoside is presented at the highest
concentration in CFE VII (isolated by Soxhlet extraction, fermented) sample.
Both extracts showed the highest cytotoxic effect on all tested cell lines.
Morphological changes and apoptotic features of HeLa cells were obtained
after exposure to CFEs III and VII at GI50 (GI50 after 48 hrs: 0.099 mg/mL; GI50
after 72 hrs: 0.185mg/mL for the CFE III, and GI50 after 48 hrs: 0.075 mg/mL;
GI50 after 72 hrs: 0.079 mg/mL for the CFE VII) as well. In conclusion, our
results indicate that in vitro antiproliferative potential of CFEs is dependent
on extraction process, extracts’ compositions, applied concentration, and
treated cells.
98
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P27
CULTURABILITY OF MULTIDRUG-RESISTANT AND DRUG-SENSITIVE STRAINS
OF Acinetobacter baumannii ON DRY PLASTIC SURFACES
Denis Juraga, Marina Matešić, Diana Jurčić-Momčilović, Ivana Gobin
Department of Microbiology and Parasitology, School of Medicine in Rijeka,
Croatia
[email protected]
Poster Abstracts
Acinetobacter baumannii ability to survive under a wide range of
environmental conditions and to persist for extended periods of time on
surfaces makes it a frequent cause of intrahospital infections. The aim of this
study was to examine the culturability of multidrug – resistant and drug –
sensitive clinical strains A. baumannii on dry plastic surfaces. Bacterial strains
used in this study were A. baumannii multidrug – resistant strain ATCC BAA –
1605 and drug – sensitive strain ATCC 19606, as well as 4 clinical isolates
(strains 771, 53154, 56781 and 54531). Bacterial inoculums were prepared in
sterile tap water and 6 x 20 μl of bacterial suspension (~108 cfu/ml) were
deposited in 96 wells plates and dried for one hour under laminar flow hood.
To test bacterial viability the Bacterial Viability Kit LIVE/DEAD® BacLight™
dying were used. At various intervals, the bacteria was rehydrated in sterile
tap water and plated on LB agar to determine the number of culturable
bacteria. All tested A. baumannii strains showed resistance to drying over a
long period. Sensitive strains of A. baumannii, ATCC 19606 and 771, were
culturable from 70 to 90 days of drying, while multidrug – resistant strain A.
baumannii ATCC BAA – 1605 was culturable after 100 days in dry conditions.
Resistant clinical isolates of A. baumannii 53154, 56781, and 54531 were
culturable after 100 days of exposure to drying. So, in hospital environment,
special attention should be paid to the presence of A. baumanii on dry
plastics surfaces.
99
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P28
CELL
LOCALIZATION
AND
SEX-DEPENDENT
EXPRESSION
OF
CHLORIDE/FORMATE EXCHANGER CFEX (Slc26a6) IN RAT KIDNEYS
1
1
2
1
Dean Karaica , Davorka Breljak , Jovica Lončar , Marija Ljubojević , Carol M.
1
1
1
1
Herak-Kramberger , Vedran Micek , Ivana Vrhovac , Jana Ivković , Ivan
2
2
2
3
Mihaljević , Petra Marić , Tvrtko Smital , Birgitta Burckhardt , Gerhard
Burckhardt3, Ivan Sabolić1
1
Poster Abstracts
Molecular Toxicology, Institute for Medical Research and Occupational Health,
Zagreb, Croatia; 2Laboratory for Molecular Ecotoxicology, Division for Marine and
Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia; 3Institute of
Systemic Physiology and Pathophysiology, Center of Physiology and Pathophysiology,
University Medical Center Göttingen, Germany
100
[email protected]
The chloride/formate exchanger CFEX, a member of the solute carrier family 26
(SLC26A6 in humans/Slc26a6 in rodents), in various mammalian organs mediates
transport of anions including chloride, bicarbonate, oxalate, formate and
hydroxyl ion. Except mice, its distribution and expression in the kidneys of other
species are poorly documented. Here we used a commercial polyclonal antipeptide antibody (CFEX-Ab) to study distribution of protein (rCFEX) along the
nephron, and its possible sex-related expression in adult male (M) and female (F)
Wistar rats. In order to validate specificity of the CFEX-Ab, we transiently
transfected the HEK293 cells with the rCFEX cDNA; total RNA was isolated from
the rat kidney, cDNA was synthesized and amplified by PCR using specific
primers, a full-length rCFEX cDNA was inserted into the pcDNA3.1/His C and used
for transformation of the competent DH5α E. coli, and the HEK293 cells were
transfected with this vector using polyethyleneimine. Specificity of the CFEX-Ab
was verified by immunofluorescence cytochemistry (IFC); the antibody strongly
stained the plasma membrane of rCFEX-transfected cells, whereas the staining
was not observed in mock-transfected cells and in the rCFEX-transfected cells
incubated with the peptide-blocked antibody. The CFEX-Ab was further
characterized by IFC on tissue cryosections and by Western blotting (WB) of
isolated total cell (TCM) or brush-border (BBM) membranes. By IFC, the CFEX-Ab
stained the BBM of proximal tubules (PT) with heterogeneous intensity (S1 ~ S2 >
S3). By WB of TCM or BBM, the CFEX-Ab labelled a single protein band of 120
kDa. The rCFEX-related staining of PT BBM and labeling of the protein band were
abolished with the immunizing peptide-blocked antibody. The intensity of rCFEXrelated staining in PT BBM, as well as the density of rCFEX-related protein band
exhibited strong sex differences, M > F. The observed expression of rCFEX protein
was downregulated by castration and unaffected by ovariectomy, whereas in the
sex hormone-treated gonadectomized males, testosterone upregulated, while
estrogen and progesterone had no effect on the renal expression of protein.
Collectively, in the rat kidneys the rCFEX protein is localized to the PT BBM
showing zonal differences (cortex outer stripe) and M-dominant expression
due to androgen stimulation.
P29
CAN ACETYLCHOLINESTERASE MUTATIONS HELP CREATE MORE EFFICIENT
REACTIVATORS FOR ORGANOPHOSPHORUS COMPOUNDS POISONING
TREATMENT?
1
1
2
2
Maja Katalinić , Goran Šinko , Florian Nachon , José Dias , Nikolina Maček
1
1
Hrvat , Zrinka Kovarik
1
Institute for Medical Research and Occupational Health, Zagreb, Croatia;
2
Département de Toxicologie, Institut de Recherche Biomédicale des Armées,
Grenoble, France
[email protected]
Ever since its discovery at the turn of the 20 th century, acetylcholinesterase
(AChE) has been a focus of investigation in biochemistry, biomedicine and
toxicology, owing to its irreplaceable role in neurotransmission.
Organophosphorus compounds (OPs) were synthesized to modulate AChE
activity as a way of efficient pest control and became a great problem due to
intentional or unintentional human poisonings. The development of more
effective treatment for OP poisoning still presents a challenge for researchers in
this field. In the 1950s, pyridinium based oximes such as 2-PAM and HI-6 were
developed as reactivators of OP-inhibited AChE and introduced into medical
practice. However, they were synthesized before any knowledge about the AChE
active site structure was gained, and are therefore not as efficient in OP-inhibited
AChE reactivation as one would expect. Today, since we possess knowledge
about the fine architecture of AChE and computational techniques, a more
rational approach in oxime design should be made a priority. Nevertheless, there
is still too little experimentally obtained kinetic data on interactions of OP-AChE
w.t./mutants with oximes to complete a high-quality structure-activity
relationship scheme. Only with such data can AChE mutations help create more
efficient reactivators for OP poisoning treatment. To meet the requirements for a
step forward in the synthesis of more efficient oximes, we bring a full-scope
interactions kinetic profile for a set of five AChE mutants with structurally related
pyridinium oximes. Results indicated residues at the choline binding site (Y337,
F338) and the acyl pocket (F295) as ones influencing the placement of oximes
into the right position for reactivation, while the amino acids at the peripheral
site (Y124, W286) dictated the binding affinity for bispyridinum oximes and
presented a limitation to their reactivation efficiency. Results also indicated that
the aromatic aspects of oximes are favourable for the formation of required
interactions but the two aromatic rings in the oximes were shown to be a
disadvantage. On the other hand, several mutants showed favourable
characteristics that could enable the development of pseudo-catalytic scavengers
for OP detoxification. By means of computational modelling we suggested
possible oxime orientations/interactions within the phosphylated AChE active
site. We can generally assert that we have obtained a clearer concept as to
where we can focus the design of oximes in the future.
This work was supported in part by the Croatian-French bilateral grant (PIs: M. Katalinić, F.
Nachon).
Poster Abstracts
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
101
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P30
IN VIVO AND IN VITRO ANALYSIS OF PLANT SERYL-tRNA SYNTHETASE
INTERACTOME
1
1
2
3
Mario Kekez , Jasmina Rokov Plavec , Nataša Bauer , Genadij Razdorov ,
4
4,5
1
Vesna Hodnik , Gregor Anderluh , Ivana Weygand-Đurašević
1
University of Zagreb, Faculty of Science, Department of Chemistry, Zagreb,
Croatia; 2University of Zagreb, Faculty of Science, Department of Biology,
3
4
Zagreb, Croatia; University of Zagreb, Zagreb, Croatia; University of
Ljubljana, Biotechnical Faculty, Department of Biology, Ljubljana,
5
Slovenia; National Institute of Chemistry, Laboratory for Molecular Biology
and Nanobiotechnology, Ljubljana, Slovenia
[email protected], [email protected]
102
Aminoacyl-tRNA synthetases (aaRS) play significant role in translation
process by binding amino acids to their cognate tRNAs. Once the tRNA is
charged, a ribosome can transfer the amino acid from the tRNA onto a
growing peptide, according to the genetic code. Beyond translation, these
enzymes can be involved in diverse cellular functions. Characterization of
these non-canonical functions broadens our knowledge in functional
proteomics. The studies of aaRS assemblies in plants are scarce, therefore
our main scientific goals were to determine and characterize potential
protein-protein interacting partners of cytosolic seryl-tRNA synthetase
(SerRS) from plant Arabidopsis thaliana. We conducted yeast-two hybrid
(Y2H) screen on cDNA libraries followed by DNA sequencing, as well as
tandem affinity purification combined with mass spectrometry analysis (TAPMS). Among several SerRS potential interacting partners revealed by Y2H
screen, BEN1, protein potentially involved in metabolism of brassinosteroid
hormones was the most promising interacting partner. Interaction of BEN1
and SerRS was also analyzed in vitro using isothermal calorimetry titration
(ITC), pull-down and surface plasmon resonance method (SPR). Probably due
to the nature of interaction we were not able to retrieve positive results
using pull-down assay and ITC, but SPR gave us positive confirmation and
information about dissociation constant. To pinpoint regions responsible for
protein-protein interaction we prepared shortened variants of both SerRS
and BEN1 proteins which will be further subjected to biophysical analysis.
Biophysical determination of possible interactions between SerRS and BEN1
variants will give us insight in additional cell functions and physiology of both
SerRS and BEN1 proteins. TAP-MS analysis of transgenic plant overexpressing
SerRS-TAP construct identified several SerRS potential interacting partners
yet to be confirmed in vitro. Revealing plant SerRS interactome has great
importance in shedding light on novel functions of SerRS beyond translation.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P31
GLYCANS ARE A NOVEL BIOMARKER OF CHRONOLOGICAL AND BIOLOGICAL
AGE
1
2
2
3
Toma Keser , Jasminka Krištić , Frano Vučković , Cristina Menni , Lucija
2
2
2
2
Klarić , Ivona Bečeheli , Maja Pučić-Baković , Mislav Novokmet , Massimo
Mangino3, Kujtim Thaqi2, Pavao Rudan4, Natalija Novokmet4, Jelena Šarac4,
Saša Missoni4, Ivana Kolčić5, Ozren Polašek5, Igor Rudan6, Harry Campbell6,
6
7
3
6
Caroline Hayward , Yurii Aulchenko , Ana Valdes , James F. Wilson , Olga
1
8
9
3
1,2
Gornik , Dragan Primorac , Vlatka Zoldoš , Tim Spector , Gordan Lauc
1
University of Zagreb, Faculty of Pharmacy and Biochemistry, A. Kovačića 1,
10000 Zagreb, Croatia; 2Genos Glycobiology Laboratory, Hondlova 2/11,
3
10000 Zagreb, Croatia; Department of Twins Research and Genetic
4
Epidemiology, Kings College London, London WC2R 2LS, UK; Institute for
5
Anthropological Research, 10000 Zagreb, Croatia; Faculty of Medicine,
6
University of Split, 21000 Split, Croatia; Centre for Population Health
Sciences, The University of Edinburgh Medical School, Edinburgh EH8 9YL, UK;
7
Institute of Cytology and Genetics SD RAS, Novosibirsk 630090, Russia;
8
9
University of Osijek School of Medicine, Osijek, Croatia; University of
Zagreb, Faculty of Science, Horvatovac 102A, 10000 Zagreb, Croatia
[email protected]
Poster Abstracts
Fine structural details of glycans attached to the conserved N-glycosylation
site significantly affect function of individual IgG molecules, but also mediate
inflammation at the systemic level. By analysing IgG glycosylation in 5117
individuals from four European populations we have revealed very complex
patterns of changes of IgG glycosylation with age. Several IgG glycans
(including FA2B, FA2G2, FA2BG2) changed considerably with age and the
combination of these three glycans can explain up to 58% of variance in
chronological age, significantly more than other markers of biological age like
telomere lengths. The remaining variance in these glycans strongly
correlated with physiological parameters associated with biological age. Thus
IgG glycosylation appears to be closely linked with both chronological and
biological age. Considering the important role of IgG glycans in inflammation,
and since the observed changes with age promote inflammation, changes in
IgG glycosylation also seem to represent a factor contributing to aging.
103
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P32
OBESITY PHENOTYPE OF RATS WITH CONSTITUTIONAL HYPERACTIVITY OF
SEROTONIN TRANSPORTER
Maja Kesić, Darko Orešković, Lipa Čičin-Šain
Laboratory of Neurochemistry and Molecular Neurobiology, Department of
Molecular Biology, Rudjer Boskovic Institute, 10000 Zagreb, Croatia
[email protected]
104
An inverse relationship between brain serotonin (5-hydroxytryptamine, 5HT)
system and food intake has been known for over 30 years. Specifically, increase
of brain 5HT activity leads to decreased food intake and weight gain, and vice
versa. The 5-HT transporter (5HTT) is an important regulator of brain 5HT
function as it is responsible for reuptake of 5HT in serotonergic nerve endings.
Studies have shown that inhibition of 5HTT reduces food intake and body weight
gain in rats and humans, but much remains to be learned about the 5HT
mechanisms underlying the regulation of obesity phenotype.
In our research we use Wistar-Zagreb 5HT (WZ-5HT) rats, an animal model with
constitutively high or low 5HTT activity (termed high-5HT and low-5HT subline),
developed in our laboratory by selective breeding of animals toward extremes of
peripheral 5HTT activity. Besides in periphery, two sublines of WZ-5HT rats differ
also in the central 5HT homeostasis, as evidenced from neurochemical and
behavioral studies.
Here, we aimed to compare phenotypes of animals from 5HT-sublines regarding
their body weight, adiposity and fat distribution, as well as their food intake.
Body weight (BW) accumulation was monitored from birth to senescence and
body mass index (BMI, ratio of body weight and body lenght2), total body fat
mass and pattern of adiposity were determined at 3 and 8 months of age.
Animals from 5HT-sublines show clear differences in obesity phenotypes.
Specifically, rats from the high-5HT subline have shown a higher body weight in
comparison to their low-5HT counterpart, and this difference is present
throughout the entire period of observation. Body mass index was also higher in
the high-5HT animals. Deposition of fat in several adipose tissue depots (visceral,
retroperitoneal, gonadal and subcutaneous) in rats from 5HT sublines showed
that the relative amount of fat pads (g of fat pad/100 g of BW) was significantly
higher in rats from the high-5HT subline.
The present data demonstrate that rats from the high-5HT subline of WZ-5HT
model show clear obesity phenotype as compared to rats from the low-5HT
subline. Results point to the fundamental role of individual variability in
endogenous serotonergic tone in the control of body weight regulation. They
also
validate
Wistar-Zagreb
5HT
rats,
with
constitutionally
upregulated/downregulated serotonin transporter, as a potential novel genetic
model in obesity research.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P33
GLOBAL HISTONE ACETYLATION IN DIABETIC EMBRYOPATHY
Marina Korolija, Mirko Hadžija, Sandra Sobočanec, Marijana Popović Hadžija
Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb 10002,
Croatia
[email protected]
Poster Abstracts
Maternal diabetes increases the rate of congenital malformations in
embryos, causing diabetic embryopathy which mostly affects heart and
neural tube (embryonic precursor of central nervous system), in both
humans and animal models. High maternal blood glucose concentration,
which is presumably the major teratogenic factor, obstructs the process of
embryonic neurulation (neural tube closure) by an unknown mechanism.
Resulting open neural tube defects arise along rostrocaudal axis of the tube,
affecting brain (exencephaly), spine (spina bifida aperta) or the entire neural
tube (craniorachischisis). It has been shown that embryos developing in
hyperglycemic milieu display disrupted gene expression pattern which seems
rather non-specific, varying markedly between different mouse strains, or
even between individual organisms of the same inbred strain. In an attempt
to approach this issue from the point of genome-wide gene regulation, we
analyzed global histone H3 and histone H4 acetylaton in 44 non-obese
diabetic mouse embryo specimens at midgestation (embryonic day 10.5).
Hypoacetylation of both H3 and H4 has been detected in embryos with spina
bifida aperta and craniorachischisis, but not exencephaly. More detailed
analysis of histone acetylation at gene promoters is needed to establish
potential relationship between histone acetylation and transcriptional
regulation in diabetic embryopathy.
105
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P34
ISOLATION AND CHARACTERIZATION OF LYSOSOMES IN NPC MODEL CELLS
Marko Kosicek, Tanja Jovic, Silva Hecimovic
Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia
[email protected]
106
Enlarged endosomes are the earliest pathological feature of Alzheimer’s
disease (AD), the most common type of dementia among elderly population.
There is increasing evidence that lipids, especially cholesterol, play important
role in pathogenesis of AD, but the exact mechanism(s) of how lipids may
modulate development of the disease and its progression is still unknown.
The link between cholesterol and pathological hallmarks of AD (such as
accumulation of Aβ peptides and endosomal/lysosomal enlargement) has
been revealed in Niemann-Pick Type C (NPC) disease, a genetic disorder of
lysosomal cholesterol accumulation. We have previously shown that an
increase of Aβ in NPC model cells is at least partly caused by sequestration of
Amyloid Precursor Protein (APP) and β-secretase (BACE1), the two key
proteins in the pathogenesis of AD, in enlarged endosomes/lysosomes.
Overall, our findings indicate that dysfunction of the membrane and protein
trafficking within the endolysosomal system plays an important role in AD
pathogenesis.
The goal of this work is to further analyse enlarged endosomes/lysosomes in
NPC model cells. Lysosomes were isolated using two approaches – by
ultracentrifugation in a sucrose density gradient and using paramagnetic
nanoparticles. In the latter method the cells were incubated for 24h in the
media containing paramagnetic iron oxide particles in water stabilised with
dextran, following few hours chase in a normal medium. The time of chase
was optimized by monitoring the uptake of fluorescein labelled dextran by
confocal microscopy. Lysosomal fractions were verified by western blot
(using Lamp1 as a positive marker, and EEA1, TfR and Rab7 as negative
markers). Lysosomal cholesterol levels were analyzed by the Amplex Red
Cholesterol Assay. Vesicular size was determined using Zetasizer (Malvern),
which showed that isolated lysosomes are enlarged in NPC model cells. After
verification of lysosomal identity and purity, activity of several lysosomal
degradation enzymes (β-N-acetylglucosaminidase and acid phosphatase), as
well as lysosomal enzymes involved in glycosphingolipid metabolism was
measured. In addition, Cathepsin b activity was monitored in situ using
MagicRed Cathepsin b substrate. Understanding lysosomal dysfunction in
NPC disease could elucidate molecular details of the link between the two
neurodegenerative disorders: a rare inherited NPC disease and the most
common AD.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P35
MACRODOMAIN PROTEIN FROM BACTERIUM Streptomyces coelicolor
Jasna Lalić1, Andreja Mikoč1, Drago Perina1, Igor Sabljić2, Bruna Pleše1, Mirna
1
1
2
3,4
4
Imešek , Helena Ćetković , Marija Luić , Roko Žaja , Ivan Ahel
1
Division of Molecular Biology, Ruđer Bošković Institute, Zagreb 10002,
Croatia; 2Division of Physical Chemistry, Ruđer Bošković Institute, Zagreb
10002, Croatia; 3Division for Marine and Environmental Research, Ruđer
4
Bošković Institute, Zagreb 10002, Croatia; Sir William Dunn School of
Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
[email protected]
Poster Abstracts
Macrodomains are evolutionary conserved structural domains that bind
ADP-ribose derivatives found in proteins with diverse cellular functions.
Some proteins from the macrodomain family (human MacroD1, MacroD2)
can hydrolyze ADP-ribosylated substrates and therefore reverse posttranslational modification - ADP-ribosylation in which an ADP-ribose moiety
from NAD+ is transferred to a target protein. ADP-ribosylation can alter the
physical and chemical properties of target proteins and controls many
important cellular processes. Bacteria, and Streptomyces in particular, are
known to utilize protein ADP-ribosylation to control a variety of important
pathways (such as morphological differentiation and antibiotic production).
We have determined crystal structure and biochemically and functionally
characterized a macrodomain protein form bacterium Streptomyces
coelicolor. This protein is a member of an uncharacterised subfamily of
macrodomain proteins. Its crystal structure revealed highly conserved
macrodomain fold. This macrodomain protein is involved in the DNA damage
response, while knockout strain shows a conditional effect on antibiotic
production. Our results give us a first insight into the molecular basis of its
action, involvement in ADP-ribosylation cycle and impact on Streptomyces
metabolism.
107
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P36
CROSS-TALK BETWEEN ESTROGEN RECEPTOR ALPHA AND Hh-Gli
SIGNALING PATHWAYS IN BREAST CANCER CELLS
1
1
2
1
Diana Trnski , Maja Sabol , Zvonimir Uzarevic , Petar Ozretic , Vesna
1
1
Musani , Sonja Levanat
1
Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54,
10000 Zagreb, Croatia; 2Faculty of Education, Josip Juraj Strossmayer
University of Osijek, Ulica cara Hadrijana bb, 31000 Osijek, Croatia;
[email protected]
108
Hedgehog-Gli (Hh-Gli) signaling pathway is one of the new molecular targets found
upregulated in breast tumors. Estrogen receptor alpha (ERα) signaling has a key role
in the development of hormone-dependent breast cancer. The aim of this study is to
determine the possible combined effects of Hh-Gli pathway inhibitor cyclopamine
with ER inhibitor tamoxifen in breast cancer therapy, and to establish potential
interactions between Hh-Gli and ER signaling in breast cancer. ER-positive MCF-7 and
ER-negative SkBr-3 breast cancer cell lines were used. Drug treatments and
Competition experiments were with: cyclopamine 0.5-7.5 μM (Toronto Research
Chemicals) and tamoxifen 1-10 μM (Toronto Research Chemicals). MTT and cell
migration assays were used. Gene expression studies with : cyclopamine (2.5 μM),
Shh protein (3 ng/ml), tamoxifen (1 μM for MCF-7 or 5 µM for SkBr-3), β-estradiol (5
nM, Sigma) or cyclopamine + tamoxifen and Transfection with GLI1 and PTCH1
silencing (Life Technologies) in MCF cells were performed, and QRT-PCR with primers
for ERα, c-MYC, RPLP0, PTCH1, SMO, GLI1, SHH, and SUFU.Immunofluorescent
staining, Immunoprecipitation and Western blot were performed with primary
antibodies: anti-Hh (Santa Cruz, sc-9024), anti-ERα (Santa Cruz, sc-8002), anti-Ptch1
(Santa Cruz, sc-6147), anti-Gli1 (Santa Cruz, sc-20687) and Actin (Santa Cruz, sc1616). Confocal images were examined using the Manders coefficient plugin of the
ImageJ software (v 1.45e). Dynabeads Protein G (Life Technologies) were coated with
anti-ERα antibody and cell lysates were immunoprecipitated as per manufacturer’s
instructions. ER-positive cells show decreased viability after treatment with
cyclopamine, a Hh-Gli pathway inhibitor, as well as after tamoxifen (an ERα inhibitor)
treatment. Simultaneous treatment with cyclopamine and tamoxifen on the other
hand, causes short-term survival of cells, and only longer treatment decreases cell
proliferation. The survival effect is strongest 48 hours after simultaneous treatment
with both inhibitors, and we found upregulated Hh-Gli signaling under these
conditions. In addition to promoting survival, the combination of these two drugs
promotes cell migration and longer treatment decreases Hh-Gli signaling as well as
ERα levels. We also show a direct interaction between Shh and ERα. Shh protein is
able to bind and activate ERα independently of the canonical Hh-Gli signaling
pathway. The mechanism which is responsible for the increased viability of ERpositive cell line after combined treatment with cyclopamine and tamoxifen is not
clear. Although Hh-Gli signaling seems to be a good potential target for breast cancer
therapy, combined treatment of cyclopamine and tamoxifen may induce an opposite
effect, providing cells with short-term survival.
P37
CHOLINE BINDING SITE MUTATIONS IMPROVE HI-6 ASSISTED
REACTIVATION OF THE VX-ACETYLCHOLINESTERASE CONJUGATE
1
2
2
1
Nikolina Maček Hrvat , Zoran Radić , Palmer Taylor , Zrinka Kovarik
1
Institute for Medical Research and Occupational Health, Zagreb, Croatia;
2
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of
California at San Diego, La Jolla, CA USA; [email protected]
Nerve agent organophosphates (OPs) represent a threatening means of
potential terrorism due to their inhibition of acetylcholinesterase (AChE, EC
3.1.1.7), which can lead to death. AChE inhibition can, among other
solutions, be counteracted by administering purified human AChE mutants to
OP exposed individuals. These mutant enzymes were designed with the aim
to accelerate reactivation and press the fast reactivation of the OP and AChE
catalytic serine conjugates. Mutants, when combined with oxime
reactivators, could act as pseudo-catalytic bioscavengers, degrading OPs with
a turnover before phosphylating native endogenous AChE. The AChE active
site is a gorge composed of catalytic triade, oxyanion whole, choline binding
site, acyl pocket, and peripheral site. We focused on the choline-binding site
which has a role in binding the choline moiety of the substrate. HI-6 is a very
effective reactivator of VX-inhibited AChE so we investigated the effect of
the introduced mutations by in vitro kinetic experiments using HI-6. Even
though Y337A was 4 times more quickly inhibited by VX and the Y337A-VX
conjugate exhibited a slightly higher binding affinity for HI-6 than wt or
Y337A/F338A, it was evident that the Y337A/F338A mutation increased the
rate of nucleophilic displacement of the phosphonyl-moiety from the active
site serine (k+2) for 5.5 and 13 fold compared to wt AChE and Y337A,
respectively. These results singled out the double mutant as a potential
pseudo-catalytic bioscavenger in cases of VX poisoning. Therefore, to test the
bioscavenger potential of Y337A/F338A in more realistic conditions, we
performed ex vivo experiments. Hydrolysis of VX was followed in human
whole blood (hWB) or hWB supplemented with Y337A or Y337A/F338A;
inhibited by tenfold higher VX concentration and subsequently treated with
1 mM HI-6. We observed that 95 % of total cholinesterase (ChE) activity was
restored within 15 min when supplementing with Y337A/F338A and in terms
of observed first order reactivation rate (kobs), three times faster than in the
case of supplementing hWB with Y337A or using only hWB when just 50 % of
total ChE activity was recovered. Consequently, we conclude that the joint
influence of the F338 and Y337 mutations of the choline binding site has a
key role in effective reactivation and that Y337A/F338A mutant could act as a
pseudo-catalytic bioscavenger candidate in VX exposure.
This work has been supported by the CounterACT Program, NIH OD and NINDS (Grants U01
NS058046 and R21NS072086) and in part by the Croatian Science Foundation (project 4307).
Poster Abstracts
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
109
Poster Abstracts
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
110
P38
REVERSIBLE INHIBITION OF CHOLINESTERASES WITH AROMATIC
N-SUBSTITUTED 2-HYDROXYIMINOACETAMIDES
1
2
2
1
Nikola Maraković , Anamarija Knežević , Vladimir Vinković , Zrinka Kovarik ,
1
Goran Šinko
1
Institute for Medical Research and Occupational Health, Ksaverska cesta 2,
Zagreb, Croatia; 2Ruđer Bošković Institute, Bijenička cesta 54, Zagreb, Croatia
[email protected]
Acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC
3.1.1.8) play an important role in the neurotransmission and
biotransformation of xenobiotics. Organophosphorus (OP) nerve agents
acting as an irreversible AChE inhibitors are a persistent threat to the general
population because of their use as warfare agents in armed conflicts and
terrorist attacks. The current therapy in cases of OP nerve agent poisoning
includes the reactivation of AChE by quaternary pyridinium oximes.
However, due to their permanent positive charge, these compounds do not
cross the blood-brain barrier and thus cannot reactivate AChE in the central
nervous system. We evaluated the affinity of AChE and BChE for novel
centrally acting reactivators prepared by introducing a phenyl ring in the
structure of a previously reported N-substituted 2-hydroxyiminoacetamide
scaffold. 1-phenyl-allylamine was prepared from cinnamyl alcohol in an
Overman reaction and an azide group was introduced via a three-step
proces. An azide group enabled us to prepare more elaborate structures by
the well-known copper-catalysed azide-alkyne cycloaddition. N-substituted
2-hydroxyiminoacetamides (CM1 – CM4) differ in their AChE peripheral site
binding moiety, which ranges from an azide group to functionalized
heterocycles connected with central N-(1-phenylpropyl)-2-hydroxyiminoacetamide scaffold via a 1,2,3-triazole ring. All four compounds reversibly
inhibited BChE with apparent inhibition constants ranging from 1,62 μmol/L
to 270 μmol/L. The inhibition potency of compounds increased in the
following order: CM1 < CM2 < CM4 < CM3. AChE was also reversibly
inhibited by all compounds with same order of inhibition potency. Apparent
inhibition constants ranged from 241 μmol/L to 600 μmol/L. All of the new
compounds displayed a higher preference for binding to BChE. CM3 showed
an approx. 150-fold higher preference for BChE. The structural diversity of
new compounds allowed us to determine the importance of ligand
peripheral site binding properties for overall stabilization and binding
affinity. Molecular docking studies were performed on all of the new
compounds to help us rationalize the observed differences between their
binding affinities. The compounds lacked permanent positive charge and
were therefore expected to cross the blood-brain barrier with greater
capacity than the quaternary pyridinium oximes in use.
This work was supported in part by the Croatian Science Foundation (project 4307).
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Deep vein thrombosis (DVT) and pulmonary embolism (PE) as the most
common forms of venous thrombosis occur as a result of the simultaneous
influence of acquired and inherited risk factors. The aim of this study was to
examine the prevalence of four genetic risk factors in patients with DVT and
patients with PE in Eastern Croatia. Factor V Leiden and prothrombin
G20210A mutation as the most common genetic prothrombotic defects,
MTHFR C677T and PAI-1 5G/4G polymorphisms that contribute to the risk of
venous thrombosis in combination with other factors were analyzed using
the real-time PCR method and melting curve analysis.
This retrospective study included 120 DVT patients and 45 PE patients
genotyped between 2005 and 2014 at the Department of Molecular
Diagnostics and Tissue Typing, Osijek University Hospital, Croatia. Allele and
genotype frequencies were analyzed by using Fisher's exact test and
compared to previously genotyped healthy Croatian controls (n=106). Factor
V Leiden polymorphism was significantly associated with DVT (p=4x10 -6,
-4
OR=10.61, 95% CI 3.10-55.77) and PE (p=2.3x10 , OR=10.52, 95% CI 2.4661.86) while prothrombin G20210A polymorphism was significantly
associated with DVT (p=0.029, OR=2.89, 95% CI 1.03-9.33). Allele and
genotype frequencies of MTHFR C677T and PAI-1 5G/4G polymorphisms did
not differ between cases and controls.
Our results confirm previously reported association of factor V Leiden with
venous thrombosis in Croatian population. Additionally, the association
between prothrombin G20210A polymorphism and DVT in Eastern Croatian
population was observed too. However, in order to strengthen the statistical
power of test larger sample size is required for both analyzed groups, DVT
and PE as well.
Poster Abstracts
P39
THE ASSOCIATION OF FACTOR V LEIDEN, PROTHROMBIN G20210A, MTHFR
C677T AND PAI-1 5G/4G POLYMORPHISMS WITH DEEP VEIN THROMBOSIS
AND PULMONARY EMBOLISM IN EASTERN CROATIA
1,2
1
1
2
Saška Marczi , Stana Tokić , Nevenka Krajina , Ljubica Glavaš-Obrovac
1
Department of Molecular Diagnostics and Tissue Typing, Osijek University
Hospital, Osijek, Croatia; 2Department of Medicinal Chemistry, Biochemistry
and Clinical Chemistry, Faculty of Medicine, University of Osijek, Osijek,
Croatia
[email protected]
111
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P40
PRE-EXPOSURE TO OLIVE OIL POLYPHENOLS EXTRACT STIMULATES LIVER
REGENERATION IN MICE VIA STRESS SENSITIVE GENES
Jelena Marinić, Dalibor Broznić, Gordana Čanadi Jurešić, Marin Tota,
Čedomila Milin
Department of Chemistry and Biochemistry, School of Medicine, University of
Rijeka, Braće Branchetta 20,
51 000 Rijeka, Croatia
[email protected]
112
Several studies addressing the role of polyphenols in tissue repair attributed
wound healing potential of these compounds to their antioxidant capacity
based on the observation that reactive oxygen species (ROS), produced in
response to tissue injury, impede or exacerbate the healing process. We
investigated the effect of pre-exposure with the olive oil polyphenols extract
(PFE) on the course of liver regeneration induced by one-thirds hepatectomy
(pH) – a process during which ROS account for the early signals involved in
the initiation of tissue mass restoration.
Prior to pH mice were administered PFE (50 mg/kg bwt; i.p.) or saline during
seven consecutive days, while controls received vehicle alone. Stress
sensitive gene expression profile along with the oxidant and electrophilic
load, involving hepatic glutathione, lipid peroxidation (LP) and serum αglutathione S-transferase (GSTα) level was determined at different time
intervals after pH. A calculated relative liver weight was used to assess the
liver mass restoration.
Although regenerating liver itself exhibited intrinsic metabolic load, pretreatment with PFE increased LP and GSTα level and promoted glutathione
depletion within the first three hours after pH, pointing to the increase in
oxidant and electrophilic load during the early phase of the liver
regeneration. These changes were accompanied by the adaptive stress
response induction, as indicated by the Nrf2, heme oxygenase-1 and γglutamylcystein synthetase gene upregulation, leading to the normalization
of parameters of oxidative stress, reduced detoxification demands and
recovery of glutathione status, with the final outcome in increased liver mass
recovery.
Pre-exposure to polyphenols extract increases oxidant and electrophilic load
and induces endogenous defense mechanism under the control of stress
response gene-profiles, thereby stimulating the development of liver
regeneration in a model of a minor tissue loss.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P41
FRACTIONATION AND CHARACTERIZATION OF MAJOR BOVINE AND GOAT
WHEY PROTEINS
1a
1
1
Andrea Markovinović , Marko Jovanović , Darko Gumbarević , Jasminka
1*
Giacometti ,
1
Department of Biotechnology, aStudent at the Department of Biotechnology
University of Rijeka, Radmile Matejčić 2, HR-51000 Rijeka, Croatia
[email protected]; *[email protected]
Poster Abstracts
Whey proteins (WP), a by-product from the cheese and curd manufacturing,
well known for their nutritional value and different functional properties, are
widely utilized in the food industry.
The use of WP preparations, rather than the individual proteins, contributes
to functional variability among commercially available WP and can limit their
applications.
Several methods for whey separation and fractionation have been proposed,
including salting out, precipitation methods, heating at low pH, filtration
techniques and chromatographic techniques such as affinity chromatography
and ion exchange chromatography using membranes, conventional and
nonconventional resins as well as size exclusion chromatography,
hydrophobic chromatography, and combination of enzymatic treatment and
membrane filtration.
The objective of this study was to (a) separate -lactoglobulin ( -Lg) and lactalbumin ( -Lac) fractions from liquid bovine (LBW) and goat whey (LGW),
and (b) characterize the separated protein fractions.
The use of anion-exchange chromatography (AEX), ultra-high performance
liquid chromatography (UHPLC-DAD) and sodium dodecyl sulphate–
polyacrylamide gel electrophoresis (SDS-PAGE) for fractionation and
characterization of major whey proteins were evaluated. Total protein
concentration (TPC) was determined by BCA method.
Our results showed high purity of -Lg and -Lac and the used methodology
may be proposed as a method of choice for the separation of proteins in
dairy industry.
113
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P42
THE EFFECT OF A PHOSPHOLIPASE C GAMMA INHIBITOR ON THE
PROLIFERATION AND PHENOTYPE OF DU145 PROSTATE CANCER CELLS
1
1
1
Angela Mastelić , Nikolina Režić-Mužinić , Anita Markotić , Vedrana Čikeš1
2
3
4
4
Čulić , Milena Vuica-Ross , Ashley Ross , David Barker , Jóhannes Reynisson
1
Department of Medical Chemistry and Biochemistry, University of Split, School
of Medicine, Split, Croatia; 2Department of Pathology and 3Department of
4
Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA; School of
Chemical Sciences, The University of Auckland, Auckland, New Zealand.
[email protected]
114
INTRODUCTION: Prostate cancer remains the second most common cause of
cancer related death among men, highlighting the need for new therapies.
Many cancer cellular functions have been discovered to be regulated by
phospholipase C (PLC) gamma activation, suggesting that it represents an
important therapeutic target for development of anticancer drugs. Here, we
investigate the influence of a newly developed, small molecule PLC gamma
inhibitor, with or without taxane therapy, on the growth and survival of subpopulations of a prostate cancer cell line.
MATERIALS AND METHODS: Cells were incubated 48h with Paclitaxel (5 nM)
and PLC gamma inhibitor (1 microM) alone or in their combination. The
viable cells were determined by the MTT assay. Flow cytometric analysis of
cells positive to anti-CD44, anti-CD54, and propidium iodide staining was
performed to characterise apoptotic Du145 sub-populations 48h after
inhibitor treatment.
RESULTS: Treatment of the DU145 prostate cancer cell line with the PLC
gamma inhibitor resulted in cell cycle arrest with minimal increase in
apoptosis. Sub-populations of prostate cancer cell lines have unique
phenotypes (with CD44+ cells being more proliferative and CD54+ cells
serving as better CD8+ T cell targets). We examined the effects of the PLC
gamma inhibitor on these subpopulations and found that exposure
decreased the percentage of both CD44+ (p=0.00007) and CD54+ (p=0.009)
sub-populations. In contrast, treatment with Paclitaxel only effected CD44+
cells (p=0.0002). Combination treatment of the PLC gamma inhibitor and
Paclitaxel however had synergistic effects on both CD44+ and CD54+ DU145
cells (p=0.005 and p=0.0002, respectively).
CONCLUSIONS: These results suggest that a combination of PLC gamma
inhibitor and Paclitaxel could be a novel strategy for the treatment of
prostate cancer.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
By-products constitute a rich but yet underutilized source of valuable
components such as phenolics, which may be applied as ingredients in the
food, feed, cosmetics, and pharmaceutical industries. Special attention is
focused on their extraction from inexpensive or residual sources from the
food and agricultural products. Potential sources for their exploitation are
plant leaves as agricultural by-products.
The growing interest in the substitution of synthetic food antioxidants and
antimicrobial agents by natural ones has fostered research on plant sources
and the screening of raw materials for identifying new substances. Phenolic
compounds are secondary metabolites of plants involved in their defenses
against various attacks such as oxidative damage and aggression by
pathogens and also potential antimicrobial agents against various pathogens.
The objective of this study is to assess the efficacy of plant leaves as residual
source as antioxidants and antimicrobial agents.
Polyphenols were extracted from several available leaves sources, such as
Olea europaea L., Vitis vinifera (two cultivars), Morus nigra and Aronia
melanocarpa and antimicrobial activity of extracts was tested against grampositive (Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC
19115) and gram-negative bacteria (Escherichia coli ATCC 25922, Yersinia
enterocolitica ATCC 9610). Extracts were characterized by the determination
of the total phenolics, the total flavonoids, DPPH and ABTS scavengers’
activity. UPLC/DAD was used for individual phenolics determination. In
general, olive, grape and mulberry leaf phenolic extracts showed strong,
while chokeberry leaf phenolic extract showed moderate antimicrobial
activity against selected food-borne pathogenic bacteria. All tested extracts
are more effective against gram-positive bacteria. The study indicates the
antimicrobial potential of these plant extracts as natural ingredients in food,
cosmetics, and other products.
Poster Abstracts
P43
ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES OF THE EXTRACTS FROM
PLANT LEAVES AS A POTENTIAL RICH SOURCES OF BIOACTIVE PHENOLICS
1
2
1
2
Sanja Milovanović , Marina Bubonja Šonje , Marko Jovanović , Maja Abram ,
2
1*
Diana Jurčić-Momčilović , Jasminka Giacometti
1
Department of Biotechnology, University of Rijeka, Radmile Matejčić 2, HR51000 Rijeka, Croatia; 2Department of Microbiology, Faculty of Medicine,
University of Rijeka, Brace Branchetta 20, 51000 Rijeka, Croatia
[email protected]; *[email protected]
115
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P44
IN VITRO AND IN VIVO ACTIVITY OF THREE NOVEL MONOMETHINE
CYANINE DERIVATIVES - MCDs
1
1
2
2
Katarina Mišković , Tatjana Belovari , Jasmina Rajc , Vatroslav Šerić , Ranko
3
3
3
1
Stojković , Ivo Piantanida , Mirela Baus Lončar , Ljubica Glavaš-Obrovac
1
Faculty of Medicine J. Huttlera 4, University of J.J.Strossmayer, Osijek,
Croatia; 2University Hospital Centar Osijek, J. Huttlera 4, Osijek, Croatia;
3
Ruđer Bošković Institute Bijenička cesta 54, Zagreb, Croatia
[email protected]
116
Most of monomethine cyanine derivatives (MCDs) interact with nucleic acid
by the minor groove DNA binding mechanism. An exception is investigated 2[(1-cyano-4-methyl-(3H)-benzothiazol-[3,
2-a]-pyrido-2-lidene)-methyl]-3methylbenzoxazole perchlorate (MCD 8) compound that interact with nucleic
acids by intercalation mechanism. In this study, biological properties and
applicative abilities of MCD derivatives were investigated. In vitro research
methodology includes antiproliferative evaluation by MTT test on SCCVII,
CT26.WT, 4T1, FsaR, and B16-F19 mouse tumour cell lines, cell cycle analysis
by flow cytometry, MCDs entry and cellular localization by fluorescent
microscopy, test of visualization of nucleic acids dyed with MCDs by gel
electrophoresis, and MCDs application as detection dyes in a real time PCR
method. Acute and chronic toxicity as well as antitumor effects were studied
on the mouse model. Obtained results are analysed by Student t-test,
multifactorial variance analysis (MANOVA) and Fisher LSD test in STATISTICA
8.0 program. The most sensitive was 4T1 mouse cell line on MCD 4 (IC 50 = 0.9
µM) and FsaR on MCD 8 (IC50 = 8.4 µM). The cycle of HeLa cell was stopped
by MCD 4 in G2/M phase, while MCD 8 stops it in S and G2/M phase. All
analysed MCDs expressed characteristic green fluorescent signal spread all
over the cell. Both MCD 4 and MCD 8 gave good results as possible detection
dyes. MCD 4 is applicable for a real time PCR technique at the final
concentration of a 0.51 µg/mL. MCD 8 (2 mg/mL) is a good choice in the
visualization of nucleic acids on agarose gels. In vivo results pointed to
stronger MCD 4 toxic effect in a chronic exposure to female in comparison to
male mice. MCD 8 regarding to MCD 4 have lower toxicity with no difference
among sexes and time of exposure. Tested compounds do not suppress the
growth of implanted mouse mammary tumour. Study resulted with two new
fluorescent monomethine cyanine dyes (MCD 4 and 8) of low toxicity with
great application potential in the field of molecular biology for live cells
denotation and biological molecular tracking.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Proper positioning and function of membrane proteins depends on the lipid
composition of the plasma membrane as result of lipid-protein interactions.
Indeed altered glycosphingolipid environment, e.g. in mice with disrupted
ganglioside synthesis, causes change in localisation of membrane proteins,
especially lipid raft proteins. We have shown that in mice lacking complex
gangliosides, sialic acid-bearing glycosphingolipids especially abundant in
mammalian brain, expression of cell adhesion molecule neuroplastin (Np) is
increased and distribution altered. Nps are important for brain function: studies
on human cognition show that a SNP in the regulatory region of the human Np
gene correlates with cortical thickness and cognitive abilities in adolescents.
Moreover, mice lacking Np display reduced number and altered function of
synapses in the hippocampus. As both Np and gangliosides are important
molecular constituents of synaptic membranes and we previously shown that
ganglioside composition affects Np localisation, it is of particular interest to
determine their specific intermolecular interactions. The aim of this study was to
answer the question whether lack of neuroplastin leads to changes in brain
ganglioside content and composition. For that purpose, we used brain tissue
(cortex, hippocampus and cerebellum) of neuroplastin knock-out (Nptn KO) mice.
The gangliosides were extracted, quantified and separated by high performance
thin layer chromatography (HPTLC). The overall ganglioside concentration as well
as appearance in HPTLC was similar in wild-type (wt) and Nptn KO mice,
indicating there is no disturbance in ganglioside composition in animals lacking
Np. However, to look for subtle differences in structural diversity of brain
gangliosides of wt and Nptn KO mice, we further analysed the extracted
gangliosides by tandem mass spectrometry and structurally characterized them
in detail. Since the ganglioside environment seems to be, apart from minor
modifications, comparable in brain of Nptn KO and wt mice, that indicates that
gangliosides influence neuroplastin, both on gene and protein expression level,
but not vice versa. That observation is an excellent starting point for clarifying
the exact relationship, particularly specific intermolecular interactions between
gangliosides and neuroplastin within the membrane.
Poster Abstracts
P45
SEARCHING FOR PROTEIN-LIPID INTERACTIONS: GANGLIOSIDE PROFILING
OF NEUROPLASTIN KNOCK-OUT MICE
1
2
2
Kristina Mlinac , Rodrigo Herrera-Molina , Angela Kolodziej , Marko
3
1
2
2
Rožman , Željka Vukelić , Karl-Heinz Smalla , Dirk Montag , Svjetlana Kalanj
Bognar1
1
School of Medicine, University of Zagreb, Croatia; 2Leibniz Institute for
3
Neurobiology, Magdeburg, Germany; Ruđer Bošković Institute, Zagreb,
Croatia; [email protected]
117
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P46
CHROMATIN REMODELING PROCESS AT THE YEAST PHO5 PROMOTER IS
ESSENTIALLY DEPENDENT ON THE ACTIVITY OF RSC REMODELING COMPLEX
1
1
2
2
Sanja Musladin , Dora Hlevnjak , Nils Krietenstein , Philipp Korber , Slobodan
1
Barbarić
1
Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology,
University of Zagreb, Croatia; 2Adolf-Butenandt-Institut, Molecular Biology,
University of Munich, Germany
[email protected]
118
The yeast PHO5 promoter was the first and still is one of the best
characterized examples of a massive chromatin transition that is an absolute
prerequisite for transcription activation. The comprehensive search for
involved cofactor(s) revealed a complex network of five remodelers from all
four major subfamilies in yeast. We showed recently that RSC, the only
remodeling complex essential for viability in yeast, is a major component of
this network. In continuation we wished to fully clarify the role of RSC,
especially if it is essential for PHO5 promoter opening. We applied new
strategies for more complete RSC ablation than the previous inactivation of
its catalytic subunit Sth1 by the temperature sensitive degron allele sth1td.
First, we combined the deletion of RSC2, encoding a subunit of a major RSC
complex isoform, with inactivation of Sth1td during PHO5 induction at the
nonpermissive temperature (37 °C). Second, we constructed a double mutant
containing a Tet-Off-promoter driven STH1 gene and the rsc2 deletion allele
and examined chromatin remodeling upon physiological induction at 30 °C. In
contrast to the sth1td single mutant, both double mutants achieved no
appreciable PHO5 promoter opening even after prolonged induction
suggesting an essential role of RSC complex. Interestingly, chromatin
remodeling at PHO8 and PHO84 promoters, coactivated with PHO5 by the
same transactivator Pho4, was not significantly affected even under such
strong ablation of RSC complex. Chromatin remodeling at a PHO5 promoter
variant activated by the non-physiological activator Gal4 was also fully
prevented in both double mutants, showing that the RSC effect is not specific
for induction through PHO signaling nor for promoter activation by the
native activator Pho4. We also demonstrated that RSC activity is not only
essential for opening but also for the maintenance of open chromatin at the
PHO5 promoter.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P47
SURVIVAL OF F. tularensis SUBSP. novicida IN DIFFERENT SPRING WATER
SAMPLES
1
1
2
3
Mateja Ozanic , Valentina Marecic , Danijela Lenac , Vanda Piskur , Marin
3
4
1
1
Glad , Majda Meden , Marin Bajek , Marina Santic
1
Department of Microbiology and Parasitology, Faculty of Medicine,
University of Rijeka, Rijeka; 2Water and Wastewater Company, Rijeka;
3
Teaching Institute of Public Health of Primorsko-Goranska County, Rijeka;
4
Water Supply System, Krk, Croatia
[email protected]
Poster Abstracts
Francisella tularensis is a gram-negative facultative intracellular bacterium
that can cause a fatal disease, tularemia, in human and animals. It resists
harsh environments, and has been shown to survive in water and mud for
more than a year. There are several records of tularemia epidemics such as
in Sweden, Spain, Finland, Kosovo and recently in Turkey, where more than
500 people were infected by water. The aim of the study was to follow
survival of F. tularensis subsp. novicida in natural spring water samples
collected from springs of Rijeka (“Zvir I” and “Zvir II”) and springs of Krk
(“Vela Fontana” and “Ponikva”). F. tularensis subsp. novicida was added to
spring water samples and growth kinetics of bacteria was determined by
plate counting method. Results were observed in correlation to
concentration of metal ions in these natural spring waters. Concentration of
metals in water was determined before adding the F. tularensis subsp.
novicida and after a period of 10 days. Our results showed that F. tularensis
subsp. novicida survives in all natural spring water samples, but the best rate
of survival was achieved in water collected from spring “Vela Fontana” where
we measured the highest concentration on metal ions. We conclude that F.
tularensis survives better in natural spring water samples with highest
concentration of metal ions especially Manganese.
119
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P48
RECOGNITION OF ALKALI-LABILE GANGLIOSIDES BY TWO-DIMENSIONAL
THIN-LAYER
CHROMATOGRAPHY
AND
IMMUNOHISTOCHEMICAL
LOCALIZATION AFTER ALKALI TREATMENT FROM THE BRAIN OF TWO
POIKILOTHERMIC FISH SPECIES
Valentina Pavić1, Elizabeta Has-Schön1, Ivan Bogut2, Marija Heffer3
1
Department of Biology, J.J. Strossmayer University, Cara Hadrijana 8/A,
2
31000 Osijek, Croatia; Department of Animal Husbandry, Faculty of
Agriculture, J.J. Strossmayer University, P. Svačića 1d, 31000 Osijek, Croatia;
3
Department of Biology, Faculty of Medicine, J.J. Strossmayer University, J.
Huttlera 4, 31000 Osijek, Croatia; [email protected]
120
The relative content of alkali-labile gangliosides and distribution of these
sphingolipids were examined in the brain of two poikilothermic fish species:
eurythermic common carp (Cyprinus carpio) and stenothermic rainbow trout
(Oncorhynchus mykiss). Animals were subjected to seasonal temperature
fluctuations in their natural ecosystem. In order to separate alkali-labile
gangliosides from alkali-stabile we have performed two-dimensional thin layer
chromatography with ammonia treatment. Rainbow trout brain in winter
showed large quantities of alkali-labile ganglioside (40.56%), while in the summer
established in a smaller proportion (33.14%). Common carp brain showed
approximately equal amounts of alkali-labile ganglioside in winter (30.86%) and
summer (30.88%). In this research for the first time immunohistochemical
staining of sections previously treated with ammonium alkali vapors in order to
determine the distribution of alkali-labile gangliosides in tissues was performed.
Our results indicate that the epitopes recognized by the monoclonal antibody in
alkali-labile gangliosides were changed with ammonia treatment and only
recognition of alkali-stabile gangliosides was present. We assumed that the alkali
treatment causes changes in ester bonds in epitope of alkali-labile gangliosides
and therefore antibody does not recognize them. Imunohistochemical staining of
sections previously treated with ammonium vapors gave insight into the
distribution of alkali-stabile gangliosides and comparison with the positive
reaction in untreated samples insight into the distribution of alkali-labile
gangliosides. Content and distribution of brain alkali-labile gangliosides changes
differently in common carp and rainbow troutMost pronounced changes were
those where anti-GT1b staining after alkali treatment of rainbow trout forebrain
was used. During the winter GT1b ganglioside has a higher proportion of alkalilabile gangliosides in telencephalon and optic tectum, while a smaller portion is
visible in the valvula and cerebellum. During the summer there was a change in
the distribution and ganglioside GT1b has a lower proportion of alkali-labile
gangliosides in the telencephalon and optic tectum, and a larger in the valvula
and cerebellum. The percentage of alkali-labile gangliosides was found to be
related to the ecophysiological state of thermal adaptation.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P49
THE STRESS REGULATED ASR PROTEIN CAN BE DETECTED IN IN VITRO
GROWN TISSUES OF THE CACTUS Mammillaria gracilis
1
1
2
1
Petra Peharec Štefanić , Tea Rogić , Dudy Bar-Zvi , Biljana Balen
1
Abscisic acid-, Stress-, and Ripening-induced (ASR) proteins are plant specific,
low molecular weight, heat-stable proteins that have a high hydrophilicity.
ASR proteins were shown to possess transcription factor and chaperon-like
activities. They are involved in regulation of sugar, branched amino acids and
cell wall metabolism, and in plant tolerance to drought and salinity. The ASR
protein family is widespread in the plant kingdom; ASR genes were cloned
from a number of gymnosperm, monocotyledon and dicotyledon plant
species. However, the model plant Arabidopsis as well as other species from
the Brassicaceae family lack ASR genes, suggesting that ASR proteins are not
ubiquitous to all plant species. Cacti plants are highly tolerant to water
stress. Thus, it was interesting to find out if they encode ASR proteins. We
analyzed two in vitro-grown tissues (callus and tumor), from the cactus M.
gracilis Pfeiff. in order to reveal if the ASR-like proteins can be found in the
member of the Cactaceae family. ASR proteins are acid soluble, and they can
be effectively purified to homogeneity on Ni-NTA-agarose, since they contain
an authentic pentahistidine sequence close to their N-termini. Therefore, we
took advantage of ASR histidine-rich tract to purify the ASR-like protein from
cactus callus and tumor tissues. Eluted proteins from Ni-NTA-agarose, were
separated by SDS-PAGE. One gel was Coomassie stained and the proteins on
the other gel were subsequently transferred onto the nitrocellulose
membrane and treated with anti-ASR1 antibody. Protein bands which
reacted with anti-ASR1 antibody on the membrane were excised from the
gels and subjected to mass spectrometry analysis after which they were
identified as ABA- and ripening-induced protein. Once we uncovered ASR-like
protein presence in cactus tissues, our aim was to determine what impact
abiotic stress has to ASR-like protein expression. For that purpose, both
callus and tumor were subjected to short-term salt- and mannitol-induced
osmotic stress and further analyzed. It was determined that salinity and
osmotic stress in cactus tissue culture caused induction in ASR-like protein
expression. ASR-like protein appeared as 15 kDa protein in both control
tissues. In response to NaCl and mannitol, one additional band of around 30
kDa appeared. The 30 kDa protein was visible in callus tissue treated with
mannitol and particularly pronounced in tumor tissue treated with both NaCl
and mannitol.
Poster Abstracts
Department of Molecular Biology, Division of Biology, Faculty of Science, University of
Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia; 2Department of Life Sciences and
The Doris and Bertie Black Center for Bioenergetics in Life Sciences, Ben-Gurion
University of the Negev, Beer-Sheva 84105, Israel; [email protected]
121
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P50
POLY(ADP-RIBOSYL)ATION IN THE RED SEAWEED Chondrus crispus
Drago Perina1, Andreja Mikoč1, Josip Ahel1, Helena Ćetković1, Roko Žaja2,3,
3
Ivan Ahel
1
Division of Molecular Biology, Ruđer Bošković Institute, Zagreb 10002,
Croatia; 2Division for Marine and Environmental Research, Ruđer Bošković
Institute, Zagreb 10002, Croatia; 3Sir William Dunn School of Pathology,
University of Oxford, South Parks Road, Oxford OX1 3RE, UK
[email protected]
Poster Abstracts
Poly(ADP-ribosyl)ation is a post-translational modification of proteins
involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR)
consists of chains of repeating ADP-ribose nucleotide units and is synthesized
by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This
modification can be removed by the hydrolytic action of poly(ADP-ribose)
glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity
of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for
the removal of terminal ADP-ribose unit and for complete reversion of
protein ADP-ribosylation. According to the phylogenetic distribution of
proteins involved in PAR metabolism, it was proposed that the last common
ancestor of all eukaryotes already possessed full set of proteins required for
reversible PAR metabolism. To check whether this set was fully functional,
we analyzed proteins from early branching eukaryotic lineage. The red algae
(Rhodophyta) are one of the oldest groups of eukaryotic algae formed during
the primary endosymbiosis event which enable the emergence of the first
photosynthetic eukaryote. The red macroalgal fossil, 1.2 billion years old,
provides the oldest evidence of multicellular, sexually reproducing
eukaryote. Recently, genome of Chondrus crispus, or Irish moss, has been
sequenced which was a prerequisite for positioning of red algae as excellent
model organisms for understanding PARP evolution. Our analyses provides
insight into the evolution of these important signaling systems, as well as
providing evidence that red algae are appropriate genetic model organisms
to study ancestral PARP(s), which are structurally and functionally similar to
the highly sophisticated multifunctional enzymes which functions are usually
associated with higher Metazoans.
122
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Superparamagnetic iron oxide (SPIO) nanoparticles are widely used for
different biomedical applications. Due to their low toxicity and outstanding
magnetic properties, they represent a promising tool for in vivo studies
involving cell labelling, tracking and imaging using medical imaging
techniques such as magnetic resonance imaging (MRI). To this aim, different
strategies are being investigated in order to achieve highly specific, efficient
and rapid internalization of SPIO nanoparticles into specific target cells. For
example, coating newly synthesized SPIO nanoparticles with biocompatible
polymers was shown to lead to increased intracellular uptake of the
nanoparticles and thus to higher cell labelling efficiency.
In this study, neural stem cells (NSCs) isolated from E14.5 mouse embryos
were labelled with poly(L-lysine)-coated (PLL) SPIO nanoparticles. These
nanoparticles were obtained by chemical coprecipitation of Fe(II) and Fe(III)
chlorides, oxidation with sodium hypochlorite to maghemite ( -Fe2O3) and its
post-synthesis coating with poly(L-lysine). Poly(L-lysine) represents promising
coating agent for transport of the SPIO nanoparticles into cells because it is
commonly used to enhance cell adhesion to the surface of a culture dish in
in vitro cell cultivation. In vitro survival and labelling efficiency of NSCs upon
labelling with PLL SPIO nanoparticles was evaluated. Furthermore, the
localization of the PLL SPIO nanoparticles within NSCs was characterized by
transmission electron microscopy (TEM). Performance of PLL SPIO
nanoparticles in all in vitro biological experiments was compared with the
commercial nanomag -D-spio nanoparticles, which are dextran iron oxide
composite nanoparticles.
Poly(L-lysine)-coated SPIO nanoparticles described in this study represent a
powerful tool for future in vivo studies of NSC behaviour after their
transplantation into mouse brain in mouse ischemic stroke model.
Acknowledgments: This study was supported by EU FP7 grant GlowBrain
(REGPOT–2012–CT2012–316120).
Poster Abstracts
P51
IN VITRO EVALUATION OF POLY(L-LYSINE)-COATED MAGHEMITE
NANOPARTICLES: APPLICATION IN BRAIN RESEARCH
1
1
2
1
Igor Pongrac , Marina Dobrivojević , Michal Babič , Marija Lovrić , Lejla
1
2
1
2
Ferhatović Hamzić , Miroslav Šlouf , Srećko Gajović , Daniel Horák
1
University of Zagreb School of Medicine, Croatian Institute for Brain
Research, Zagreb, Croatia; 2Institute of Macromolecular Chemistry, Academy
of Sciences, Prague, Czech Republic
[email protected]
123
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P52
INVESTIGATIONS OF THE KEY BINDING INTERACTIONS OF NOVEL
IMIDAZOLE- AND BENZIMIDAZOLE-BASED OXIMES WITHIN THE ACTIVE SITE
OF BUTYRYLCHOLINESTERASE
Ines Primožič, Srđanka Tomić, Tomica Hrenar
Department of Chemistry, Faculty of Science, University of Zagreb, HR-10 000
Zagreb, Croatia
[email protected]
124
Organophosphorus compounds can act as cholinesterase inhibitors and thus
are used as pesticides, insecticides and nerve agents (soman, sarin, tabun,
VX). The best antidotes for organophosphorus poisoning are oximes which
antidotal properties are related to their ability to reactivate phosphorilated
acetylcholinesterase (AChE, EC 3.1.1.7). Butyrylcholinesterase (BChE, EC
3.1.1.8) can be used for the treatment of organophosphorus poisoning as a
stoichiometric bioscavenger. Applied with the BChE reactivator would make
this enzyme even better treatment drug. [1, 2] Since there is still no single,
broad-spectrum compound suitable as antidote for treatment of poisoning
with various organophosphorus agents, search for more efficient oximes and
better understanding of their interactions with both cholinesterases are
needed. Therefore, a series of novel imidazole- and benzimidazole-2aldoximes were tested as potential reactivators of paraoxone, tabun and VX
inhibited human serum BChE. Imidazole and bezimidazole-2-aldoximes were
alkylated with different alkyl, alkenyl as well as arylalkyl groups and 34 new
compounds were prepared. All prepared oximes inhibited human BChE
reversibly, and the inhibition potency was nanomolar (ponetial pretreatment drugs) to micromolar. Reactivation power was also related to the
structure of substituents, percentage varied from ten to ninety. To explain
differences in inhibition and oximes reactivation potency, conformational
analysis, molecular modelling and docking studies were carried out.
Orientations of all studied oximes in the active site of human BChE have been
proposed by flexible ligand docking and subsequent QM/QM studies.
Analyses of the obtained complexes revealed the presence of numerous
hydrogen bonds and close contacts between oximes and residues of the
active site. Calculated interaction energies predicted correctly the relative
order of the inhibition potency of compounds as well the most probable
orientation of the best reactivators which can result in an attack on the
phosphorus atom of VX and tabun-phosphorylated human BChE.
[1] Radić Z., Dale T., Kovarik Z., Berend S., Garcia E., Zhang L., Amitai G., Green C.,
Radić B., Duggan B. M., Ajami D., Rebek, J. Jr., Taylor, P. Biochem J 450 (2013) 231242.
[2] Radić, Z., Sit R. K., Kovarik Z., Berend S., Garcia E., Zhang L., Amitai G., Green C.,
Radić B., Fokin V. V., Sharpless K. B., Taylor P. J Biol Chem 287 (2012) 11798-11809.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Aminoacyl-tRNA synthetases (aaRSs) play a critical role in translation by
catalyzing the ligation of their cognate amino acids and tRNAs to establish
the genetic code. Beside their canonical role in translation, aaRSs participate
in various cellular processes, including response to stress conditions.
Research on the role of aaRSs in stress-responses in plants is very scarce. One
proteomic investigation showed upregulation of Arabidopsis seryl-tRNA
synthetase (SerRS) in the early response of A. thaliana cells to cadmium
exposure. To further investigate the role of SerRS in the stress, we examined
growth of Arabidopsis transgenic seedlings overexpressing SerRS. We tested
different stress media that induce ionic stress, osmotic stress and stress
imposed by heavy metals. Influence of the plant hormones, abscisic acid and
brassinosteroids, was also investigated. Our results show that transgenic
seeds germinate earlier and that in most cases transgenic seedlings grow
better on stress media compared to wild type seedlings. This confirms that
enhanced expression of SerRS plays a role in the response of plant to various
stress conditions. Stress related function of SerRS may be linked to its cellular
localization. To determine subcellular destination of the SerRS protein and
targeting properties of its basic C-terminus we prepared three fusion
constructs: GFP-SerRS, SerRS-GFP and GFP-C terminus. All constructs were
transiently transformed into heterologous epidermal onion cells and
homologous Arabidopsis leaves protoplasts. GFP fluorescence was examined
under confocal microscope. For all constructs cytosolic localization was
observed. In addition, some onion cells showed accumulation of full-length
GFP-SerRS or SerRS–GFP fusion proteins in the nucleus. In Arabidopsis
protoplasts nuclear localization was observed only in the case of full-length
GFP-SerRS fusion protein. In both onion cells and Arabidopsis protoplasts
SerRS C-terminus did not exhibit targeting properties of nuclear localization
signals. Because GFP-localization experiments gave ambiguous results we
performed immunoblot analysis of cytosolic and nuclear fraction of
Arabidopsis leaves. Western blot analysis performed so far indicates cytosolic
localization of Arabidopsis SerRS protein.
Poster Abstracts
P53
Arabidopsis thaliana SERYL-tRNA SYNTHETASE PARTICIPATES IN CELLULAR
STRESS RESPONSE
1
1
2
1
Jasmina Rokov Plavec , Mario Kekez , Nataša Bauer , Ela Šarić , Ivana
1
Weygand-Đurašević
1
Department of Chemistry, Faculty of Science, University of Zagreb,
Horvatovac 102a, 10 000 Zagreb, Croatia; 2Department of Biology, Faculty of
Science, University of Zagreb, Horvatovac 102a, 10 000 Zagreb, Croatia
[email protected]
125
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P54
THE EFFECT OF 17Β-ESTRADIOL ON THE EXPRESSION OF DIPEPTIDYL
PEPTIDASE III AND HEME OXYGENASE 1 IN LIVER OF CBA/H MICE
1
1
1
2
Željka Mačak Šafranko , Sandra Sobočanec , Ana Šarić , Nina Jajčanin Jozić ,
1
2
Tihomir Balog , Marija Abramić
1
Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia;
2
Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute,
Zagreb, Croatia
[email protected]
Poster Abstracts
17β-estradiol (E2) has well established cardioprotective, antioxidant and
neuroprotective role and exerts vast range of biological effects in both males
and females. In this study we examined the effect of E2 on the expression of
dipeptidyl peptidase III (DPP III), the protease involved in Nrf2-Keap1
signaling pathway, along with the expression of known antioxidant enzyme
heme oxygenase 1 (HO-1) in the liver of adult CBA mice of both sexes. Also,
we determined the lipid oxidative damage in all experimental groups.
Ovariectomy markedly diminished expression of both DPP III and HO-1
proteins. E2 implementation abolished this effect, and even increased these
proteins levels above the control. A significant enhancement in DPP III
protein content was found in E2-treated males as well. In females depleted
of E2, increased lipid peroxidation was determined. Decrease in the
expression of HO-1 gene, but not of the DPP III gene, measured by the realtime PCR, was detected in the liver of ovariectomized female mice. Obtained
results, for the first time reveal that E2 influences the protein level of DPP III
in vivo. These results additionally confer new insights into complexity of
protective action of this hormone.
126
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Phytoplasmas (genus 'Candidatus Phytoplasma') are wall-less bacteria
belonging to the class Mollicutes together with mycoplasmas, spiroplasmas
and acholeplasmas which are all believed to share a common
Bacillus/Clostridium-like ancestor. These endocellular pathogens have a
unique life-style as they have hosts from two kingdoms – Plantae (plants)
and Animalia (insects), and need both for their survival and dispersal in
nature. Numerous plant species worldwide, including a range of
economically important crops, are affected by phytoplasmoses resulting in
serious yield losses. Axenic cultivation of these bacteria is still challenging
and not common. However, a number of genome drafts as well as four fully
sequenced and annotated phytoplasma genomes are available so far which
started a new era in functional and comparative genomics research.
Phytoplasmas possess relatively small genomes that experienced specific
gene losses and gains through their dynamic co-evolution with plant and
insect hosts. In spite of being small and reduced, their genomes are repeatrich and often contain multicopy genes. One of those multicopy genes is the
ssb gene. SSB proteins, essential for cell survival, are found in all domains of
life as well as in viruses. In phytoplasma genomes, the number of SSB protein
genes is variable among different species with only one ssb gene of different
evolutionary origin located in a specific genomic neighbourhood. In this
comparative study, all ssb gene sequences available from sequenced
phytoplasma genomes were analyzed together with the “original” ssb gene
amplified and sequenced from a number of new 'Ca. P. asteris' and 'Ca. P.
solani' isolates. Phylogenetic analyses have shown that all the “original” ssb
genes from different phytoplasma species clustered together. Unlike the
other, shorter ssb gene copies of undetermined functionality, the single ssb
gene copy from 'Ca. P. mali' genome was phylogenetically closer to the
“original” ones which was in accordance with its' different genome
organization and stability. Southern blot analyses have confirmed the
presence of one “original” ssb gene in the genomes of ‘Ca. P. asteris’ isolates.
Surprisingly, in the genome of ‘Ca. P. solani’ isolate, another copy of the
“original” ssb gene was repeatedly detected suggesting that it could be a part
of extrachromosomal element characteristic of the species which is known to
possess the largest and supposedly the most unstable genome among all
phytoplasmas.
Poster Abstracts
P55
COMPARATIVE ANALYSIS OF SSB GENES FROM PHYTOPLASMA GENOMES
AND THEIR POTENTIAL ROLE IN GENOME INSTABILITY
Martina Šeruga Musić, Anamarija Slović, Marija Pinterić
Department of Biology, Faculty of Science, University of Zagreb, Zagreb,
Croatia; [email protected]
127
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P56
RECA730 SUPPRESSES UV SENSITIVE PHENOTYPE IN RECA LOADING
MUTANTS OF Escherichia coli
Ana Šimatović, Ignacija Vlašić, Krunoslav Brčić-Kostić
Ruđer Bošković Institute, Zagreb, Croatia
[email protected]
Poster Abstracts
Homologous recombination is a process important in the repair of double
strand breaks (DSB), single strand gaps (SSG) and collapsed replication forks.
The central part of the recombination process is binding of RecA protein to
ssDNA, i. e., production of RecA filament. Depending on the type of lesion,
there are two pathways that operate in Escherichia coli: RecBCD and RecF. In
wild-type (wt) Escherichia coli strains, DSBs are processed by the RecBCD
pathway, where the same enzyme performs and coordinates all activities
necessary for RecA filament formation: helicase, nuclease and RecA loading.
SSGs utilize the RecF recombination pathway where the functions are
provided by different proteins (RecQ helicase, RecJ nuclease and RecFOR
complex which provides RecA loading activity) [1]. These pathways are
interchangeable in a recB1080 mutant which is nuclease and RecA loading
deficient [2].
We have studied a specific recA mutant named recA730 (RecAE38K) which
encodes a form of RecA protein that is able to achieve binding to ssDNA
without the help of RecFOR loading mediators [3]. We tested weather
recA730 mutation can suppress UV deficient phenotype of recB1080 mutants
and its derivates. The results show that the recA730 mutation suppresses
DNA repair deficiency in cells where both mechanisms for RecA filament
formation are inactivated by mutations in genes for mediator proteins
involved in RecA loading. In contrast, the suppression of DNA repair and
recombination is not possible in cells where the defect is at the level of
nuclease activity [4].
128
[1] Kowalczykowski S.C., (2000) Initiation of genetic recombination and
recombination-dependent replication. Trends Biochem. Sci. 25,156-165.
[2] Ivančić-Baće, I., Peharec, P., Moslavac, S., et al. (2003) RecFOR function is
required for DNA repair and recombination in a RecA loading-deficient recB
mutant of Escherichia coli. Genetics 163, 485-494.
[3] Wang, T.C.V., Chang, H.Y., Hung, J.L. (1993) Cosuppression of recF, recR
and recO mutations by mutant recA alleles in Escherichia coli cells. Mutat.
Res. 294, 157-166.
[4] Vlašić, I. et al. (2011) The recA730 dependent suppression of
recombination deficiency in RecA loading mutants of Escherichia coli. Res.
Microbiol. 162: 262-269.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P57
EVALUATION OF BUTYRYLCHOLINESTERASE STEREOSELECTIVITY IN
INTERACTION WITH ENANTIOMERS OF ETHOPROPAZINE
1
1
2
Goran Šinko , Nikola Maraković , Jure Stojan
1
Institute for Medical Research and Occupational Health, POB 291, HR-10001
Zagreb, Croatia; 2Institute of Biochemistry, Medical Faculty, University of
Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia
[email protected]
Poster Abstracts
Stereoselectivity of biological macromolecules originates from chiral amino
acids which are building blocks for enzymes and other proteins. It is usual for
enzyme to show stereoselectivity in interaction with chiral substrate or
inhibitor. We studied butyrylcholinesterase (BChE, EC 3.1.1.8)
stereoselectivity in interaction with enantiomers of ethopropazine. BChE is
related to acetylcholinesterase (AChE, EC 3.1.1.7) which is involved in
neurotransmission and they share 54% of sequence homology. BChE is
involved in hydrolysis of various esters and xenobiotics which makes it useful
in prodrug conversion, i.e. bambuterol used in the treatment of asthma.
Ethopropazine is a chiral drug used in treatment of Parkinson’s disease in
form of a racemate, an equimolar mixture of individual enantiomers. We
performed a series of BChE activity measurements in which we used low,
mid and high substrate concentrations (100 µM, 1 mM and 100 mM) and
performed BChE inhibition with addition of racemic and enantiomeric pure
ethopropazine into the reaction mixture to evaluate BChE stereoselectivity.
We evaluated stereoselectivity of a free enzyme and different enzymesubstrate complexes by using various substrate concentrations. Different
BChE-substrate complexes occur at high substrate concentration well above
KM value for acetylthiocholine (0.79 mM, 25°C). It is known that temperature
may have a significant effect on enzyme structure dynamics. To test link
between BChE structure dynamics and stereoselectivity we performed
activity measurements in the absence and presence of ethopropazine at 12,
20, 25, 30 and 37 °C. Our results will give more insight into BChE
stereoselectivity with purpose of future drug design especially for chiral
drugs or prodrugs.
129
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P58
THE PILOT STUDY OF VIROIDS IN ASYMPTOMATIC Solenostemon
scutellarioides (L.) CODD PLANTS
Dijana Škorić, Silvija Černi, Karlo Jezernik
Department of Biology, Faculty of Science, University of Zagreb, Zagreb,
Croatia
[email protected]
130
Solenostemon scutellarioides (L.) Codd is a new name of the ornamental
plant Coleus blumei Benth., well known worldwide for its brightly coloured
leaves and medicinal value owing to its phenolic content. It is also a main
host of six Coleus blumei viroids (CbVds) species (CBVd-1 to -6). CbVd-1
occurs globally. CbVd-2 and -4 have been reported only in Germany, CbVd-3
in Germany and China, and CbVd-5 and -6 only in Southeast Asia.
Interestingly, CbVds are transmissible not only by vegetative propagation and
mechanically like other viroids, but also by seeds. Viroids are noncoding
small covalently closed circular RNA (cccRNA) molecules independently
parasitizing in plants. Most of the viroids, including CbVds, belong to the
family Pospiviroidae having over 50% internal base pairing in their cccRNAs,
Central Conserved Region (CCR), asymmetric rolling–circle replication
catalysed by cellular RNA polymerase II in nucleus and no ribozyme activity.
The aim of this study was to investigate the occurrence of Pospiviroidae in a
small population of asymptomatic S. scutellarioides plants grown in the
Botanical garden of the Faculty of Science, Zagreb. Leaf and stem tissue from
ten plants were used to compare three methods for obtaining viroid RNAs.
Generic primers covering CCR of all six CbVds were used in RT-PCR based
detection procedure. Although the method using microliter quantities of
crude sap can be useful for preliminary detection, and the total RNA after
RNeasy kit (Qiagen) extraction was acceptable too, the best results were
obtained from small RNA templates after procedure encompassing phenolchloroform
extraction,
LiCl-fractionation
and
CF-11
cellulose
chromatography. Amplicons of approx. 250 and 360 bp were obtained from
5, and 2, out of ten plants, respectively. As direct sequencing of PCR products
did not yield clear results due to the quasispecies nature of viroids, an
amplicon of each size was AT-cloned. After SSCP-screening, all identified
variants in each viroid’s population structure were sequenced. For the first
time in Croatia, CbVd-1 and CbVd-3 were identified with variants of 249-251
and 361-362 nucleotides, respectively. The incidence levels of 50% for CbVd1 and 20% for CbVd-3 are high for such a small population of investigated
plants but those values are within reported incidence ranges. Also, finding
CbVd-3 the second time in Europe and in 2 out of only 10 plants suggests
CbVd-3 could be more widespread than assumed.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P59
THE TRANSPORT ABILITY OF PROTECTIVE BIOMOLECULE QUERCETIN
THROUGH Arabidopsis thaliana IS IMPROVED BY THE RARE-EARTH
ELEMENT EUROPIUM
1
2
2
1
Ivana Šola , Ivo Piantanida , Ivo Crnolatac , Gordana Rusak
1
Department of Biology, Faculty of Science, University of Zagreb, 10000
Zagreb, Croatia; 2Division of Organic Chemistry and Biochemistry, Ruđer
Bošković Institute, 10 000 Zagreb, Croatia
[email protected]
Poster Abstracts
Quercetin (Q) is a plant polyphenol from the group of flavonoids with strong
antioxidative, antiviral and antibacterial activities, yet its ability of a longdistance movement through a plant is quite limited. Moreover, the mobility
of exogenously applied Q through an Arabidopsis thaliana plant is minimal or
not possible at all. Recently, it was discovered that biological properties of
free flavonoids can be improved by their chelation with metals. In last years
it was proven that a rare-earth metal europium (Eu(III)) is naturally present in
some plants, though in small quantities and, more interesting, can affect
their physiological processes. Accordingly, we tested whether Eu(III) could
bind to flavonoid Q, and if so would and how affect the transport ability of Q
through Arabidopsis. Our in vitro results showed that Eu(III) binds to Q, and
at the ratio rQ/Eu(III) = 0.2 complex stoichiometry 1/1 (Q/Eu(III)) reaches its
maximal concentration with 95% of the total concentration of UV/Vis
absorbing species in the solution. This complex stoichiometry was therefore
used in further in vivo examinations, and the results showed that the longdistance transport of Q through Arabidopsis root could be stimulated by
complexation with Eu(III). During transport, the Q/Eu(III) complex got
degraded and therefore enabled efficient, slow, release of protective
metabolite at the distal site, offering enhanced Q delivery. The
spectrophotometric data suggested, one of possible reasons of Q/Eu(III)
degradation could be the interaction of the complex with double stranded
RNAs present in Arabidopsis. Since Q itself has very limited transportation
ability, yet positively affects plant defence responses, its improved longdistance transportation could lead to a better protection of plant tissues.
Accordingly, our results suggest that the rare-earth element Eu(III) can
improve the biological relevance of protective biomolecule Q.
131
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P60
SUBSTRATE-INDUCED CONFORMATIONAL CHANGES OF THE ADENYLATION
DOMAIN FROM TYROCIDINE SYNTHETASE 1 PROBED BY INTRINSIC TRP
FLUORESCENCE
Matilda Šprung, Viljemka Bučević-Popović, Barbara Soldo, Stjepan Orhanović,
Maja Pavela-Vrančič
Faculty of Natural Sciences, Department of Chemistry, University of Split, N.
Tesle 12, 21000 Split, Croatia; [email protected]
132
Nonribosomal peptide synthetases (NRPS) are large multifunctional enzymes
that catalyse the synthesis of nonribosomal peptides (NRP) known for a
broad range of pharmaceutical properties such as antimicrobial,
immunosuppressive and antitumor activity. With overwhelming reports of
bacterial resistance, it is of fundamental importance to gain more insight into
structural and functional properties of these megastructures. Biosynthesis of
NRP starts with the adenylation (A) domain that activates a specific amino
acid substrate in form of aminoacyl-adenylate with concomitant release of
pyrophosphate. The activated amino acid is subsequently transferred via the
peptidyl carrier protein (PCP) domain to the neighbouring active site of the
condensation (C) domain that finally catalyses peptide bond formation.
Crystallographic studies on various NRPS A-domains showed that these
enzymes undergo extensive structural rearrangements during the catalytic
cycle. Two catalytically distinct conformations are reported for A-domains:
the first being implied in adenylate formation, and the second in transfer of
the activated amino acid onto the PCP-domain. Here we report the first
extensive study on fluorescence properties of a representative A-domain
from tyrocidine synthetase 1 (TycA-A). TycA-A comprises five potentially
fluorescent Trp residues designated W227, W301, W323, W376 and W406,
respectively. Individual Trp accessibility surface area (ASA) and their
structural position were assessed based on a structural model of the TycA-A
protein in both conformations. To resolve which Trp contributes most to the
emission spectrum of TycA-A, single point mutants bearing Trp to Phe
substitutions were constructed. Mutant proteins were tested for thermal
and structural stability using the thermal shift assay and limited proteolysis.
Acrylamide quenching was employed to probe accessibility of individual Trp
residues upon substrate binding to mutant and wild type protein,
respectively. Our results show that among five Trp residues only W227
reports to substrate binding. Conformational changes upon non-cognate
amino acid binding were also probed by acrylamide quenching in a mutant
protein bearing only W227. This showed that the extent of quenching is
more evident with non-cognate substrates, indicating a suboptimal overall
conformation with concomitant hydrolysis of the aminoacyl-adenenylate.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P61
IGG FC N-GLYCOSYLATION PROFILING BY NANO-LC-ESI-QTOF-MS OF
GLYCOPEPTIDES
1
1
2
Jerko Štambuk , Maja Bučić Baković , Maurice H. J. Selman , Manfred
2
1,3
Wuhrer , Gordan Lauc
1
Genos Ltd, Zagreb, Croatia; 2Biomolecular Mass Spectrometry Unit,
Department of Parasitology, Leiden University Medical Center, Leiden, The
3
Netherlands; Department of Biochemistry and Molecular Biology, Faculty of
Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia
[email protected]
Poster Abstracts
Glycosylation is the most common post-translational modification of proteins
that has a crucial role in their regulation and structure as well as in
modulation of variety of biological functions. Immunoglobulin G glycans are
important for binding to Fc receptors and complement activation. Changes in
glycosylation pattern like addition or removal of a single monosaccharide
residue from IgG glycans can completely alter its biological function. In order
to fully understand the role of protein glycosylation it is necessary to use
novel approach such as site-specific structural characterization.
In our study we conducted a subclass specific analysis of IgG glycosylation
pattern comprised of individuals from two Croatian Adriatic islands, Vis and
Korčula. IgG was isolated from human plasma by protein G affinity
chromatography and trypsinized to obtain glycopeptide fragments. Nano-LCESI-MS method was used for fast and detailed subclass specific IgG Fc Nglycosylation profiling in human plasma from a total of 1747 individuals. The
results we obtained show significant differences in glycome composition
between analysed individuals, revealing changes in subclass specific IgG
galactosylation, sialylation and incidence of bisecting N-acetylglucosamine.
These results may indicate that given changes in IgG Fc N-glycosylation
profile may contribute to the human phenotypic variability.
133
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P62
THE EFFECT OF PROTEOLYTIC PROCESSING ON THE LOCALIZATION AND
PHYSIOLOGICAL ACTIVITY OF THE Saccharomyces cerevisiae CELL WALL
PROTEIN Scw4p
Renata Teparić, Antonija Grbavac, Sandra Kunštek, Vladimir Mrša
Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology,
University of Zagreb, Zagreb, Croatia
[email protected]
Poster Abstracts
Yeast cell wall contains proteins that are noncovalently (Scw-proteins) or
covalently (Ccw-proteins) bound to β-1,3-glucan, either through GPI-anchors
and β-1,6-glucan, or by alkali labile ester linkages between γ-carboxyl groups
of glutamic acid and hydroxyl groups of glucoses (Pir-proteins). It was
previously shown that one of the most abundant Scw protein, Scw4p is partly
also covalently linked to the cell wall and that non-covalently bound Scw4p
underwent the proteolytic processing, while the covalently bound Scw4p was
not processed. Such finding indicates that the proteolytic processing might
determine the localization of these two forms of the protein. Proteolytic
enzymes which might have a role in processing of Scw4p are Kex2p and a
family of aspartic proteases called yapsins. To get a better insight in the
processing of Scw4p, different mutations were introduced to SCW4 in the
region of predicted processing sites. On the other hand mutant strain was
constructed missing all yapsin proteases and Kex2p (5yps∆kex2). Native and
mutated forms of Scw4p were expressed in kex2 yeast strain, strain with all
yapsin genes disrupted (5yps∆) and strain with all proteases disrupted
(5yps∆kex2) and Scw4p processing was examined. Furthermore, phenotypes
of strains overproducing native and genetically modified Scw4p forms were
examined.
134
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
The seronegative spondyloarthropathies (SpA) are a diverse family of chronic
rheumatoid disorders characterized by rheumatoid factor autoantibody
negative serum, joint inflammation and extra-articular manifestations
including psoriasis, uveitis and inflamatory bowel disease. The clinical
subsets of SpA include ankylosing spondylitis (AS), reactive arthritis (ReA),
psoriatic arthritis (PsA), entheropathic arthritis (EA) and undifferentiated SpA
(UdSpA). Human leukocyte antigen-B27 (HLA-B27) gene is the most
commonly accounted HLA locus in the genetic background of SpA. However,
its overall genetic contribution to disease susceptibility varies between the
SpA subsets as well as between ethnic groups and populations. In order to
determine HLA-B27-associated risk for SpA in Eastern Croatian population,
patients with AS (n=7), PsA (n=15), ReA (n=23), UdSpA (n=27) and
rheumatoid arthritis (n=16) were typed for HLA-B27 locus using sequencespecific primer PCR methodology (SSP-PCR). Antigen frequency was
compared to data obtained from previously typed, unrelated, healthy
Croatian controls (n=210) by using Fisher exact test and odds ratio. The
frequency of HLA-B27 antigen was significantly associated with the risk for
UdSpA [25.9% in patients vs. 10.0% in controls, P=0.025 OR=3.15, 95%
confidence interval (1.06-9.1)], and particularly with AS susceptibility [71.4%
vs. 10%, P=0.0003 OR=22.5 95% CI (3.53-180.4)]. No significant association
was revealed between B27 and other tested SpA clinical subsets, or RA.
These observations suggest an important role for B27 in genetic risk
assessment for UdSpA forms and AS development in our population.
However, the occurrence of PsA, ReA and RA appears to be under the
influence of non-B27 predisposing factors, both within and outside of the
major histocompatibility complex. Sample size enlargement and HLA class I
loci typing are necessary for determining form-specific SpA genetic variants.
Poster Abstracts
P63
THE
INFLUENCE
OF
HLA-B27
IN
PREDISPOSITION
TO
SPONDYLOARTHROPATIES AMONG EASTERN CROATIANS
1
2,4
3,4
4
Stana Tokić , Marija Glasnović , Mario Štefanić , Ljubica Glavaš-Obrovac ,
1,4
Saška Marczi
1
Department of Molecular Diagnostics and Tissue Typing, Osijek University
Hospital, J.Huttlera 4, 31000 Osijek, Croatia; 2Clinic for Internal Medicine,
3
Osijek University Hospital, J. Huttlera 4, 31000 Osijek, Croatia; Clinical
Institute of Nuclear Medicine and Radiation Protection, Osijek University
4
Hospital, J.Huttlera 4, 31000 Osijek, Croatia; Faculty of Medicine, University
of Osijek, J. Huttlera 4, 31000 Osijek, Croatia
[email protected]
135
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P64
PROTECTIVE EFFECT OF POLYPHENOLS FROM CHOKEBERRY JUICE AND
POWDER (Aronia melanocarpa) ON OXIDATIVE STRESS AND
HYPERCHOLESTEROLEMIA IN C57BL MICE
1
1
2
Mandica-Tamara Tolić , Lana Nikolić , Domagoj Đikić , Ines Panjkota
1
3
1
Krbavčić , Nada Vahčić , Irena Landeka
1
Laboratory for Food Chemistry and Biochemistry, Faculty of Food Technology
and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb,
Croatia; 2Department of Animal Physiology, Faculty of Science, University of
3
Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia; Laboratory for Food
Quality Control, Faculty of Food Technology and Biotechnology, University of
Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia.
[email protected]
136
The potential hypolipidemic effect of chokeberry juice and powder (Aronia
melanocarpa) was studied in a mice model of dietary-induced
hypercholesterolemia. Animals were organized in groups (N=7/group)
according to diet. For four weeks they were fed in the following order: 1.
normal diet (ND, control); 2. ND+Chokeberry juice; 3. ND+Quercetin; 4.
ND+Powder (Aronia melanocarpa); 5. Cholesterol rich diet (CH); 6.
CH+Chokeberry juice; 7. CH+Quercetin; 8. CH+Powder (Aronia melanocarpa).
Lipid profile, transaminases as aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) were measured in serum in addition to the activity of
the antioxidant enzymes catalase (CAT), glutathione (GSH), superoxide
dismutase (SOD) and malondialdehyde in the liver after the treatment
period. Hypercholesterolemia and hypertriglyceridemia were established as
a consequence of the cholesterol-rich diets. Hypercholesterolemic diet
significantly increased (P<0.05) TC, TAG and LDL in comparison with normal
diet. Treatment with chokeberry juice, powder (Aronia melanocarpa) or
quercetin in hypercholesterolemic group significantly decreased TC, TAG and
LDL in comparison with the hypercholesterolemic group (P<0.05) while HDL
statistically significant magnified (P<0.05). The activities of hepatic
superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT) were
found particularly increased in groups with chokeberry juice and powder
treatment. These effects might be attributed to the high natural presence of
antioxidant polyphenols. The consumption of powder (Aronia melanocarpa)
with a hypercholesterolemic diet improved the lipidemic profile, reduced
lipid peroxidation and increases antioxidant enzymes, suggesting that
powder (Aronia melanocarpa) might contribute to a reduction of
cardiovascular risk.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
The yeast Saccharomyces cerevisiae is a useful model for studying the
influence of different stress factors to the cells. It is known that metalinduced stress is mostly oxidative stress. The mechanism by which yeast cells
combat the toxicity of pesticides is less known. The aim of this work was to
analyse and compare the influence of pesticide imidacloprid and heavy metal
lead to the proteome of Saccharomyces cerevisiae W303 yeast. During
cultivation of the yeast biomass, defined amount of imidacloprid (0.2
mmol/L) or lead (10 ppm) was added to the growth media and their
influence to the plasma membrane, mitochondrion, cytosol with microsomes
and on the whole yeast cells has been monitored. Crude organelles were
isolated after enzymatic disruption of the yeast’s cell wall. Proteins from the
whole cells and isolated organelles were separated using 2D electrophoresis,
stained using Mortz's silver staining method and processed using UVISpot
program. Change in proteins expression was monitored through the change
in their volume. Defined number of proteins was analysed using MS MALDITOF/TOF technique and identified using nrNCBI database. In the samples of
cytosol with microsomes, plasma membrane and mitochondrion treated
either with lead or imidacloprid, an increase in a total number of detected
proteins, comparing nontreated samples, has occurred. No significant change
in expression of proteins was observed in treated samples of plasma
membrane while in treated samples of mitochondrion, enzyme succinate
dehidrogenase shown significant increase in expression. In the samples of
cytosol with microsomes, some proteins, like those involved in processes of
carbohydrate metabolic pathways (particularly GAPDH) and enzyme Cu,Zn
superoxide dismutase responsible for protection of yeast from oxidative
stress, have increased their activity. The highest, significant increment in the
expression in both treated samples of cytosol with microsomes, disulphide
isomerase, the enzyme that catalyzes the formation and breakage of
disulfide bonds between cysteine residues within proteins as they fold, was
noticed. The involvement of Cu,Zn superoxide dismutase in yeast cell's
response treated with imidacloprid imply the possibility that imidacloprid
provoke oxidative stress in the yeast cells as the lead do.
Poster Abstracts
P65
COMPARATIVE PROTEOME ANALYSIS OF YEAST’S ORGENELLES TREATED
WITH LEAD OR IMIDACLOPRID
1
1
2
2
Ana Vida , Iva Justinić , Gordana Čanadi Jurešić , Čedomila Milin
1
2
School of Medicine, University of Rijeka, Rijeka, Croatia; Department of
Chemistry and Biochemistry, School of Medicine, University of Rijeka, Rijeka,
Croatia.
[email protected]; [email protected]; [email protected]
137
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Poster Abstracts
P66
MITE-LIKE ELEMENTS WITH INTERNAL TANDEM REPEATS IN THE PACIFIC
OYSTER Crassostrea gigas (Thunberg 1796)
Tanja Vojvoda Zeljko, Robert Bakarić, Miroslav Plohl
Ruđer Bošković Institute, Zagreb, Croatia
[email protected]
138
The Pacific oyster Crassostrea gigas (Thunberg, 1793) is a marine bivalve
(class Bivalvia) known as commercially the most important oyster species in
the world. Recently sequenced genome of this species showed abundance of
repetitive sequences that comprise ~36% of the genome and include some
transposable elements (TEs) still being active. MITE (miniature invertedrepeat transposable element) elements were estimated to constitute 8,8% of
the genome.
Recent research performed in our laboratory shows that the clam Donax
trunculus genome is populated by MITE-related interspersed repetitive DNA
sequences that bear short arrays of tandem repeats, connecting in this way
satellite DNAs and TEs (Genome Biol. Evol. 5:2549). The described MITE
elements are characterized by a modular structure composed of the left
conserved region, followed by a spacer sequence of different length (app.
70-160 bp), internal tandem repeats (1-6), an imperfect microsatellite repeat
motif (ACRG)n and a conserved right region. Imperfect inverted repeats (app.
10-20 bp) are located at terminal and subterminal positions and elements
are flanked with two-nucleotide target site duplications.
Here we report three new elements which correspond to the described
modular structure. Elements named Cragi 1, Cragi 2 and Cragi 3 are found by
bioinformatic analysis of the assembled genome of the oyster C. gigas.
Although Cragi elements share a similar microsatellite motif, their sequences
are different in all other modules – the left and right flanking sequences, and
the internal tandem repeats. The number of internal tandem repeats is
variable within an element, and in Cragi elements can be up to 12, what is
twice more than in previously described MITE–like elements. While Cragi 1
internal tandem repeats show 80% sequence similarity with the highly
abundant Cg170 satellite DNA already described in C. gigas, internal repeats
of the other two elements do not reveal similarity with any known satellite
DNA. Further research is in progress to resolve if Cragi 2 and Cragi 3 repeats
can be present as autonomous units repeated in tandem and form arrays
typical for satellite DNAs. It was also noted that all three whole-length Cragi
elements show a high degree of sequence similarity with eukaryotic rollingcircle transposons known as Helitrons. Here we propose that Helitron-related
TEs bearing internal tandem repeats might be an initial form from which
satellite DNA spread begins.
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
Oxidative stress represents one of the key pathophysiological mechanisms in
liver disease associated with obesity. Due to its significant role in disease
development, increased oxidative stress remains a potential attractive target
for prevention and therapy of adverse high fat diet and ovariectomy impact.
Intake of phytochemical-rich food or supplements could reduce the impact
of high fat diet and estrogen deficiency on oxidative stress. The aim of this
study was to estimate the impact of high fat diet and ovariectomy on the
oxidative and antioxidative status in the rat liver, as well as the effect of
cereal selenized onion biscuit supplements, containing bioactive compounds
with antioxidative properties, on oxidative damage in the rat liver. Fortyeight female Wistar rats were included in the study and divided into six
groups: sham operated and ovariectomized rats that received either
standard diet, high fat diet, or high fat diet supplemented with cereal
selenized onion biscuits. Liver oxidative damage was determined by lipid
peroxidation levels expressed in terms of thiobarbituric acid reactive
substances (TBARS), while liver antioxidative status was determined by
catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST),
glutathione reductase (GR) activities, and glutathione (GSH) content. High fat
diet significantly increased TBARS content in the liver compared to standard
diet. Furthermore, high fat diet decreased the activities of CAT, GR, and GST,
as well as the content of GSH. Selenized onion biscuits increased GR activity
in sham operated rats, and CAT activity in ovariectomized group of rats. No
interaction effect on oxidative/antioxidative status was observed between
high fat diet and ovariectomy. Feeding rats with high fat diet resulted with
decreased antioxidative defense and increased oxidative stress. Bioactive
compounds of cereal selenized onion biscuits showed potential to attenuate
the adverse impact of high fat diet on antioxidative status.
Poster Abstracts
P67
SELENIZED ONION BISCUITS IN ATTENUATING OXIDATIVE STRESS, INDUCED
BY HIGH FAT DIET, IN THE LIVER OF OVARIECTOMISED RATS
1
1
1
1
Rosemary Vuković , Senka Blažetić , Ana Vuković , Kristina Vuković , Martina
1
2
3
2
1
Varga , Marta Balog , Zora Krivošíková , Marija Heffer , Elizabeta Has-Schön
1
Department of Biology, J. J. Strossmayer University of Osijek, Osijek, Croatia;
2
Department of Medical Biology, School of Medicine, J. J. Strossmayer
3
University of Osijek, Osijek, Croatia; Faculty of Medicine, Slovak Medical
University in Bratislava, Bratislava, Slovakia.
[email protected]
139
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
P68
REVERSIBLE DISSOCIATION OF THE YEAST V-ATPASE ANALYZED UNDER IN
VIVO CONDITIONS
Katharina Tabke, Andrea Albertmelcher, Olga Vitavska, Markus Huss, HansPeter Schmitz, Helmut Wieczorek
Department of Biology/Chemistry, University of Osnabrück, 49069
Osnabrück, Germany
[email protected]
Poster Abstracts
Proton transport by V-ATPases is regulated via the reversible dissociation of
the V1VO holoenzyme into its peripheral, catalytic V 1 domain (consisting of
subunits A, B, C, D, E, F and G) and its membrane bound, proton
translocating VO domain (consisting of subunits a, c, c’, c’’, d, and e). The
nutrient dependent dissociation had been first detected in the midgut
epithelium of moulting or starving tobacco hornworms (Manduca sexta) and
shortly afterwards in glucose deprived yeast cells (Saccharomyces cerevisiae).
Since reversible dissociation so far had been analyzed mostly in vitro, we
tested this phenomenon under in vivo conditions. We used living yeast cells
with V-ATPase subunits fused to fluorescent proteins and found that, in
contrast to earlier findings using in vitro techniques, not the whole V1
domain with its seven different subunits, but only the V 1 subunit C was
released into the cytosol after withdrawal of extracellular glucose.
Disassembly but not reassembly depended on an intact microtubular system.
Results from overlay blots, pull-down assays and bimolecular fluorescence
complementation support the assumption that the V 1 subunit C directly
interacts with microtubules without involvement of linker proteins.
140
Author Index
Abram, Maja: P43
Abramić, Marija: P54
Agić, Dejan: P4
Ahel, Ivan: P35, P50
Ahel, Josip: P50
Albertmelcher, Andrea: P68
Amić, Dragan: P4
Anderluh, Gregor: P30
Antolović, Roberto: P6
Anzai, Naohiko: P6
Apáti, Ágota: L17
Arikala, Hema M.: SP1
Aulchenko, Yurii: P31
Baati, Rachid: SP8
Babič, Michal: P51
Babić, Ana: P1
Bajek, Marin: P47
Bakarić, Robert: P66
Balen, Biljana: P49
Balen, Daniela: P6
Balog, Marta: P2, P67
Balog, Tihomir: P54
Barbarić, Slobodan: P46
Barišić, Karmela: P16
Barker, David: P42
Bartölke, Rabea: L15
Bar-Zvi, Dudy: P49
Batičić Pučar, Lara: P3, P14
Bauer, Nataša: L10, P30, P53
Baus Lončar, Mirela: P9, P44
Bečeheli, Ivona: P31
Belovari, Tatjana: P44
Belužić, Robert: P23
Bešlo, Drago: P4
Biđin, Siniša: SP11
Bielen, Ana: SP11, P18
Bisio, Alessandra: SP7
Blazevic, Sofia: SP2
Blažeković, Robert: P2
Blažetić, Senka: L3, P2, P67
Bogut, Ivan: P48
Bolt, Edward L.: SP5
Bosak, Anita: P5
Bosch, Thomas C. G.: L13
Brady, Nathan: L1
Brčić-Kostić, Krunoslav: P56
Breljak, Davorka: P6, P7, P28
Broznić, Dalibor: P40
Brüning, Jens C: L16
Bruvo-Mađarić, Branka: SP11
Brzica, Hrvoje: P6
Bubonja Šonje, Marina: P43
Bučević-Popović, Viljemka: P8, P60
Bučić Baković, Maja: P61
Buday, László: L2
Bujak, Maro: P9
Buljević, Sunčica: P3, P14
Burckhardt, Birgitta C.: P6, P28
Burckhardt, Gerhard: P6, P28
Bušić, Valentina: P10
Campbell, Harry: P31
Chen, Kevin: SP1
Coelho, Miguel: L14
Colletier, Jacques-Philippe: SP8
Crnković, Goranka: P11
Crnolatac, Ivo: P59
Cvetanović, Aleksandra: P26
Čanadi Jurešić, Gordana: P40, P65
Čaušević, Mirsada: P12
Čermak, Stjepko: P12
Černi, Silvija: P58
Čičin-Šain, Lipa: P32
Čikeš-Čulić, Vedrana: P42
Čulić, Ognjen: P16
Ćetković, Helena: P23, P35, P50
Ćurković-Perica, Mirna: SP4
Dabelić, Sanja: P16
de Sousa, Julien: SP8
Degoricija, Marina: P8
Delaš, Ivančica: P13
Detel, Dijana: P3, P14
Dias, José: P29
Dobrivojević, Marina: P51
Domazet-Lošo, Tomislav: L13
Dominko, Kristina: P15
Dražić, Tonko: P13
Author Index
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
143
Author Index
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
144
Drenjančević, Ines: P9
Dulić, Morana: SP6
Dulić, Vjekoslav: SP10
Dumić, Jerka: P16, P17
Dutra-Clarke, Marina: SP1
Đelmiš, Josip: P13
Đikić, Domagoj: P15, P64
Đikić, Ivan: L1
Erdei, Zsuzsa: L17
Erjavec, Igor: SP2
Faix, Jan: L9
Ferhatović Hamzić, Lejla: P51
Filić, Vedrana: L9
Filić, Želimira: P18
Fokin, Valery V.: L8
Franjević, Damjan: P19
Fučić, Aleksandra: L12
Gajović, Srećko: P51
Garaj-Vrhovac, Vera: P20
Gašo-Sokač, Dajana: P10
Gazić Smilović, Ivana: P5
Gerić, Marko: P20
Giacometti, Jasminka: P41, P43
Glad, Marin: P47
Glasnović, Marija: P63
Glavaš-Obrovac, Ljubica: P26, P39,
P44, P63
Gobin, Ivana: P1, P11, P27
Goldstein, Pavle: SP11
Gornik, Olga: P31
Gould, Sven: P19
Grbavac, Antonija: P62
Gros, Katarina: SP3, P21
Grubič, Zoran: SP3, P21
Grubor, Mirko: P22
Gruić-Sovulj, Ita: SP6
Gulin, Maja: P24
Gumbarević, Darko: P41
Hadžija, Mirko: P33
Hagos, Yohannes: P6
Hamacher-Brady, Anne: L1
Harcet, Matija: P23
Has-Schön, Elizabeta: P48, P67
Hayward, Caroline: P31
Hećimović, Silva: L4, P12, P15, P34
Heffer, Marija: L3, P2, P48, P67
Hegedüs, Csilla: L17
Heinisch, Jürgen: L15
Henjakovic, Maja: P6
Herak Bosnar, Maja: P23
Herak-Kramberger, Carol M.: P28
Herrera-Molina, Rodrigo: P45
Herron, Paul: P18
Hlevnjak, Dora: P46
Hodnik, Vesna: P30
Horák, Daniel: P51
Horvat, Anđela: SP10
Horvatić, Anita: P9
Hranilovic, Dubravka: SP2
Hrenar, Tomica: P52
Huss, Markus: P68
Imešek, Mirna: P35
Inga, Alberto: SP7
Ismail, Said I.: P8
Ivančić Baće, Ivana: SP5
Ivić, Nives: P18
Ivković, Jana: P28
Jaing, Crystal J.: P8
Jajčanin Jozić, Nina: P54
Jakimowicz, Dagmara: P18
Janušić, Renato: P20
Jean, Ludovic: SP8
Jelenčić, Vedrana: L16, P24
Jelić, Dubravko: L7
Jezernik, Karlo: P58
Ježić, Marin: SP4
Jovanović, Marko: P25, P41, P43
Jovic, Tanja: P34
Jukić, Marijana: P26
Juraga, Denis: P27
Jurak, Igor: L5
Juranić, Martina: L10
Juras, Josip: P13
Jurčić-Momčilović, Diana: P27, P43
Justinić, Iva: P65
Kalafatić, Mirjana: P19
Kalanj Bognar, Svjetlana: P45
Karaica, Dean: P6, P7, P28
Katalinić, Maja: P10, P29
Katanić, Zorana: SP4
Kekez, Mario: P30, P53
Keser, Toma: P31
Kesić, Maja: P32
Klarić, Lucija: P31
Kliachyna, Maria: SP8
Klimovich, Alexander: L13
Knežević, Anamarija: P5, P38
Koepsell, Hermann: P7
Kolčić, Ivana: P31
Kolodziej, Angela: P45
Kopačin, Tomislav: P9
Korber, Philipp: P46
Korolija, Marina: P33
Kosicek, Marko: P34
Kovačević, Goran: P19
Kovarik, Zrinka: L8, P5, P29, P37,P38
Krajina, Nevenka: P39
Kraus, Ognjen: P6
Kraushar, Mathew L.: SP1
Krietenstein, Nils: P46
Krištić, Jasminka: P31
Krivošíková, Zora: P67
Krstin, Ljiljana: SP4
Kučić, Natalia: P3
Kunštek, Sandra: P62
Labak, Irena: P2
Lalić, Jasna: P35
Landeka, Irena: P64
Lauc, Gordan: P31, P61
Leljak-Levanić, Dunja: L10
Lenac, Danijela: P47
Levanat, Sonja: SP7, P36
Lončar, Jovica: SP12, P28
Lorenzon Paola: SP3
Lovrić, Marija: P51
Lučić, Bono: P4
Luić, Marija: P18, P35
Ljubojević, Marija: P6, P28
Mačak Šafranko, Željka: P54
Maček Hrvat, Nikolina: L8, P29, P37
Majsec, Kristina: SP5
Malenica Staver, Mladenka: P1, P11
Malnar, Martina: P15
Mandelboim, Ofer: L16
Mangino, Massimo: P31
Manjasetty, Babu A.: P18
Maraković, Nikola: P38, P57
Marc, Janja: P17
Marczi, Saška: P39, P63
Marecic, Valentina: P47
Marić, Petra: P28
Marinić, Jelena: P40
Marinković, Mija: L1
Marinović, Maja: L9
Markotić, Anita: P42
Markovinović, Andrea: P41
Marš, Tomaž: SP3, P21
Martin, William: PL7, P19
Mastelić, Angela: P42
Matešić, Marina: P27
Matkovič, Urška: SP3, P21
McLoughlin, Kevin S.: P8
Meden, Majda: P47
Mendrila, Davor: L16
Menni, Cristina: P31
Micek, Vedran: P6, P28
Mihalj, Martina: P9
Mihaljevic-Peles, Alma: P22
Mihaljević, Branka: P9
Mihaljević, Ivan: SP12, P28
Mijakovic, Ivan: PL3
Mikoč, Andreja: P23, P35, P50
Milin, Čedomila: P40, P65
Milovanović, Sanja: P43
Missoni, Saša: P31
Miš, Katarina: SP3, P21
Mišković, Katarina: P9, P26, P44
Mlinac, Kristina: P45
Montag, Dirk: P45
Mrša, Vladimir: P62
Muck-Seler, Dorotea: P22
Musani, Vesna: SP7, P36
Author Index
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
145
Author Index
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
146
Musladin, Sanja: P46
Mustapic, Maja: P22
Nachon, Florian: SP8, P29
Nikolić, Lana: P64
Novak Nakir, Ivana: L1
Novokmet, Mislav: P31
Novokmet, Natalija: P31
Opačak-Bernardi, Teuta: SP9
Orešković, Darko: P32
Orhanović, Stjepan: P60
Ozanic, Mateja: P47
Ozretić, Petar: SP7, P36
Panjkota Krbavčić, Ines: P64
Paradžik, Martina: P8
Paradžik, Tina: SP11, P18
Parato, Giulia: SP3
Pavela-Vrančič, Maja: P60
Pavić, Valentina: P48
Pecht, Israel: PL2
Peharec Štefanić, Petra: P49
Perina, Drago: P23, P35, P50
Pernjak Pugel, Ester: P14
Perona, John J.: SP6
Peter-Katalinić, Jasna: P25
Petrik, Jozsef: P16
Pezer Sakač, Željka: SP11
Piantanida, Ivo: P44, P59
Pinterić, Marija: P55
Pirkmajer, Sergej: SP3, P21
Piskur, Vanda: P47
Pivac, Nela: P22
Pleše, Bruna: P35
Plohl, Miroslav: P66
Podbregar, Matej: SP3, P21
Pohlentz, Gottfried: P25
Polašek, Ozren: P31
Polić, Bojan: L16, P24
Poljak, Igor: SP4
Pongrac, Igor: P51
Pongracz, Judit E.: L11
Popovic, Marta: SP12
Popović Hadžija, Marijana: P33
Primorac, Dragan: P31
Primožič, Ines: P52
Prunk, Mateja: P17
Pučić-Baković, Maja: P31
Punda Polić, Volga: P8
Radić, Zoran: L8, P37
Radović, Nikola: P6
Rajc, Jasmina: P44
Rajević, Nives: P19
Rasin, Mladen-Roko: SP1
Raucher, Drazen: SP9
Razdorov, Genadij: P30
Renard, Pierre-Yves: SP8
Reynisson, Jóhannes: P42
Režić-Mužinić, Nikolina: P42
Rigling, Daniel: SP4
Rogić, Tea: P49
Rokov Plavec, Jasmina: P30, P53
Ross, Ashley: P42
Rožman, Marko: P45
Rudan, Igor: P31
Rudan, Pavao: P31
Ruiz-Trillo, Innaki: P23
Rusak, Gordana: P59
Ryu, Jung Su: SP9
Sabljić, Igor: P35
Sabol, Maja: SP7, P36
Sabolić, Ivan: P6, P7, P28
Sagud, Marina: P22
Salopek-Sondi, Branka: L6
Santic, Marina: P47
Santoni, Gianluca: SP8
Sarkadi, Balázs: L17
Schleiff, Enrico: PL6
Schmitz, Hans-Peter: P68
Selman, Maurice H. J.: P61
Sever, Sanja: PL5
Sit, Rakesh K.: L8
Slade, Neda: SP10
Slović, Anamarija: P55
Smalla, Karl-Heinz: P45
Smital, Tvrtko: SP12, P28
Sobočanec, Sandra: P33, P54
Soldo, Barbara: P60
Soreq, Hermona: PL1
Spector, Tim: P31
Sprinzl, Mathias: PL4
Starčević, Vito: P13
Stillman, Althea: SP1
Stojan, Jure: P57
Stojković, Ranko: P44
Svob Strac, Dubravka: P22
Šarac, Jelena: P31
Šarčević, Božena: P20
Šarić, Ana: P54
Šarić, Ela: P53
Šerić, Vatroslav: P44
Šeruga Musić, Martina: P55
Šestan, Marko: L16
Šimatović, Ana: P56
Šinko, Goran: P29, P38, P57
Šitum, Marijan: P8
Škorić, Dijana: P58
Šlouf, Miroslav: P51
Šola, Ivana: P59
Šprung, Matilda: P60
Štambuk, Jerko: P61
Štefanić, Mario: P63
Štimac, Davor: L16
Šupraha Goreta, Sandra: P16
Švarc-Gajić, Jaroslava: P26
Tabke, Katharina: P68
Tartaro Bujak, Ivana: P9
Taylor, Palmer: L8, P37
Teparić, Renata: P62
Terzić, Janoš: L1, P8
Thaqi, Kujtim: P31
Theurich, Sebastian: L16
Tokić, Stana: P39, P63
Tolić, Iva: L14
Tolić, Mandica-Tamara: P64
Tolušić Levak, Maja: P9
Tomić, Srđanka: P52
Tota, Marin: P40
Trnski, Diana: SP7, P36
Turk Wensveen, Tamara: L16
Tyldesley-Worster, Richard: P25
Uzarevic, Zvonimir: P36
Vahčić, Nada: P64
Valdes, Ana: P31
Valentić, Sonja: L16
Varga, Martina: P67
Varljen, Jadranka: P3, P14
Vida, Ana: P65
Viljetić, Barbara: SP1, P2
Vinković, Vladimir: P5, P38
Vitavska, Olga: L15, P68
Vlašić, Ignacija: P56
Vojvoda Zeljko, Tanja: P66
Vrhovac, Ivana: P6, P7, P28
Vučinić, Srđan: P9
Vučković, Frano: P31
Vuica-Ross, Milena: P42
Vujaklija, Dušica: SP11, P18
Vujaklija, Ivan: SP11
Vukelić, Željka: P45
Vukicevic, Slobodan: SP2
Vuković, Ana: P67
Vuković, Kristina: P67
Vuković, Rosemary: P2, P67
Weber, Igor: L9
Weik, Martin: SP8
Wensveen, Felix M.: L16, P24
Weygand-Đurašević, Ivana: P30, P53
Wieczorek, Helmut: L15, P68
Wijeratne, Sagara H.R.: SP1
Wilson, James F.: P31
Wuhrer, Manfred: P61
Wunderlich, F. Thomas: L16
Zivkovic, Maja: P22
Zoldoš, Vlatka: P31
Zorbaz, Tamara: P16
Zorić, Arijana: SP10
Žaja, Roko: SP12, P35, P50
Žunec, Suzana: L8
Author Index
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
147
List of
Participants
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Ambramović, Denis, Crux d.o.o., II Poljanice 8, 10000 Zagreb,
[email protected]
Babić, Ana, School of Medicine, University of Rijeka, Croatia,
[email protected]
Balog, Marta, Faculty of Medicine, J.J. Strossmayer University Osijek, J.
Hutlera 4, 31000 Osijek, Croatia, [email protected]
Balog, Tihomir, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb,
Croatia, [email protected]
Barišić, Karmela, Faculty of Pharmacy and Biochemistry, University of
Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia, [email protected]
Batičić Pučar, Lara, University of Rijeka, School of Medicine, Department
of Chemistry and Biochemistry, Braće Branchetta 20, 51000 Rijeka,
Croatia, [email protected]
Bešlo, Drago, Faculty of Agriculture in Osijek, J.J. Strossmayer University
Osijek, Kralja Petra Svačića 1d, 31107 Osijek, Croatia, [email protected]
Bielen, Ana, Laboratory for Biology and Microbial Genetics, Department
for Biochemical Engineering, Faculty of Food Technology and
Biotechnology, University of Zagreb, Pierrotijeva 6, 10000 Zagreb,Croatia,
[email protected]
Blažetić, Senka, J.J. Strossmayer University of Osijek, Department of
Biology, Cara Hadrijana 8A, 31000 Osijek, Croatia,
[email protected]
Borovac, Josip Anđelo, School of Medicine, University of Split, Šoltanska 2,
21000 Split, Croatia, [email protected]
Bosak, Anita, Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10000 Zagreb, Croatia, [email protected]
Breljak, Davorka, Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10000 Zagreb, Croatia, [email protected]
Bučević-Popović, Viljemka, Department of Chemistry, Faculty of Science,
University of Split,Teslina 12, 21000 Split, Croatia, [email protected]
Buday, László, Institute of Enzymology, Research Centre for Natural
Sciences, Hungarian Academy of Sciences, Magyar Tudósokkörútja 2.,
1117 Budapest, Hungary, [email protected]
Bujak, Maro, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb,
Croatia, [email protected]
Bušić, Valentina, J.J. Strossmayer University of Osijek, Faculty of Food
Technology, Kuhačeva 20, 31000 Osijek, Croatia, [email protected]
Carević, Andreja, HEBE d.o.o., Bukovčeva 2, 21000 Split,
[email protected]
Crnković, Goranka, School of Medicine, University of Rijeka, Braće
Branchetta 20, 51000 Rijeka, Croatia, [email protected]
List of Participants
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
151
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
List of Participants
32.
152
33.
34.
35.
Čanadi Jurešić, Gordana, Department of Chemistry and Biochemistry,
School of Medicine, University of Rijeka, Braće Branchetta 20, 51000
Rijeka, Croatia, [email protected]
Čermak, Stjepko, Ruđer Bošković Institute, Division of Molecular
Medicine, Bijenička cesta 54, 10000 Zagreb, Croatia,
[email protected]
Ćurković-Perica, Mirna, University of Zagreb, Faculty of Science,
Department of Biology, Marulićev trg 9a, 10000 Zagreb, Croatia,
[email protected]
Dananić, Mario , Alphachrom d.o.o., Karlovačka cesta 24, 10000 Zagreb,
Croatia, [email protected]
Delaš, Ivančica, Department of Chemistry and Biochemistry, School of
Medicine, University of Zagreb, Šalata 3, 10000 Zagreb, Croatia,
[email protected]
Detel, Dijana, University of Rijeka, School of Medicine, Department of
Chemistry and Biochemistry, Braće Branchetta 20, 51000 Rijeka, Croatia,
[email protected]
Dias, José, Département de Toxicologie - Pôle NRBC, Institut de Recherche
Biomédicale des Armées, BP73, 91223 Brétigny sur Orge, France,
[email protected]
Domazet-Lošo, Tomislav, Laboratory of Evolutionary Genetics, Ruđer
Bošković Institute and Catholic University of Croatia, Bijenička cesta 4,
10000 Zagreb, Croatia, [email protected]
Dominko, Kristina, Ruđer Bošković Institute, Bijenička cesta 54, 10000
Zagreb, Croatia, [email protected]
Dulić, Morana, Department of Chemistry, Faculty of Science, University of
Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia, [email protected]
Dumić, Jerka, University of Zagreb, Faculty of Pharmacy and Biochemistry,
Department of Biochemistry and Molecular Biology, Ante Kovačića 1,
10000 Zagreb, Croatia, [email protected]
Filić, Želimira, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb,
Croatia, [email protected]
Franjević, Damjan, Faculty of Science, University of Zagreb, Rooseveltov
trg 6, 10000 Zagreb, Croatia, [email protected]
Fučić, Aleksandra, Institute for Medical Research and Occupational
Health, Ksaverska c. 2, 10000 Zagreb, Croatia, [email protected]
Fulgosi, Hrvoje, Division of Molecular Biology, Ruđer Bošković Institute,
Bijenička 54, 10000 Zagreb, Croatia, [email protected]
Gerić, Marko, Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10000 Zagreb, Croatia, [email protected]
Giacometti, Jasminka, Department of Biotechnology, University of Rijeka,
Radmile Matejčić 2, 51000 Rijeka, Croatia, [email protected]
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
Glavaš-Obrovac, Ljubica, Faculty of Medicine, J.J. Strossmayer University
of Osijek, Huttlerova 4, 31000 Osijek, Croatia, [email protected]
Gobin, Ivana, University of Rijeka, School of Medicine, Braće Branchetta
20, 51000 Rijeka, Croatia, [email protected]
Granić, Ivica, Sartorius Croatia - Libra elektronik d.o.o., Savska 45ª, 10 290
Zaprešić, Croatia, [email protected]
Gros, Katarina, University of Ljubljana, Faculty of Medicine, Institute of
Pathophysiology, Zaloška 4, 1001 Ljubljana, Slovenia,
[email protected]
Grubor, Mirko, Ruđer Bošković Institut, Bijenička cesta 54, 10000 Zagreb,
Croatia, [email protected]
Hečimović, Silva, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb,
Croatia, [email protected]
Heffer, Marija, Faculty of Medicine, J.J. Strossmayer University of Osijek,
Huttlerova 4, 31000 Osijek, Croatia, [email protected]
Herak Bosnar, Maja, Ruđer Bošković Institute, Bijenička 54, 10002 Zagreb,
Croatia, [email protected]
Hranilović, Dubravka, University of Zagreb, Faculty of Science, Division of
Biology, Department of Animal Physiology, Rooseveltov trg 6, 10000
Zagreb, Croatia, [email protected]
Ivančić Baće, Ivana, Faculty of Science, Horvatovac 102a, 10000 Zagreb,
Croatia, [email protected]
Jelenčić, Vedrana, School of Medicine, University of Rijeka, Braće
Branchetta 20, 51000 Rijeka, Croatia, [email protected]
Jelić, Dubravko, Fidelta Ltd., Prilaz baruna Filipovića 29, 10000 Zagreb,
Croatia, [email protected]
Jerončić, Ana, University of Split, School of Medicine, Šoltanska 2, 21000
Split, Croatia, [email protected]
Jovanović, Marko, Department of Biotechnology, University of Rijeka,
Radmile Matejčić 2, 51000 Rijeka, Croatia, [email protected]
Jukić, Marijana, J.J. Strossmayer University of Osijek, Faculty of Medicine,
J. Huttlera 4, 31000 Osijek, Croatia, [email protected]
Juraga, Denis, School of Medicine, University of Rijeka, Braće Branchetta
20, 51000 Rijeka, Croatia, [email protected]
Jurak, Igor, Department of Biotechnology, University of Rijeka, R. Matejčić
2, 51000 Rijeka, Croatia, [email protected]
Justinić, Iva, School of Medicine, University of Rijeka, Braće Branchetta 20,
51000 Rijeka, Croatia, [email protected]
Kalanj Bognar, Svjetlana, School of Medicine, University of Zagreb, Šalata
3, 10000 Zagreb, Croatia, [email protected]
Karaica, Dean, Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10000 Zagreb, Croatia, [email protected]
List of Participants
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
153
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
List of Participants
71.
154
72.
73.
Katalinić, Maja, Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10000 Zagreb, Croatia, [email protected]
Kekez, Mario, Department of Chemistry, Faculty of Science, University of
Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia,
[email protected]
Keser, Toma, Faculty of Pharmacy and Biochemistry, University of Zagreb,
Ante Kovačića 1, 10000 Zagreb, Croatia, [email protected]
Kesić, Maja, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb,
Croatia, [email protected]
Korolija, Marina, Ruđer Bošković Institute, Bijenička 54, 10000
Zagreb,Croatia, [email protected]
Košiček, Marko, Division of Molecular Medicine, Ruđer Bošković Institute,
Bijenička 54, 10000 Zagreb, Croatia, [email protected]
Kovarik, Zrinka, Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10000 Zagreb, Croatia, [email protected]
Kućan, Željko, Croatian Academy of Sciences and Arts, Trg Nikole Šubića
Zrinskog 11, 10000 Zagreb, Croatia, [email protected]
Labak, Irena, Department of Biology, J.J. Strossmayer University in Osijek,
Ulica cara Hadrijana 8A, 31000 Osijek, Croatia, [email protected]
Lalić, Jasna, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb,
Croatia, [email protected]
Landeka Jurčević, Irena, University of Zagreb, Faculty of Food Technology
and Biotechnology, Laboratory for Food Chemistry and Biochemistry,
10000 Zagreb, Croatia, [email protected]
Leljak-Levanić, Dunja, University Zagreb, Faculty of Science, Department
of Molecular Biology, Horvatovac 102a, 10000 Zagreb, Croatia,
[email protected]
Levanat, Sonja, Laboratory for Hereditary Cancer, Division of Molecular
Medicine, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia,
[email protected]
Maček Hrvat, Nikolina, Institute for Medical Research and Occupational
Health, Ksaverska cesta 2, 10000 Zagreb, Croatia, [email protected]
Maraković, Nikola, Institute for Medical Research and Occupational
Health, Ksaverska cesta 2, 10001 Zagreb, Croatia, [email protected]
Marczi, Saška, Department of Molecular Diagnostics and Tissue Typing,
Osijek University Hospital, Osijek, Croatia, J. Huttlera 4, 31000 Osijek,
Croatia, [email protected]
Marčac Grahek, Tatjana, Kemomed d.o.o., Kališka 9, 4000 Kranj, Slovenia,
[email protected]
Marinić, Jelena, Department of Chemistry and Biochemistry, School of
Medicine, University of Rijeka, Braće Branchetta 20, 51000 Rijeka, Croatia,
[email protected]
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
Markovinović, Andrea, Department of Biotechnology, University of Rijeka,
Radmile Matejčić 2, 51000 Rijeka, Croatia,
[email protected]
Maršić, Tereza, INEL - medicinska tehnika d.o.o., Buzinski prilaz 32, 10010
Zagreb, [email protected]
Martin, Stanislav, LKB Vertriebs G.m.b.H, Nova Cesta 103, 10000 Zagreb,
Croatia, [email protected]
Martin, William, Institute of Molecular Evolution at Heinrich-HeineUniversität Düsseldorf, Düsseldorf, Germany, [email protected]
Mastelić, Angela, Department of Medical Chemistry and Biochemistry,
University of Split, School of Medicine, Šoltanska 2, 21000 Split, Croatia,
[email protected]
Matek Sarić, Marijana, University of Zadar, Department of Health Studies,
Splitska 1, 23000 Zadar, Croatia, [email protected]
Mihaljević, Ivan, Ruđer Bošković Institute, Bijenička cesta 54, 10000
Zagreb, Croatia, [email protected]
Mijaković, Ivan, SysBio, Chalmers University of Technology, Kemivägen 10,
41296 Göteborg, Sweden, [email protected]
Milovanović, Sanja, Department of Biotechnology, University of Rijeka,
Radmile Matejčić 2, 51000 Rijeka, Croatia, [email protected]
Miš, Katarina, University of Ljubljana, Faculty of Medicine, Institute of
Pathophysiology, Zaloška 4, 1000 Ljubljana, Slovenia,
[email protected]
Mišković Katarina, Faculty of Medicine, J.J. Strossmayer University of
Osijek, Josipa Huttlera 4, 31000 Osijek, Croatia, [email protected]
Mlinac, Kristina, School of Medicine, University of Zagreb, Šalata 3, 10000
Zagreb, Croatia, [email protected]
Mraković, Jasminka, Merck d.o.o., Andrije Hebranga 32, 10000 Zagreb,
Croatia, [email protected]
Mrša, Vladimir, Laboratory of Biochemistry, Faculty of Food Technology
and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb,
Croatia, [email protected]
Musladin, Sanja, Laboratory of Biochemistry, Faculty of Food Technology
and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb,
Croatia, [email protected]
Nachon, Florian, Institut de Recherche Biomédicale des Armées, BP73,
91223 Brétigny-sur-Orge, France, [email protected]
Novak Nakir, Ivana, University of Split, School of Medicine, Šoltanska 2,
21000 Split, Croatia, [email protected]
Opačak-Bernardi, Teuta, Faculty of Medicine, J.J. Strossmayer University
of Osijek, Huttlerova 4, 31000 Osijek, Croatia, [email protected]
List of Participants
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
155
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
List of Participants
104.
156
105.
106.
Orhanović, Stjepan, University of Split, Faculty of Natural Sciences,
Department of Chemistry, Teslina 12, 21000 Split, Croatia, [email protected]
Ozretić, Petar, Laboratory for Hereditary Cancer, Division of Molecular
Medicine, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia,
[email protected]
Ožanič, Mateja, Department of Microbiology and Parasitology, Faculty of
Medicine, University of Rijeka, Braće Branchetta 20, 51000 Rijeka,
[email protected]
Pavela-Vrančič, Maja, University of Split, Faculty of Natural Sciences,
Department of Chemistry, Teslina 12, 21000 Split, Croatia,
[email protected]
Pavić, Valentina, Department of Biology, J.J. Strossmayer University of
Osijek, Cara Hadrijana 8/A, 31000 Osijek, Croatia,
[email protected]
Pecht, Israel, The Weizmann Institute of Science, Rehovot, Israel,
[email protected]
Peharec Štefanić, Petra, Department of Molecular Biology, Division of
Biology, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000
Zagreb, Croatia, [email protected]
Perina, Dragutin, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb,
Croatia, [email protected]
Polić, Bojan, Dept. Histology & Embryology, Faculty of Medicine,
University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia,
[email protected]
Pongrac, Igor, EU FP7 project GlowBrain, Croatian Institute for Brain
Research, School of Medicine, University of Zagreb, Šalata 12, 10000
Zagreb, Croatia, [email protected]
Pongracz, Judit, Department of Pharmaceutical Biotechnology, University
of Pecs, 12 Szigeti, 7624 Pecs, Hungary, [email protected]
Primozič, Ines, Department of Chemistry, Faculty of Science, University of
Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia,
[email protected]
Radić, Zoran, Skaggs School of Pharmacy and Pharmaceutical Sciences,
University of California at San Diego, La Jolla, CA 92093-0650, USA, 9500
Gilman drive, 92093 San Diego, United States of America,
[email protected]
Rokov Plavec, Jasmina, Chemistry Department, Faculty of Science,
University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia,
[email protected]
Sabolić, Ivan, Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10000 Zagreb, Croatia, [email protected]
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
122.
123.
Salopek-Sondi, Branka, Ruđer Bošković Institute, Bijenička cesta 54, 10000
Zagreb, Croatia, [email protected]
Sarkadi, Balazs, Hung. Acad. Sci., Research Center for Natural Sciences,
Dioszegi 64, 1113 Budapest, Hungary, [email protected]
Schleiff, Enrico, Cluster of Excellence Frankfurt & Center for Membrane
Proteomics, Buchmann Institute of Molecular Life Sciences, Department of
Biosciences, Molecular Cell Biology, Goethe University, D-60438 Frankfurt
am Main, Germany, [email protected]
Sever, Sanja, Nephrology Division, Massachusetts General Hospital and
Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA,
[email protected]
Slade, Neda, Ruđer Bošković Institute, Division of Molecular Medicine,
Bijenička 54, 10000 Zagreb, Croatia, [email protected]
Sobočanec, Sandra, Ruđer Bošković Institute,Bijenička cesta 54, 10000
Zagreb, Croatia, [email protected]
Soreq, Hermona, The Hebrew University of Jerusalem, Safra Campus-Givat
Ram, 91904 Jerusalem, Israel, [email protected]
Sprinzl, Mathias, Laboratorium für Biochemie, Universität Bayreuth,
Bayreuth, Germany, [email protected]
Sumpor, Danijel, Biosistemi d.o.o., Pijavišće 32, 10090 Zagreb, Croatia,
[email protected]
Šeruga Musić, Martina, Dept. of Biology, Faculty of Science, University of
Zagreb, Marulićev trg 9A, 10000 Zagreb, Croatia,
[email protected]
Šestan, Marko, Department of Histology and Embryology, School of
Medicine, University of Rijeka, Braće Branchetta 20, 51000 Rijeka, Croatia,
[email protected]
Šimac, Brankica, Diagnostica Skalpeli d.o.o., Ljudevita Juraka 24, 10090
Zagreb, Croatia, [email protected]
Šimatović, Ana, Ruđer Bošković Institute, Bijenička cesta 54, 10000
Zagreb, Croatia, [email protected]
Šinko, Goran, Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10000 Zagreb, Croatia, [email protected]
Škorić, Dijana, University of Zagreb, Faculty of Science, Department of
Biology, Marulićev trg 9a, 10000 Zagreb, Croatia,
[email protected]
Šola, Ivana, Department of Biology, Faculty of Science, University of
Zagreb, Marulićev trg 9a, 10000 Zagreb, Croatia, [email protected]
Šprung, Matilda, University of Split, Faculty of Natural Sciences,
Department of Chemistry, Teslina 12, 21000 Split, Croatia,
[email protected]
List of Participants
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
157
HDBMB2014 „The Interplay of Biomolecules“
24-27 September 2014, Zadar, Croatia
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
List of Participants
139.
158
140.
141.
Štambuk, Jerko,Genos d.o.o., I Ferenščica 68, 10000 Zagreb, Croatia,
[email protected]
Teparić, Renata, Laboratory of Biochemistry, Faculty of Food Technology
and Biotechnology, University of Zagreb, Pierottiejva 6, 10000 Zagreb,
Croatia, [email protected]
Terzić, Janoš, School of Medicine, University of Split, Šoltanska 2, 21000
Split, Croatia, [email protected]
Tokić, Stana, Department of Molecular Diagnostics and Tissue Typing,
Osijek University Hospital, J. Huttlera 4, 31000 Osijek, Croatia,
[email protected]
Tolić, Iva, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia,
[email protected]
Tolić, Mandica Tamara, Laboratory for Food Chemistry and Biochemistry,
Faculty of Food Technology and Biotechnology, University of Zagreb,
Pierottijeva 6, Zagreb, Croatia, [email protected]
Valentić, Sonja, School of Medicine, University of Rijeka, Braće Branchetta
20, 51000 Rijeka, Croatia, [email protected]
Varljen, Jadranka, University of Rijeka, School of Medicine, Department of
Chemistry and Biochemistry, Braće Branchetta 20, 51000 Rijeka, Croatia,
[email protected]
Vida, Ana, School of Medicine, University of Rijeka, Braće Branchetta 20,
51000 Rijeka, Croatia, [email protected]
Viljetić, Barbara, Faculty of Medicine Osijek, J.J. Strossmayer University of
Osijek, J. Huttlera 4, 31000 Osijek, Croatia and RWJ Medical School,
Rutgers University, USA, [email protected]
Vinter, Adrijana, Fidelta d.o.o., Prilaz B. Filipovića 29, 10000 Zagreb,
Croatia, [email protected]
Vitale, Ljubinka, Horvatovac 17, 10000 Zagreb, Croatia, [email protected]
Vitavska, Olga, University of Osnabrück, Barbarastr. 11, 49076 Osnabrück,
Germany, [email protected]
Vojvoda Zeljko, Tanja, Ruđer Bošković Institute, Bijenička cesta 54, 10000
Zagreb, Croatia, [email protected]
Vujaklija, Dušica, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb,
Croatia, [email protected]
Vuković, Rosemary, J.J. Strossmayer University of Osijek, Department of
Biology, Cara Hadrijana 8/A, 31000 Osijek, Croatia,
[email protected]
Weber, Igor, Ruđer Bošković Institute, Division of Molecular Biology,
Bijenička 54, 10000 Zagreb, Croatia, [email protected]
Wieczorek, Helmut, University of Osnabrück, Barbarastr. 11, 49076
Osnabrück, Germany, [email protected]
Biosistemi d.o.o.
Diagnostica skalpeli d.o.o
Eli Lilly (Suisse) S.A.
FEBS
Fidelta d.o.o.
Gorea Plus d.o.o.
HEBE d.o.o.
INEL - medicinska tehnika d.o.o.
Kandit d.o.o.
KEFO d.o.o.
Kemomed d.o.o.
Koestlin d.d.
Labena d.o.o.
LKB Vertriebs Ges.M.B.H.
Maraska d.d.
MERCK d.o.o.
Obrnuta faza j.d.o.o.
Sartorius Croatia-Libra elektronik
d.o.o.
UMNA
Zadar County

PDF version of Agenda. - Interdisciplinary Management Research

• Croatian Delegation in the Economic Visit to the

Seget Donji

here - TEPSA

Survival Guide Zagreb

CURRICULUM VITAE

gala concert in zagreb - Croatian Scholarship Fund

Zagreb

incroatia where to go, what to see with nautical charts, north part

Northern Dalmatia