Báo cáo khoa học: POSTER PRESENTATIONS P1 Functional Genomics, Proteomics and Bioinformatics docx

POSTER PRESENTATIONS
P1 Functional Genomics, Proteomics and Bioinformatics
P1–1
The influence of the HERV-K LTR on KIAA1245
subfamily gene expression
N. Abrarova, E. Stoukacheva, T. Vinogradova and E. Sverdlov
Laboratory of structure and function of human genes, Institute of
Bioorganic Chemistry, Moscow, RUSSIA
Long terminal repeats (LTR) of endogenous retroviruses com-
prise about 8% of human genome. Typical LTR contains a set
of regulatory elements: promoters, enhancers, polyadenilation
sites, which can take part in neighbouring genes expression regu-
lation. Earlier, we have described a subfamily of closely related
genes highly similar to the KIAA1245 mRNA, part of which
contains a HERV-K LTR in their structure, whereas the other
lacks it. Using this subfamily as a model for studying regulation
of gene transcription, we showed that LTR serves as an alterna-
tive promoter and possess high enhancer activity. Genes that
contain LTR differ from genes lacking it in cell type specificity of
transcription. We generated a series of successive 5¢- and 3¢-dele-
tion mutants with a 200 bp increment, and also two middle vari-
ants, 200 and 400 bp in length (Mid200 and Mid400,
respectively). To determine promoter activity of LTR and its
fragments they were cloned into pGL3-BV vector, containing
luciferase reporter gene. The full-sized LTR demonstrated high
promoter activity in Tera1 and NT2/D1 cell lines. To determine
the enhancer activity LTR and subfragments were cloned in
pGL3-PV vector, which has SV40 promoter in its structure
besides luciferase gene. Transient transfections demonstrated that
Mid200 fragment of the LTR exhibits high cell type specific
enhancer activity. In Tera1 cell line it was comparable to the

activity of universal SV40 enhancer. This fact allows to suggest
that enhancer is localized in this region of the LTR. The analysis
of transcription of KIAA1245 subfamily genes have shown that
LTR-lacking gene Al592309 is not transcribed in Tera1 and
NT2/D1 cell lines, whereas LTR-containing genes are transcribed
in these cell lines. Thus, we may suggest that insertion of the
LTR in second intron of KIAA1245 genes may lead to change in
cell type specificity of their transcription. This fact allows to sug-
gest the role of the LTR in evolution of KIAA1245 subfamily
genes.
P1–2
Study of DNA interactions with cerium (III),
lanthanum (III) and gadolinium (III) ions by
using of Raman spectroscopy
V. Kohoutkova
1
, P. Babula
1
, R. Opatrilova
1
, O. Vrana
2
,
V. Adam
3
, J. Zehnalek
3
and R. Kizek
3
1

Department of Natural Drugs, University of Veterinary and
Pharmaceutical Sciences, Brno, CZECH REPUBLIC,
2
Institute of
Biophysics, Academy of Sciences of the Czech Republic, Brno,
CZECH REPUBLIC,
3
Department of Chemistry and Biochemis-
try, Mendel University of Agriculture and Forestry, Brno, CZECH
REPUBLIC
The biological properties of the lanthanides, based on their simi-
larity to calcium, have stimulated research into their therapeutic
application. Up-to-date we have been successfully using at least
two pharmaceuticals cerium nitrate as a topical cream with silver
sulfadiazene for the treatment of burn wounds and lanthanum
carbonate as a phosphate binder for the treatment of hyperphos-
phatemia. Lanthanides3+ compounds have also been investi-
gated for their anti-cancer potential. Clinical reports suggested
that several compounds such as cerium (III) iodide posed cyto-
toxic effect, however the biochemical mechanism of their action
is still unclear (1–3). Therefore, we aim our attention on study of
interactions DNA with inorganic salts of cerium (III), lanthanum
(III) and gadolinium (III). Raman spectroscopy was employed to
characterize the perturbations to DNA conformation induced in
DNA by three inorganic salts of the above mentioned lantha-
nides (2). All studied lanthanides coordinated to N7 of two
neighbouring guanine bases, as this nitrogen does not form H
bonds with other bases, in the same or in opposite DNA strands.
The results of the present work demonstrate that Raman spec-
troscopy represents a suitable tool to provide insights into struc-

tural factors involved in the mechanisms underlying antitumor
effects of drugs.
Acknowledgement: This work was supported by GA CR 522/
07/0692.
References:
1. Fricker SP. Chem. Soc. Rev. 2006; 35(6): 524–533.
2. Vrana O et al., J. Struct. Biol. 2007; 159(1): 1–8.
3. Babula P et al., Curr. Pharm. Anal. 2009; 5(1): 47–68.
P1–3
Real-time polymerase chain reaction with
electrochemical detection for detection
pandemic viruses
V. Adam
1
, D. Huska
1
, S. Krizkova
1
, J. Hubalek
2
and R. Kizek
1
1
Department of Chemistry and Biochemistry, Mendel University of
Agriculture and Forestry in Brno, Brno, CZECH
REPUBLIC,
2
Department of Microelectronics, Brno University of
Technology, Brno, CZECH REPUBLIC
Viral infections pose a threat for man kind. Diagnostic systems

focus on the direct cultural proof of identity. Almost all systems
use polymerase chain reaction. Real-time polymerase chain reac-
tion is based on the polymerase chain reaction and routinely used
to amplify and simultaneously quantify a targeted DNA molecule.
Fluorescent dyes intercalating with double-stranded DNA and
modified DNA oligonucleotide probes fluorescing when hybridized
with a complementary DNA are used for real time quantification
of the amplicons. The process of DNA quantification has demands
on robust instrument and on high cost chemicals. Electrochemical
detection is low cost and sensitive alternative method to detect
nucleic acids (1, 2). The main aim of this work is to propose novel
electrochemical detector for monitoring PCR. Miniaturized poten-
tiostat with nanotubes-based screen-printed electrodes was used
for detection of specific sequence of severe acute respiratory syn-
drome, avian influenza virus and Ebola virus amplified by poly-
merase chain reaction. The amplicons were obtaining after 2, 4, 6,
8, 10, 15, 20, 25, 30 and 35 cycles, whereas the amount of amplified
DNA was between 10 and 100 pg. The amplicons obtained even
after two cycles were detectable by using the electrochemical
instrument. Moreover we compared the results with agarose gel
electrophoresis. The lowest detectable amount of DNA was
obtained after 15 PCR cycles.
Acknowledgement: This work was supported by GA AV
KAN208130801.
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 95
References:
1. Palecek E et al. Anal. Chim. Acta. 2002; 496(1): 73–83.
2. Huska D et al. Electrophoresis 2008; 29(24): 4964–4971.
P1–4

Spatial distribution of heavy metals in healthy
and melanoma animal tissues
V. Adam
1
, O. Zitka
1
, D. Huska
1
, S. Krizkova
1
, J. Strnadel
2
,
V. Horak
2
, T. Vaculovic
3
, K. Novotny
3
, V. Kanicky
3
, J. Kaiser
4
and R. Kizek
1
1
Department of Chemistry and Biochemistry, Mendel University of
Agriculture and Forestry in Brno, Brno, CZECH
REPUBLIC,
2

Academy of Sciences of the Czech Republic, Insti-
tute of Animal Physiology and Genetics, Libechov, CZECH
REPUBLIC,
3
Department of Chemistry, Masaryk University,
Brno, CZECH REPUBLIC,
4
Institute of Physical Engineering,
Brno University of Technology, Brno, CZECH REPUBLIC
Levels of some of heavy metals are monitored and their altera-
tions can be used as diagnostic markers. Nevertheless, spatial dis-
tribution of the essential metals in animal tissues is still not clear.
The aim of this work is detection of copper and zinc in healthy
and tumour tissues of miniature pigs by using of the laser
induced breakdown spectroscopy (LIBS). Concentration of essen-
tial-heavy-metal-transporting protein metallothionein (MT) was
determined by the Brdicka reaction. Tissue cryosections (thick-
ness 40 lm) were obtained from the MeLiM strain of miniature
pigs with hereditary melanoma, particularly from healthy skin,
cutaneous nodular melanomas and metastases in the liver, spleen
and lymph nodes. Using LIBS we measured maps of spatial dis-
tribution of the essential heavy metals in cryosections and found
that the maps of healthy and tumour cryosection markedly dif-
fered. The highest content of MT was determined in the tumours
localised on the back of animals and was nearly 500 lgofMT
per gram of tissue. The MT levels determined in metastases in
spleen, lungs and liver were within the range from 40 to 160 lg/g
of the tissue, the average level was 110 ± 40 lg/g of the tissue.
Acknowledgements: This work was supported by the GA AS
CR (IAA401990701) and by the Ministry of Education, Youth

and Sport CR (2B08063) and Liga proti rakovine Praha (2009).
References:
1. Krizkova S et al. Sensors 2008; 8(5): 3106–3122.
2. Kaiser J et al. Spectrochim. Acta Part B 2009; 64(1): 67–73.
P1–5
Chromatin accessibility of MHCII locus and
enhanceosome stability determine
transcriptional competence through mitosis
P. Arampatzi, M. Gialitakis, T. Makatounakis and
J. Papamatheakis
Department of Biology, Institute of Molecular Biology and
Biotechnology, Heraklion, GREECE
During mitosis, transcription factors and RNA polymerase II are
displaced from mitotic chromatin, which is condensed and tran-
scription is interrupted. However specific gene regions are sug-
gested to be bookmarked by TBP and histone modifications for
rapid reactivation after cell division. The MHCII locus is trans-
criptionally activated by the assembly of an enhanceosome
(MCE) which recruits the master regulator, CIITA. We found
that endogenous or GFP-fusions subunits of the MCE remain
associated and dynamically exchanged on mitotic chromosomes.
Chromatin immunoprecipitation assays show significant occu-
pancy by the MCE, CIITA and general transcriptional machinery
at various phases of the cell cycle including mitosis in B lym-
phoid cells. Mitotic maintenance of the MCE does not depend
on CIITA expression and correlates with a chromatin state that
is fully or partly maintained in mitotic lymphoid or non lym-
phoid cells, respectively. Monitoring of newly synthesized RNA
shows reduced transcriptional activity of the DRa gene in mito-
sis. Constitutive expression of exogenous CIITA is sufficient to

fully support mitotic expression in lymphoid but not in epithelial
cells. Using an inducible system we show that CIITA synthesised
in cells arrested in mitosis rescues MHCII transcription to asyn-
chronous levels in lymphoid cells demonstrating full expression
competence of DRa gene. These results show there are two pat-
terns of mitotic deficiency of gene expression: in B lymphoid cells
low but significant MHCII transcription can be fully rescued, as
opposed to epithelial cells that show minimal mitotic transcrip-
tion may due to enhanced chromatin condensation.
P1–6
Associations between PTGS1 gene mutations
and aspirin resistance in patients with
congenital heart diseases
I. Coskun
1
, Y. Atay
1
, A. Berdeli
2
, M. Mecidov
1
, A. Atay
3
,
M. F. Ayik
1
, T. Yagdi
1
and E. A. Alayunt
1

1
Cardiovascular surgery, Ege University, izmir, TURKEY,
2
Pediatric Molecular Medicine Laboratory, Ege University, izmir,
TURKEY,
3
Department of Clinical Biochemistry, Ataturk Training
Hospital, izmir, TURKEY
Introduction: This study was aimed to evaluate that frequency
and existance of PTGS1 gene mutation which coded cyclooxy-
genase-1 (COX-1) enzyme and associations with aspirin resistance
in patients with congenital heart diseases (CHD) in Turkish pop-
ulation.
Methods: It was included 90 patients with CHD treated by
aspirin after their operations. 24 healthy participants were
enrolled as control group. Direct DNA coding sequence method
was used for analysing PTGS1 gene mutation. PCR amplification
was made by using specific oligo primers (MWG) and AmpliTaq
Gold PCR kits for four exons. After capillary gel electrophoresis,
nucleotide sequences of PTGS1 gene was obtained and compared
with cDNA Genbank (NM_000962) and protein (NP_000953)
sequences to determine single- nucleotide polymorphisms and
amino acid mutations.
Results: W8R missense aminoacid mutation was the most fre-
quent mutation (83.3%) in exon-2. P188P sinonym aminoacid
polimorphism (34%) and D189E amino acid mutation (20%)
were other mutations detected in exon-6 in patient group. In con-
trol group, W8R missense aminoacid mutation (83.3%) in exon-
2, Q41Q sinonim aminoacid mutation (8.3%) in exon-3, D189E
missense aminoacid mutation (12.5%) in exon-6 were found.

Nine new missence amino acid mutations (L13P; L15V; P18R;
C58T; I45M; R53S; D189E; F200L; R239L) and eight sinonym
amino acid mutations (L23L; P39P; R53R; G44G; G62G;
K185K; P18P) were detected. It was observed the association
between aspirin resistance and missence amino acid mutations in
patients.
Conclusion: Genetic variations in PTGS-1 may affect aspirin
resistance in CHD. Detecting these variations may help to predict
drug responce and to select optimal therapy and dosage regi-
mens. This is the first study in our country dealing with PTGS1
gene mutation and polymorphism which coded COX-1 enzyme
and association with aspirin resistance in CHD.
Abstracts Poster Presentations
96 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
P1–7
Analysis of the ID1 gene expression level and
promoter methylation in patients with benign
and malignant thyroid nodules
A. Banaszewska
1
, M. Piechota
1
, K. Drobnik
2
, K. Ziemnicka
2
,
K. Ziemnicki
1
and R. Plewa

1
1
Department of Animal Physiology and Development, Adam
Mickiewicz University, Poznan, POLAND,
2
Department of
Endocrinology Metabolism and Internal Disease, University of
Medical Science, Poznan, POLAND
Inhibitor of DNA binding (ID1) has been proposed to serve a
dual function as a regulator of cell proliferation and differentia-
tion in both normal and pathological conditions associated with
cancer development. ID is a transcription family of factors lack-
ing basic DNA binding domain, which form inactive heterodi-
mers with basic helix-loop-helix transcription factors and inhibits
expression of tissue specific genes and cells differentiation. Recent
research shows, that ID1 mRNA level correlates with malignant
thyroid carcinoma phenotype. Thus, dual role of ID1 gene in
benign and malignant thyroid nodules was studied. ID1 mRNA
level of 33 patients was investigated in human papillary thyroid
carcinoma (PTC), non-toxic nodular goiter, toxic nodular goiter
and nodular goiter with Hashimoto disease using Real time PCR
and Delta Delta Ct method to estimate relative amount of ID1
mRNA. Furthermore, genomic DNA was extracted and ID1 pro-
moter methylation was examined in the majority of patients spec-
imens. Six of seven PTC patients samples were found to have
barely detectable ID1 mRNA level. Expression of ID1 was higher
in non-toxic nodular goiter and in nodular goiter with Hashimoto
disease in comparison to PTC and toxic nodular goiter
(P < 0.05). No hypermethyleted ID1 gene promoter was found
in the majority of analyzed cases. In our study no significant

association was observed between ID1 gene expression and
malignant thyroid nodules. This findings indicate that ID1 could
not be a marker of aggressive phenotype in PTC.
P1–8
TNF alpha -308 polymorphism and matrix
metalloproteinase-9 level in children with
juvenile idiopathic arthritis threated with
etanercept
J. Basic
1
, D. Pavlovic
1
, T. Jevtovic-Stoimenov
1
, J. Vojinovic
2
,
M. Marinkovic
1
and A. Veljkovic
1
1
Medical Faculty, Institute of Biochemistry, Nis, SERBIA,
2
Clinic
of Pediatrics, Clinical Centre Nis, Nis, SERBIA
Juvenile idiopathic arthritis (JIA) is the most common form of
persistent arthritis in children. Tumor necrosis factor-a (TNF- a)
is likely to have a primary role in the pathogenesis of JIA,
including matrix metalloproteinase-9 (MMP-9) production. The

aim of this study was to investigate G-A -308 single nucleotide
polymorphism (SNP) in the promoter region of TNF a gene, as
well as to evaluate the eventual influence of etanercept (TNF
receptor II-Fc fusion protein) on MMP-9 level in children with
JIA. A total of 42 patients with JIA were screened for the poly-
morphism using the PCR-RFLP method. Plasma MMP-9 level
was determined before starting etanercept therapy and 12 months
after, using Elisa kit. The genotype -308GG was present in 13
(31%) patients and the -308AA genotype was present in two
(4.7%) patients. Twenty-seven (64.3%) patients were heterozy-
gous. MMP-9 level in patients with the genotype -308GG was
significantly decreased after 1 year of treatment with etanercept
compared to the value before (P = 0.01). On the other hand,
there were no significant decrease of MMP-9 levels after treat-
ment in patients with the genotype -308GA and -308 AA
(P = 0.147; P = 0.126). We discussed decrease of MMP-9 level
in children with -308GG genotype as a possible consequence of
better response to etanercept treatment compared to A allele car-
riers.
P1–9
Screening of Three Exons of the RET Proto-
oncogene in Turkish Patients with Papillary
Thyroid Carcinoma
N. S. Bayramci
1
, L. Acik
2
, L. Y. Koc
3
, G. Vural

4
, M. Kilic
5
,
M. Tez
5
, M. Koc
5
and N. Ercakmak
4
1
Faculty of Education, Department of Science Education, Bayburt
University, Bayburt, TURKEY,
2
Faculty of Arts and Science,
Department of Biology, Gazi University, Ankara, TURKEY,
3
Faculty of Science, Department of Biology, Ankara University,
Ankara, TURKEY,
4
Nuclear Medicine Department, Dr. Abdurrah-
man Yurtaslan Ankara Oncology Education and Research Hospi-
tal, Ankara, TURKEY,
5
5th Surgery, Ankara Numune Education
and Research Hospital, Ankara, TURKEY
Different types of tumors can develop in thyroid gland cells. Thy-
roid carcinoma represents the most frequent form of cancer of
the thyroid glands, with prevalence in Europe of about 3 per
100 000. PTC is the most frequent histotype of differentiated thy-

roid cancer. The RET proto-oncogene, localized to chromosome
subband 10q11.2, comprises 21 exons, which encodes the protein
RET, a receptor tyrosine kinase (RTK) expressed in derivatives
and tumors of neural crest origin. The RET proto-oncogene is
involved in the genesis of papillary thyroid carcinoma. To deter-
mine the mutation frequency of RET proto-oncogene in Turkish
papillary thyroid carcinoma patients, we conducted this retro-
spective study. Eighty-two patients with PTC were screened for
mutations in exon 10, 11 and 13 using PCR and sequence analy-
sis. A negative control was included in each amplification analy-
sis. Twelve of the patients exhibited mutation, 24 of them
showed SNP. Twelve different mutations located in exon 11 and
three different mutations in exon 13. 12.19% of the patients had
mutation in exon 11. 3.65% of the patients had mutation in exon
13. Twenty-four of the patients exhibited SNP (29.26%). We
found the SNP at codons 631 (1.21%), 769 (1.21%), 691 (3.65%)
in exon 11 and at codons 769 (24.39%) and 763 (1.21%) in exon
13. The most frequent SNP that is found in patients with PTC
(24.3%) involve codon 769 (exon 13) in the tyrosine kinase
domain. The common mutation found in patients involves codon
630 (four patients) in the extracellular domain of RET encoded
by exon 11, less frequently, mutation occurred codons 765, and
770 and 795 (exon 13) in the tyrosine kinase domain. The muta-
tions were found in 41.46% of all thyroid carcinoma patients.
The numbers of mutations were higher in exon 11 than exon 13.
In our series, we detected that four patients in 82 of all PTC
patients presented Cys630Ser (TGC fi AGC, 5.9%) is the most
common mutation at codon 630 in exon 11. In addition to that,
Cys630Ser mutation, is associated with medullary thyroid carci-
noma, however in our genetic testing it was diagnosed on four

patients with papillary thyroid carcinoma. Therefore, the rela-
tionship between Cys630Ser variation and development of PTC
should be further explored. All of the investigated polymor-
phisms are silent mutations, leave out codon 691 polymorphism.
We observed the codon 691 polymorphism in three patients
(GGT/AGT) of all 82 PTC patients. In the 13rd exon region of
the RET proto-oncogene, we found that these possible mutations,
which expressed as an alteration in the form of TCC (serine)
replacement with TGC (sistein) at codon 765, CGA (arginine)
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 97
replacement with CAA (glutamic acid) at codon 770. The other
mutation in exon 13 was at codon 770, Arg replacement with
Glu. The large number of SNP at codon 769 in exon 13 with
PTC patients was interesting and it should be further explored.
P1–10
Metabolomic profiling of serum from obese
adult females infected by Chlamydophila
pneumoniae
G. Bazylak
1
, C. Ludwig
2
, E. Fagadar-Cosma
3
and
U. L. Gunther
2
1
Department of Pharmaco-Bromatology & Molecular Nutrition,

Faculty of Pharmacy Collegium Medicum Nicolaus Copernicus
University, Bydgoszcz, POLAND,
2
HWB NMR, CR UK Institute
for Cancer Studies, University of Birmingham, Edbagston Birming-
ham, UK,
3
Department of Organic Chemistry, Institute of Chemis-
try Timisoara of Romanian Academy, Timisoara, ROMANIA
Chlamydophila (Chlamydia) pneumoniae is a very common path-
ogen causing an acute respiratory diseases and multidirectional
inflammation progress during a past and persistent infections in
humans of all age. Recently, significant associations between C.
pneumoniane infection and obesity indices have been observed
on results of epidemiological studies in various populations from
several north Europe countries. However, until now the number
of NMR- or MS-based metabolomic and proteomic studies on
specific biomarkers related with variety of nutrition disorders,
including obese subjects, is still scarce. Thus, the goal of our
studies was to find by use of the 1D- and 2D-1H-NMR proce-
dures the range of characteristic, specific and unique low weight
metabolites (proteins) in serum samples from wide group of over-
weight and obese adult females with increased hormonal activity
of adipose tissue, and which indicating past (or persistent) infec-
tion by C. pneumoniae as diagnosed by introductory serological
immunosorbent assay. Proposed here 1H-NMR methodology
should be effective for profiling changes in the serum metabolo-
me (proteome) of subjects with obesity and its associated implica-
tions with bacterial infection and disorders of immunological
status, thus enabling future early diagnosis, risk assessment and

advanced treatment of infection related obesity in humans.
Acknowledgement: Supported by the grant from EU NMR
project – Contract No. RII3-026145.
P1–11
The study on Fnr-type transcription regulators
functions in bacterium Paracoccus
denitrificans using proteomics, transcriptomics
and bioinformatics tools
P. Bouchal
1
, I. Struharova
1
, E. Budinska
2
, O. Sedo
3
,
T. Vyhlidalova
1
, Z. Zdrahal
3
, R. van Spanning
4
and I. Kucera
1
1
Department of Biochemistry, Faculty of Science, Masaryk Univer-
sity, Brno, CZECH REPUBLIC,
2
Institute of Biostatistics and

Analyses, Masaryk University, Brno, CZECH REPUBLIC,
3
Department of Experimental Biology, Masaryk University, Brno,
CZECH REPUBLIC,
4
Department of Molecular Cell Physiology,
Vrije Universiteit Faculty of Earth and Life Sciences, Amsterdam,
THE NETHERLANDS
Paracoccus denitrificans is a facultatively autotrophic soil bacte-
rium which responds to decreasing level of oxygen in the environ-
ment by a switch from aerobic to anaerobic (denitrifying) growth
mode. In our work, we analyzed roles of fnr-type transcription
regulators FnrP, NNR and NarR (with described key roles as
sensors for oxygen, nitrate and nitrous oxide) using proteomics,
transcriptomics and bioinformatics tools to consider their roles in
the adaptation processes at global level. Protein compositions of
four P. denitrificans strains (wild type, FnrP-, NNR- and NarR-
mutants grown aerobically, semiaerobically and semiaerobically
with nitrate) were analyzed using set of 36 large format 2-D
PAGE gels, their statistical evaluation and MS/MS protein iden-
tification of more than 500 proteins. Expression differences of
key up-/down-regulated gene products were validated using qRT-
PCR. Comparative proteomic analysis revealed, besides other
results, differences between strains in the expression of three key
denitrification enzymes (nitrate reductase, nitrite reductase,
nitrous oxide reductase), OmpW and UspA proteins. These find-
ings are in agreement with known mechanisms and/or confirmed
the positions of proposed fnr binding sites in their promoters.
Detection of additional group of up-/down- regulated proteins
without fnr binding sites in their promoters (e.g. SDR dehydro-

genases, TonB receptors) indicates involvement of additional reg-
ulatory mechanisms into the adaptation processes studied.
Evidences for potential roles of transcription regulators of other
families (e.g. LysR, MarR and two-domponent regulators) are
discussed.
Acknowledgements: We are thankful to Czech Science Foun-
dation (grant No. 203/07/P0471) and to Czech Ministry of Edu-
cation (grant No. MSM0021622413) for financial support.
P1–12
Analysis of proteome alteration in tomato
roots after Meloidogyne hapla infection
A. Obrepalska-Steplowska
1
, K. Nowaczyk
1
, M. Luczak
2
,
M. Figlerowicz
2
, R. Dobosz
3
and M. Budziszewska
1
1
Interdepartmental Laboratory of Molecular Biology, Institute of
Plant Protection, Poznan, POLAND,
2
Polish Academy of Sci-
ences, Institute of Bioorganic Chemistry, Poznan, POLAND,

3
Department of Zoology, Institute of Plant Protection, Poznan,
POLAND
Root-knot nematodes from the genus Meloidogyne are parasitic
to many economically important crop plants and cause great
losses in the crop production. Out of fifteen European species,
eight were found in Poland; among them the most common and
widely distributed is M. hapla, parasiting on dicotyledonous
plants. During the initial stage of infection characteristic root
galls form on the roots of susceptible plant hosts. As a result of
interaction between parasite and the host, in the vascular cylinder
of the root permanent feeding sites are formed with specialized
multinucleated feeding cells (giant cells). The redifferentiation of
root cells and development of metabolically active giant cells is
induced by the nematode. The mechanism of giant cells forma-
tion and the role of plant proteins in this processs remain
unclear. Our attempt was to analyse the changes in the plant pro-
teome in the early stage of M. hapla infection. Tomato plants
were grown in isolated conditions and infected with M. hapla.
After isolation of proteins from roots of healthy and infected
plants, as well as from nematodes, two-dimensional electrophore-
sis was performed. The results of experiments were compared to
ensure elimination of nematode proteins from further analysis.
As some qualitative and quantitative changes were observed, cho-
sen proteins were analysed using mass spectrometry. The decrease
of cytosolic and mitochondrial dehydrogenases of cellular respi-
ration and electron transport chain was observed as well as for
proteins related to cytoskeleton (actins), relevant to changes in
the cell’s shape and wound response (annexin). The qualitative
changes are related with proteasome endopeptidase complex,

absent in infected root cells. Nematode proteins secreted during
Abstracts Poster Presentations
98 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
infection had been the subject of many recent investigations. But,
as the relationship between these parasites and the host is very
close, determination of proteins engaged on the both sides of the
interaction can give us the insight to the mechanism of plant sub-
ordination to the parasite.
P1–13
Determination of chromosomal regions
affecting some production traits in F
2
intercross chickens
Z. Bulut
1
, E. Kurar
2
, Y. Ozsensoy
2
, M. Nizamlioglu
1
, M. Garip
3
,
A. Yilmaz
3
, T. Caglayan
3
, S. Dere
3

, V. Kurtoglu
4
and
M. Dogan
1
1
Faculty of Veterinary Medicine, Department of Biochemistry, Sel-
cuk University, Konya, TURKEY,
2
Faculty of Veterinary Medi-
cine, Department of Genetics, Selcuk University, Konya,
TURKEY,
3
Faculty of Veterinary Medicine, Department of Zoo-
technics, Selcuk University, Konya, TURKEY,
4
Faculty of Veteri-
nary Medicine, Department of Animal Nutrition, Selcuk
University, Konya, TURKEY
In this study, a F
2
level Denizli X White Leghorn population
(n = 441) was used to dissect chromosomal regions, which have
an affect on body weight and egg yield. DNA samples were iso-
lated from all F
0
,F
1
and F
2

animals using a standard phenol/
chloroform method. For chromosomal level screening, a total of
99 markers which were suitable for genome searching were ampli-
fied using Polymerase Chain Reaction (PCR). The resulting PCR
products were separated by capillary electrophoresis by using
Beckman Coulter CEQ-8000 Genetic Analysis System and geno-
types were identified at each marker loci. QTL Express Program
was used for QTL data analysis. In study, Quantitative Trait
Loci (QTL) were identified on chicken chromosome 1 (GGA1),
GGA2, GGA4, GGA8 and sex chromosome (GGAZ) for hatch-
ing weight, body weight at different ages and egg yield. There is
a need for narrowing these QTL regions by typing new markers
in these intervals and for identifying genes that have affect on
these economically important traits.
P1–14
Investigation of osmoprotection and cold
adaptation of halomonas halophilia DSMZ
4770 by proteomics
S. Ceylan
1
, B. Akbulut
1
, D. Kazan
1
and D. Kazan
2
1
Engineering Faculty, Bioengineering Department, Marmara
University, Istanbul, TURKEY,
2

TUBITAK Marmara Research
Center, Genetic Engineering and Biotechnology Institute, Kocaeli,
TURKEY
Halophiles are an important group of microorganisms that can
adapt to extremely saline environments. Since, they have the abil-
ity to function under extreme saline conditions, halophilic micro-
organisms are used at different industrial processes such as in the
production of protein/enzyme stabilizer metabolites (e.g. betain,
ectoin), in the removal of pollution in salinity effluents and in the
production of stable enzymes. Proteomics is the large-scale study
of proteins, particularly their structures and functions. Proteins
are vital parts of living organisms, as they are the main compo-
nents of the physiological metabolic pathways of cells. By under-
standing metabolic pathways of moderately halophilic
microorganisms it will be possible to modulate their metabolism
to enhance the production of their industrially important prod-
ucts. In this study osmoprotection and cold adaptation metabo-
lism of moderately halophilic Halomonas halophilia was
investigated. Halomonas halophilia was grown on different salt
concentrations (5% NaCL, -20% NaCl) and different tempera-
tures (19–37°C). Growth at these conditions was investigated and
microorganisms were harvested at the end of exponential phase.
Afterwards, whole cell proteins of the DSMZ 4770 were
extracted and analyzed by proteomic tools. Two DE maps are
prepared according to tube gel systems with NEPHGE technique
in the first dimension and normal SDS-PAGE in the second
dimension. Protein profiles between normal and stress conditions
were determined and compared. Protein expression differences
were determined by MALDI-Tof Mass Spectrometer (Waters/Mi-
cromass). Proteins that involved in the osmoprotection and cold

adaptation were determined.
P1–15
Molecular typing of Staphylococcus aureus
strains from ovine mastitis by pulsed-field gel
electrophoresis and polymerase chain reaction
A. Ciftci
1
, E. E. Onuk
2
, A. Findik
1
, T. Yildirim
3
and
M. Unlu Sogut
4
1
Veterinary Faculty, Microbiology, Ondokuz Mayis University,
Samsun, TURKEY,
2
Veterinary Faculty, Diseases and Clinical
Sciences, Ondokuz Mayis University, Samsun, TURKEY,
3
Science Faculty, Biology, Amasya University, Amasya, TURKEY,
4
Medicine Faculty, Microbiology, Ondokuz Mayis University,
Samsun, TURKEY
Staphylococcus aureus is one of the most important etiological
agents of ovine mastitis. In order to develop effective control mea-
sures for mastitis, it is important to type S. aureus strains that

have considerable genetic heterogeneity. In the current study, 47
S. aureus strains isolated from ovine mastitis were typed by poly-
merase chain reaction (PCR) based on coagulase (coa) and protein
A (spa) polymorphisms and by pulsed-field gel electrophoresis
(PFGE). Eight different coa-types and four spa-types were identi-
fied by PCR. While the most prevalent coa-type was CG2
(42.56%), the spa-types, S4 and S1, were the most commonly
observed (44.68% and 38.29%, respectively). Nineteen different
pulsotypes were identified, and 12 of these were represented by a
single isolate. Pulsotypes J and K were predominant, and each
represented nine isolates (19.14%). All isolates belonging to J and
K pulsotypes were CG2. While all nine isolates belonging to J
pulsotype were S4, all isolates in K pulsotype were S1. Although
PFGE was found to be the best discriminatory technique for dis-
tinguishing strains, coa- and spa-types were found to be in correla-
tion with PFGE types and can be used for quick, preliminary
epidemiologic studies for detecting strains that may cause mastitis.
P1–16
Development and optimization of multiplex
polymerase chain reaction for identification
of Flavobacterium psychrophilum, Yersinia
ruckeri and Aeromonas salmonicida subsp.
salmonicida
E. E. Onuk
1
, A. Ciftci
2
, A. Findik
2
and Y. Durmaz

3
1
Veterinary Faculty, Fisheries and Fish Diseases, Ondokuz Mayis
University, Samsun, TURKEY,
2
Veterinary Faculty, Microbiology,
Ondokuz Mayis University, Samsun, TURKEY,
3
Veterinary Con-
trol and Research Enstitute, Fisheries and Fish Diseases, Samsun,
TURKEY
Bacterial cold water, enteric red mouth and frunculosis are the
common bacterial diseases of fish worldwide. The etiologic agents
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 99
of these diseases are Flavobacterium psychrophilum, Yersinia
ruckeri and Aeromonas salmonicida subsp. salmonicida,
respectively. In this study, a Multiplex Polymerase Chain Reac-
tion (M-PCR) method which can identify these fish pathogens,
Aeromonas salmonicida subsp. salmonicida, Flavobacterium psy-
chrophilum and Yersinia ruckeri, simultaneously was developed
and optimized. It was observed that M-PCR developed in this
study, with YER8/10-Fer3/4-FP1/3 primer pairs, occured neither
false specific nor nonspecific amplification. The detection limits
of M-PCR method using DNA extract from dilutions of pure
cultures of bacteria were detected as 35 pg for Y. ruckeri and
F. Psychrophilum and 70 pg for A. salmonicida subsp. salmonicida.
It was determined that 15 CFU Y. ruckeri and F. psychrophilum
and 30 CFU A. salmonicida subsp. salmonicida could be detected
by M-PCR developed using genomic DNA extracted from dilu-

tions of suspensions. The detection limits in the presence of tissue
debris were detected as 125 CFU for Y. ruckeri ve F. psychrophi-
lum and 250 CFU for A. salmonicida subsp. salmonicida. In con-
clusion, we decided that M-PCR method developed and
optimised in this study could be used for accurate and rapid
identification of these bacteria.
P1–17
Evaluation of protein profiles of Enterococcus
faecalis strains in relation to antigenic
glycoproteins
G. Ciftci
1
and H. Uysal
2
1
Veterinary Faculty, Biochemistry, Ondokuz Mayis University,
Samsun, TURKEY,
2
Veterinary Faculty, Biochemistry, Ankara
University, Ankara, TURKEY
The aim of this research is to determine the specific antigenic gly-
coproteins and comparative analysis of antigenic protein profiles
of Enterococcus faecalis (E. faecalis) strains distinguished with
aggregation substance (AS), gelatinase and cytolysine. For the
determination of the whole cell protein profiles of E. faecalis, the
Sodium Dodecyl Polyacrylamide Gel Analysis (SDS-PAGE) was
performed. All the E. faecalis strains showed protein bands
between the sizes of 29 and 165 kDa. The protein bands of 44
and 83 kDa were determined as major band in E. faecalis
OG1RF (gelatinase positive) and E. faecalis OG1X (pAM9058)

(AS positive). On the other hand; E. faecalis OG1X (pAM944)
(cytolisine positive) strain also showed a 43 kDa major band.
For the antigenic characterization, the immunblot analysis was
performed. All of the three strains of E. faecalis showed common
antigenic protein bands which were at the size of 165, 35 ve
32 kDa. The other antigenic protein bands of E. faecalis OG1X
(pAM944), E. faecalis OG1RF and E. faecalis OG1X
(pAM9058) were determined as 85, 65, 55, 39 ve 29 kDa, 83, 75
ve 45 kDa and 111, 98, 83, 75, 55, 45, 29 kDa in sizes, respec-
tively. The Glycan Detection Kit from Roche Diagnostics and
Periodic Acid Schiff (PAS) stainings were used for the investiga-
tion of glycoproteins of E. faecalis. After the PAS staining, no
glycoprotein band was shown as the method has low sensitivity.
When the Glycan Detection Kit used, E. faecalis OG1RF and
E. faecalis OG1X (pAM9058) strains showed two glycoprotein
bands which were at the size of 111 and 39 kDa. Moreover,
E. faecalis OG1X (pAM944) strain was also showed some glyco-
protein bands which were 111, 106 and 39 kDa in size. In conclu-
sion; no difference was determined for the protein profiles among
the strains of Enterococcus faecalis which have AS, gelatinase
and cytolisine. The antigenic profiles of the glycoproteins and the
other proteins of E. faecalis was found as varied among these
strains. It was the first study in the literature that suggested E.
faecalis strains include the glycoproteins.
P1–18
RAPD-PCR analysis of the genus apodemus
KAUP, 1829 (Mammalia:Rodentia) in Turkey
R. Colak, G. Olgun, I. Kandemir, N. Yigit and E. Colak
Faculty of Science, Biology, Ankara University, Ankara,
TURKEY

The aim of the present study is to survey genetic structure based
on DNA markers and to make contribution to the taxonomic
status, population genetics of the Genus Apodemus in Turkey. In
order to analysis genetic variation in Apodemus species, a Ran-
domly Amplified Polymorphic DNA (RAPD) marker system was
used. A total of 82 specimens (22 A. iconicus,26A. flavicollis,7
A. sylvaticus,15A. uralensis,4A. agrarius and 8 A. mystacinus)
collected from 16 locations in Turkey were used. The estimates
of NEI’s (1972) standart genetic identity and standart genetic dis-
tance were calculated to show the genetic relationships between
studied populations. All estimations were calculated with the
POPGENE software. The 60 RAPD markers were tested in
Apodemus, 11 ones yielded 101 polymorphic DNA bands.
Genetic differentiation values, H = 0.0721 (P = 16.83%), were
the lowest in A. agrarius. A. uralensis has H = 0.2303
(P = 77.23%), being the highest value between Apodemus spe-
cies. According to Nei (1972), A. agrarius and A. mystacinus are
the least similar to each other (I = 0.6256), and A. iconicus and
A. uralensis are the nearest to each other (I = 0.9406). Phyloge-
netic relationships between Apodemus species were shown with
UPGMA dendogram constructed based genetic distance values.
P1–19
What does it take to make an activated
cortical domain within a plant cell?
F. Cvrckova
1
, R. Bezvoda
1
and V. Zarsky
2

1
Faculty of Science, Department of Plant Physiology, Charles
University, Prague, CZECH REPUBLIC,
2
Faculty of Science,
Department of Plant Physiology and Laboratory of Cell Biology,
Institute of Experimental Botany ASCR, Charles University,
Prague, CZECH REPUBLIC
Exocytosis is central to plant cell morphogenesis. A single cell
may possess multiple plasmalemma domains performing localized
exocytosis-driven expansion (‘activated cortical domains’, ACDs).
Several large families of paralogous regulatory proteins might be
involved in generating the diversity of ACDs within a cell, such
as e.g. small GTPases of the Rop family and their interactors, su-
bunits of the exocyst complex, actin-nucleating FH2 proteins,
receptor-like kinases, or enzymes locally modifying membrane
composition (the ‘candidate set’ genes). Rhizodermis of Arabidop-
sis thaliana consists of two cell types that differ by a single ACD
– trichoblasts carrying root hairs, and atrichoblasts. We used
publicly available cell-type specific transcriptome data to identify
genes specifically induced in trichoblasts compared to atricho-
blasts (i), and to examine their relationship to over 60 ‘candidate
set’ genes (ii), as well as to known genes whose mutations exhibit
root hair-specific phenotypes (iii). In addition, we have searched
for homologues of all three gene classes in the published soybean
Abstracts Poster Presentations
100 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
root hair proteome (iv; Brechenmacher et al., 2009). We found
only limited overlap among the gene classes (i–iv), with most
‘candidate set’ genes exhibiting <5· induction in trichoblasts

compared to atrichoblasts and low protein abundance in root
hairs; very few class iii genes were found in either the transcrip-
tomic or proteomic set. Thus, unlike forward or reverse genetics,
transcriptomics and proteomics alone are unlikely to identify
genes involved in plant ACD specification.
Acknowledgement: This work was supported by the
MSM0021620858, LC06004 and LC06034 projects.
P1–20
Drug resistant MCF-7 cells possessed
epithelial-mesenchymal transition related gene
expression pattern
O. D. Iseri
1
, M. Demirel Kars
1
, F. Arpaci
2
, C. Atalay
3
, I. Pak
4
and U. Gu
¨
ndu
¨
z
1
1
Department of Biological Sciences, Middle East Technical
University, Ankara, TURKEY,

2
Department of Oncology, Gu
¨
lhane
Military Medical Academy, Ankara, TURKEY,
3
Department of
General Surgery, Ankara Oncology Hospital, Ankara, TURKEY,
4
Department of Pathology, Ankara Oncology Hospital, Ankara,
TURKEY
Background: Epithelial-mesenchymal transition (EMT) is a
developmental process by which cells of epithelial origin lose epi-
thelial characteristics, and acquire a mesenchymal phenotype.
EMT, an indicator of poor prognosis, may also occur during
tumor progression. Altered expression levels of Snail family tran-
scription factors (Snail, Slug and Twist) are the key regulatory
elements of EMT. Slug gene is one of the downstream mediators
of transforming growth factor beta1 (TGFB1) signaling.
Objectives: In the present study evaluation of expression of
genes related to EMT was aimed in docetaxel and doxorubicin
resistant MCF-7 breast carcinoma cells in order to assess their
involvement in drug resistance.
Methods: Resistant sublines (MCF-7/DOC and MCF-7/DOX)
were developed by docetaxel and doxorubicin applications in
dose increments and development of resistance was confirmed by
XTT proliferation assays. RNA was isolated from sensitive and
resistant cells and cDNA microarray analysis was performed
using Affymetrix
Ò

Human Genome U133 Plus 2.0 Arrays in
duplicate experiments. GeneSpring GX 7.3.1 Software was used
for data analysis. Immunocytochemistry was also performed to
determine relevant protein expression.
Results: XTT demonstrated that MCF-7/DOC and MCF-7/
DOX were resistant to docetaxel and doxorubicin, respectively.
According to data analysis, estrogen receptor a was drastically
downregulated in MCF-7/DOC and MCF-7/DOX. It is known
as a transcriptional repressor of the Slug. Therefore, decreased
ERa and increased Slug expression could cause transcriptional
activation of key regulatory elements of EMT. In addition,
increased expression levels of TGF beta receptor2 (TGFBR2)
together with SMAD3 might have stimulated EMT in resistant
cells. Furthermore, amplified epidermal growth factor signaling
via epidermal growth factor receptor1 upregulation might have
enhanced the pathways leading to EMT. Slug upregulation
caused cadherin switch in resistant cells i.e. E-cadherin and occlu-
din were downregulated, and N-cadherin was upregulated. N-
cadherin associates with the fibroblast growth factor receptor1
(FGFR1) leading to increased FGFR1 expression at the cell sur-
face which was also upregulated in resistant cells. Immunocyto-
chemistry results confirmed microarray expression analysis.
Conclusion: This report demonstrates that in docetaxel and
doxorubicin resistant cells EMT process was induced indicating a
possible relationship of this process and drug resistance.
P1–21
Characterization and whole genome
sequencing of Arthrobacter
phenanthrenivorans, a new phenanthrene
degrading bacterium

C. Drainas
1
, A. Kallimanis
1
, K. Kavakiotis
1
, K. Mavromatis
2
,
N. C. Kyrpides
2
and A. I. Koukkou
1
1
Chemistry, University of Ioannina, Ioannina, GREECE,
2
Genome
Biology Program, DOE-Joint Genome Institute, Walnut Creek,
CA, USA
Several bacterial strains were isolated from a creosote polluted
site at Perivleptos (Epirus, Greece), based on their ability to uti-
lize various polycyclic aromatic hydrocarbons as a sole carbon
and energy source. One of them, Sphe3, was proved to be effi-
cient degrader of phenanthrene, a polycyclic aromatic hydrocar-
bon (PAH). Biochemical tests and 16S rDNA analysis showed
that Sphe3 belonged to the genus Arthrobacter comprising a
novel species named Arthobacter phenanthrenivorans sp. nov.
Whole genome sequencing of the isolated organism revealed that
it had a single circular chromosome of 4.25 Mb and two circular
plasmids of 190 and 94 kb, respectively. A total of 4288 genes

were predicted in the Sphe3 genome. With BLAST searches using
peptide fragment sequences from a purified 1-H-2-N dioxygenase
activity determined by Mass Spectrometry, we identified two
encoding genes with over 90% homology to each other at the
nucleotide level, one (diox1) located on the 190 kb plasmid and
the other (diox2) on the chromosome of the Sphe3 genome. Fur-
thermore, BLAST analysis of the 190 kb plasmid revealed that it
additionally contained several genes involved in phenanthrene
degradation. These findings revealed novelties that confirm the
extended biodiversity of PAH catabolic genes.
P1–22
Stress response to aromatic compounds in
Acinetobacter radioresistens S13
P. Fattori, M. Zapponi, C. Lamberti, A. Pessione, R. Mazzoli,
C. Giunta and E. Pessione
University of Turin, Human and Animal Biology, Turin, ITALY
Acinetobacter radioresistens S13 is a strain selected for its ability
in phenol degradation. It is also able to degrade other aromatic
compounds (i.e., Benzoate) through the b-ketoadipate pathway
(1). Comparative proteomics alkaline studies on bacterium grown
either on phenol or benzoate as sole carbon source reveal a dif-
ferent response to stress induced by these two aromatic com-
pounds. Phenol is a solvent able to solubilize outer membrane
lipoproteins, lipooligosaccharides (LOS) and phosphatidyletha-
nolamine, damaging cell wall (2). This compound induces an over
expression of two proteases (ClpX and Serine protease) and of
two RNA polymerase modulator factors (NusA and Rho)
suggesting a gene modulation based on rE regulation. Instead
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 101

benzoate induces an alternative phosphorylation-dependent
stress-sensing response, maintained by a two-component regula-
tory system, which consist of a membrane-embedded sensory
kinase and a response regulator (3). The kinase auto-phosphory-
lates a conserved histidine on the receipt of a stress-signal before
transferring the phosphoryl group to an invariant aspartate in its
cognate response regulator and in doing so activates its latent
biological function (4).
References:
1. Mazzoli R, Pessione E, Giuffrida MG, Fattori P. et al. Degra-
dation of aromatic compounds by Acinetobacter radioresistens
S13: growth characteristics on single substrates and mixtures.
Arch. Microbiol. 2007; 188(1): 55–68.
2. Mrozik A, Piotrowska-Seget Z & Labuzek S. Cytoplasmatic
bacterial membrane responses to environmental perturbations.
Polish J Environ. Studies 2004; 13(5): 487–494.
3. Bekker M, Teixeira de Mattos MJ & Hellingwerf KJ. The role
of two-component regulation systems in the physiology of the
bacterial cell. Sci. Prog. 2006; 89: 213–242.
4. Mizuno T. His-Asp phosphotransfer signal transduction.
J. Biochem. (Tokyo) 1998; 123: 555–563.
P1–23
Identification of a(1,6)fucosylated proteins as
biomarkers for human colorectal carcinoma
L. M. Romay, S. V. Portela, R. F. Poceiro, E. G. Martı
´
n and
A. F. Briera
Department of Biochemistry Genetics and Immunology, University
of Vigo, Vigo, SPAIN

a(1,6)-fucose residues within the N-glycan core structures are
commonly observed in glycoproteins and are often altered under
pathological conditions. This type of fucosylation is product of
a(1,6)fucosyltransferase [a(1,6)FT] activity and is regarded as an
important manner of posttranslational modification and func-
tional regulation of glycoproteins. Our previous studies showed
that a(1,6)fucosyltransferase activity and expression are altered in
human colorectal cancer (CRC). Therefore, the a(1,6)fucosylation
of some glycoproteins might be implicated in the development
and progression of colorectal carcinomas, being potential bioindi-
cators for the diagnosis and prognosis or targets for CRC ther-
apy. In the present study we have employed a Lens culinaris
agglutinin lectin (LCA) chromatography combined with 1-DE
separation and followed by MALDI-MS/MS analysis to identify
distinctive core fucosylation patterns in five healthy and tumour
tissue samples from CRC patients. Indeed, a lectin and western
blotting methodology was performed to validate our preliminary
results. We demonstrated that colorectal tumour tissues have
higher levels of a(1,6)fucosylation compared to healthy ones.
Besides, among the several protein bands observed after the lectin
chromatography and the electrophoretic separation, we selected
the bands with a higher difference of intensity between healthy
and tumour specimens. Two proteins were identified as possible
biomarkers for CCR, the heat shock protein gp96 precursor and
the Fc binding protein receptor. The western blot analysis con-
firmed the enhanced expression of heat shock protein gp96 pre-
cursor and the diminution of Fc binding protein receptor levels
in tumour tissues compared to healthy ones. In conclusion, these
glycoproteins could be considered as candidates for future studies
focused on their function in colorectal tumourigenesis and their

potential clinical usefulness.
P1–24
Molecular typing and methicillin resistance
profile of Staphylococcus aureus strains
isolated from the noses of healthy dogs
A. Findik
1
, N. Akan
2
, E. E. Onuk
3
, D. Cakiroglu
4
and A. Ciftci
1
1
Veterinary Faculty, Microbiology, Ondokuz Mayis University,
Samsun, TURKEY,
2
Veterinary Faculty, Ondokuz Mayis
University, Samsun, TURKEY,
3
Veterinary Faculty, Diseases and
clinical sciences, Ondokuz Mayis University, Samsun, TURKEY,
4
Veterinary Faculty, Internal Medicine, Ondokuz Mayis
University, Samsun, TURKEY
It was aimed to detect the carriage of methicillin-resistant Staph-
ylococcus aureus (MRSA) strains in the nasal flora of healthy
dogs and to genotype these S. aureus isolates. The swabs were

taken from the nasal region of 80 dogs examined in the veteri-
nary clinics for the check-up in Samsun. The methicillin resis-
tance profile of 80 isolates which were identified as S. aureus
were analysed phenotypically and genotypically. Agar Disc Diffu-
sion Test was performed to determine the methicillin resistance
phenotype of these S. aureus strains using the oxacillin antibiotic
discs (5 g) and all the 80 strains were found as sensitive to methi-
cillin. In Polymerase Chain Reaction (PCR) targetting nuc, mecA
and fem genes, three strains (3.75%) was found to possess mecA.
In the same analysis none of these strains were positive for fem
gene. The PCR targetting coa and spa genes was performed for
molecular typing of the S. aureus strains. Nine different coa
genes and three different spa genes were determined according to
the polymorphism of these genes. In conclusion, the determining
of the nasal carriage of different genotypes of S. aureus in
healthy dogs was considered as a basic finding for both the char-
acterization of nasal isolates of S. aureus and molecular epidemi-
ologic researches. In these strains, the detection of mecA gene,
accepted as ‘Gold Standart’ for methicillin resistance, was evalu-
ated as an important finding for community health.
P1–25
Detection of methicillin resistance and slime
factor production of Staphylococcus aureus in
bovine mastitis
A. Ciftci
1
, A. Findik
1
, E. E. Onuk
2

and S. Savasan
3
1
Veterinary Faculty, Microbiology, Ondokuz Mayis University,
Samsun, TURKEY,
2
Veterinary Faculty, Diseases and Clinical
Sciences, Ondokuz Mayis University, Samsun, TURKEY,
3
Veterinary Faculty, Microbiology, Adnan Menderes University,
Samsun, TURKEY
Mastitis is the most prevalent and costly disease of dairy cattle.
Among the pathogens that cause mastitis, S. aureus is the most
important species and the hardest to eliminate with antibiotics
because of its multiple antibiotic resistance. It was aimed to
detect methicillin resistant and slime producing S. aureus in the
case of bovine mastitis. The triplex PCR was optimized targetting
16S rRNA, nuc and mecA for detection Staphylococcus species,
S. aureus and methicillin resistance, respectively. For detection of
slime producing, it was performed a PCR assay targetting icaA
and icaD genes. In this study, 59 strains were detected as S. aur-
eus by both conventional tests and PCR. Among the 13 S. aureus
strains of them were found as methicillin resistant phenotypically
and 4 (30.7%) were positive for mecA gene. Although 22 of 59
(37.2%) S. aureus isolates were slime-producing in Congo Red
Agar in PCR analysis, only 15 of 59 were positive for both icaA
and icaD genes. 16 of 59 strains were positive for icaA and 38 of
them were positive for icaD gene. We found only two of 59
strains were positive for both methicillin resistance and slime pro-
ducing, phenotypically.

Abstracts Poster Presentations
102 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
P1–26
Immobilization of acid phosphatase to
magnetic particles and their use for
dephosphorylation of proteins
J. Frydlova
1
, L. Kubosek
1
, L. Kubosek
2
, Z. Kucerova
1
,
M. Ticha
1
and M. Ticha
3
1
1st Faculty of Medicine, Institute of Pathophysiology and CEH,
Charles University in Prague, Praha 2, CZECH REPUBLIC,
2
Faculty of Chemical Technology, Department of Biological and
Biochemical Sciences, University of Pardubice, Pardubice, CZECH
REPUBLIC,
3
Faculty of Science, Department of Biochemistry,
Charles University in Prague, Praha 2, CZECH REPUBLIC
In proteomics studies, the presence of phosphate group in a pro-

tein or in a peptides obtained by proteolytic digestion is often
confirmed by the comparison of mass spectra of peptides/proteins
before and after the phosphatase treatment. A loss of phosphate
groups results in a shift of peptide in mass spectrum. Generally,
the use of enzyme immobilized to magnetic carriers has several
advantages as compared with an application of soluble enzyme
forms: an increased stability of enzymes, a possibility of direct
use of enzyme reaction products for MALDI-TOF MS and an
easy manipulation. Contrary to proteolytic enzymes, only limited
information is available on properties of immobilized phosphata-
ses. In the present study, acid phosphatase from potato was
immobilized to glyoxal 4% agarose magnetic particles (20–
75 lm) via its free amino groups. The following properties of
immobilized acid phosphatase were compared with those of the
soluble enzyme: the pH dependence of the enzyme activity, the
effect of the presence of cofactor (Mg
2+
), the effect of tempera-
ture and further storage and operational stability. A reusability
of the immobilized phosphatase was also examined. Both forms
of acid phosphatase were used for dephosphorylation of porcine
pepsin A, bovine a-casein and chicken ovalbumin.
Acknowledgements: This work was supported by the Ministry
of Education, Youth and Sports of the Czech Republic (grant
MSM 0021620806 and project CEH LC 06044) and by the Czech
Science Foundation (grant 203/09/0857).
P1–27
Deletions with the presence of two repetitive
DNA sequences in CEBPA gene
O. Fuchs

1
, A. Kostecka
1
, D. Provaznikova
1
and J. Filkukova
2
1
Department of Cell Physiology, Institute of Hematology and
Blood Tranfusion, Prague 2, CZECH REPUBLIC,
2
Department
of Molecular Genetics, Institute of Hematology and Blood
Tranfusion, Prague 2, CZECH REPUBLIC
C/EBPalpha (CCAAT/enhancer binding protein alpha) belongs
to family of leucine zipper transcription factors and it is neces-
sary for transcriptional control of granulocyte and adipocyte dif-
ferentiation, glucose metabolism and lung development. C/
EBPalpha is encoded by an intronless gene that is 2783 bp long
and maps to human chromosome 19q13.1. Up to now, CEBPA
gene mutations were detected only in patients with acute myeloid
leukemia (AML), in patients wih myelodysplastic syndrome
(MDS), in multiple myeloma and non-Hodgkin
´
s lymphoma
patients. We detected 14 various heterozygous CEBPA gene dele-
tions with the presence of two repetitions in CEBPA gene in
AML patients, MDS patients, non-Hodgkin
´
s lymphoma patient,

patients with peripheral artery disease, ischemic heart disease,
hyperlipidemia and type 2 diabetes. These mutations are charac-
teristic by the loss of one from these two same repetitions on the
ends of deleted sequence. Two most frequent repetitions included
in these deletions in CEBPA gene are GCCAAGCAGC (508–
517_907–916, GenBank Accession No. NM_004364.2) and CGC-
GAG (493–498_865–870, GenBank Accession No.
NM_004364.2). We found both these most frequent deletions in
CEBPA gene in one young man (38 years old) after coronary
thrombosis (heart attack). The same deletions were detected in
his son (15 years old). In many cases (13 patients with AML,
MDS, peripheral artery disease, heart disease, hyperlipidemia and
type 2 diabetes) more than one type of these deletions was
observed. These findings suggest that observed CEBPA gene dele-
tions of this type are likely the result of unequal mitotic cross-
over. We observed also in several patients in addition to these
deletions other mutation which was created probably by mecha-
nism of replication slippage in the repetitive sequence (for exam-
ple 68dupl in addition to 912_929del or 311_313del in addition
to 44_694del, in both cases GenBank Accession No.
NM_04364.2).
Acknowledgements: This work was supported by the Internal
Grant Agency of the Ministry of Health of the Czech Republic
(VZ 00023736).
P1–28
Study of thermal tolerance in Cronobacter
strains
J. Gajdosova
1
, K. Kunikova

1
, I. Turcovsky
2
, E. Kaclikova
2
and
H. Drahovska
1
1
Department of Molecular Biology, Comenius University,
Bratislava, SLOVAK REPUBLIC,
2
Department of Microbiology,
Food Research Institute, Bratislava, SLOVAK REPUBLIC
Cronobacter spp., formerly named as Enterobacter sakazakii,is
an opportunistic pathogen associated with sporadic cases of
severe infections in neonates causing meningitis, necrotizing
enterocolitis and sepsis. This organism is widely distributed in
environment and rehydrated infant formula is the most common
source of infection. Cronobacter was shown to be particularly tol-
erant to osmotic stress and desiccation and some strains of this
genus are also tolerant to elevated temperatures. Considering
infant formula is not a sterile product, thermotolerant strains of
Cronobacter spp. represent increased risk to survive during infant
formula reconstitution. In our study, the genetic variability of 76
food isolates and 23 Cronobacter collection strains was measured
by AFLP and 16S rRNA sequencing. Within AFLP profiles, a
great variability was observed, the similarity among strains
reached values 50–100%. Strains were clustered into 46 different
clusters at the similarity level of 90%. Six main groups (desig-

nated A-F) were clearly distinguished at the 75% similarity level;
strain grouping was in concordance with their species identifica-
tion and biochemical properties. Test of survival at 58°C sepa-
rated strains into two groups; D values (decreasing of bacterial
counts in one order) of thermosensitive strains fell within the
range 17–50 s; on the other hand eleven strains (nine Cr. sak-
azakii and two to Cr. malonaticus) were assessed as thermotoler-
ant because their D values reached from 100 to more than 300 s.
Thermotolerant strains were also positive for PCR thermotoler-
ance marker homologous to a hypothetical protein Mfla_1165
from thermotolerant bacterium Methylobacillus flagellatus KT
(Williams et al. 2005). 5.5 kbp DNA fragment around thermotol-
erance marker was sequenced in strain Cr. sakazakii ATCC 2954.
The region possessed 94% similarity with M. flagellatus KT com-
plete genome including genes Mfla_1162 – Mfla_1168. Cloning of
2950 bp long part of Cr. sakazakii thermotolerance region into
pGEM plasmid resulted in 3–5 times increased survival of
Escherichia coli laboratory strain at 58°C. Our results have
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 103
shown that this genetic region can be important in response to
several stress conditions and may contribute to increased envi-
ronmental resistance of Cronobacter strains.
P1–29
Mitochondrial proteome changes during
prereplicative phase of liver regeneration
A. Gnoni
1
, R. Mangiullo
1

, R. Schiavone
2
, S. Vilella
2
, S. Papa
1,3
and F. Zanotti
1,3
1
Department of Medical Biochemistry, Physics and Biology,
University of Bari, ITALY,
2
Department of Biological and Envi-
ronmental Sciences and Technologies, Laboratory of Comparative
Physiology, University of Salento, Lecce, ITALY,
3
Institute of
Biomembranes and Bioenergetics, CNR, Bari, ITALY
Introduction: The liver is usually a quiescent organ, but it is
able to regenerate itself and replace lost tissue, after transplanta-
tion and upon the loss of cells, following chemical and viral
injury, and partial hepatectomy (PH). Liver regeneration is
mainly divided in two phases: the prereplicative phase, during
which the liver’s energy demand increases, and the replicative
phase, during which increased DNA synthesis and mitosis occurs.
In the prereplicative phase (0–24 h after PH), mitochondria
show, a decrease in oxidative phosphorylation capability and
production of oxygen free radicals (1). We applied a proteomic
approach to mitochondria isolated 6 h after PH, to characterize
mitochondrial proteins that are involved in the prereplicative

phase of liver regeneration.
Methods: Rats were subjected to PH at 6 h and mitochondria
were isolated. Mitochondria from sham-operated rats were used
as controls. Mitochondrial protein expression pattern were stud-
ied by 2-DE electrophoresis, and subsequent spots identification
was performed by nanoLC-ESI MS/MS analysis.
Results: Compared to the sham-operated control group, 1 pro-
tein was up-regulated and 13 proteins were down-regulated at
6 h. We identified eight differentially expressed proteins that were
associated with lipid metabolism, the OXPHOS system, biotrans-
formation and other metabolic pathways. Among these, Mn-
superoxide dismutase, was down-regulated 6 h after PH. SOD2 is
a matrix mitochondrial enzyme that catalyzes superoxide radicals
dismutation.
Reference:
1. Guerrieri F et al., Free Radic. Biol. Med. 1999; 26: 34–41.
P1–30
Evolutionary dynamics of the Arabidopsis
formin family
M. Grunt and F. Cvrckova
Faculty of Science, Plant Physiology, Charles University, Prague,
CZECH REPUBLIC
Formins (FH2 proteins) are a family of actin-organizing proteins
exhibiting interesting evolutionary dynamics, with variable
domain composition in some lineages including plants. Seed
plants, in particular angiosperms, exhibit an extraordinary diver-
sity of formins, which can be divided into two distinct classes on
the basis of sequence of the conserved actin-nucleating FH2
domain, as well as domain composition (1). The genome of the
common laboratory model, Arabidopsis thaliana, encodes 11

Class I formins and up to 12 Class II formins, which appears to
be especially prone to gene duplication. Since the draft genome
sequence of a closely related Arabidopsis species, A. lyrata,
became available recently, we have used this sequence to identify
members of the A. lyrata formin family, and compared them
with their A. thaliana homologues. While we could easily distin-
guish orthologues of each of the Class I formins, the situation is
somewhat less clear in case of Class II proteins, consistent with
gene duplications taking place even after separation of the thali-
ana and lyrata lineages. Nevertheless, even one of the loci sus-
pected to be pseudogenes in A. thaliana has an orthologue A.
lyrata, suggesting that it might be a functional gene.
Acknowledgement: This work was supported by the
MSM0021620858 and LC06004 projects.
Reference:
1. Grunt et al., BMC Evol. Biol. 2008; 8:115.
P1–31
Impaired function of mitochondrial ATP
synthase depending upon 9205delTA mutation
load in ATP6 gene of mtDNA
K. Hejzlarova, P. Jesina, V. Kaplanova, Z. Drahota, M. Kalous
and J. Houstek
Department of Bioenergetics, Institute of Physiology AS CR v.v.i.,
Prague 4, CZECH REPUBLIC
Maternally transmitted disorders of mitochondrial ATP synthase
are typically caused by heteroplasmic missense point mutations
in mtDNA encoded subunit 6. In contrast, unique ATP synthase
disorder due to microdeletion 9205delTA in ATP6/COX3 gene is
associated with altered splicing of ATP6-COX3 mRNA and
diminished synthesis of subunit 6 (F

o
-a). Up to now, only two
patients with 9205delTA mutation have been found with very dif-
ferent phenotypes and mutation load. To investigate phenotypic
expression of the mutation, we have prepared transmitochondrial
cybrids with varying mutation load (50–100%) and studied the
relationship between heteroplasmy, mitochondrial respiration,
ATP production and F
o
-a subunit content. Heteroplasmy in cell
lines was analyzed by PCR/RFLP using NsiI restriction endonu-
clease and digitonin-permeabilised cells were used for measuring
oligomycin-sensitive, ADP-stimulated oxygen consumption. In
parallel, ATP production was determined in DMSO-quenched
samples by a luciferin-luciferase reaction. To evaluate how the
mutation load affects on biosynthesis of F
o
-a subunit, patient cy-
brids were analyzed by SDS-PAGE and WB using specific anti-
bodies to ATP synthase subunits F
o
-a and F
1
-alpha. We have
found that the decrease in ATP production and in ADP-stimu-
lated respiration as well as the loss of subunit F
o
-a content exert
similar threshold dependce on increasing mutation load. Pro-
nounced decrease in all parameters was observed when mutation

load reached about 80%. In contrast, near-linear relationship
was found between ATP production, respiration and loss of F
o
-a
subunit. In conclusion, our results demonstrate, that similarly as
ATP6 missense mutations, 9205delTA biochemical phenotype
exhibits distinct threshold effect that originates from a gene-pro-
tein level.
P1–32
Localisation of Pcb antenna complexes in
photosynthetic prokaryote Prochlorothrix
hollandica
M. Herbstova and F. Vacha
Institute of Plant Molecular Biology, Photosynthesis, Ceske Bude-
jovice, CZECH REPUBLIC
A filamentous freshwater phytoplankton Prochlorothrix hollandic-
a belongs to an unusual group of cyanobacteria called prochloro-
phytes. Typical cyanobacteria organise photosynthetic light-
harvesting complexes into so-called phycobilisomes where no
chlorophyll is bound. Prochlorophyta lost the ability to create
Abstracts Poster Presentations
104 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
phycobilisomes, but on the other hand as chloroplasts of high
plants and green algae it can synthesize chlorophyll (Chl) a and
b bound to the Pcb proteins (PcbA, PcbB, PcbC). They are simi-
lar to the IsiA, a chl a-binding protein induced in response to an
iron deficiency in many of cyanobacteria and to the CP43 protein
forming the Chl
a inner antenna protein of photosystem II. An
aim of this work was to identify and localise Pcb antenna com-

plexes in P. hollandica. The PcbA and PcbB antenna polypeptides
are genetically closely related to each other, the third PcbC pro-
tein is divergent from the first two. This fact can indicate their
different function in association with photosynthetic complexes.
The PcbA and PcbB proteins seems to function as mobile
unbound antenna of both photosystems in contrast to PcbC pro-
teins that may be bound to the photosystem I. Solubilization of
thylakoid membranes resulted in a separation of seven zones on
the sucrose density gradient. They were characterised by the
absorption and fluorescence emission spectroscopy. Protein com-
position was determined by SDS-PAGE and the Pcb proteins
was detected by western blotting. Pigment-protein complexes
were separated by colourless native electrophoresis (CN-PAGE).
Subunit protein composition of complexes was resolved in a sec-
ond dimension by SDS-PAGE and Pcb proteins were identified
by immunoblot.
P1–33
Selection of reference genes for gene
expression studies in Bombus terrestris
D. Hornakova
1
, P. Matouskova
1
, J. Kindl
2
, I. Valterova
2
and
I. Pichova
1

1
Institute of Organic Chemistry and Biochemistry Czech Academy
of Sciences, Biochemistry, Prague 6, CZECH REPUBLIC,
2
Institute of Organic Chemistry and Biochemistry Czech Academy
of Sciences, Chemistry of Natural Products, Prague 6, CZECH
REPUBLIC
Bumblebees are studied as important agricultural pollinators.
They use pheromones for many life-important activities, such as
mating, foraging, finding the way to food sources. In sexual com-
munication males mark a territory using pheromone to attract
females for mating. The marking sex pheromone is synthesized in
the cephalic region of the labial gland. The pathway and enzymes
involved in pheromone biosynthesis in Bombus terrestris are not
known. Previous experiments showed that qualitative and quanti-
tative changes in composition and amount of the labial gland
secretion depend on the age of bumblebee (1). To determine,
which enzymes participate in pheromone biosynthesis, in tissue
restructuring and in programmed cell death of the secretory cells,
we will analyze the gene expression profile in the labial gland and
fat body at different points in the bumblebee’s life cycle. No ref-
erence genes for qPCR quantification of gene expression in Bom-
bus terrestris have been characterized. We have evaluated nine
potential reference genes [arginine kinase (AK), 18S rRNA, elon-
gation factor 1a (EF), b-Actin, glyceraldehyde-3P-dehydrogenase,
a-tubulin, ribosomal protein L13, acidic ribosomal protein P2,
and phospholipase A2 (PA2)] in the labial gland and the fat body
of B. terrestris. Sequences of AK, 18S rRNA, EF were publicly
accessible. Remaining six candidate gene fragments were cloned
using degenerate primers. Using geNorm and NormFinder Excel

based software we found that AK and PA2 genes are the most
stable in both tissues from B. terrestris and can be used as refer-
ence genes for gene expression profiling.
Reference:
1. S
ˇ
obotnı
´
k et al., J. Insect. Physiol. 2008; 54: 204–214.
P1–34
Comparative analysis of proteome changes in
contrasting flax cultivars upon cadmium
exposure
J. Hradilova
1
, P. Rehulka
2
, H. Rehulkova
2
, M. Vrbova
3
,
M. Griga
3
and B. Brzobohaty
1
1
Laboratory of Plant Molecular Biology, Institute of Biophysics
AS CR, Mendel University of Agriculture and Forestry in Brno,
Brno, CZECH REPUBLIC,

2
Department of Proteomics and
Glycomics, Institute of Analytical Chemistry of the AS CR, Brno,
CZECH REPUBLIC,
3
Plant biotechnology department,
AGRITEC Ltd., Brno, CZECH REPUBLIC
Cadmium (Cd) is classified as a serious pollutant due to its high
toxicity and carcinogenicity, and widespread presence in environ-
ment. Phytoremediation represents an effective low-cost technol-
ogy for removal of pollutants from contaminated soils. Flax
belongs to crops with significant phytoremediation potential. Pre-
vious investigation revealed significant differences in Cd accumu-
lation and tolerance among commercial flax cultivars with cv.
Jitka being superior to cv. Ta
´
bor in respect of tolerance to
increased Cd content in soil and plant tissues. Here significant
changes in expression of 14 proteins (related to disease/defence,
metabolism, protein destination and storage, signal transduction,
energy and cell structure) were detected by image and mass spec-
trometric analysis of two-dimensionally separated proteins
extracted from Cd treated cell suspension cultures derived from
the two contrasting cultivars. Importantly, up-regulation by Cd
of two of them, ferritin and glutamine synthetase, was found
only in cv. Jitka. This might infer increased Cd detoxification in
cv. Jitka via binding to ferritin, and low-molecular thiol peptides
as glutamine synthetase is an important enzyme in glutathione
biosynthesis. The identified proteins could turn useful in marker
assisted breeding for Cd tolerance, and development of trans-

genic flax lines possessing enhanced Cd accumulation and toler-
ance for phytoremediation of Cd contaminated soils.
Acknowledgements: This work was supported by grants
1M06030 and MSM 2678424601 (Ministry of Education of the
Czech Republic), and AV0Z50040507, AV0Z50040702 and
AV0Z40310501 (Academy of Sciences of the Czech Republic).
P1–35
Intergenic co-transcription, erratic alternative
splicing and recruitment of transposable
element sequences into transcripts of
testis-specific retrogenes
C J. Huang
1
, W. Y. Lin
2
, C. M. Chang
3
and K. B. Choo
2
1
Department of Animal Science, School of Agriculture, Chinese
Culture University, Taipei, TAIWAN,
2
Taipei Veterans General
Hospital, Department of Medical Research and Education, Taipei,
TAIWAN,
3
Graduate Institute of Biotechnology, Chinese Culture
University, Taipei, TAIWAN
It is a puzzle why mammalian genomes are heavily burdened

with highly repetitive relics of ancestral transposable elements
(TEs); the biological meaning of TE in the genome remains lar-
gely elusive. We have previously reported a cluster of retrogenes
in the rat genome two of which, Rtdpoz-T1 and -T2, are testis-
specific. In the course of investigation of the structure of T1/T2
transcripts expressed in the embryo, the exons of these retroge-
nes, some duplicated as cassettes of 2–3 exons, are found to be
deeply embedded within a dense minefield of up to 70% occu-
pancy of LINE1 and ERV sequences. Furthermore, the retroge-
nes are seemingly regulated by a single promoter and T1-T2
chimerism is also observed. To explain the novel features of the
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 105
T1/T2 transcripts, we propose a co-transcription model in which
transcripts of the downstream gene, T1, are highly complex as a
result of rogue recruitment of TE remnants sequences to the
untranslated regions, in particular the 5¢-UTR, due to unruly
alternative splicing of some extended transcriptional read-through
primary transcript(s). TE invasion of 5¢-UTR results in upstream
ORFs with experimentally demonstrated deleterious effects on
translation. Our findings expand the list of possible biological
functions attributable to transposable elements in the mammalian
genome.
P1–36
Identification of novel cancer-associated
molecules
A. Chernenko
1
, M. Reinman
2

, P. R. Karhemo
1
, S. Goodison
3
,
K. Takkinen
2
and P. Laakkonen
1
1
Faculty of Medicine, Institute of Biomedicine, University of
Helsinki, Helsinki, FINLAND,
2
VTT Technical Research Centre
of Finland, Biotechnology, Espoo, FINLAND,
3
Department of
Surgery, University of Florida, Jacksonville, FL, USA
Tumor metastasis is the most serious challenge for cancer treat-
ment, since the metastatic dissemination of tumour, rather than
primary tumours, is responsible for most cancer deaths. In our
study we are aiming to discover the metastasis-promoting mole-
cules and construct the antibodies against these molecules. We
are using two subclones of the MDA-MB-435 human melanoma
cell line as a model for the metastatic spread of cancer. One cell
clone metastasizes consistently to the lungs whereas the other,
equally tumorigenic, fails to metastasize to any distal site in athy-
mic mice. In order to find new metastasis-associated markers we
panned these cell lines with phage-displayed single-chain variable
fragment (scFv) antibody library. Several clones of scFv-phages

were identified to bind differently to cells with metastatic and
non-metastatic phenotype, as judged by FACS analysis. The
selected scFvs recognize antigens on the cell surface and some of
them are able to internalize. We are currently trying to identify
the antigens for selected antibodies by immunoprecipitation from
the metastatic cells lysates using soluble biotinylated scFvs and
scFv-expressing phages. After immunoprecipitation the putative
antigens will be analyzed with MALDI-TOF mass-spectrometry.
Next we are planning to study the role of the discovered mole-
cules in tumour metastasis. For further studies the antibodies can
be produced and purified in IgG or IgM format. The results of
our study might have the direct clinical implication, since the
revealed metastasis-associated molecules might be used as drug
targets and the created human antibodies can be used to inhibit
metastatic process in cancer patients.
P1–37
Characterization of the barrier activity of Wari
insulator in D. melanogaster
D. Chetverina, M. Erokhin and P. Georgiev
Department of the Control of Genetic Processes, Institute of Gene
Biology, Moscow, RUSSIA
Background: Insulators are thought to isolate independent tran-
scriptional units from cross-reaction with neighboring regulatory
sequences specifically blocking the activity of an enhancer. Insu-
lators operate in a position-dependent manner, preventing the
activity of enhancers only when inserted between these regulatory
elements and a promoter. It was shown that at least some of
insulators have a barrier activity: they can block the activity of
silencers and prevent the spread of repressive chromatin. The
detailed mechanism of insulator action is currently unknown.

Methods: Plasmid construction, germ line transformation,
genetic crosses, X-ChIP.
Results: We have tested the ability of the recently described
Wari insulator to block the repression of the mini-white and yel-
low genes by the Ubx PRE. We show that Wari insulator can
block the activity ofPRE-mediated silencing. The distance
between Wari insulator and a protected promoter is important
for the effective blocking from PRE-mediated silencing. We
found that part of Wari insulator (368 bp) which is minimal for
enhancer-blocking activity could be shortened in case of the bar-
rier activity. Moreover, we show with X-ChIP that proteins
CP190 and Mod(mdg4)-67.2 interact with Wari insulator. In
addition, we investigated that mutations in mod(mdg4)-67.2 and
e(y)2 genes affect the ability of Wari insulator to protect gene
expression from PRE-mediated silencing, but not its ability to
block activation by enhancer.
Conclusions: Wari insulator can protect yellow and mini-white
genes from PRE-mediated silencing. It does not require
Mod(mdg4)-67.2 or E(y)2 for its enhancer-blocking activity, but
these proteins are important for its barrier activity.
P1–38
Abstract withdrawn
P1–39
A multiple sclerosis risk-associated SNP
influences splicing of the myelin
oligodendrocyte glycoprotein gene
C. Jensen and J. Rubio
Howard Florey Institute, Neurogenetics, Parkville, Vic.,
AUSTRALIA
Multiple sclerosis (MS) is a complex autoimmune disease charac-

terised by T and B-cell mediated demyelinating lesions in the cen-
tral nervous system (CNS). An auto-antigen in MS is the myelin
oligodendrocyte glycoprotein (MOG), a CNS restricted protein
expressed on the outer cell membrane of oligodendrocytes that
can be used to induce the MS-like condition, experimental auto-
immune encephalitis (EAE), in laboratory animals. There have
been conflicting reports in regard to the association between com-
mon variation in the MOG gene and susceptibility to MS. In this
study we investigated some of the expression characteristics of
MOG in post-mortem human MS and control brain tissue in
order to try to elucidate the cause of an association we observed
between the single nucleotide polymorphism, rs3130253G>A
(V145I), and susceptibility to MS. We found that the risk-associ-
ated allele, rs3130253A, was associated with a small shift in
expression towards the MOG alpha1 and/or MOG alpha4 splice
variants. We also demonstrated that neither rs3130253G>A
(exon 3) or other common exonic variations in the MOG gene
[rs282857766 (exon3, V142L) and rs3130250G>A (exon 1, S5S)]
were associated with allele specific changes in overall MOG
expression. In conclusion, subtle changes in MOG isoform selec-
tion, but not expression levels, appear to be associated with
rs3130253G>A, which in turn could influence the function of
MOG and the pathogenesis of MS.
Abstracts Poster Presentations
106 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
P1–40
FATT mouse: a novel leptin receptor mutant
C. Jensen
1
, H. Chen

2
, M. Morris
2
, L. Johnson
1
, R. Burfoot
1
,
K. Simpson
3
, G. Smyth
3
, K. Walder
4
, S. Foote
3
, J. Hollis
5
,
B. Oldfield
5
and J. Rubio
6
1
Howard Florey Institute, Neurogenetics, Parkville, Vic.,
AUSTRALIA,
2
Department of Pharmacology, University of
Melbourne, Parkville, Vic., AUSTRALIA,
3

Genetics and
Bioinformatics Division, The Walter and Eliza Hall Institute of
Medical Research, Parkville, Vic., AUSTRALIA,
4
Metabolic
Research Unit, School of Exercise and Nutrition Sciences, Deakin
University, Geelong, Vic., AUSTRALIA,
5
Department of
Physiology, Monash University, Clayton, Vic., AUSTRALIA,
6
Neurogenetics Laboratory, Howard Florey Institute, Parkville,
Vic., AUSTRALIA
A spontaneous nucleotide substitution 1279 G>A was observed
in the leptin receptor (lepr) gene [NM_146146] in mice of the 129/
SvEvTac strain. Mutant mice (designated FATT) carrying two
copies of the mutation (Lepr
FATT
)became hyperphagic and obese
post-weaning. In exon 7 of lepr, adjacent to the splice donor ‘GT’
consensus sequence in intron 7, the Lepr
FATT
mutation correlated
with the production of two alternatively spliced transcripts in the
hypothalamus of obese mice; 1) intron 7 sequence remaining in
the transcript, 2) exon 7 sequence (predicted immunoglobulin-like
domain) discretely removed with the spliced variant remaining in
frame. While there were a number of phenotypic similarities with
other leptin signalling deficient mutant strains such as the leptin
deficient ‘ob’ mouse and the leptin receptor deficient ‘db’ mouse

(similarities include hyperphagia, obesity, hyperleptinaemia, insu-
lin resistance, infertility and increased respiratory exchange ratio)
there were also distinct differences, (principally being increased
body length, and minimal changes in the lymphoid tissues, in con-
trast to shorter body length and compromised lymphatic tissues
in db mice). Phenotypic characteristics that differentiated the
Lepr
FATT
mutant from the ‘ob’ and ‘db’ mutants remained after
backcrossing onto a C57/Bl6 genetic background. Given the
unique site of the mutation, its effect on Lepr splicing and this
unique combination of phenotypic characteristics it is likely that
this leptin receptor mutant will prove useful in elucidating further
the signalling mechanisms of the leptin receptor and their down
stream consequences.
P1–41
Contact of medical steel and parylene with
endothelial cells induces changes in gene
expression profile
H. Jerczynska
1
, P. Komorowski
2
, A. Nosal
2
, H. Szymanowski
2
,
M. Gazicki-Lipman
2

, B. Walkowiak
2
and Z. Pawlowska
1
1
Department of Molecular and Medical Biophysics, Medical
University in Lodz, Lodz, POLAND,
2
Institute of Materials
Science and Engineering, Technical University of Lodz, Lodz,
POLAND
Biomaterials can be used successfully to replace many parts of
living systems in the human body. Material selection and bio-
compatibility are critical issues in today’s biomedical implants
applications. Biomaterials research is an exciting and rapidly
growing field, the results of such studies should be undertaken to
determine accurately the safety and performance of the implants.
Currently the material of choice for thousands of medical appli-
cations are polymers since they can be prepared in a variety of
forms (solids, fibers, fabrics, films and gels) and they can be eas-
ily fabricated giving various shapes and structures. Additionally,
they are easily functionalized and, depending on the polymeric
building blocks, may also be degraded in the body after a desired
period. However, biomaterials used for medical implants or
instruments production can cause numerous adverse effects. They
may cause changes in gene expression and protein profile of the
cells which were in contact with applied material. The aim of this
study was to evaluate an influence of medical steel AISI 316L
and the medical steel coated with poly-para-xylylene (parylene)
on a cell cycle and apoptosis of human endothelial cells. The

results demonstrated that interactions between cells and studied
materials induce a number of changes in the gene expression
profile.
Acknowledgement: Supported by project No P01/0001/SPB-
PSS/2008a.
P1–42
Tumor necrosis factor, tumor necrosis factor
receptors type 1 and 2, lymphotoxin-a gene
polymorphism in lymphoproliferative diseases
in Serbian population
T. Jevtovic
1
, G. Kocic
1
, D. Pavlovic
1
, N. Tosic
2
, S. Aveic
2
,
G. Marjanovic
3
, L. Macukanovic-Golubovic
3
and V. Djordjevic
1
1
Institute for Biochemistry, Medical Faculty University of Nis,
Nis, SERBIA,

2
Department for Molecular Biology, Institute of
Molecular Genetics and Genetic Engineering, Belgrade, SERBIA,
3
Institute of Hematology and Immunology, Clinical Centre Nis,
Nis, SERBIA
Tumor necrosis factor-a and lymphotoxin-a are involved in the
pathogenesis of established lymphoproliferative diseases. TNF
molecules bind to TNFRI and TNFRII. TNFRI is the major
mediator of the TNF pro-apoptotic and prolifferative effects and
TNFRII might potentiate this effects. In this study, we have
analysed polymorphism in TNF gene -308 G/A, in LT-a gene
+250G/A, one polymorphisms in TNFRI gene (TNFRI + 36A/
G SNP) and one polymorphism in TNFRII gene
(TNFRII + 676 T/G). All these polymorphisms were studied in
patients with chronic lymphocytic leukemia’s (CLL), patients
with non-Hodkin’s lymphoma (NHL) and in healthy controls.
The present study was undertaken to investigate the genetic asso-
ciation of these polymorphisms with lymproproliferative disease
development. Genotyping was performed by PCR-RFLP analy-
sis. The study provided evidence of the influence of TNFG/A
genotype and A alleles in the susceptibility to NHL, since the
association of LT-aG/G genotype with CLL was observed. High-
producing TNF-a -308/LT-a +250 heterozygous haplotype is
associated with high NHL incidence. No significant differences in
allele frequencies of TNFR1 polymorphism were found between
the patients with lymphoproliferative disease and healthy individ-
uals. In a group of healthy individuals the study has revealed for
the first time significantly higher TNFRI G/G genotype com-
pared to the patients with lymphoproliferative disease

(v
2
= 5.66; P = 0.017). Also, we reported the implication of
TNFRII T allele in NHL pathogenesis, respectively (v
2
= 10.77;
P = 0.001; Mantel-Haenszel: v
2
= 10.64; P = 0.0011). Our data
showed that TNFRII T676G polymorphisms have an important
role in NHLs pathogenesis but not in CLL patients. A/A poly-
morphism in TNFRI is associated with CLL and NHL patients
in Serbian population.
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 107
P1–43
Genetic evaluation of the human oral cancer
via systematic DNA resequencing approach
M. C. Kao
1
, M. H. Yeh
2
, T. M. Shieh
3
, S. F. Chen
3
, M. H. Tsai
4
and Y. S. Lee
5

1
Biological Science and Technology, China Medical University,
Taichung, TAIWAN,
2
National Defense Medical Center, Graduate
Institute of Life Sciences, Taipei, TAIWAN,
3
Department of
Dental Hygiene, China Medical University, Taichung, TAIWAN,
4
Department of Otolaryngology, China Medical University Hospi-
tal, Taichung, TAIWAN,
5
Office of Research & Development,
China Medical University, Taichung, TAIWAN
Oral cancer is of worldwide occurrence and concerned globally,
approximately 500 000 new cases of oral and pharyngeal cancers
are diagnosed annually. The development of oral cancer is a mul-
tistep process. It has a late onset and shows a poorly clinical out-
come. Early detection of precancerous lesions or premalignant
conditions exhibiting transformation potential will lead to a con-
servative intervention and efficient therapy. Therefore, under-
standing the molecular mechanisms of oral carcinogenesis is
necessary to improve early prognosis and even implant an early
detection protocol for the oral cancer. In this research, we glob-
ally search for human oral cancer altered genes via a comprehen-
sive Real-Time RT-PCR (qPCR) and DNA resequencing
strategy. To date, we have assayed both tumor and their adjacent
normal tissues from 60 oral cancer patients by qPCR and identi-
fied 20 differential expression genes in human oral tumors. Fur-

thermore, we used DNA resequencing approach to determine the
spectrum and extent of altered somatic mutations in these 20
genes, four mutation sites were identified within three genes
(MMP1, PTN and BMP3). We propose these 20 genes and the
mutations may have potential as biomarkers for early diagnosis
of the oral cancer. In addition, they may be used as a potential
targets for anti-cancer drug design and beneficial to the clinical
treatment of oral carcinomas.
P1–44
SDS-PAGE characterization of the proteins and
glycoproteins in thumb pad secretion of the
frog (Rana ridibunda)
E. Kaptan and S. Bolkent
Faculty of Science, Department of Biology, Istanbul University,
Istanbul, TURKEY
Generally, frogs exhibit cyclic reproductive activity throughout
1 year. Their primary and secondary sexual characteristics show
structural and biochemical changes during this cycle. Thumb pad
is an androgen-dependent secondary sexual characteristic playing
a role in amplexus. In this study, we focused on documenting
protein and glycoprotein profile of its secretion and determining
the variability among those which obtained from different sea-
son. For this propose, we composed of four groups namely;
active, prehibernating, hibernating and posthibernating. The
secretion of thumb pad isolated from all groups and determina-
tion of protein and glycoprotein concentrations were performed
by method of Lowry and Dubois, respectively. Protein and glyco-
proteins of secretion content were separated using sodium dode-
cyl sulphate polyacrylamide gel electrophoresis. Prior to scan and
analyze of the gels using densitometer, they were stained with

Coomassie blue and glycoprotein detecting kit, respectively. A
total of 22 bands were determined, ranging from 156 to 16 kDa
protein. Of these bands, five protein (66, 32, 25, 224 and 23 kDa)
were detected to be glycolyzed. In addition to these protein and
glycoprotein pattern of the secretion, they showed seasonal
changes. Protein band numbers and their optic density were high
in posthibernation period. Glycoprotein band numbers and their
density were also exhibited same aspect as proteins. These find-
ings point out that thumb pad secretion is abounded in terms of
protein content and the changes about protein and glycoprotein
profiles might be related to seasonal reproductive activity. This
means that they can be dependent on androgenic hormones.
P1–45
Comparison of protein and mRNA expression
levels of titin isoforms in cardiac muscles of
hibernating ground squirrels
E. Karaduleva, I. Vikhlyantsev, J. Shumilina and Z. Podlubnaya
Laboratory of Structure and Function of Muscle Proteins, Insitute
of Theoretical and Experimental Biophysics, Pushchino, RUSSIA
Titin is giant elastic protein of vertebrate skeletal and cardiac
muscles. Titin- encoding gene located in the chromosome 2
(region 2q31) consists of 363 exons encoding 4200 kDa protein
(38138 amino acid residues). Alternative splicing of titin elastic
area in an I-zone of sarcomere is a basis of a variety of titin iso-
forms. Cardiac titin is expressed in two isoforms: short N2B
(3000 kDa) and long N2BA (3200–3400 kDa) with up to seven
variants of alternative splicing. Research of protein regulation on
transcriptional and translational levels is of particular interest for
description of muscle functional state. In this work we carried
out the investigation of changes in titin isoform composition in

myocardium of ground squirrels in various stages of hibernation-
al cycle. We have selected this model because hibernation is a
unique model of adaptation to stress conditions, immobilization
and heart function depression. The results of qRT-PCR revealed
the decrease of mRNA level both for N2BA and N2B titin iso-
forms in heart of hibernating ground squirrels as compared to
their levels in the heart of summer active animals. Using electro-
phoresis and western blotting we have shown the decrease in
overall amount of titin with respect to heavy myosin chains in
different heart chambers of ground squirrels upon hibernation in
comparison with summer active animals (ð‡0,998). The ratio of
long N2BA to N2B titin isoforms in the heart of hibernating
ground squirrels was increased approximately two-fold
(P < 0.0005). However this trend was not revealed for the
mRNA levels of corresponding isoforms. This discrepancy in
protein and mRNA levels may be considered as the posstran-
scriptional regulation of titin isoforms’ expression. The overall
decrease in mRNA level may be explained by repressed transcrip-
tion or mRNA degradation in the cell during hibernation. To
understand the mechanisms regulating titin isoform composition
upon hibernation these investigations are to be extended.
Acknowledgement: This work was supported by grant RFBR
07-04-00479.
P1–46
Uniform reporting standards for enzyme
activity data
C. Kettner
Funding Department, Beilstein-Institut, Frankfurt am Main,
GERMANY
The access to the essential background information on enzymo-

logical experiments is of paramount impact for the understanding
of both the experimental data themselves and their interpretation
Abstracts Poster Presentations
108 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
under consideration of the broader context. However, scientists
who are evaluating these data for pathway research be it as in sil-
ico modellers or as experimentalists often are missing full details
of the assay conditions to be able to refer the enzyme activity
data presented to the materials and methods applied. Further-
more, they are often are faced with the problem of a broad range
of method-specific enzyme data ranges associated with individual
methods found in databases or in the scientific literature. Modern
experimental techniques provide apparently endless opportunities
to generate huge amounts of enzyme structure and activity data.
These technological advances have resulted in increased accuracy
of measurement and analysis methods and given rise to large
amounts of data. But this increasing rate of production exacer-
bates the problem of fragmented descriptions of materials and
methods which leads to incomparable and incomplete data. It
appears to be perfectly obvious that these problems could be
addressed by adopting a set of reporting standards. Since the
need for standardization is widely recognized by the scientific
community, the STRENDA Commission (1,2), founded and sup-
ported by the Beilstein-Institut, proposed guidelines for reporting
minimum information on both the experimental design and the
obtained functional data. These guidelines resulted as checklists
from consultation with the community and were embedded in the
international checklists development project MIBBI (3). A piv-
otal achievement of the cooperation with a number of biochemi-
cal journals was the adoption of these guidelines in their

instructions for authors last year. Further journals will follow
these flagships in due course. It is hoped that future publications
will more readily yield the sort of information that researchers
hope to find. The guidelines and further aims of STRENDA will
be presented in detail.
References:
1. Apweiler R et al. (2005) The importance of uniformity in
reporting protein-function data. TRENDS in Biochemical
Sciences 30(1):11–12.
2. Alberty R et al. (2009) Standards for reporting enzyme data:
the STRENDA consortium. submitted.
3. Taylor CF et al. (2008) Promoting coherent minimum report-
ing guidelines for biological and biomedical investigations: the
MIBBI project. Nature Biotechnology 26(8):889–896.
P1–47
Characterization of cytochrome CYP52 and
NADPH-P450 reductase from Candida albicans
D. Kim, H. G. Park, Y. R. Lim, C. Y. Eun and S. Han
Biological Sciences, Konkuk University, Seoul, SOUTH-KOREA
Candida albicans is a major pathogenic fungus that causes oppor-
tunistic oral and vaginal infections in humans. It contains ten
putative cytochromes P450 (CYPs) and an NADPH-P450 reduc-
tase (NPR) genes coding for enzymes that appear to play impor-
tant roles in fungal survival and virulence. Here, we report the
characterization of CYP52, a putative alkane/fatty acid hydroxy-
lase and NPR, an essential reduction partner for CYP52. The
recombinant CYP52 protein was expressed and was purified. The
purified protein primarily w-hydroxylated lauric acid to give
12-hydroxylauric acid, but to a lesser extent also catalyzed (w-1)-
hydroxylation. The regioselectivity of CYP52 fatty acid hydroxyl-

ation can be attributed to the presence of a narrow channel that
restricts access of terminal atom for oxidation even though this
narrow channel. As an essential enzyme for P450 reaction, the
recombinant NPR protein from C. albicans was also expressed
and was purified. C. albicans NPR protein showed a strong
NADPH-dependent reduction activity of nitrotetrazolium blue
chloride. This study of functional characterization of P450s and
NPR from C. albicans could provide the critical understanding of
its role in the processing of pathogenesis.
Acknowledgements: This study is supported by a grant of the
Korea Healthcare technology R&D Project, Ministry of Health
& Welfare, A084005.
P1–48
Electrochemical and computational study of
doxorubicin interactions with DNA
D. Huska
1
, V. Adam
1
, J. Burda
2
, J. Hrabeta
3
, T. Eckschlager
3
,
P. Babula
4
, R. Opatrilova
4

, L. Trnkova
5
, M. Stiborova
6
and
R. Kizek
1
1
Department of Chemistry and Biochemistry, Mendel University of
Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC,
2
Department of Chemical Physics and Optics, Charles University,
Prague, CZECH REPUBLIC,
3
Department of Paediatric Haema-
tology and Oncology, Charles University, Prague, CZECH
REPUBLIC,
4
Department of Natural Drugs, University of Veteri-
nary and Pharmaceutical Sciences, Brno, CZECH REPUBLIC,
5
Department of Chemistry, Masaryk University, Brno, CZECH
REPUBLIC,
6
Department of Biochemistry, Charles University,
Prague, CZECH REPUBLIC
Anthracyclines including doxorubicin belong to antibiotics pro-
duced by Streptomyces peucetius subsp. cesius. There is compelling
evidence that cellular DNA is the primary target for them. It is not
surprise that cell protective mechanisms cause resistance on the

treatment with these drugs. The main aim is to study intercalation
of doxorubicin into DNA in vitro by using of square wave voltam-
metry. The electrochemical method is further utilized for detection
of doxorubicin-DNA adducts in neuroblastoma cells. Intercalated
doxorubicin reduced observed dsDNA cytosine and adenine sig-
nal, but also provided new signal of the cytostatic at -0.35 ±
0.12 V (n = 8). For more comprehensive understanding of the
intercalation, computational experiments using design for testabil-
ity approach with correction on dispersion interaction were per-
formed. Several molecular structures were explored and clear
preference for very stable stacked structures was observed. In addi-
tion, we detected doxorubicin-DNA adducts in doxorubicin trea-
ted neuroblastoma cells. We observed significant (a = 0.05)
difference in DNA signals after intercalation of doxorubicin
between resistant and sensitive cells. Resistant cells showed negligi-
ble amount of adducts with the increasing concentration of the
drug compared to resistant cells. The suppressed formation of ad-
ducts can be related to presence of other mechanisms to withstand
the effect of the cytostatic. Therefore we determined low-molecu-
lar-mass thiols (glutathiones and its derivatives) and found the
increased thiols level in resistant cells compared to sensitive ones.
Acknowledgement: This work was supported by GA AV
IAA401990701.
References:
1. Krizkova S. et al., Electroanalysis 2007; 19(2–3): 331–338.
2. Huska D. et al., Electroanalysis 2009; 21(3–5): 487–494.
P1–49
Respiratory supercomplexes in COX-deficient
mammalian mitochondria
N. Kovarova

1
, P. Pecina
1
, J. Houstek
1
, C. Dell‘Agnello
2
and
M. Zeviani
2
1
Bioenergetics, Institute of Physiology Academy of Sciences of the
Czech Republic, Prague, CZECH REPUBLIC,
2
Unit of Molecular
Neurogenetics, National Neurological Institute, Milan, ITALY
Respiratory chain enzyme complexes are assembled into higher
structural units, respiratory supercomplexes. In mammalian mito-
chondria they are mainly composed of complexes I, III, and IV.
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 109
Isolated deficiency of complex IV represents frequent cause of
mitochondrial disease and we investigated how alterations of
cytochrome c oxidase (COX) content influence the supercomplex
assembly and occurrence in mitochondria from different tissues.
We used mice model with isolated COX defect due to SURF1
gene knock-out (SURF1
-/-
) that prevents synthesis of Surf1
assembly protein, and leads to varying degree of COX deficiency

in mouse tissues. For comparison we analysed COX-deficient fi-
broblasts mitochondria of a patient carying mutation in the
SURF1 gene. Digitonin-solubilised protein complexes were analy-
sed by 2D electrophoresis (BN-PAGE/SDS-PAGE or BN-
PAGE/BN-PAGE) and Western blotting using specific antibodies
to subunits of complex I, III and IV. Results of our experiments
revealed presence of two types of supercomplexes, I-III
2
and III
2
-
IV, in mitochondria from liver, brain, muscle and heart from
control and COX-deficient mouse. In mouse fibroblasts we also
detected I-III
2
-IV supercomplex and the same pattern was found
in human fibroblasts. In the latter case all complex I signal was
present in I-III
2
-IV supercomplex. Decrease of COX content in
mitochondria from both COX-deficient mouse and from the
patient markedly decreased the assembly of COX-containing su-
percomplexes, whereas the content of complex I and III in super-
complexes was unchanged. Our study demonstrates significant
tissue- and species-specific differences in the assembly and com-
position of the respiratory supercomplexes in both control and
COX-deficient mammalian mitochondria.
P1–50
Characterization of a putative threonine
phosphatase in Mycobacterium bovis BCG

P. C. Lee, F. M. Oh and A. A. M. Faik
School of Science and Technology, Universiti Malaysia Sabah,
Kota Kinabalu Sabah, MALAYSIA
Mycobaterial ‘eukaryotic-like’ serine/threonine protein kinases
coupled with their phosphatases are involved in intracellular sur-
vival and host-pathogen interaction. Understanding these kinases
and phosphatases will provide clues to facilitate the development
of specific protein kinase or phosphatase inhibitors, as well as
drugs that modulate the activities of these kinases and phosphata-
ses. We report here the cloning and characterization of a putative
threonine phosphatase, Mbpp from Mycobacterium bovis BCG
1173P2. The Mbpp gene was cloned by PCR in pET42a (+) and
expressed as His-tagged recombinant protein in Escherichia coli as
inclusion bodies. The recombinant protein was purified by using
high concentration of urea and refolded by dialysis. Mbpp showed
distinct phosphatase activity toward p-nitrophenyl phosphate with
optimal temperature 55°C and pH 8.0. The phosphatase activity of
Mbpp was strictly Mn
2+
dependent, a characteristic that is found
in other serine/threonine phosphatases. Three-dimensional struc-
tural analysis revealed three Mn
2+
ion binding sites at the active
domain of the phosphatase. We detected strong phosphatase activ-
ity toward phosphothreonine peptide but not phosphorserine or
phosphotyrosine, with a K
m
value of 1.34 ± 0.074 mM. The phos-
phatase activity was not inhibited by known protein phosphatase

inhibitors such as okadaic acid and sodium orthovanadate, which
are known to be specific inhibitors of eukaryotic-like phosphatases
indicating Mbpp is a threonine phosphatase. Bioinformactics anal-
yses revealed eleven universally conserved motifs that are well-
known characteristic of PP2C and showed 99% amino acid
sequence identity to Mstp, a serine/threonine phosphatase of
Mycobacterium tuberculosis which is present only in slow growing
mycobacterial species that might regulate cell division.
P1–51
N-linked glycosylation of the human ovarian
carcinoma SKOV3 cell line
E. Machado
1
, S. Kandzia
2
, P. Altevogt
3
, H. S. Conradt
2
and
J. Costa
1
1
ITQB, Laboratory of Glycobiology, Oeiras, PORTUGAL,
2
GlycoThera, GmbH, Hannover, GERMANY,
3
Division of Cellular
Immunology, German Cancer Research Center, Heidelberg,
GERMANY

Ovarian carcinoma is the leading cause of death from gynecologi-
cal cancers in many Western countries. Aberrant glycosylation is
an important aspect in malignant transformation, normally asso-
ciated with increased expression of membrane glycoproteins,
appearance of novel or truncated oligosaccharides, fucosylated
glycoconjugates and abnormal types of terminal oligosaccharides
containing antigenic determinants. The SKOV3 ovarian carci-
noma cell line has low amounts of Lewis carbohydrate epitopes
at the cell surface, synthesized by a1,3/4- fucosyltransferases
(FUTs) (1). The stable overexpression of several FUTs in the
SKOV3 cell line resulted in de novo synthesis of Lewis carbohy-
drate determinants. Over expression of FUT4, FUT5, FUT6,
FUT7 and FUT9 originated de novo expression of Le
x
and/or
sLe
x
epitopes, observed on the plasma membrane and intracellu-
lar compartments. These findings were consistent with the prefer-
ential specificity of the enzymes towards type 2 (Galb1,4GlcNAc)
acceptors. Only overexpression of FUT3, with predominant a1,4-
FUT activity, produced the type 1 (Galb1,3GlcNAc) related car-
bohydrate sLe
a
. The N-linked oligosaccharides from total glyco-
proteins of SKOV3 cell line was further characterized by high
performance anion exchange chromatography with pulsed amper-
ometric detection and matrix assisted laser desorption/ionization
time-of-flight mass spectrometry. The results showed that SKOV3
cells were rich in unprocessed oligomannose structures, with low

peripheral sialic acid levels. Furthermore, the N-glycosylation of
a recombinant secretory glycoprotein, erythropoietin, stably pro-
duced from this cell line was characterized.
Acknowledgement: This work will contribute to the knowledge
of the glycosylation profile of human ovarian cancer cell lines.
Reference:
1. Escrevente C, Machado E. et al., Int J Oncol 2006; 29: 557–
566.
P1–52
Quantification of point mutations ratio with a
one-tube single nucleotide primer extension
followed by reverse phase HPLC
T. Majerova
1
, M. Ingr
2
, J. Dostal
1
and J. Konvalinka
1
1
Biochemistry and Moleular Biology, Institute of Organic Chemis-
try and Biochemistry, Prague, CZECH REPUBLIC,
2
Faculty of
Science, Biochemistry, Charles University, Prague, CZECH
REPUBLIC
SNP (single nucleotide polymorphism) quantification in DNA
pools could be important for a rapid analysis of allele distribu-
tion in populations, including determination of gene variants

among individuals, monitoring drug resistance development and
prognosis of tumor growth. Although many methods of SNP
detections are available, precise SNP quantification is still trou-
blesome. In this work, we demonstrate a method for accurate
quantification of the SNP ratio in mixtures of two synthetic oli-
gonucleotides possessing guanine (G) or adenine (A) at a defined
position. This method is based on a one-tube single base primer
extension with fluorescently labeled nucleotides followed by
reverse phase HPLC and fluorescence detection of the primer
Abstracts Poster Presentations
110 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
extension products. In our experiments, we addressed problems
of the elimination of errors arising due to competition of two
templates differing by a single base and problems with unknown
concentration of the template DNA. In the presence of compet-
ing template we obtained a lower yield of the extension product
of interest. To solve this problem, we prepared a set of serial
dilutions from the sample of interest and from a reference sample
containing templates at a defined ratio. We correlated the ratio
of mutations in the unknown and the reference sample. Conse-
quently, we eliminated the need for the determination of absolute
DNA concentration in the reference sample and in the sample of
interest.
P1–53
Porential role of tumor necrosis factor alpha
gene polymorphism in autoimmune disease
M. Marinkovic
1
, T. Jevtovic-Stoimenov
1

, S. Stojanovic
2
,
M. Todorovic
1
, J. Basic
1
, A. Veljkovic
1
, T. Cvetkovic
1
and
D. Pavlovic
1
1
Medical Faculty, Institute of Biochemistry, Nis, SERBIA,
2
Medical Faculty, Clinic of Rheumatology, Nis, SERBIA
Introduction: Tumor necrosis factor alpha (TNF-alpha) plays a
key role in immune regulation, inflammation and autoimmunity.
It has been shown that this polymorphism is associated with vari-
ety of inflammatory and autoimmune diseases, such as, rheuma-
toid arthritis (RA) and systemic lupus erythematosus (SLE).
Considering the results of association of TNF-a -308 G/A poly-
morphism with RA and SLE, which are inconsistent, the aim of
this study was to examine the possible association between TNF-
a -308 G/A gene promoter polymorphism and RA and SLE.
Materials and Methods: Polymorphism of TNF-a -308 was
analyzed in RA patients (n = 76), SLE patients (n = 23) and
healthy controls (n = 34). Single nucleotide polymorphism

(SNP) was determined by PCR-RFLP method. The results were
analyzed using chi-squared test.
Results and Discussion: The observed genotype distribution in
RA patients did not show significant differences compared to
control group of healthy individuals. A higher frequency of het-
erozygous TNF G/A was observed in the group of SLE patients-
compared to controls (73.9% versus 41.2%, v
2
= 5.93,
P < 0.01). Significantly lower frequency of G/G genotype was
found in SLE patients compared to controls (8.7% versus 55.8%,
v
2
= 13.13, P < 0.001). No differences in the distribution of
TNF G and TNF A alleles were observed in group of RA
patients compared to control group. Contrary, there was signifi-
cant difference in the TNF G and A allele between SLE patients
and control individuals (v
2
= 11.32, P < 0.001). Hence, these
results suggest immunogenetic association involving polymor-
phism of TNF-a gene, serving as paracrine and autocrine growth
factors, which can contribute to the pathogenesis of SLE but not
of RA.
P1–54
RNase R is the main enzyme involved in the
poly(A)-dependent degradation of rpsO mRNA
J. M. Andrade
1
, E. Hajnsdorf

2
,P.Re
´
gnier
2
and C. M. Arraiano
1
1
Instituto de Tecnologia Quı
´
mica e Biolo
´
gica, Universidade Nova
de Lisboa, Oeiras, PORTUGAL,
2
Institut de Biologie Physico-Chi-
mique, CNRS UPR 9073, Paris, FRANCE
Polyadenylation is a universal post-transcriptional RNA modifi-
cation. It provides a single-stranded region downstream of termi-
nation hairpin allowing exonuclease binding and RNA
degradation. TherpsO mRNA encoding for the ribosomal protein
S15, has been widely used as a model of study for polyadenyla-
tion-mediated mRNA turnover. Escherichia coli RNase II and
PNPase are two major degradative exonucleases involved in the
control of mRNA stability. However, in the absence of these two
exonucleases the polyadenylated rpsO mRNA is still very effi-
ciently degraded. Data suggested that at least one yet unknown
poly(A)-dependent ribonuclease is involved in this complex regu-
lation. In this work, we demonstrate that the exonuclease RNase
R is the major enzyme responsible for the poly(A)-dependent

degradation of the rpsO mRNA. Moreover, RNase R is shown
to have a more relevant effect on rpsO mRNA stability than
PNPase, which was so far characterized as the main exonuclease
involved in the poly(A)-metabolism, either acting as a free
enzyme or in complex. In the absence of RNase R, the rpsO
transcript presents longer poly(A) tails and has higher stability.
The RNase R/polyadenylation pathway may be more general-
ized, as a 3¢ rpsT mRNA fragment whose degradation is highly
dependent on polyadenylation is also stabilized in an rnr single
mutant. All these results highlight the importance of bacterial
RNase R in the post-transcriptional control of gene expression
establishing an important parallel with its eukaryotic counter-
parts, Rrp44/Dis3, present in exosomes, which are also key play-
ers involved in the metabolism of polyadenylated RNA.
P1–55
An in vitro fish system to unravel bone-related
mechanisms of BMP2
C. Marques, V. Laize
´
and M. L. Cancela
CCMAR, F C M A, Faro, PORTUGAL
Mechanisms of tissue mineralization are complex and controlled
by several proteins involved in cell differentiation and extracellu-
lar matrix synthesis. Bone morphogenetic protein 2 (BMP2) was
primarily identified for its osteogenic properties and several
human bone-related diseases have been associated with BMP2
defect. Although numerous studies have been performed in mam-
mals to unravel BMP2 mechanisms of action, those are still not
fully understood. Fish have recently emerged as a suitable model
to study vertebrate biology and a suitable alternative to mamma-

lian systems. We propose to develop a fish-based in vitro cell sys-
tem to study bone-related mechanisms of BMP2, in particular its
interaction with matrix Gla protein (MGP), a physiological
inhibitor of calcification. BMP2 gene expression was profiled by
real-time PCR in a variety of seabream cell lines capable of in vi-
tro mineralization and shown to be differentially regulated in
osteoblast- and chondrocyte-like cells. Transcriptional regulation
of seabream BMP2 gene expression was later evaluated using
reporter vectors containing gene promoter and shown to be
under the control of various mineralization-related transcription
factors, in particular osteoblast-specific factor runx2. Cellular
clones, where BMP2 gene expression has been altered, have been
constructed, validated by Western blot analysis using antibodies
developed in mammals, then evaluated for mineralization
capacity using mineralization-specific stains and for mineraliza-
tion-related gene expression by real-time PCR. In future experi-
ments, BMP2 regulatory network will be assessed using recently
developed seabream microarray hybridized with cDNA prepared
from cellular clones, and BMP2-MGP interaction will be evalu-
ated in a co-culture system of clones overexpressing seabream
BMP2 and/or MGP cDNAs. Results obtained within the scope
of this work are preliminary but should give new and interesting
insights on bone-related mechanisms of BMP2 in vertebrates.
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 111
P1–56
Characterization of Bv8 gene promoter and its
regulation by transcription factors
S. Marsango, R. Miele, M. Bonaccorsi di Patti and D. Barra
Department of Biochemisty sciences ‘A. Rossi Fanelli’, Sapienza

University of Rome, Rome, ITALY
Bv8 is a small protein with a molecular mass close to 8 kDa
secreted by frog skin (1). Homologs of Bv8 can be found in
fishes, amphibians, reptiles, birds and mammals (2). Two mam-
malian orthologs of Bv8, prokineticin 1 and prokineticin 2 (PKs),
are the natural ligands for two G-protein-coupled receptors, PK-
R1 and PK-R2. Bv8/prokineticins and their receptor expression
is restricted to specific endothelial cells of steroidogenic glands,
central nervous system, peripheral blood leukocytes and cells of
the innate immune system. These proteins are involved in hema-
topoiesis and in inflammatory/immunomodulatory processes act-
ing like chemokines (3). The capacity of the immune system to
respond appropriately and eradicate microbial infections depends
on the production of peptide and small protein mediators,
chemokines and antimicrobial peptides, many of which are
induced by contact with pathogens or inflammatory stimuli (4).
Recently, a high degree of similarity was evidenced among defen-
sins, prokineticins and chemokines, either in structure, size, sig-
nalling or biological activities (3). In mammals, the synthesis of
mediators of innate immunity, like chemokines, antimicrobial
peptides and cytokines, as well as effectors of adaptative immu-
nity are regulated by the same mechanism that involve the activa-
tion and nuclear translocation of NF-jB and NFAT (5). Here
we report the organization of the amphibian Bv8 gene. Analysis
of the promoter sequence permits identify several putative tran-
scription factor binding sites like AP1, NF-kB and NFAT. Func-
tional analysis of Bv8 promoter by one-hybrid assay in yeast
indicates that these transcription factors are involved in the regu-
lation of Bv8 expression.
References:

1. Mollay C, Wechselberger C, Mignogna G, Negri L, Melchiorri
P, Barra D & Kreil G. Eur. J. Pharmacol. 1999; 374: 189–196.
2. Kaser A, Winklmayr M, Lepperdinger G & Kreil G. EMBO
Rep. 2003; 4: 469–73.
3. Monnier J, Samson M. FEBS J. 2008; 275: 4014–4021.
4. Negri L, Lattanzi R, Giannini E & Melchiorri P. Curr. Neuro-
pharmacol. 2006; 4: 207–215.
5. Serfling E, et al., Int. J. Biochem. Cell. Biol. 2004; 36: 1166–
1670.
P1–57
InterPro: deciphering the function and
classification of novelproteins
J. McDowall and S. Hunter
EMBL-EBI, Sequence Databases, Hinxton, UK
With increasing numbers of novel sequences being identified from
genome-sequencing projects, there is a dire need for elucidating
functional information that goes beyond the capabilities of exper-
imental work alone. Computer algorithms that take into account
sequence identity, structural similarity and phylogenetic tree dis-
tribution are invaluable for protein function prediction. InterPro
( is an open-source protein
resource combining ten major signature databases (PROSITE,
PRINTS, PFAM, PRODOM, SMART, TIGRFAMs, PIR-
SUPERFAMILY, PANTHER, GENE3D, SUPERFAMILY)
into one powerful, easy-to-use diagnostic tool. InterPro capitalis-
es on the strengths of each member database to provide compre-
hensive annotation on function, structure, splice variation and
hierarchical classification of proteins from all organisms, includ-
ing metagenomic projects. With 3 60 000 signatures, InterPro
provides annotation for 3 80% of UniProt sequences. Novel

sequences/genomes can be queried through InterProScan. Protein
signatures model amino acid conservation in families or domains,
then predict these classifications/features in uncharacterised pro-
teins. By linking related signatures together, InterPro places them
within a hierarchical classification scheme which reflects their
underlying evolutionary relationships. As such, one can address
issues such as the co-evolution of domains, the functional diver-
gence of proteins based on domain composition and their point
of divergence between different species. InterPro provides a direct
comparison of protein signatures with structural data, incorpo-
rating >14 000 structures from PDB with their classification in
SCOP and CATH, as well as >1 650 000 homology models from
SWISS-MODEL and MODBASE. Each protein signature has an
abstract, references, GO mapping, taxonomy, and links to rele-
vant external databases. With its multi-layered annotation, Inter-
Pro is a valuable resource for biologists and bioinformaticians
alike.
P1–58
Revealing of potential FoxA target genes,
involved in proliferation control
N. Ershov, T. Merkulova, L. Bryzgalov, M. Pakharukova,
D. Oschepkov and V. Kaledin
Institute of Cytology and Genetics SB RAS, Laboratory of gene
expression control, Novosibirsk, RUSSIA
FoxA transcription factors are known to be critical to liver cell dif-
ferentiation. We demonstrated a strong association between he-
patocarcinogenic activity of a number of compounds and their
ability to reduce FoxA DNA-binding activity in the liver. Thus rat-
specific hepatocarcinogen 3¢-methyl-4-dimethylaminoazobenzene
significantly reduced FoxA DNA-binding activity only in rats,

while mouse-specific ortho-aminoazotoluene – only in mice. Diet-
hylnitrosamine, which is highly carcinogenic for the liver of both
rats and mice inhibited FoxA activity in both species, while non-
carcinogenic 4¢-methyl-4-dimethylaminoazobenzene was ineffective
both in rats and mice. With the aim to elucidate the mechanism of
potential FoxA tumorsuppressor action we searched for their tar-
get genes related to proliferation control. First we examined the
published data on global gene profiling in mouse liver (a high level
of FoxA) and kidneys (FoxA are absent). Then the search for
FoxA binding sites was carried out by computer-assisted SIT-
ECON method in regulatory regions of 40 differentially expressed
genes involved in the proliferation control. 11 genes containing
putative sites organized as TTTG repeats (3 to six-fold) were dis-
covered. FoxA binding to these sites was confirmed by EMSA
using GST-fusion protein comprising the FoxA 2 DNA-binding
domain. The effect of OAT administration (leading to reduction of
FoxA activity) on the expression of six genes containing confirmed
FoxA binding sites was studied by real-time PCR. We found that
the mRNA levels for Cul2 and CDC73 genes increased dramati-
cally after OAT administration.
Acknowledgement: The work was supported by a grant from
the RFBR (07-04-00441).
Abstracts Poster Presentations
112 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
P1–59
Characterization of a putative carbohydrate
catabolic pathway of the megaplasmid pAO1
of Arthrobacter nicotinovorans
M. Mihasan
1

, V. Artenie
1
and R. Brandsch
2
1
University Alexandru Ioan Cuza, Faculty of Biology, Biochemistry
and Molecular Biology Department, Iasi, ROMANIA,
2
Institute
for Biochemistry and Molecular Biology, Center for Biochemistry
and Molecular Cell Research Albert Ludwigs University, Freiburg,
GERMANY
The gram positive soil bacterium Arthrobacter nicotinovorans car-
ries the pAO1 catabolic megaplasmid which enables it to grow
on nicotine. Besides the well-characterized pathway for nicotine
degradation, pAO1 carries a gene cluster of a hypothetical path-
way for carbohydrate utilization. The cluster consists of ORFs of
a transcriptional regulator, of a sugar ABC-transporter, and of
several putative dehydrogenases and oxidoreductases. A. nicoti-
novorans lacking a functional pAO1 plasmid was, as shown here,
unable to degrade melibiose and inuline out of 21 carbohydrates
tested. We previously established that the pAO1 orf39 gene
encodes an aldehyde-dehydrogenase. Here we focused on the
orf40 gene, encoding a putative oxido-reductase, in an attempt to
further characterize this sugar catabolic pathway. The gene was
cloned, expressed and the 45 kDa His-tagged ORF40 protein
purified. The native molecular mass of 163 kDa determined by
GPC indicated that the protein was a tetramer in solution. Metal
content analysis showed that the enzyme binds 2Zn
2+

atoms/pro-
tein monomer. The enzyme contained the amino acid sequence
A
119
GKHIFTEKP
128
similar to the consensus sequence of sugar
dehydrogenases. A tridimensional model of the enzyme was gen-
erated by the EasyPred3D and used for in silico docking experi-
ments using Chimera/Antechamber/Dock6/ZINC suite. 27 out of
29 tested sugars were found to bind to the model forming two
binding clusters. One of the clusters, consisting of 23 sugars was
located close to the E
128
KP
130
motif and would indicate the puta-
tive catalytic site. The binding energy values suggested the fol-
lowing order of substrate preference: D-tagatose, D-psicose,
L-sorbose, D-xylose. In conclusion, the ORF40 protein belongs
to sugar dehydrogenases. It might be the starting point of the
catabolic pathway by producing sugar aldehydes which may be
oxidized in a following step to acids by the aldehyde dehydroge-
nase of ORF39.
P1–60
Recognition of non-canonical DNA structures
in genomic DNA sequences by p53 proteins
L. Navratilova
1
, M. Brazdova

1
, V. Tichy
1
, M. Fojta
1
, M. Lexa
2
,
I. Kyjovsky
1
, W. Deppert
3
and E. Palecek
1
1
Department of Biophysical Chemistry and Molecular Oncology,
Institute of Biophysics AS CR v.v.i., Brno, CZECH REPUBLIC,
2
Faculty of Informatics, Masaryk University, Brno, CZECH
REPUBLIC,
3
Heinrich-Pette-Institute, Tumor Virology, Hamburg,
GERMANY
The human genome contains a chromosomal DNA that exists
primarily as a right-handed double helix (B-DNA, canonical
DNA), but DNA can also contain the formation of unusual
DNA structures, such as cruciforms, triplexes, quadruplexes,
unwound DNA, hairpins and others. These non-canonical DNAs
(non-B DNAs) are associated with regulatory sequences of genes,
recognition sites of protein and occurre in different cellular pro-

cesses. Tumor suppressor protein p53 (wtp53) and its mutant
alternatives (mutp53 proteins) are able to recognize several spe-
cific DNA motifs (p53 consensus sequence for wtp53) and struc-
tural DNA motifs (DNA hairpins) as well as supercoiled DNA
(scDNA). In our work we gained a library of genomic fragments
naturally bound by mutant p53 proteins and a library of plas-
mids containing repeat DNA for non-B DNA structures forma-
tion (e.g., triplex, DNA cruciform). Genomic DNA bound by
mutp53 was isolated by specific chromatin imunoprecipitation
(genome-wide ChIP-cloning). We combined molecular (detection
by enzymatic and chemical probing) and computational
approaches to identify non-canonical DNA structures (cruciforms
and triplex DNA) as binding sites for p53 proteins. Electropho-
retic mobility shift assay (EMSA) was used for confirmation of
the specific binding of p53 proteins to DNA sequences. Our data
suggest that binding of p53 proteins to non-B DNA sequences is
thought to be the important molecular mechanism in human can-
cerogenesis.
Acknowledgements: This work was supported by EC (FP6-
mutp53, QLGA-CT-2001-52001 and MERG-6-CT-2005-014875),
MEYSCR (1K04119 and LC06035), GA ASCR (IAA500040701,
204/06/P369 and 204/08/1560).
Further reading:
M., Quante, T., Togel, L., Walter, K., Loscher, C., Tichy, V.,
Cincarova, L., Deppert, W., Tolstonog, G.: Modulation of gene
expression in U251 glioblastoma cells by binding of mutant p53
R273H to intronic and intergenic sequences, Nucleic Acids Res.
2009; 37: 1486–1500.
P1–61
Development of Denizli x White Leghorn F

2
population for quantitative trait loci mapping
M. Nizamlioglu
1
, M. Garip
2
, A. Yilmaz
2
, T. Caglayan
2
,
E. Kurar
3
, Z. Bulut
1
, V. Kurtoglu
4
, S. Dere
2
, Y. Ozsensoy
3
and
M. Dogan
1
1
Faculty of Veterinary Medicine, Department of Biochemistry,
Selcuk University, Konya, TURKEY,
2
Faculty of Veterinary
Medicine, Department of Zootechnics, Selcuk University, Konya,

TURKEY,
3
Faculty of Veterinary Medicine, Department of
Genetics, Selcuk University, Konya, TURKEY,
4
Faculty of
Veterinary Medicine, Department of Animal Nutrition, Selcuk
University, Konya, TURKEY
Development of gene maps for human and animals is critical for
identification of genomic regions and gene(s) that have an effect
on human and animal diseases and animal production traits.
Gene level dissection of diseases and these traits allows diagnosis,
protection of diseases and development of novel medical treat-
ments. The genetic level knowledge also can be utilized in animal
breeding programs to improve animal health, production effi-
ciency and product quality. Poultry production is an important
sector in agriculture for obtaining economical animal originated
foods. Chicken is also accepted and extensively used as an excel-
lent model organism in genetic studies. In this study, five Denizli
males and eleven White Leghorn hens were mated to generate F
1
population. Fourteen F
1
males and 58 F
1
females were crossed to
generate five full-sib F
2
families including 441 birds. Body
weights at hatching, 3, 6, 9, 12, 16, 24, 28 and 32 weeks of age,

egg yield and egg weight phenotypic data were recorded. Blood
and tissue samples were collected from all F
0
,F
1
and F
2
animals
and DNA samples were isolated for mapping QTL analysis. Den-
izli X White Leghorn F
2
population is available scientific com-
munity for gene and QTL mapping studies.
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 113
P1–62
Posterior polymorphous corneal dystrophy –
copy number, gene expression and candidate
gene analyses within the PPCD1 candidate
region on chromosome 20p11.2
L. Noskova
1
, P. Liskova
2
, V. Stranecky
1
, H. Hartmannova
1
,
R. Ivanek

3
, K. Jirsova
4
, S. Merjava
4
, M. Filipec
5
and S. Kmoch
1
1
First Faculty of Medicine, Institute of Inherited Metabolic Disor-
ders, Charles University, Prague 2, CZECH REPUBLIC,
2
Labo-
ratory of Biology and Patology of the Eye and Department of
Oftalmology, General Teaching Hospital and Charles University,
Prague 2, CZECH REPUBLIC,
3
Academy of Sciences, Institute
of Molecular Genetics, Prague, CZECH REPUBLIC,
4
Laboratory
of Biology and Patology of the Eye and Ocular Tissue Bank, Gen-
eral Teaching Hospital and Charles University, Prague 2, CZECH
REPUBLIC,
5
Department of Oftalmology, General Teaching
Hospital and Charles University, Prague 2, CZECH REPUBLIC
Posterior polymorphous corneal dystrophy (PPCD) is a geneti-
cally heterogeneous autosomal dominant disorder characterised

by epithelisation of the endohelium and irregular thickening of
Descemet’s membrane. It often leads to irreversible corneal
edema and requires corneal transplantation. The genetic hetero-
geneity of PPCD is currently known to be represented by three
loci on chromosomes 20, 1, and 10 and several disease causing
genes (COL8A2, ZEB1/TCF8) have been identified. We have pre-
viously shown linkage in two Czech PPCD1 families to chromo-
some 20p11.2. To further narrow the PPCD1 candidate interval
we used Affymetrix Genome-Wide Human SNP Array 6.0 and
genotyped family members demonstrating critical recombination
events by STR analysis. Haplotype analysis narrowed the critical
region to a 2.1 Mb interval delimited by markers D20S114 and
rs7509232. In parallel we used the genotyping data, assesed copy
number status and found no indication of microdeletions within
the candidate region. We have also manufactured custom oligo-
nucleotide array and analysed expression changes of all genes
located within the candidate region in samples of corneal tissues
obtained from patients undergoing corneal transplantation and
controls. This analysis showed significantly reduced amounts of
destrin (DSTN) transcript in corneal tissues of four patients com-
pared to healthy control tissue. However, subsequent sequencing
analysis of the coding and promotor regions of DSTN did not
reveal any potential disease causing mutation. As no mutations
were found by sequencing of genomic coding regions of other
genes in candidate region, we decided to sequence whole 2.1 Mb
candidate region by combination of NimbleGen Sequence Cap-
ture array and next-gen sequencing approach.
P1–63
Sorption of phosphopeptides to metal chelate
complexes immobilized to magnetic particles

and to metal oxides
L. Novotna
1
, T. Emmerova
1,2
, J. Vavrova
1
, M. Ticha
1,2
and
Z. Kucerova
1
1
1st Faculty of Medicine Charles University in Prague, Institute
of Pathophysiology and CEH, Praha 2, CZECH REPUBLIC,
2
Faculty of Science Charles University in Prague, Department of
Biochemistry, Praha 2, CZECH REPUBLIC
Protein phosphorylation is one of the most important posttrans-
lational modifications of proteins. The system of protein phos-
phorylation-dephosphorylation belongs to the major cellular
signaling mechanisms. Moreover, anomalous phosphorylation of
proteins can be also related to development of cancer or to meta-
bolic diseases. Processes involving phosphoproteins are dynamic
and this fact affects their low abundance and relatively short
half-life. To understand these processes, structural studies on
involved phosphoproteins is desired. Despite recent advances in
instruments and methods used for the characterization of phos-
phoproteins, the effective separation or enrichment of phospho-
peptides and phosphoproteins remains to be fully solved. The

subject of the present study was to compare the efficiency of two
procedures used for the separation or enrichment of phosphopep-
tides from the tryptic digest of phosphoprotein: (i) sorption of
phosphopeptides to different metal ions chelates immobilized to
magnetic particles or (ii) sorption to titanium dioxide or zirco-
nium oxide. Bovine a-casein was chosen as a model phosphopro-
tein. The adsorbed phosphopeptides from the tryptic a-casein
digest were analyzed by matrix-assisted laser desorption/ioniza-
tion time-of-flight/time-of-flight mass spectrometry (MALDI-
TOF/TOF MS). Conditions for the phosphopeptide adsorption
and following desorption were optimized.
Acknowledgements: This work was supported by the Ministry
of Education, Youth and Sports of the Czech Republic (grant
MSM 0021620806 and project CEH LC 06044) and by the Czech
Science Foundation (grant 203/09/0857).
P1–64
Functional analyses of Lupinus luteus
cyclophiline promoter activities
K. Nuc
1
, P. Nuc
2
and R. Slomski
1
1
Poznan University of Life Sciences, Biochemistry and Biotechnol-
ogy, Poznan, POLAND,
2
Adam Mickiewicz University, Institute
of Molecular Biology and Biotechnology, Poznan, POLAND

Cyclophilins (CyPs) constitute a large class of ubiquitous immu-
nophilins with peptidyl-prolyl cis-trans isomerase activity. It has
been shown that isomerization around Xaa-Pro bonds is one of
the most rate limiting steps in protein folding and this process
can be accelerated by the PPIase activity of the CyPs. We have
analyzed a promoter region of the cytosolic cyclophilin from
Lupinus luteus (EMBL/GenBank/DDBJ AF178458, LCyp). The
expression of CyP genes depends on cis elements in their pro-
moter regions which promote interaction of appropriate tran-
scription factors in response to different stimuli. To investigate
the regulation of its expression, deletion promoter fragments were
fused to the uidAint (GUS) reporter gene and the constructs were
introduced into Lotus japonicus via Agrobacterium rhizogenes
transformation. We have observed different patterns of the b-glu-
curonidase expression in L. japonicus roots and nodules. Only
the largest 1054 bp promoter fragment causes expression of the
b-glucuronidase in nodules suggesting that the nodule specific cis
regulating element is located in the region spanning nucleotides -
1054 to -845 indicating to 5¢AAAGAT 3¢motif. We have also
analyzed the influence of different abiotic treatments on the b-
glucuronidase expression level in L. luteus leaves after agroinfil-
tration with Agrobacterium tumefaciens carrying analyzed pro-
moter fragments. Fluorometric study of these plants revealed
that LCyp promoter is responsive to plant hormones such as
ABA, IAA and BAP as well as to salt, but not to cold stress.
Abstracts Poster Presentations
114 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
P1–65
Quantitative and temporal proteome analysis
of gamma-Tocotrienol treated androgen-

unresponsive prostate cancer cells delineates a
novel caspase – independent programmed cell
death pathway
T. Roumeliotis
1
, A. Evdokiou
2
, P. Vraka
3
, L. Loizou
4
, S. Garbis
1
and A. D. Odysseos
4
1
Center of Basic Research – Biotechnology Division, Biomedical
Research Foundation of the Academy of Athens, Athens,
GREECE,
2
Department of Orthopaedics and Trauma, University
of Adelaide, Adelaide, Australia
3
Department of Chemistry, Uni-
versity of Cyprus, Nicosia, CYPRUS,
4
EPOS-Iasis, Biomedical
Research, Nicosia, CYPRUS
Proteomic approaches have a considerable potential in the drug
development process providing a meand of obtaining a detailed

molecular signature of drug action, since analysis of gene expres-
sion at the mRNA level alone can not adequately predict protein
expression or functional states. The overall objective of this study
has been to investigate the feasibility of using an LC-MS based
quantitative proteomic approach for the evaluation of c-Tocotrie-
nol (c-T), a potent medicinal agent based on the time dependent
differential expression of proteins involved in target pathways.
Combining temporal and quantitative proteomics with molecular
imaging, biochemical, enzymatic and immunoprecipitation
approaches, we have revealed a new panel of proteins under c-T
regulation interwoven in a novel caspase-independent programmed
cell death pathway. Significantly modulated protein targets cross-
sectioning redox and programmed cell death pathways through (i)
heat shock proteins in the Nrf-2-mediated transcriptional control
axis and (ii) the G2/M transition check point of the cell cey le pro-
tein networl, converge to the MAPK cascade and activate a novel
PARP-modulated caspase independent programmed cell death sig-
naling pathway. Increased levels of calcium-dependent proteases
(caplains) in treated cells are characteristically associated with
upregulation of total PARP and apoptotic chromatin condensa-
tion inducer-1. Probing against total adn cleaved PARP revealed a
temporal pattern of cleavage and activation while nuclear staining
disclosed chromatin condensation confirming early apoptosis.
Reactive nitrogen species, inherent to the androgen-independent
phenotype of DU-145 cells and additional components of the mito-
chondrial axis drive peroxynitrite mediated calcium-dependent
mitochondrial dysfunction and elicit features of early apoptosis
with enhancement of caplain activation, whereas the anchorage
dependence of these cells is also highly suggestive of anoikis-resem-
bling cell death. Significant elevation of caspase-degradable La-

mins A nd B2 further reflects the low degree of implication of the
caspase cascade while marked upregulation of active components
of the mitochondrial pathway such as VDAC I–III accenuates
mitochondrial membrane permeability. Conclusively, this
approach delineates a novel caspase independent programmed cell
death signaling pathway elicited aby a natural, minimally toxic
agent and defines reliable biomarkers of efficacy.
P1–66
Electrophoretic analysis of esterase and
superoxide dismutase enzymes of the wheat
bug, eurygaster maura
(heteroptera:scutellaridae) populations in the
Central Anatolia
S. Tuna, G. Olgun and R. Colak
Faculty of Science, Ankara University, Biology, Ankara, TURKEY
In this study, a total of 194 Eurygaster maura specimens were
used from 10 locations in central Anatolia. Esterase and superox-
ide dismutase enzymes were studied using starch gel electrophore-
sis and native-polyacrilamide gel electrophoresis (N-PAGE). A
total of 16 esterase activity bands were observed. Only Est-1
locus distinguished among esterases to be thought encoding by a
lot of loci. In the bug populations studied, three allele (A, B, C)
were recognized in this locus. In the starch gels examined of
superoxide dismutase enzymes were determined one cathodal
locus. All specimens studied were homozygous for the same allele
in this locus. It was determined genetic identity and genetic dis-
tance of bug populations by BIOSYS-1 computer program,
according to allele frequencies of two loci (EST-1 and SOD)
observable. On the basis of genetic relationship of ten Eurygaster
maura populations based on Nei’s Genetic Distance of two

enzyme loci, ten populations were divided into two main clusters
and two subgroups in each cluster; 1: Kirikkale (subgroup1),
Beynam, Haymana, Afyon, Konya and Yunak (subgroup2), 2:
Aksaray (subgroup1) and Ayas, Kirsehir and Beypazari (sub-
group2).
P1–67
Associations between serum linolenic acid/
alpha-linolenic acid ratio and blood gene
expression profiles
K. S. Olsen
1
, C. G. Fenton
2
, E. Anderssen
3
, L. Froyland
4
,
R. H. Paulssen
2
and E. Lund
1
1
Institute of Community Medicine, University of Tromso, Tromso,
NORWAY,
2
Microarray Resource Centre, University of Tromso,
Tromso, NORWAY,
3
Department of Cancer Research and Molec-

ular Medicine, NTNU, Trondheim, NORWAY,
4
National Institute
of Nutrition and Seafood Research, Bergen, NORWAY
Nutrition is one of the major modifiable risk factors for athero-
sclerosis and certain cancers. Linoleic acid (LA, 18:2n-6) and
alpha-linoleic acid (ALA, 18:3n-3) are essential to humans, and
modulate immune response by giving rise to pro- (LA) or anti-
(ALA) inflammatory signaling molecules. Despite the well-known
metabolism of fatty acids, the molecular and physiolocigal mech-
anisms of these dietary risk-modifying factors need further atten-
tion. In the context of a large, representative cohort of
postmenopausal Norwegian women, we have explored the associ-
ation between serum LA/ALA ratio and blood gene expression
profiles. Samples from 286 women were successfully analyzed for
expression profiles in PAXgene-stored whole blood using
ABI1700 microarrays, and for fatty acid content in citrate-buf-
fered plasma using rapid gas chromatography. After data prepro-
cessing including adjustment for technical variability,
differentially expressed genes were identified using R software
and Bioconductor packages. In women with a high LA/ALA
ratio, we revealed a pro-inflammatory expression profile that
includes genes related to Fc epsilon signaling, phosphatidyl inosi-
tol signaling, cytokine production, arachidonic acid metabolism
and generation of eicosanoids. Strikingly, we identified the
increased expression of the key pro-inflammatory enzyme 5-lipox-
ygenase (5-LO) in women with a high LA/ALA ratio. These pre-
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 115
liminary results lead us to conclude that dietary fatty acids do

have an effect on the blood trancriptome, and that an unfavour-
able LA/ALA ratio could be related to a pro-inflammatory gene
expression profile. Further analyses will be conducted to reveal
the complex effects of the lipidome on the blood transcriptome.
P1–68
Proteomic approach to elucidate antimicrobial
effect of Papaver polychaetum alkaloids
K. Dilek
1
, S. A. Berna
1
and U. Caglayan
2
1
Department of Bioengineering, Marmara University, Istanbul,
TURKEY,
2
Department of Pharmacognosy, Istanbul University,
Istanbul, TURKEY
Although bacteria have evolved numerous mechanisms to fight
against antimicrobial agents, drug-resistant pathogens are on the
rise. In the past few decades, this has led many research groups
to medicinal plants for a search of novel antimicrobial agents.
Long before mankind discovered the existence of microbes, the
idea that certain plants had healing potential was well accepted
and plants were used to treat common infectious diseases. The
endemic plant Papaver polychaetum, from the genus of poppies,
yields berberine as its major alkaloid. Berberine is a plant alka-
loid with a long history of medicinal use in both Chinese and Ay-
urvedic medicine. It has demonstrated significant antimicrobial

activity against different organisms including fungi and is rela-
tively nontoxic to humans. However the antimicrobial mode of
action of berberine is not clear. The aim of this work was to
investigate the antimicrobial property of the alkaloid extract of
the P. polychaetum against E. coli K12. For this purpose, proteo-
mic approach was used which involved the selection of up and
down regulated proteins on 2-dimensional polyacrylamide gels
and their subsequent identification by MALDI-tof analysis. The
common feature of the identified proteins was that they were
DNA-binding. This approach enabled the elucidation of regula-
tion of protein expression levels in the model microorganism E.
coli K12 in the presence of the antimicrobial agent. The search
for novel substances involves understanding of the molecular
mechanisms of action of the drug/drug candidates and the related
bacterial response. The results of this study will help to identify
targets for future pro-drug design.
Further reading:
Musumeci R, Speciale A, Costanzo R, Annino A, Ragusa S,
Rapisarda A, Pappalardo MS & Iauk L. Berberis aetnensis C.
Presl. extracts: antimicrobial properties and interaction with
ciprofloxacin. Int. J. Antimicrob. Agents 2003; 22: 48–53.
Rios JL & Recio MC. Medicinal plants and antimicrobial activ-
ity. J. Ethnopharmacol 2005; 100: 80–84.
Stermitz FR, Lorenz P, Tawara JN, Zenewicz LA & Lewis K.
Synergy in a medicinal plant: antimicrobial action of berberine
potentiated by 5¢-methoxyhydnocarpin, a multidrug pump
inhibitor. PNAS 2000; 97(4): 1433–1437.
P1–69
Computational analysis of 5¢ and 3¢
untranslated regions of breast cancer genes

BRCA1 and BRCA2
P. Ozretic, M. L. Cvok, V. Musani, M. Cretnik and S. Levanat
Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb,
CROATIA
Untranslated regions (UTRs) are parts of the mature mRNA
located before the start codon (5¢ UTR) and after the stop codon
(3¢ UTR). They are transcribed with the coding region but they
are not translated. Several regulatory roles have been assigned to
the untranslated regions, including mRNA’s localization and sta-
bility, and translational efficiency. These functions depend on the
sequence and the structure of the UTR. BRCA1 and BRCA2 are
the major hereditary breast/ovarian cancer predisposing genes
and their mutations increase the risk of developing cancer. Inter-
pretation of sequence variants found in genetic testing is the
major concern for BRCA genes, especially for risk assessment in
genetic counseling. From different repositories (dbSNP, BIC,
kConFab), we collected BRCA1 and BRCA2 sequence variants
that were found within 5¢ and 3¢ UTRs and we analyzed, using
different on-line tools like UTResource and Transterm, their
potential functional significance that could be expressed by dis-
rupting or creating putative regulatory elements. As it is known
that the function of non-coding RNAs (ncRNAs) greatly depends
on their secondary structure, we analyzed how these non-coding
sequence variants could have impact on the predicted secondary
structure. By analyzing changes in the predicted secondary struc-
tures of the 5¢ and 3¢ UTRs from the patients with breast/ovarian
cancer we tried to find out if this approach could be used for
assessing clinical significance in cancer etiology.
P1–70
Phylogenetic and bioinformatic analysis of

Glutathione S-Transferase Tau from Pinus
brutia
E. Oztetik
1
, M. Aydin
2
and F. Kockar
2
1
Anadolu University, Biology, Eskisehir, TURKEY,
2
Balikesir
University, Biology, Balikesir, TURKEY
Glutathione S-Transferases (GSTs, EC.2.5.1.18) are widespread
and complex enzyme superfamily that play important roles in
detoxification and stress tolerance in plants. Plant GSTs are
divided into four classes (Phi, Tau, Zeta and Theta), among
which Tau is the most numerous one. It has been shown that
plant Tau class GSTs could play essential roles in plant develop-
ment and in buffering environmental and biotic stresses. Even,
there are many studies on GSTs in plants, which have been
focused generally on agricultural plants, there is no such an
information considering the molecular characterization of GSTs
in gymnosperms. The definition of detoxification enzymes like
GSTs in perennial conifers, a large group of gymnosperms, is
very important for their adaptations to several environmental
stresses during their long lifespans. The present study reports the
phylogenetic and bioinformatic analysis of GST gene (PbGST-
Tau) from a conifer, Pinus brutia, which is widely distributed in
the north-eastern Mediterranean area, including Turkey. The

PbGST-Tau gene encodes a protein of 228 amino acid residues
with a calculated molecular mass of 26.37 kDa. The sequence
comparisons of PbGST-Tau gene to other plant GST-Tau genes
(Pinus densata, Pinus tabulaeformis, Pinus yunnanennis, Oryza sa-
tiva japonica, Aegilops tauschii and Ginko biloba) were performed
at the amino acid level by using Bioedit programme. The phylo-
genetic tree was also constructed by using Neighbor-Joining/UP-
GMA method. The analyses indicated that PbGST-Tau is placed
with other three GSTs in Tau class from Pinus tabulaeformis, Pi-
nus densata and Pinus yunnanensis, with 10.82%, 11.45% and
11.18% sequence diversity, respectively. The current difficulties
for the comprehensive characterization of GST family in conifers
originates from the insufficiency of comprehensive genome infor-
mation on conifers in the literature. As the described members of
the GST gene family in conifers are increased, the understanding
for diversity and evolution of the GST classes would also be
advanced.
Abstracts Poster Presentations
116 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
P1–71
Human Accelerated Region 20 (HAR20)
operates as an enhancer: functional study of a
HAR20 SNP associated with diabetes and
cardiovascular disease
S. Pampı
´
n
1
, L. Parnell
2

, J. M. Ordovas
2
and
J. C. Rodriguez-Rey
1
1
Universidad de Cantabria, Biologı
´
a Molecular, Santander,
SPAIN,
2
Tufts University, Jean Mayer USDA HNRCA, Boston
MA, USA
Comparative genomics has made possible the discovery of human
sequences which have evolved very rapidly since the recent
human/non-human primate divergence. These sequences have
been termed Human Accelerated Regions (HARs). Except for
HAR1, which encodes a small RNA, the function of HARs is
unknown. The fact that many map in non-coding sequences sug-
gests that sequence differences between humans and other pri-
mates drive altered the regulation of genes involved in the
determination of human-specific characteristics. HAR20 is a
263 pb sequence located within the second intron of PPARCG1A
(PCG1a). Changes in PCG1a expression have been associated
with diabetes, and more recently a SNP (rs10028665 C>T)
located within HAR20 has been associated with diabetes and car-
diovascular disease. These results prompted us to investigate (i) if
HAR20 is a regulatory region and (ii) if the change due to
rs10028665 results in a functional change in its properties. To test
if HAR20 has enhancer-like characteristics, we carried out repor-

ter gene assays in HepG2 cells by cloning both variants of
HAR20 into the pGL3-promoter vector. In our assays, HAR20
highly increases the promoter strength, suggesting that it has
expression-regulatory properties. Moreover, C allele produces an
increase of 40% in the enhancer activity compared to T allele.
Subsequent EMSA analysis has shown binding differences
between both variants, indicating that this SNP could alter the
PCG1a expression. We conclude that HAR20 is a regulator of
gene expression and that rs10028665 is at least responsible for
part of the variability of PCG1a expression associated with dia-
betes and cardiovascular disease.
P1–72
Study of cellular prion protein (PrPc) role in
erythroid differentiation in vitro
M. Panigaj
1
, H. Glierova
1
, A. Simkova
2
and K. Holada
1
1
1st Faculty of Medicine, Institute of Immunology and Microbiol-
ogy, Prague 2, CZECH REPUBLIC,
2
Faculty of Science, Section
of Biology, Prague 2, CZECH REPUBLIC
Despite its significant medical importance, involvement of PrPc
in cell biology is still subject of discussion. Connection between

PrPc/ prion pathogenesis and erythropoesis was already shown.
Our pilot study of erythroid differentiation in murine erythroleu-
kemia cells (MEL), indicates regulation of PrPc expression as
shown by qRT-PCR and FACS analysis. Creation of stable cell
line expressing shRNAmir (LP1, LP2) which downregulates PrPc
(3 90% inhibition) shows similar level of total hemoglobine con-
tent and normal expression pattern of AHSP and hemoglobine-a
in all lines during erythroid differentiation. Interestingly we
found higher expression of c-myb in LP1 and LP2 cells and its
more profound downregulation after inducing to differentiation.
Previous reports showed that neither physiological nor enhanced
expression altered percentage of apoptotic MEL cells during dif-
ferentiation. However, even opposite approach- downregulation
of PrPc probably do not lead to sensitization of cells to apopto-
sis. It is tempting to speculate that if under normal conditions all
lines shows equal differentiation, then they could react differen-
tially after exposure to external stress. We induced stress condi-
tions by 3 various treatments- elevated incubation temperature
(40°C), oxidative stress by presence of H
2
O
2
(500 lM) and Stau-
rosporin (125 nM). But even such conditions do not altered level
of apoptosis/ necrosis in lines with inhibited PrPc. Finally, we
introduce the new cell culture model for study of PrPc function
in erythroid differentiation. Initial observations shows that direct
cytoprotection is not mode of PrPc action in studied process, but
it may play role in cell cycle which is subject of undergoing
research.

Acknowledgements: This work was supported by GAUK
203429, Grants of Czech Science Fundation 310/08/0878.
P1–73
Comparative exosomal and cellular genomic
profiling of human colorectal cancer cells
K. S. Park
1
, J. H. Cho
2
, J. H. Kim
1
, D. Hwang
2
and Y. S. Gho
1
1
Department of Life Science, POSTECH, Pohang,
SOUTH-KOREA,
2
School of Interdisciplinary Bioscience and
Bioengineering, POSTECH, Pohang, SOUTH-KOREA
Various cancer cells, including colorectal cancer (CRC) cells,
release exosomes into the surrounding tissues and peripheral cir-
culation. As exosomes are thought to be involved in tumor pro-
gression, we hypothesized that CRC cells may secrete exosomes
carrying mRNA that causes epigenetic reprogramming of tumor
microenvironmental cells. Here, we present a global comparative
exosomal and cellular transcriptomic analysis of human SW480
CRC cells. We identified 11 327 exosomal and 11 624 cellular
mRNAs, and 241 and 1461 of these were overexpressed in exo-

somes and in cells, respectively. The exosomal and cellular
mRNAs were similarly enriched in tumorigenesis-related biologi-
cal processes. Furthermore, exosome-enriched mRNAs were
involved in the cell cycle, whereas cell-enriched mRNAs were
involved in angiogenesis. Our study demonstrates that the exoso-
mal and cellular mRNAs of human CRC cells are similar to each
other at the global level, in terms of intensities and biological
processes, and that exosomal mRNA may be involved in tumor
progression via the horizontal transfer of mRNA to target cells.
This information will help elucidate the pathological functions of
tumor-derived exosomes and will aid in the development of meth-
ods using exosomal mRNAs to determine the diagnosis and
prognosis of CRC.
P1–74
Proteomic approach for detection of
biomarkers in rheumatoid arthritis
M. Park, S. Chung, T. Kim and J. Lee
Department of Internal Medicine, Yonsei University College of
Medicine, Seoul, SOUTH-KOREA
Background: Rheumatoid arthritis is an autoimmune disease
characterized by the sequestration of various leukocyte subpopu-
lations within both the developing pannus and synovial space.
There is a strong demand to identify the specific antigens or pep-
tides of rheumatoid synovitis for application as diagnostic bio-
markers for diagnosis of RA in early stages and as potential
therapeutic targets. Proteomics can be defined as the qualitative
and quantitative comparison of proteomes under different condi-
tions to further unravel biological processes.
Objective: In this study, we aimed to identify and analyze the
potential peptides expressed in the rheumatoid synovitis using

proteomics-based approach.
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 117
Methods: We enrolled synovial fluids of 10 patients with rheu-
matoid arthritis and we also enrolled those of 10 patients with
osteoarthritis as controls. The synovial fluids were fractionated
into peptide and protein fractions; the former was subjected to
one-dimensional high-performance liquid chromatography
(HPLC) and the latter fractions were analysed and identified by
ion-trap mass spectrometry. Then, the latter was subjected to 2D
electrophoresis and differentially displayed protein spots were
identified by mass spectrometry.
Results: Several protein spots were significantly up-regulated in
synovial fluids from patients with rheumatoid arthritis, compared
to those from patients with osteoarthritis. The up-regulated spots
were identified as leucine-rich alpha2-glycoprotein, serum amy-
loid A (SAA) protein precursor, fibrinogen Fragment D, and
fibronectin.
Conclusion: These data indicated that application of proteomics
platform could facilitate analyzing and identifying protein bio-
markers differentially existed in diseases.
P1–75
Proteomic analysis to identify molecular
targets of low temperature sensing
S. Park and M. Jang
Department of Applied chemistry, Dongduk Women’s University,
Seoul, KOREA
Transient receptor potential (TRP) channels are important for
our sensory systems, responding to touch, taste, osmolarity, pain,
temperature and other stimuli. The somatosensory system detects

changes in ambient temperature using these channels. Intracellu-
lar signaling of cold sensing in mammals is not completely under-
stood, although significant progress has been made recently with
the cloning of two cold-activated ion channels, TRPM8 and
TRPA1. To understand the molecular basis of cold receptor-
induced intracellular signaling, it is important to identify the
expression or function of proteins which are modified by low
temperature sensing. Little is known about the protein products
produced by cells exposed to low temperature sensing since most
studies have been performed at the electrophysiological effect of
low temperature sensing. In the present study, we analyzed
molecular modifications induced by low temperature sensing in
TRPM8 expressed cells using a proteomic approach. This meth-
odology provides important qualitative information on post-
translational modifications to each protein and quantitative data
on protein expressions in response to a particular stimulus. This
information is particularly important because it provides data on
early cellular events, such as, on the stimulus and signaling cas-
cades triggered independently of protein neosynthesis. We identi-
fied proteins that sensitively reacted to low temperature sensing
by using two-dimensional gel electrophoresis and matrix-assisted
laser desorption ionization time-of-flight-MS.
P1–76
Mitochondrial DNA content and expression of
genes involved in maintenance and replication
of mtDNA during human fetal development
M. Pejznochova, M. Magner, M. Tesarova, H. Hansikova and
J. Zeman
1st Faculty of Medicine, pediatric, Prague 2, CZECH
REPUBLIC

The inadequate efficiency of mitochondrial biogenesis leads to
low energy production which may play a crucial role both in the
fetal development and neonatal morbidity. The aim of our study
was to characterize the changes in expression of genes involved
in the regulation and maintenance of mtDNA content (PGC1,
NFR1, TFAM, POLG). DNA and RNA were isolated from 26
pairs of liver and muscle tissue samples obtained at autopsy from
miscarriages after informed consent, between 13th and 28th week
of gestation. The mtDNA amount and gene expression levels
were analyzed by the real-time PCR method using SybrGreen I.
In both fetal liver and muscle tissues, mtDNA content and
TFAM expression levels were increasing with onward fetal devel-
opment (mtDNA: r = 0.50; P < 0.01; respectively r = 0.62;
P < 0.01); (TFAM: r = 0.56; P < 0.01; respectively r = 0.61;
P < 0.01). On the other hand, POLG expression level was
increasing only in fetal liver tissue (r = 0.54; P < 0.01). NRF1
was unchanged in both fetal tissues and PGC1 was slightly rising
between 13th and 28th week of gestation in liver tissue
(r = 0.42; P < 0.05). Our results showed that POLG expression
varies between two different tissues during gestation and proba-
bly is not associated with mtDNA content as tightly as TFAM
expression. The increase of PGC1 transcript level is statistically
significant in liver tissue and we suggest that it could bear evi-
dence of tissue specific expression or regulation. In fetal liver, ris-
ing PGC1 expression positively affects the binding of NRF1 to
its specific sites on TFAM promoter. In conclusion, this study
described mainly the increasing trends of gene expression in the
pathway leading from expression of PGC1 to mtDNA content
changes in fetal liver and muscle tissues during second trimester
of gestation. According to our results, we suppose that the mito-

chondrial proliferation is growing up between 13th and 28th
week of gestation and therewithal it is the first hallmark of pre-
pare for postnatal adaptation on transcriptional level.
Acknowledgement: This work was supported by grant
GAUK25755707, IGAMZ-NR9410,GACR305/08/H037.
P1–77
DNA methylation in LDL receptor and
Apolipoprotein B100 genes promoter in
patients with hypercholesterolemia
M. Piechota
1
, A. Banaszewska
1
, E. Guzniczak
2
, R. Link
2
,
G. Rosinski
1
, T. Siminiak
2
and R. Plewa
1
1
Department of Animal Physiology and Development, Adam Mick-
iewicz University, Poznan, POLAND,
2
Cardiac and Rehabilitation
Hospital Kowanowko, Poznan University of Medical Sciences,

Poznan, POLAND
The DNA methylation is an epigenetic modification present in
Prokaryotic and Eukaryotic genomes. The CpG alterations lead
to selective utilization of gene information and are responsible
for tissue specific gene expression, embryonic development, geno-
mic imprinting and X-chromosome inactivation. Hypermethyla-
tion of promoters is usually associated with silencing of the gene
expression. Disorders of the methylation pattern may contribute
to autoimmune disease development, cancer as well as metabolic
and cardiovascular diseases. Atherosclerosis is a metabolic dis-
ease causes by the accumulation of low-density lipoprotein cho-
lesterol (LDL) in the walls of arteries. The reduce expression of
LDLR or/and ApoB100 can influence on decrease the synthesis
of proteins and the accumulation LDL-cholesterol in the walls
and advance atherosclerosis disease. The purpose of the study
was to evaluate LDLR and ApoB100 expression level and the
methylation status in patients with hypercholesterolemia. The
investigated group consisted of 25 patients with no mutation in
LDLR exons. Expression of LDLR was significantly lower and
average 24–45% then in the healthy individuals. The analysis of
CpG methylation pattern within the LDLR and ApoB100 pro-
moters revealed higher methylation level in 11 patients in com-
parison to the healthy controls. In four patients, the methyl
Abstracts Poster Presentations
118 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies
group was present at seven cytosine (C) residues of LDLR, while
in seven almost all cytosines were methylated of ApoB100. The
elevated level of the CpG methylation in LDLR or ApoB100 pro-
moters in 11 patients with hipercholesterolemia can decrease the
gene expression and could be the cause of the atherosclerosis

symptoms.
P1–78
Characterization of the non-coding MicA RNA
in Escherichia coli: mutational analysis shows
that 3¢ end controls stability of this sRNA
V. Pobre, J. M. Andrade and C. M. Arraiano
Instituto de Tecnologia Quı
´
mica e Biolo
´
gica, Universidade Nova de
Lisboa, Oeiras, PORTUGAL
Small non-coding RNAs (sRNAs) are an expanding class of cell
regulators. Changes in sRNA levels can potentially affect the
genetic pathways assisted by these tiny regulatory molecules.
sRNAs usually act by an antisense mechanism and bind target
mRNAs through their 5¢ end, inhibiting translation and promot-
ing mRNA decay. sRNA stability is mainly believed to rely on
its 5¢ end as the degradation of some sRNAs was shown to be
coupled with the endonucleolytic inactivation of their target
mRNAs. We have previously shown that 3¢–5¢ exonucleolytic
degradation is a major regulatory pathway controlling the levels
of the small non-coding MicA RNA, an important regulator of
outer membrane protein expression. In this study, we have now
characterized the RNA determinants involved in the stability of
MicA and further analysed how this may influence the expression
of its targets. We constructed several plasmids harbouring either
the wild-type or mutated MicAs and transformed delmicA cells.
Remarkably, mutations in the 5¢ region (responsible for the base-
pairing of MicA with its target mRNAs) showed a much less

drastic effect rather than mutations in the 3¢ which resulted in
the pronounced destabilization of this sRNA. Binding of Hfq
RNA chaperone and the presence of two stable stem-loops at the
3¢ end are shown to be important determinants of sRNA stabil-
ity. Moreover, we demonstrate in vivo that the most 3¢ nucleo-
tides display an essential role in the establishment of MicA RNA
levels, highlighting the important role of the 3¢ extremity in
sRNA expression.
P1–79
The first signs of blood coagulation pathway
evolution by bioinformatic studies of
Branchiostoma genome
M. Ponczek
Department of General Biochemistry, Institute of Biochemistry,
Lodz, POLAND
Blood clotting and fibrinolysis proteins of mammals and other
higher vertebrates appear to be a result of gene duplications,
divergence and domain shifting. Extrinsic pathway, important in
physiologic coagulation in human, looks to be simpler in lam-
preys comparing to Gnathostomata. On the other hand, intrinsic
pathway, significant in pathological thrombus formation, evolved
relatively early in mammals as divergence of factor XI after
plasma prekallikrein duplication. The availability of sequenced
Branchiostoma floridae genome enables reconstruction of early
stages of blood coagulation evolution employing accessible bioin-
formatic tools. Branchiostoma seems to have orthologs of pro-
thrombin, but with no GLA or kringle domains. Genes coding
peptides with FRED domains related to fibrinogen chains are
present. Ferroxidase ‘A’ domain, typical for coagulation factors
V, VIII, hephaestin and ceruloplasmin, occurs in four genes, but

all are more related to ceruloplasmin and hephaestin. Factors IX
an X could not be detected what seems to be in agreement with
lack of their cofactors – factors VIII and V.
P1–80
Database of the secondary structures of HIV-1
genome control elements
A. Potyahaylo, I. Kolomiets, M. Zarudnaya and D. Hovorun
Department of Molecular and Quantum Biophysics, Institute of
Molecular Biology and Genetics NAS of Ukraine, Kiev, UKRAINE
Multiple steps in replication cycle of human immunodeficiency
virus type 1 (HIV-1) causing AIDS illness are directed by control
elements located both in untranslated and translated regions of
HIV-1 genome, among them are TAR, PolyA, PBS, DIS, SD,
Psi, RRE and others. A high heterogeneity of HIV-1 genome sig-
nificantly impedes AIDS diagnostics, and on other hand it
enables to use phylogenetic approach to study mechanisms of
virus replication. To assist in analyzing of phylogenetic and sec-
ondary structure data we have created database of Control Ele-
ments Secondary Structures of HIV-1 genome (CESSHIV-1). At
present database contains secondary structures predicted by the
mfold program (Zuker, 2003) of the region encompassing DIS,
SD and Psi hairpins of 5¢-UTR for about 1300 HIV-1 isolates
selected from NCBI (GenBank). The database has been devel-
oped to assist in analyzing of frequencies of different variants of
control elements, influence of mutations on the secondary struc-
ture, dependencies of secondary structures of control elements on
subtype and geographical region, evolution of control elements.
In particular we found tolerant and intolerant combinations of
base changes in DIS, SD and Psi hairpins. We present the
schemes of hypothetical transitions between all frequent variants

of DIS, SD and Psi hairpins via single base change. The authors
believe CESSHIV-1 database to be a useful guide for a wide
range of researchers and physicians engaged in HIV-1 science.
P1–81
Proteomic and biochemical analysis of
14-3-3-binding proteins indicates new roles of
14-3-3 proteins in apoptosis
M. Pozuelo Rubio
Andalusian Center for Molecular Biology and Regenerative
Medicine (CABIMER), Senalizacio
´
n Celular 4, Sevilla, SPAIN
14-3-3 is a ubiquitous protein that in many cases had been
involved in antiapoptotic signals in cells, promoting cell survival
by association with proapoptotic proteins. Here, a proteomic and
biochemical analysis was used to identify apoptotic-related 14-3-
3e-binding proteins aimed to have a comprehensive knowledge of
survival functions of 14-3-3e isoform in cells. Tandem affinity
purification, LC-MS/MS analysis and biochemical validation
were combined to characterize 46 proteins associated with 14-3-
3e which binding status is regulated by C2-ceramide-induced
apoptosis in HeLa cells. Among this pool of proteins, 16 of them
were directly implicated in the apoptotic process comprising pro-
teins related with regulation of intermediate filament integrity
and cell shrinkage during apoptosis as Desmin or Vasodilatador-
stimulated phosphoprotein (VASP), oncogenic or death promot-
ers such as Nucleophosmin (B23) or Receptor interacting protein
3 (RIP3), and regulation of DNA double-strand breaks repair in
early stages of apoptosis as DNA protein kinase (DNA-PKcs).
The functional diversity of theses identified proteins suggests that

14-3-3e regulates the apoptotic process through new mechanisms
in addition to others previously characterized. The majority of
Poster Presentations Abstracts
FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 119