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

Kucera1 1Department of Biochemistry, Faculty of Science, Masaryk Univer-sity, Brno, CZECH REPUBLIC,2Institute of Biostatistics and Analyses, Masaryk University, Brno, CZECH REPUBLIC, 3De

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 Kohoutkova1, P Babula1, R Opatrilova1, O Vrana2,

V Adam3, J Zehnalek3and R Kizek3

1

Department of Natural Drugs, University of Veterinary and

Pharmaceutical Sciences, Brno, CZECH REPUBLIC,2Institute of

Biophysics, Academy of Sciences of the Czech Republic, Brno,

CZECH REPUBLIC,3Department of Chemistry and

Biochemis-try, Mendel University of Agriculture and ForesBiochemis-try, 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 phatemia Lanthanides3+ compounds have also been investi-gated for their anti-cancer potential Clinical reports suggestedthat several compounds such as cerium (III) iodide posed cyto-toxic effect, however the biochemical mechanism of their action

hyperphos-is still unclear (1–3) Therefore, we aim our attention on study ofinteractions DNA with inorganic salts of cerium (III), lanthanum(III) and gadolinium (III) Raman spectroscopy was employed tocharacterize the perturbations to DNA conformation induced inDNA by three inorganic salts of the above mentioned lantha-nides (2) All studied lanthanides coordinated to N7 of twoneighbouring guanine bases, as this nitrogen does not form Hbonds 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 antitumoreffects 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 Adam1, D Huska1, S Krizkova1, J Hubalek2and R Kizek1

1Department of Chemistry and Biochemistry, Mendel University ofAgriculture and Forestry in Brno, Brno, CZECH

REPUBLIC,2Department of Microelectronics, Brno University ofTechnology, Brno, CZECH REPUBLIC

Viral infections pose a threat for man kind Diagnostic systemsfocus on the direct cultural proof of identity Almost all systemsuse 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 andmodified DNA oligonucleotide probes fluorescing when hybridizedwith 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 Electrochemicaldetection is low cost and sensitive alternative method to detectnucleic acids (1, 2) The main aim of this work is to propose novelelectrochemical detector for monitoring PCR Miniaturized poten-tiostat with nanotubes-based screen-printed electrodes was usedfor 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 amplifiedDNA was between 10 and 100 pg The amplicons obtained evenafter two cycles were detectable by using the electrochemicalinstrument Moreover we compared the results with agarose gelelectrophoresis The lowest detectable amount of DNA wasobtained after 15 PCR cycles

Acknowledgement: This work was supported by GA AVKAN208130801

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 Adam1, O Zitka1, D Huska1, S Krizkova1, J Strnadel2,

V Horak2, T Vaculovic3, K Novotny3, V Kanicky3, J Kaiser4

and R Kizek1

1

Department of Chemistry and Biochemistry, Mendel University of

Agriculture and Forestry in Brno, Brno, CZECH

REPUBLIC,2Academy of Sciences of the Czech Republic,

Insti-tute of Animal Physiology and Genetics, Libechov, CZECH

REPUBLIC,3Department of Chemistry, Masaryk University,

Brno, CZECH REPUBLIC,4Institute 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 lg of MT

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 pancy by the MCE, CIITA and general transcriptional machinery

occu-at various phases of the cell cycle including mitosis in B phoid cells Mitotic maintenance of the MCE does not depend

lym-on CIITA expressilym-on and correlates with a chromatin state that

is fully or partly maintained in mitotic lymphoid or non phoid cells, respectively Monitoring of newly synthesized RNAshows reduced transcriptional activity of the DRa gene in mito-sis Constitutive expression of exogenous CIITA is sufficient tofully support mitotic expression in lymphoid but not in epithelialcells Using an inducible system we show that CIITA synthesised

lym-in cells arrested lym-in mitosis rescues MHCII transcription to chronous levels in lymphoid cells demonstrating full expressioncompetence of DRa gene These results show there are two pat-terns of mitotic deficiency of gene expression: in B lymphoid cellslow but significant MHCII transcription can be fully rescued, asopposed to epithelial cells that show minimal mitotic transcrip-tion may due to enhanced chromatin condensation

asyn-P1–6 Associations between PTGS1 gene mutations and aspirin resistance in patients with

congenital heart diseases

I Coskun1, Y Atay1, A Berdeli2, M Mecidov1, A Atay3,

M F Ayik1, T Yagdi1and E A Alayunt1

Introduction: This study was aimed to evaluate that frequencyand 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 ulation

pop-Methods: It was included 90 patients with CHD treated byaspirin after their operations 24 healthy participants wereenrolled as control group Direct DNA coding sequence methodwas used for analysing PTGS1 gene mutation PCR amplificationwas made by using specific oligo primers (MWG) and AmpliTaqGold PCR kits for four exons After capillary gel electrophoresis,nucleotide sequences of PTGS1 gene was obtained and comparedwith cDNA Genbank (NM_000962) and protein (NP_000953)sequences to determine single- nucleotide polymorphisms andamino acid mutations

Results: W8R missense aminoacid mutation was the most quent mutation (83.3%) in exon-2 P188P sinonym aminoacidpolimorphism (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-

fre-2, Q41Q sinonim aminoacid mutation (8.3%) in exon-3, D189Emissense 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 sinonymamino acid mutations (L23L; P39P; R53R; G44G; G62G;K185K; P18P) were detected It was observed the associationbetween aspirin resistance and missence amino acid mutations inpatients

Conclusion: Genetic variations in PTGS-1 may affect aspirinresistance in CHD Detecting these variations may help to predictdrug responce and to select optimal therapy and dosage regi-mens This is the first study in our country dealing with PTGS1gene mutation and polymorphism which coded COX-1 enzymeand association with aspirin resistance in CHD

Analysis of the ID1 gene expression level and

promoter methylation in patients with benign

and malignant thyroid nodules

A Banaszewska1, M Piechota1, K Drobnik2, K Ziemnicka2,

K Ziemnicki1and R Plewa1

1Department of Animal Physiology and Development, Adam

Mickiewicz University, Poznan, POLAND,2Department 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 condidifferentia-tions associated with

cancer development ID is a transcription family of factors

lack-ing basic DNA bindlack-ing 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 Basic1, D Pavlovic1, T Jevtovic-Stoimenov1, J Vojinovic2,

M Marinkovic1and A Veljkovic1

1

Medical Faculty, Institute of Biochemistry, Nis, SERBIA,2Clinic

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 ment in patients with the genotype -308GA and -308 AA(P = 0.147; P = 0.126) We discussed decrease of MMP-9 level

treat-in children with -308GG genotype as a possible consequence ofbetter 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 Bayramci1, L Acik2, L Y Koc3, G Vural4, M Kilic5,

M Tez5, M Koc5and N Ercakmak4

1Faculty of Education, Department of Science Education, BayburtUniversity, Bayburt, TURKEY,2Faculty of Arts and Science,Department of Biology, Gazi University, Ankara, TURKEY,

3Faculty of Science, Department of Biology, Ankara University,Ankara, TURKEY,4Nuclear Medicine Department, Dr Abdurrah-man Yurtaslan Ankara Oncology Education and Research Hospi-tal, Ankara, TURKEY,55th Surgery, Ankara Numune Educationand Research Hospital, Ankara, TURKEY

Different types of tumors can develop in thyroid gland cells roid carcinoma represents the most frequent form of cancer ofthe thyroid glands, with prevalence in Europe of about 3 per

Thy-100 000 PTC is the most frequent histotype of differentiated roid cancer The RET proto-oncogene, localized to chromosomesubband 10q11.2, comprises 21 exons, which encodes the proteinRET, a receptor tyrosine kinase (RTK) expressed in derivativesand tumors of neural crest origin The RET proto-oncogene isinvolved in the genesis of papillary thyroid carcinoma To deter-mine the mutation frequency of RET proto-oncogene in Turkishpapillary thyroid carcinoma patients, we conducted this retro-spective study Eighty-two patients with PTC were screened formutations 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 themshowed SNP Twelve different mutations located in exon 11 andthree different mutations in exon 13 12.19% of the patients hadmutation in exon 11 3.65% of the patients had mutation in exon

thy-13 Twenty-four of the patients exhibited SNP (29.26%) Wefound 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 kinasedomain 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 tions were found in 41.46% of all thyroid carcinoma patients.The numbers of mutations were higher in exon 11 than exon 13

muta-In our series, we detected that four patients in 82 of all PTCpatients presented Cys630Ser (TGCfi AGC, 5.9%) is the mostcommon 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 fourpatients with papillary thyroid carcinoma Therefore, the rela-tionship between Cys630Ser variation and development of PTCshould 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 ofthe 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)

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 Bazylak1, C Ludwig2, E Fagadar-Cosma3and

U L Gunther2

1Department of Pharmaco-Bromatology & Molecular Nutrition,

Faculty of Pharmacy Collegium Medicum Nicolaus Copernicus

University, Bydgoszcz, POLAND,2HWB NMR, CR UK Institute

for Cancer Studies, University of Birmingham, Edbagston

Birming-ham, UK,3Department 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 (proteometabolo-me) 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 Bouchal1, I Struharova1, E Budinska2, O Sedo3,

T Vyhlidalova1, Z Zdrahal3, R van Spanning4and I Kucera1

1Department of Biochemistry, Faculty of Science, Masaryk

Univer-sity, Brno, CZECH REPUBLIC,2Institute of Biostatistics and

Analyses, Masaryk University, Brno, CZECH REPUBLIC,

3Department of Experimental Biology, Masaryk University, Brno,

CZECH REPUBLIC,4Department of Molecular Cell Physiology,

Vrije Universiteit Faculty of Earth and Life Sciences, Amsterdam,

THE NETHERLANDS

Paracoccus denitrificansis 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 inthe adaptation processes at global level Protein compositions offour P denitrificans strains (wild type, FnrP-, NNR- and NarR-mutants grown aerobically, semiaerobically and semiaerobicallywith nitrate) were analyzed using set of 36 large format 2-DPAGE gels, their statistical evaluation and MS/MS protein iden-tification of more than 500 proteins Expression differences ofkey up-/down-regulated gene products were validated using qRT-PCR Comparative proteomic analysis revealed, besides otherresults, differences between strains in the expression of three keydenitrification enzymes (nitrate reductase, nitrite reductase,nitrous oxide reductase), OmpW and UspA proteins These find-ings are in agreement with known mechanisms and/or confirmedthe positions of proposed fnr binding sites in their promoters.Detection of additional group of up-/down- regulated proteinswithout 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 otherfamilies (e.g LysR, MarR and two-domponent regulators) arediscussed

Acknowledgements: We are thankful to Czech Science dation (grant No 203/07/P0471) and to Czech Ministry of Edu-cation (grant No MSM0021622413) for financial support

Foun-P1–12 Analysis of proteome alteration in tomato roots after Meloidogyne hapla infection

A Obrepalska-Steplowska1, K Nowaczyk1, M Luczak2,

M Figlerowicz2, R Dobosz3and M Budziszewska1

1Interdepartmental Laboratory of Molecular Biology, Institute ofPlant Protection, Poznan, POLAND,2Polish Academy of Sci-ences, Institute of Bioorganic Chemistry, Poznan, POLAND,

3Department 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 greatlosses in the crop production Out of fifteen European species,eight were found in Poland; among them the most common andwidely distributed is M hapla, parasiting on dicotyledonousplants During the initial stage of infection characteristic rootgalls form on the roots of susceptible plant hosts As a result ofinteraction between parasite and the host, in the vascular cylinder

of the root permanent feeding sites are formed with specializedmultinucleated feeding cells (giant cells) The redifferentiation ofroot cells and development of metabolically active giant cells isinduced by the nematode The mechanism of giant cells forma-tion and the role of plant proteins in this processs remainunclear Our attempt was to analyse the changes in the plant pro-teome in the early stage of M hapla infection Tomato plantswere grown in isolated conditions and infected with M hapla.After isolation of proteins from roots of healthy and infectedplants, as well as from nematodes, two-dimensional electrophore-sis was performed The results of experiments were compared toensure elimination of nematode proteins from further analysis

As some qualitative and quantitative changes were observed, sen proteins were analysed using mass spectrometry The decrease

cho-of cytosolic and mitochondrial dehydrogenases cho-of cellular ration and electron transport chain was observed as well as forproteins related to cytoskeleton (actins), relevant to changes inthe cell’s shape and wound response (annexin) The qualitativechanges are related with proteasome endopeptidase complex,absent in infected root cells Nematode proteins secreted during

respi-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 F2

intercross chickens

Z Bulut1, E Kurar2, Y Ozsensoy2, M Nizamlioglu1, M Garip3,

A Yilmaz3, T Caglayan3, S Dere3, V Kurtoglu4and

M Dogan1

1Faculty of Veterinary Medicine, Department of Biochemistry,

Sel-cuk University, Konya, TURKEY,2Faculty of Veterinary

Medi-cine, Department of Genetics, Selcuk University, Konya,

TURKEY,3Faculty of Veterinary Medicine, Department of

Zoo-technics, Selcuk University, Konya, TURKEY,4Faculty of

Veteri-nary Medicine, Department of Animal Nutrition, Selcuk

University, Konya, TURKEY

In this study, a F2 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 F0, F1 and F2 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 Ceylan1, B Akbulut1, D Kazan1and D Kazan2

1

Engineering Faculty, Bioengineering Department, Marmara

University, Istanbul, TURKEY,2TUBITAK 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 tures (19–37C) Growth at these conditions was investigated andmicroorganisms were harvested at the end of exponential phase.Afterwards, whole cell proteins of the DSMZ 4770 wereextracted and analyzed by proteomic tools Two DE maps areprepared according to tube gel systems with NEPHGE technique

tempera-in the first dimension and normal SDS-PAGE tempera-in the seconddimension Protein profiles between normal and stress conditionswere determined and compared Protein expression differenceswere determined by MALDI-Tof Mass Spectrometer (Waters/Mi-cromass) Proteins that involved in the osmoprotection and coldadaptation were determined

P1–15 Molecular typing of Staphylococcus aureus strains from ovine mastitis by pulsed-field gel electrophoresis and polymerase chain reaction

A Ciftci1, E E Onuk2, A Findik1, T Yildirim3and

M Unlu Sogut4

1Veterinary Faculty, Microbiology, Ondokuz Mayis University,Samsun, TURKEY,2Veterinary Faculty, Diseases and ClinicalSciences, Ondokuz Mayis University, Samsun, TURKEY,

S aureusstrains isolated from ovine mastitis were typed by merase chain reaction (PCR) based on coagulase (coa) and protein

poly-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 commonlyobserved (44.68% and 38.29%, respectively) Nineteen differentpulsotypes were identified, and 12 of these were represented by asingle isolate Pulsotypes J and K were predominant, and eachrepresented nine isolates (19.14%) All isolates belonging to J and

K pulsotypes were CG2 While all nine isolates belonging to Jpulsotype were S4, all isolates in K pulsotype were S1 AlthoughPFGE 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, preliminaryepidemiologic 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 Onuk1, A Ciftci2, A Findik2and Y Durmaz3

1

Veterinary Faculty, Fisheries and Fish Diseases, Ondokuz MayisUniversity, Samsun, TURKEY,2Veterinary Faculty, Microbiology,Ondokuz Mayis University, Samsun, TURKEY,3Veterinary Con-trol and Research Enstitute, Fisheries and Fish Diseases, Samsun,TURKEY

Bacterial cold water, enteric red mouth and frunculosis are thecommon bacterial diseases of fish worldwide The etiologic agents

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 Psychrophilumand 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-lumand 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 Ciftci1and H Uysal2

1Veterinary Faculty, Biochemistry, Ondokuz Mayis University,

Samsun, TURKEY,2Veterinary 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 faecalisOG1X (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 thesestrains It was the first study in the literature that suggested E.faecalisstrains 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 ColakFaculty 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 taxonomicstatus, population genetics of the Genus Apodemus in Turkey Inorder to analysis genetic variation in Apodemus species, a Ran-domly Amplified Polymorphic DNA (RAPD) marker system wasused A total of 82 specimens (22 A iconicus, 26 A flavicollis, 7

A sylvaticus, 15 A uralensis, 4 A 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 tance were calculated to show the genetic relationships betweenstudied populations All estimations were calculated with thePOPGENE software The 60 RAPD markers were tested inApodemus, 11 ones yielded 101 polymorphic DNA bands.Genetic differentiation values, H = 0.0721 (P = 16.83%), werethe 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 arethe least similar to each other (I = 0.6256), and A iconicus and

dis-A uralensisare the nearest to each other (I = 0.9406) netic relationships between Apodemus species were shown withUPGMA dendogram constructed based genetic distance values

Phyloge-P1–19 What does it take to make an activated cortical domain within a plant cell?

F Cvrckova1, R Bezvoda1and V Zarsky2

1

Faculty of Science, Department of Plant Physiology, CharlesUniversity, Prague, CZECH REPUBLIC,2Faculty 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 cellmay possess multiple plasmalemma domains performing localizedexocytosis-driven expansion (‘activated cortical domains’, ACDs).Several large families of paralogous regulatory proteins might beinvolved in generating the diversity of ACDs within a cell, such

as e.g small GTPases of the Rop family and their interactors, bunits of the exocyst complex, actin-nucleating FH2 proteins,receptor-like kinases, or enzymes locally modifying membranecomposition (the ‘candidate set’ genes) Rhizodermis of Arabidop-sis thalianaconsists of two cell types that differ by a single ACD– trichoblasts carrying root hairs, and atrichoblasts We usedpublicly available cell-type specific transcriptome data to identifygenes specifically induced in trichoblasts compared to atricho-blasts (i), and to examine their relationship to over 60 ‘candidateset’ genes (ii), as well as to known genes whose mutations exhibitroot hair-specific phenotypes (iii) In addition, we have searchedfor homologues of all three gene classes in the published soybean

su-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 Iseri1, M Demirel Kars1, F Arpaci2, C Atalay3, I Pak4

and U Gu¨ndu¨z1

1

Department of Biological Sciences, Middle East Technical

University, Ankara, TURKEY,2Department of Oncology, Gu¨lhane

Military Medical Academy, Ankara, TURKEY,3Department 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 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 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 chemistry results confirmed microarray expression analysis.Conclusion: This report demonstrates that in docetaxel anddoxorubicin resistant cells EMT process was induced indicating apossible relationship of this process and drug resistance

Immunocyto-P1–21 Characterization and whole genome sequencing of Arthrobacter

phenanthrenivorans, a new phenanthrene degrading bacterium

C Drainas1, A Kallimanis1, K Kavakiotis1, K Mavromatis2,

N C Kyrpides2and A I Koukkou1

1Chemistry, University of Ioannina, Ioannina, GREECE,2GenomeBiology Program, DOE-Joint Genome Institute, Walnut Creek,

CA, USASeveral bacterial strains were isolated from a creosote pollutedsite at Perivleptos (Epirus, Greece), based on their ability to uti-lize various polycyclic aromatic hydrocarbons as a sole carbonand 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 showedthat Sphe3 belonged to the genus Arthrobacter comprising anovel 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 circularplasmids of 190 and 94 kb, respectively A total of 4288 geneswere predicted in the Sphe3 genome With BLAST searches usingpeptide fragment sequences from a purified 1-H-2-N dioxygenaseactivity determined by Mass Spectrometry, we identified twoencoding genes with over 90% homology to each other at thenucleotide level, one (diox1) located on the 190 kb plasmid andthe other (diox2) on the chromosome of the Sphe3 genome Fur-thermore, BLAST analysis of the 190 kb plasmid revealed that itadditionally contained several genes involved in phenanthrenedegradation These findings revealed novelties that confirm theextended 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 PessioneUniversity of Turin, Human and Animal Biology, Turin, ITALYAcinetobacter radioresistens S13 is a strain selected for its ability

in phenol degradation It is also able to degrade other aromaticcompounds (i.e., Benzoate) through the b-ketoadipate pathway(1) Comparative proteomics alkaline studies on bacterium growneither 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 membranelipoproteins, lipooligosaccharides (LOS) and phosphatidyletha-nolamine, damaging cell wall (2) This compound induces an overexpression of two proteases (ClpX and Serine protease) and oftwo RNA polymerase modulator factors (NusA and Rho)suggesting a gene modulation based on rE regulation Instead

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 Studies2004; 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 Findik1, N Akan2, E E Onuk3, D Cakiroglu4and A Ciftci1

1Veterinary Faculty, Microbiology, Ondokuz Mayis University,Samsun, TURKEY,2Veterinary Faculty, Ondokuz MayisUniversity, Samsun, TURKEY,3Veterinary Faculty, Diseases andclinical sciences, Ondokuz Mayis University, Samsun, TURKEY,

4Veterinary Faculty, Internal Medicine, Ondokuz MayisUniversity, Samsun, TURKEY

It was aimed to detect the carriage of methicillin-resistant ylococcus aureus (MRSA) strains in the nasal flora of healthydogs and to genotype these S aureus isolates The swabs weretaken 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 aureuswere analysed phenotypically and genotypically Agar Disc Diffu-sion Test was performed to determine the methicillin resistancephenotype of these S aureus strains using the oxacillin antibioticdiscs (5 g) and all the 80 strains were found as sensitive to methi-cillin In Polymerase Chain Reaction (PCR) targetting nuc, mecAand fem genes, three strains (3.75%) was found to possess mecA

Staph-In the same analysis none of these strains were positive for femgene The PCR targetting coa and spa genes was performed formolecular typing of the S aureus strains Nine different coagenes and three different spa genes were determined according tothe polymorphism of these genes In conclusion, the determining

of the nasal carriage of different genotypes of S aureus inhealthy 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 Ciftci1, A Findik1, E E Onuk2and S Savasan3

1

Veterinary Faculty, Microbiology, Ondokuz Mayis University,Samsun, TURKEY,2Veterinary Faculty, Diseases and ClinicalSciences, Ondokuz Mayis University, Samsun, TURKEY,

S aureusand methicillin resistance, respectively For detection ofslime producing, it was performed a PCR assay targetting icaAand icaD genes In this study, 59 strains were detected as S aur-eusby both conventional tests and PCR Among the 13 S aureusstrains of them were found as methicillin resistant phenotypicallyand 4 (30.7%) were positive for mecA gene Although 22 of 59(37.2%) S aureus isolates were slime-producing in Congo RedAgar in PCR analysis, only 15 of 59 were positive for both icaAand icaD genes 16 of 59 strains were positive for icaA and 38 ofthem were positive for icaD gene We found only two of 59strains were positive for both methicillin resistance and slime pro-ducing, phenotypically

Immobilization of acid phosphatase to

magnetic particles and their use for

dephosphorylation of proteins

J Frydlova1, L Kubosek1, L Kubosek2, Z Kucerova1,

M Ticha1and M Ticha3

11st Faculty of Medicine, Institute of Pathophysiology and CEH,

Charles University in Prague, Praha 2, CZECH REPUBLIC,

2Faculty of Chemical Technology, Department of Biological and

Biochemical Sciences, University of Pardubice, Pardubice, CZECH

REPUBLIC,3Faculty 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 (Mg2+), 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 Fuchs1, A Kostecka1, D Provaznikova1and J Filkukova2

1Department of Cell Physiology, Institute of Hematology and

Blood Tranfusion, Prague 2, CZECH REPUBLIC,2Department

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 repetidele-tions 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

NM_004364.2) We found both these most frequent deletions inCEBPA gene in one young man (38 years old) after coronarythrombosis (heart attack) The same deletions were detected inhis son (15 years old) In many cases (13 patients with AML,MDS, peripheral artery disease, heart disease, hyperlipidemia andtype 2 diabetes) more than one type of these deletions wasobserved 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 thesedeletions 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 InternalGrant Agency of the Ministry of Health of the Czech Republic(VZ 00023736)

P1–28 Study of thermal tolerance in Cronobacter strains

J Gajdosova1, K Kunikova1, I Turcovsky2, E Kaclikova2 and

H Drahovska1

1

Department of Molecular Biology, Comenius University,Bratislava, SLOVAK REPUBLIC,2Department of Microbiology,Food Research Institute, Bratislava, SLOVAK REPUBLICCronobacter spp., formerly named as Enterobacter sakazakii, is

an opportunistic pathogen associated with sporadic cases ofsevere infections in neonates causing meningitis, necrotizingenterocolitis and sepsis This organism is widely distributed inenvironment and rehydrated infant formula is the most commonsource of infection Cronobacter was shown to be particularly tol-erant to osmotic stress and desiccation and some strains of thisgenus are also tolerant to elevated temperatures Consideringinfant formula is not a sterile product, thermotolerant strains ofCronobacterspp represent increased risk to survive during infantformula reconstitution In our study, the genetic variability of 76food isolates and 23 Cronobacter collection strains was measured

by AFLP and 16S rRNA sequencing Within AFLP profiles, agreat variability was observed, the similarity among strainsreached values 50–100% Strains were clustered into 46 differentclusters 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 58C sepa-rated strains into two groups; D values (decreasing of bacterialcounts in one order) of thermosensitive strains fell within therange 17–50 s; on the other hand eleven strains (nine Cr sak-azakiiand 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_1165from 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 intopGEM plasmid resulted in 3–5 times increased survival ofEscherichia coli laboratory strain at 58C Our results have

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 Gnoni1, R Mangiullo1, R Schiavone2, S Vilella2, S Papa1,3

and F Zanotti1,3

1

Department of Medical Biochemistry, Physics and Biology,

University of Bari, ITALY,2Department of Biological and

Envi-ronmental Sciences and Technologies, Laboratory of Comparative

Physiology, University of Salento, Lecce, ITALY,3Institute 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 propro-teins 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

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 issomewhat less clear in case of Class II proteins, consistent withgene 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 theMSM0021620858 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 Kalousand J Houstek

Department of Bioenergetics, Institute of Physiology AS CR v.v.i.,Prague 4, CZECH REPUBLIC

Maternally transmitted disorders of mitochondrial ATP synthaseare typically caused by heteroplasmic missense point mutations

in mtDNA encoded subunit 6 In contrast, unique ATP synthasedisorder due to microdeletion 9205delTA in ATP6/COX3 gene isassociated with altered splicing of ATP6-COX3 mRNA anddiminished synthesis of subunit 6 (Fo-a) Up to now, only twopatients with 9205delTA mutation have been found with very dif-ferent phenotypes and mutation load To investigate phenotypicexpression of the mutation, we have prepared transmitochondrialcybrids with varying mutation load (50–100%) and studied therelationship between heteroplasmy, mitochondrial respiration,ATP production and Fo-a subunit content Heteroplasmy in celllines was analyzed by PCR/RFLP using NsiI restriction endonu-clease and digitonin-permeabilised cells were used for measuringoligomycin-sensitive, ADP-stimulated oxygen consumption Inparallel, ATP production was determined in DMSO-quenchedsamples by a luciferin-luciferase reaction To evaluate how themutation load affects on biosynthesis of Fo-a subunit, patient cy-brids were analyzed by SDS-PAGE and WB using specific anti-bodies to ATP synthase subunits Fo-a and F1-alpha We havefound that the decrease in ATP production and in ADP-stimu-lated respiration as well as the loss of subunit Fo-a content exertsimilar threshold dependce on increasing mutation load Pro-nounced decrease in all parameters was observed when mutationload reached about 80% In contrast, near-linear relationshipwas found between ATP production, respiration and loss of Fo-asubunit In conclusion, our results demonstrate, that similarly asATP6 missense mutations, 9205delTA biochemical phenotypeexhibits 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 VachaInstitute of Plant Molecular Biology, Photosynthesis, Ceske Bude-jovice, CZECH REPUBLIC

A filamentous freshwater phytoplankton Prochlorothrix

hollandic-abelongs to an unusual group of cyanobacteria called phytes Typical cyanobacteria organise photosynthetic light-harvesting complexes into so-called phycobilisomes where nochlorophyll is bound Prochlorophyta lost the ability to create

prochloro-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 Hornakova1, P Matouskova1, J Kindl2, I Valterova2and

I Pichova1

1

Institute of Organic Chemistry and Biochemistry Czech Academy

of Sciences, Biochemistry, Prague 6, CZECH REPUBLIC,

2Institute 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 Sˇobotnı´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 Hradilova1, P Rehulka2, H Rehulkova2, M Vrbova3,

M Griga3and B Brzobohaty1

1Laboratory of Plant Molecular Biology, Institute of Biophysics

AS CR, Mendel University of Agriculture and Forestry in Brno,Brno, CZECH REPUBLIC,2Department of Proteomics andGlycomics, Institute of Analytical Chemistry of the AS CR, Brno,CZECH REPUBLIC,3Plant biotechnology department,AGRITEC Ltd., Brno, CZECH REPUBLIC

Cadmium (Cd) is classified as a serious pollutant due to its hightoxicity and carcinogenicity, and widespread presence in environ-ment Phytoremediation represents an effective low-cost technol-ogy for removal of pollutants from contaminated soils Flaxbelongs 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 toincreased Cd content in soil and plant tissues Here significantchanges 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 proteinsextracted from Cd treated cell suspension cultures derived fromthe two contrasting cultivars Importantly, up-regulation by Cd

of two of them, ferritin and glutamine synthetase, was foundonly 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 glutathionebiosynthesis The identified proteins could turn useful in markerassisted 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 grants1M06030 and MSM 2678424601 (Ministry of Education of theCzech Republic), and AV0Z50040507, AV0Z50040702 andAV0Z40310501 (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 Huang1, W Y Lin2, C M Chang3and K B Choo2

1Department of Animal Science, School of Agriculture, ChineseCulture University, Taipei, TAIWAN,2Taipei Veterans GeneralHospital, Department of Medical Research and Education, Taipei,TAIWAN,3Graduate Institute of Biotechnology, Chinese CultureUniversity, Taipei, TAIWAN

It is a puzzle why mammalian genomes are heavily burdenedwith 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 specific In the course of investigation of the structure of T1/T2transcripts expressed in the embryo, the exons of these retroge-nes, some duplicated as cassettes of 2–3 exons, are found to bedeeply 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-T2chimerism is also observed To explain the novel features of the

testis-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 Chernenko1, M Reinman2, P R Karhemo1, S Goodison3,

K Takkinen2and P Laakkonen1

1

Faculty of Medicine, Institute of Biomedicine, University of

Helsinki, Helsinki, FINLAND,2VTT Technical Research Centre

of Finland, Biotechnology, Espoo, FINLAND,3Department 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 molemole-cules 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 athy-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 Thedetailed 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 describedWari insulator to block the repression of the mini-white and yel-low genes by the Ubx PRE We show that Wari insulator canblock the activity ofPRE-mediated silencing The distancebetween Wari insulator and a protected promoter is importantfor the effective blocking from PRE-mediated silencing Wefound that part of Wari insulator (368 bp) which is minimal forenhancer-blocking activity could be shortened in case of the bar-rier activity Moreover, we show with X-ChIP that proteinsCP190 and Mod(mdg4)-67.2 interact with Wari insulator Inaddition, we investigated that mutations in mod(mdg4)-67.2 ande(y)2 genes affect the ability of Wari insulator to protect geneexpression from PRE-mediated silencing, but not its ability toblock activation by enhancer

Conclusions: Wari insulator can protect yellow and mini-whitegenes from PRE-mediated silencing It does not requireMod(mdg4)-67.2 or E(y)2 for its enhancer-blocking activity, butthese 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 RubioHoward Florey Institute, Neurogenetics, Parkville, Vic.,AUSTRALIA

Multiple sclerosis (MS) is a complex autoimmune disease terised by T and B-cell mediated demyelinating lesions in the cen-tral nervous system (CNS) An auto-antigen in MS is the myelinoligodendrocyte glycoprotein (MOG), a CNS restricted proteinexpressed on the outer cell membrane of oligodendrocytes thatcan be used to induce the MS-like condition, experimental auto-immune encephalitis (EAE), in laboratory animals There havebeen conflicting reports in regard to the association between com-mon variation in the MOG gene and susceptibility to MS In thisstudy we investigated some of the expression characteristics ofMOG in post-mortem human MS and control brain tissue inorder to try to elucidate the cause of an association we observedbetween 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 inexpression towards the MOG alpha1 and/or MOG alpha4 splicevariants 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 MOGexpression In conclusion, subtle changes in MOG isoform selec-tion, but not expression levels, appear to be associated withrs3130253G>A, which in turn could influence the function ofMOG and the pathogenesis of MS

FATT mouse: a novel leptin receptor mutant

C Jensen1, H Chen2, M Morris2, L Johnson1, R Burfoot1,

K Simpson3, G Smyth3, K Walder4, S Foote3, J Hollis5,

B Oldfield5and J Rubio6

1Howard Florey Institute, Neurogenetics, Parkville, Vic.,

AUSTRALIA,2Department of Pharmacology, University of

Melbourne, Parkville, Vic., AUSTRALIA,3Genetics and

Bioinformatics Division, The Walter and Eliza Hall Institute of

Medical Research, Parkville, Vic., AUSTRALIA,4Metabolic

Research Unit, School of Exercise and Nutrition Sciences, Deakin

University, Geelong, Vic., AUSTRALIA,5Department 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 (LeprFATT)became hyperphagic and obese

post-weaning In exon 7 of lepr, adjacent to the splice donor ‘GT’

consensus sequence in intron 7, the LeprFATTmutation 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

LeprFATTmutant 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 Jerczynska1, P Komorowski2, A Nosal2, H Szymanowski2,

M Gazicki-Lipman2, B Walkowiak2and Z Pawlowska1

1

Department of Molecular and Medical Biophysics, Medical

University in Lodz, Lodz, POLAND,2Institute 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 ily fabricated giving various shapes and structures Additionally,they are easily functionalized and, depending on the polymericbuilding blocks, may also be degraded in the body after a desiredperiod However, biomaterials used for medical implants orinstruments production can cause numerous adverse effects Theymay cause changes in gene expression and protein profile of thecells which were in contact with applied material The aim of thisstudy was to evaluate an influence of medical steel AISI 316Land the medical steel coated with poly-para-xylylene (parylene)

eas-on a cell cycle and apoptosis of human endothelial cells Theresults demonstrated that interactions between cells and studiedmaterials induce a number of changes in the gene expressionprofile

Acknowledgement: Supported by project No PSS/2008a

P01/0001/SPB-P1–42 Tumor necrosis factor, tumor necrosis factor receptors type 1 and 2, lymphotoxin-a gene polymorphism in lymphoproliferative diseases

in Serbian population

T Jevtovic1, G Kocic1, D Pavlovic1, N Tosic2, S Aveic2,

G Marjanovic3, L Macukanovic-Golubovic3and V Djordjevic1

1Institute for Biochemistry, Medical Faculty University of Nis,Nis, SERBIA,2Department for Molecular Biology, Institute ofMolecular Genetics and Genetic Engineering, Belgrade, SERBIA,

3Institute of Hematology and Immunology, Clinical Centre Nis,Nis, SERBIA

Tumor necrosis factor-a and lymphotoxin-a are involved in thepathogenesis of established lymphoproliferative diseases TNFmolecules bind to TNFRI and TNFRII TNFRI is the majormediator of the TNF pro-apoptotic and prolifferative effects andTNFRII might potentiate this effects In this study, we haveanalysed polymorphism in TNF gene -308 G/A, in LT-a gene+250G/A, one polymorphisms in TNFRI gene (TNFRI + 36A/

(TNFRII + 676 T/G) All these polymorphisms were studied inpatients with chronic lymphocytic leukemia’s (CLL), patientswith 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 diseasedevelopment Genotyping was performed by PCR-RFLP analy-sis The study provided evidence of the influence of TNFG/Agenotype and A alleles in the susceptibility to NHL, since theassociation of LT-aG/G genotype with CLL was observed High-producing TNF-a -308/LT-a +250 heterozygous haplotype isassociated with high NHL incidence No significant differences inallele frequencies of TNFR1 polymorphism were found betweenthe patients with lymphoproliferative disease and healthy individ-uals In a group of healthy individuals the study has revealed forthe first time significantly higher TNFRI G/G genotype com-pared to the patients with lymphoproliferative disease(v2= 5.66; P = 0.017) Also, we reported the implication ofTNFRII T allele in NHL pathogenesis, respectively (v2= 10.77;

P= 0.001; Mantel-Haenszel: v2= 10.64; P = 0.0011) Our datashowed that TNFRII T676G polymorphisms have an importantrole in NHLs pathogenesis but not in CLL patients A/A poly-morphism in TNFRI is associated with CLL and NHL patients

in Serbian population

Genetic evaluation of the human oral cancer

via systematic DNA resequencing approach

M C Kao1, M H Yeh2, T M Shieh3, S F Chen3, M H Tsai4

and Y S Lee5

1Biological Science and Technology, China Medical University,

Taichung, TAIWAN,2National Defense Medical Center, Graduate

Institute of Life Sciences, Taipei, TAIWAN,3Department of

Dental Hygiene, China Medical University, Taichung, TAIWAN,

4Department of Otolaryngology, China Medical University

Hospi-tal, Taichung, TAIWAN,5Office 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 concentradetermina-tions 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 theirdensity were also exhibited same aspect as proteins These find-ings point out that thumb pad secretion is abounded in terms ofprotein content and the changes about protein and glycoproteinprofiles might be related to seasonal reproductive activity Thismeans 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 PodlubnayaLaboratory of Structure and Function of Muscle Proteins, Insitute

of Theoretical and Experimental Biophysics, Pushchino, RUSSIATitin is giant elastic protein of vertebrate skeletal and cardiacmuscles 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 elasticarea 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 sevenvariants of alternative splicing Research of protein regulation ontranscriptional and translational levels is of particular interest fordescription of muscle functional state In this work we carriedout the investigation of changes in titin isoform composition inmyocardium of ground squirrels in various stages of hibernation-

al cycle We have selected this model because hibernation is aunique model of adaptation to stress conditions, immobilizationand heart function depression The results of qRT-PCR revealedthe decrease of mRNA level both for N2BA and N2B titin iso-forms in heart of hibernating ground squirrels as compared totheir levels in the heart of summer active animals Using electro-phoresis and western blotting we have shown the decrease inoverall amount of titin with respect to heavy myosin chains indifferent heart chambers of ground squirrels upon hibernation incomparison with summer active animals (ð‡0,998) The ratio oflong N2BA to N2B titin isoforms in the heart of hibernatingground squirrels was increased approximately two-fold(P < 0.0005) However this trend was not revealed for themRNA levels of corresponding isoforms This discrepancy inprotein and mRNA levels may be considered as the posstran-scriptional regulation of titin isoforms’ expression The overalldecrease in mRNA level may be explained by repressed transcrip-tion or mRNA degradation in the cell during hibernation Tounderstand the mechanisms regulating titin isoform compositionupon hibernation these investigations are to be extended.Acknowledgement: This work was supported by grant RFBR07-04-00479

P1–46 Uniform reporting standards for enzyme activity data

C KettnerFunding Department, Beilstein-Institut, Frankfurt am Main,GERMANY

The access to the essential background information on logical experiments is of paramount impact for the understanding

enzymo-of both the experimental data themselves and their interpretation

under consideration of the broader context However, scientists

who are evaluating these data for pathway research be it as in

sil-icomodellers 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 albicansis 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 ofits role in the processing of pathogenesis

Acknowledgements: This study is supported by a grant of theKorea Healthcare technology R&D Project, Ministry of Health

& Welfare, A084005

P1–48 Electrochemical and computational study of doxorubicin interactions with DNA

D Huska1, V Adam1, J Burda2, J Hrabeta3, T Eckschlager3,

P Babula4, R Opatrilova4, L Trnkova5, M Stiborova6and

5

Department of Chemistry, Masaryk University, Brno, CZECHREPUBLIC,6Department of Biochemistry, Charles University,Prague, CZECH REPUBLIC

Anthracyclines including doxorubicin belong to antibiotics duced by Streptomyces peucetius subsp cesius There is compellingevidence that cellular DNA is the primary target for them It is notsurprise that cell protective mechanisms cause resistance on thetreatment with these drugs The main aim is to study intercalation

pro-of doxorubicin into DNA in vitro by using pro-of square wave metry The electrochemical method is further utilized for detection

voltam-of doxorubicin-DNA adducts in neuroblastoma cells Intercalateddoxorubicin 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 theintercalation, computational experiments using design for testabil-ity approach with correction on dispersion interaction were per-formed Several molecular structures were explored and clearpreference 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 doxorubicinbetween resistant and sensitive cells Resistant cells showed negligi-ble amount of adducts with the increasing concentration of thedrug compared to resistant cells The suppressed formation of ad-ducts can be related to presence of other mechanisms to withstandthe effect of the cytostatic Therefore we determined low-molecu-lar-mass thiols (glutathiones and its derivatives) and found theincreased thiols level in resistant cells compared to sensitive ones.Acknowledgement: This work was supported by GA AVIAA401990701

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 Kovarova1, P Pecina1, J Houstek1, C Dell‘Agnello2and

M Zeviani2

1Bioenergetics, Institute of Physiology Academy of Sciences of theCzech Republic, Prague, CZECH REPUBLIC,2Unit of MolecularNeurogenetics, National Neurological Institute, Milan, ITALYRespiratory chain enzyme complexes are assembled into higherstructural units, respiratory supercomplexes In mammalian mito-chondria they are mainly composed of complexes I, III, and IV

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

SURF1gene Digitonin-solubilised protein complexes were

analy-sed by 2D electrophoresis (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-III2and III2

-IV, in mitochondria from liver, brain, muscle and heart from

control and COX-deficient mouse In mouse fibroblasts we also

detected I-III2-IV supercomplex and the same pattern was found

in human fibroblasts In the latter case all complex I signal was

present in I-III2-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 55C and pH 8.0 The phosphatase activity of

Mbpp was strictly Mn2+dependent, a characteristic that is found

in other serine/threonine phosphatases Three-dimensional

struc-tural analysis revealed three Mn2+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 Kmvalue of 1.34 ± 0.074 mM The

phos-phatase activity was not inhibited by known protein phosphos-phatase

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 tuberculosiswhich 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 Machado1, S Kandzia2, P Altevogt3, H S Conradt2and

J Costa1

1ITQB, Laboratory of Glycobiology, Oeiras, PORTUGAL,

2GlycoThera, GmbH, Hannover, GERMANY,3Division of CellularImmunology, German Cancer Research Center, Heidelberg,GERMANY

Ovarian carcinoma is the leading cause of death from cal cancers in many Western countries Aberrant glycosylation is

gynecologi-an importgynecologi-ant aspect in maligngynecologi-ant trgynecologi-ansformation, normally ciated with increased expression of membrane glycoproteins,appearance of novel or truncated oligosaccharides, fucosylatedglycoconjugates and abnormal types of terminal oligosaccharidescontaining antigenic determinants The SKOV3 ovarian carci-noma cell line has low amounts of Lewis carbohydrate epitopes

asso-at the cell surface, synthesized by a1,3/4- fucosyltransferases(FUTs) (1) The stable overexpression of several FUTs in theSKOV3 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 Lex and/orsLexepitopes, 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 sLea The N-linked oligosaccharides from total glyco-proteins of SKOV3 cell line was further characterized by highperformance anion exchange chromatography with pulsed amper-ometric detection and matrix assisted laser desorption/ionizationtime-of-flight mass spectrometry The results showed that SKOV3cells were rich in unprocessed oligomannose structures, with lowperipheral sialic acid levels Furthermore, the N-glycosylation of

a recombinant secretory glycoprotein, erythropoietin, stably duced from this cell line was characterized

pro-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 Majerova1, M Ingr2, J Dostal1and J Konvalinka1

1

Biochemistry and Moleular Biology, Institute of Organic try and Biochemistry, Prague, CZECH REPUBLIC,2Faculty ofScience, Biochemistry, Charles University, Prague, CZECHREPUBLIC

Chemis-SNP (single nucleotide polymorphism) quantification in DNApools could be important for a rapid analysis of allele distribu-tion in populations, including determination of gene variantsamong individuals, monitoring drug resistance development andprognosis of tumor growth Although many methods of SNPdetections are available, precise SNP quantification is still trou-blesome In this work, we demonstrate a method for accuratequantification of the SNP ratio in mixtures of two synthetic oli-gonucleotides possessing guanine (G) or adenine (A) at a definedposition This method is based on a one-tube single base primerextension with fluorescently labeled nucleotides followed byreverse phase HPLC and fluorescence detection of the primer

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 Marinkovic1, T Jevtovic-Stoimenov1, S Stojanovic2,

M Todorovic1, J Basic1, A Veljkovic1, T Cvetkovic1and

D Pavlovic1

1Medical Faculty, Institute of Biochemistry, Nis, SERBIA,

2Medical 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 TNF-and RA TNF-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%, v2= 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%,

v2= 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 (v2= 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 Andrade1, E Hajnsdorf2, P Re´gnier2and C M Arraiano1

1Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova

de Lisboa, Oeiras, PORTUGAL,2Institut 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 RNAdegradation TherpsO mRNA encoding for the ribosomal proteinS15, has been widely used as a model of study for polyadenyla-tion-mediated mRNA turnover Escherichia coli RNase II andPNPase are two major degradative exonucleases involved in thecontrol of mRNA stability However, in the absence of these twoexonucleases the polyadenylated rpsO mRNA is still very effi-ciently degraded Data suggested that at least one yet unknownpoly(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)-dependentdegradation of the rpsO mRNA Moreover, RNase R is shown

to have a more relevant effect on rpsO mRNA stability thanPNPase, which was so far characterized as the main exonucleaseinvolved in the poly(A)-metabolism, either acting as a freeenzyme or in complex In the absence of RNase R, the rpsOtranscript 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 highlydependent on polyadenylation is also stabilized in an rnr singlemutant All these results highlight the importance of bacterialRNase R in the post-transcriptional control of gene expressionestablishing 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

by several proteins involved in cell differentiation and lar matrix synthesis Bone morphogenetic protein 2 (BMP2) wasprimarily identified for its osteogenic properties and severalhuman bone-related diseases have been associated with BMP2defect Although numerous studies have been performed in mam-mals to unravel BMP2 mechanisms of action, those are still notfully understood Fish have recently emerged as a suitable model

extracellu-to study vertebrate biology and a suitable alternative extracellu-to lian systems We propose to develop a fish-based in vitro cell sys-tem to study bone-related mechanisms of BMP2, in particular itsinteraction with matrix Gla protein (MGP), a physiologicalinhibitor of calcification BMP2 gene expression was profiled byreal-time PCR in a variety of seabream cell lines capable of in vi-tro mineralization and shown to be differentially regulated inosteoblast- and chondrocyte-like cells Transcriptional regulation

mamma-of seabream BMP2 gene expression was later evaluated usingreporter vectors containing gene promoter and shown to beunder the control of various mineralization-related transcriptionfactors, in particular osteoblast-specific factor runx2 Cellularclones, where BMP2 gene expression has been altered, have beenconstructed, validated by Western blot analysis using antibodiesdeveloped in mammals, then evaluated for mineralizationcapacity 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 recentlydeveloped seabream microarray hybridized with cDNA preparedfrom cellular clones, and BMP2-MGP interaction will be evalu-ated in a co-culture system of clones overexpressing seabreamBMP2 and/or MGP cDNAs Results obtained within the scope

of this work are preliminary but should give new and interestinginsights on bone-related mechanisms of BMP2 in vertebrates

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 translocaactiva-tion 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

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

(http://www.ebi.ac.uk/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 andhierarchical classification of proteins from all organisms, includ-ing metagenomic projects With 3 60 000 signatures, InterProprovides annotation for 3 80% of UniProt sequences Novelsequences/genomes can be queried through InterProScan Proteinsignatures model amino acid conservation in families or domains,then predict these classifications/features in uncharacterised pro-teins By linking related signatures together, InterPro places themwithin a hierarchical classification scheme which reflects theirunderlying evolutionary relationships As such, one can addressissues 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 directcomparison of protein signatures with structural data, incorpo-rating >14 000 structures from PDB with their classification inSCOP and CATH, as well as >1 650 000 homology models fromSWISS-MODEL and MODBASE Each protein signature has anabstract, 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 bioinformaticiansalike

P1–58 Revealing of potential FoxA target genes, involved in proliferation control

N Ershov, T Merkulova, L Bryzgalov, M Pakharukova,

D Oschepkov and V KaledinInstitute of Cytology and Genetics SB RAS, Laboratory of geneexpression control, Novosibirsk, RUSSIA

FoxA transcription factors are known to be critical to liver cell ferentiation We demonstrated a strong association between he-patocarcinogenic activity of a number of compounds and theirability to reduce FoxA DNA-binding activity in the liver Thus rat-specific hepatocarcinogen 3¢-methyl-4-dimethylaminoazobenzenesignificantly 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 bothrats and mice inhibited FoxA activity in both species, while non-carcinogenic 4¢-methyl-4-dimethylaminoazobenzene was ineffectiveboth in rats and mice With the aim to elucidate the mechanism ofpotential FoxA tumorsuppressor action we searched for their tar-get genes related to proliferation control First we examined thepublished data on global gene profiling in mouse liver (a high level

dif-of FoxA) and kidneys (FoxA are absent) Then the search forFoxA binding sites was carried out by computer-assisted SIT-ECON method in regulatory regions of 40 differentially expressedgenes involved in the proliferation control 11 genes containingputative sites organized as TTTG repeats (3 to six-fold) were dis-covered FoxA binding to these sites was confirmed by EMSAusing GST-fusion protein comprising the FoxA 2 DNA-bindingdomain The effect of OAT administration (leading to reduction ofFoxA activity) on the expression of six genes containing confirmedFoxA binding sites was studied by real-time PCR We found thatthe mRNA levels for Cul2 and CDC73 genes increased dramati-cally after OAT administration

Acknowledgement: The work was supported by a grant fromthe RFBR (07-04-00441)

Characterization of a putative carbohydrate

catabolic pathway of the megaplasmid pAO1

of Arthrobacter nicotinovorans

M Mihasan1, V Artenie1and R Brandsch2

1University Alexandru Ioan Cuza, Faculty of Biology, Biochemistry

and Molecular Biology Department, Iasi, ROMANIA,2Institute

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-novoranslacking 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

orf40gene, 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 2Zn2+

atoms/pro-tein monomer The enzyme contained the amino acid sequence

A119GKHIFTEKP128 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 E128KP130motif 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 Navratilova1, M Brazdova1, V Tichy1, M Fojta1, M Lexa2,

I Kyjovsky1, W Deppert3and E Palecek1

1Department of Biophysical Chemistry and Molecular Oncology,

Institute of Biophysics AS CR v.v.i., Brno, CZECH REPUBLIC,

2Faculty of Informatics, Masaryk University, Brno, CZECH

REPUBLIC,3Heinrich-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 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 fragmentsnaturally 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 bymutp53 was isolated by specific chromatin imunoprecipitation(genome-wide ChIP-cloning) We combined molecular (detection

spe-by enzymatic and chemical probing) and computationalapproaches to identify non-canonical DNA structures (cruciformsand triplex DNA) as binding sites for p53 proteins Electropho-retic mobility shift assay (EMSA) was used for confirmation ofthe specific binding of p53 proteins to DNA sequences Our datasuggest that binding of p53 proteins to non-B DNA sequences isthought to be the important molecular mechanism in human can-cerogenesis

Acknowledgements: This work was supported by EC 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)

(FP6-Further reading:

M., Quante, T., Togel, L., Walter, K., Loscher, C., Tichy, V.,Cincarova, L., Deppert, W., Tolstonog, G.: Modulation of geneexpression in U251 glioblastoma cells by binding of mutant p53R273H to intronic and intergenic sequences, Nucleic Acids Res.2009; 37: 1486–1500

P1–61 Development of Denizli x White Leghorn F2

population for quantitative trait loci mapping

M Nizamlioglu1, M Garip2, A Yilmaz2, T Caglayan2,

E Kurar3, Z Bulut1, V Kurtoglu4, S Dere2, Y Ozsensoy3 and

M Dogan1

1Faculty of Veterinary Medicine, Department of Biochemistry,Selcuk University, Konya, TURKEY,2Faculty of VeterinaryMedicine, Department of Zootechnics, Selcuk University, Konya,TURKEY,3Faculty of Veterinary Medicine, Department ofGenetics, Selcuk University, Konya, TURKEY,4Faculty ofVeterinary Medicine, Department of Animal Nutrition, SelcukUniversity, Konya, TURKEY

Development of gene maps for human and animals is critical foridentification 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 animalbreeding programs to improve animal health, production effi-ciency and product quality Poultry production is an importantsector in agriculture for obtaining economical animal originatedfoods Chicken is also accepted and extensively used as an excel-lent model organism in genetic studies In this study, five Denizlimales and eleven White Leghorn hens were mated to generate F1

population Fourteen F1males and 58 F1females were crossed togenerate five full-sib F2 families including 441 birds Bodyweights at hatching, 3, 6, 9, 12, 16, 24, 28 and 32 weeks of age,egg yield and egg weight phenotypic data were recorded Bloodand tissue samples were collected from all F0, F1and F2animalsand DNA samples were isolated for mapping QTL analysis Den-izli X White Leghorn F2 population is available scientific com-munity for gene and QTL mapping studies

Posterior polymorphous corneal dystrophy –

copy number, gene expression and candidate

gene analyses within the PPCD1 candidate

region on chromosome 20p11.2

L Noskova1, P Liskova2, V Stranecky1, H Hartmannova1,

R Ivanek3, K Jirsova4, S Merjava4, M Filipec5and S Kmoch1

1First 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,3Academy of Sciences, Institute

of Molecular Genetics, Prague, CZECH REPUBLIC,4Laboratory

of Biology and Patology of the Eye and Ocular Tissue Bank,

Gen-eral Teaching Hospital and Charles University, Prague 2, CZECH

REPUBLIC,5Department 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 Novotna1, T Emmerova1,2, J Vavrova1, M Ticha1,2and

Z Kucerova1

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 cellularsignaling mechanisms Moreover, anomalous phosphorylation ofproteins can be also related to development of cancer or to meta-bolic diseases Processes involving phosphoproteins are dynamicand this fact affects their low abundance and relatively shorthalf-life To understand these processes, structural studies oninvolved phosphoproteins is desired Despite recent advances ininstruments and methods used for the characterization of phos-phoproteins, the effective separation or enrichment of phospho-peptides and phosphoproteins remains to be fully solved Thesubject of the present study was to compare the efficiency of twoprocedures used for the separation or enrichment of phosphopep-tides from the tryptic digest of phosphoprotein: (i) sorption ofphosphopeptides to different metal ions chelates immobilized tomagnetic 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-caseindigest 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 adsorptionand following desorption were optimized

Acknowledgements: This work was supported by the Ministry

of Education, Youth and Sports of the Czech Republic (grantMSM 0021620806 and project CEH LC 06044) and by the CzechScience Foundation (grant 203/09/0857)

P1–64 Functional analyses of Lupinus luteus cyclophiline promoter activities

K Nuc1, P Nuc2and R Slomski1

1054 to -845 indicating to 5¢AAAGAT 3¢motif We have alsoanalyzed 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 revealedthat LCyp promoter is responsive to plant hormones such asABA, IAA and BAP as well as to salt, but not to cold stress

Quantitative and temporal proteome analysis

of gamma-Tocotrienol treated

androgen-unresponsive prostate cancer cells delineates a

novel caspase – independent programmed cell

death pathway

T Roumeliotis1, A Evdokiou2, P Vraka3, L Loizou4, S Garbis1

and A D Odysseos4

1Center of Basic Research – Biotechnology Division, Biomedical

Research Foundation of the Academy of Athens, Athens,

GREECE,2Department of Orthopaedics and Trauma, University

of Adelaide, Adelaide, Australia3Department of Chemistry,

Uni-versity of Cyprus, Nicosia, CYPRUS,4EPOS-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 ColakFaculty of Science, Ankara University, Biology, Ankara, TURKEY

In this study, a total of 194 Eurygaster maura specimens wereused 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) Atotal of 16 esterase activity bands were observed Only Est-1locus distinguished among esterases to be thought encoding by alot of loci In the bug populations studied, three allele (A, B, C)were recognized in this locus In the starch gels examined ofsuperoxide dismutase enzymes were determined one cathodallocus All specimens studied were homozygous for the same allele

in this locus It was determined genetic identity and genetic 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 Eurygastermaura populations based on Nei’s Genetic Distance of twoenzyme loci, ten populations were divided into two main clustersand 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)

dis-P1–67 Associations between serum linolenic acid/ alpha-linolenic acid ratio and blood gene expression profiles

K S Olsen1, C G Fenton2, E Anderssen3, L Froyland4,

R H Paulssen2and E Lund1

1

Institute of Community Medicine, University of Tromso, Tromso,NORWAY,2Microarray Resource Centre, University of Tromso,Tromso, NORWAY,3Department of Cancer Research and Molec-ular Medicine, NTNU, Trondheim, NORWAY,4National Institute

of Nutrition and Seafood Research, Bergen, NORWAYNutrition is one of the major modifiable risk factors for athero-sclerosis and certain cancers Linoleic acid (LA, 18:2n-6) andalpha-linoleic acid (ALA, 18:3n-3) are essential to humans, andmodulate immune response by giving rise to pro- (LA) or anti-(ALA) inflammatory signaling molecules Despite the well-knownmetabolism 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 ofpostmenopausal Norwegian women, we have explored the associ-ation between serum LA/ALA ratio and blood gene expressionprofiles Samples from 286 women were successfully analyzed forexpression profiles in PAXgene-stored whole blood usingABI1700 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 softwareand Bioconductor packages In women with a high LA/ALAratio, we revealed a pro-inflammatory expression profile thatincludes genes related to Fc epsilon signaling, phosphatidyl inosi-tol signaling, cytokine production, arachidonic acid metabolismand generation of eicosanoids Strikingly, we identified theincreased expression of the key pro-inflammatory enzyme 5-lipox-ygenase (5-LO) in women with a high LA/ALA ratio These pre-

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 Dilek1, S A Berna1and U Caglayan2

1

Department of Bioengineering, Marmara University, Istanbul,

TURKEY,2Department 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

coliK12 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 bility, and translational efficiency These functions depend on thesequence and the structure of the UTR BRCA1 and BRCA2 arethe major hereditary breast/ovarian cancer predisposing genesand their mutations increase the risk of developing cancer Inter-pretation of sequence variants found in genetic testing is themajor concern for BRCA genes, especially for risk assessment ingenetic counseling From different repositories (dbSNP, BIC,kConFab), we collected BRCA1 and BRCA2 sequence variantsthat were found within 5¢ and 3¢ UTRs and we analyzed, usingdifferent on-line tools like UTResource and Transterm, theirpotential functional significance that could be expressed by dis-rupting or creating putative regulatory elements As it is knownthat the function of non-coding RNAs (ncRNAs) greatly depends

sta-on their secsta-ondary structure, we analyzed how these nsta-on-codingsequence variants could have impact on the predicted secondarystructure By analyzing changes in the predicted secondary struc-tures of the 5¢ and 3¢ UTRs from the patients with breast/ovariancancer we tried to find out if this approach could be used forassessing clinical significance in cancer etiology

P1–70 Phylogenetic and bioinformatic analysis of Glutathione S-Transferase Tau from Pinus brutia

E Oztetik1, M Aydin2and F Kockar2

in gymnosperms The definition of detoxification enzymes likeGSTs in perennial conifers, a large group of gymnosperms, isvery important for their adaptations to several environmentalstresses during their long lifespans The present study reports thephylogenetic and bioinformatic analysis of GST gene (PbGST-Tau) from a conifer, Pinus brutia, which is widely distributed inthe north-eastern Mediterranean area, including Turkey ThePbGST-Tau gene encodes a protein of 228 amino acid residueswith a calculated molecular mass of 26.37 kDa The sequencecomparisons of PbGST-Tau gene to other plant GST-Tau genes(Pinus densata, Pinus tabulaeformis, Pinus yunnanennis, Oryza sa-tiva japonica, Aegilops tauschiiand Ginko biloba) were performed

at the amino acid level by using Bioedit programme The genetic tree was also constructed by using Neighbor-Joining/UP-GMA method The analyses indicated that PbGST-Tau is placedwith other three GSTs in Tau class from Pinus tabulaeformis, Pi-nus densata and Pinus yunnanensis, with 10.82%, 11.45% and11.18% sequence diversity, respectively The current difficultiesfor the comprehensive characterization of GST family in conifersoriginates from the insufficiency of comprehensive genome infor-mation on conifers in the literature As the described members ofthe GST gene family in conifers are increased, the understandingfor diversity and evolution of the GST classes would also beadvanced

Human Accelerated Region 20 (HAR20)

operates as an enhancer: functional study of a

HAR20 SNP associated with diabetes and

cardiovascular disease

S Pampı´n1, L Parnell2, J M Ordovas2and

J C Rodriguez-Rey1

1Universidad de Cantabria, Biologı´a Molecular, Santander,

SPAIN,2Tufts 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 Panigaj1, H Glierova1, A Simkova2and K Holada1

11st Faculty of Medicine, Institute of Immunology and

Microbiol-ogy, Prague 2, CZECH REPUBLIC,2Faculty 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 tially after exposure to external stress We induced stress condi-tions by 3 various treatments- elevated incubation temperature(40C), oxidative stress by presence of H2O2(500 lM) and Stau-rosporin (125 nM) But even such conditions do not altered level

differen-of apoptosis/ necrosis in lines with inhibited PrPc Finally, weintroduce the new cell culture model for study of PrPc function

in erythroid differentiation Initial observations shows that directcytoprotection is not mode of PrPc action in studied process, but

it may play role in cell cycle which is subject of undergoingresearch

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 Park1, J H Cho2, J H Kim1, D Hwang2and Y S Gho1

1Department of Life Science, POSTECH, Pohang,SOUTH-KOREA,2School of Interdisciplinary Bioscience andBioengineering, POSTECH, Pohang, SOUTH-KOREAVarious 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 exosomescarrying mRNA that causes epigenetic reprogramming of tumormicroenvironmental cells Here, we present a global comparativeexosomal and cellular transcriptomic analysis of human SW480CRC cells We identified 11 327 exosomal and 11 624 cellularmRNAs, and 241 and 1461 of these were overexpressed in exo-somes and in cells, respectively The exosomal and cellularmRNAs were similarly enriched in tumorigenesis-related biologi-cal processes Furthermore, exosome-enriched mRNAs wereinvolved in the cell cycle, whereas cell-enriched mRNAs wereinvolved in angiogenesis Our study demonstrates that the exoso-mal and cellular mRNAs of human CRC cells are similar to eachother at the global level, in terms of intensities and biologicalprocesses, and that exosomal mRNA may be involved in tumorprogression via the horizontal transfer of mRNA to target cells.This information will help elucidate the pathological functions oftumor-derived exosomes and will aid in the development of meth-ods using exosomal mRNAs to determine the diagnosis andprognosis of CRC

P1–74 Proteomic approach for detection of biomarkers in rheumatoid arthritis

M Park, S Chung, T Kim and J LeeDepartment of Internal Medicine, Yonsei University College ofMedicine, Seoul, SOUTH-KOREA

Background: Rheumatoid arthritis is an autoimmune diseasecharacterized 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 potentialtherapeutic targets Proteomics can be defined as the qualitativeand quantitative comparison of proteomes under different condi-tions to further unravel biological processes

Objective: In this study, we aimed to identify and analyze thepotential peptides expressed in the rheumatoid synovitis usingproteomics-based approach

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 26pairs of liver and muscle tissue samples obtained at autopsy frommiscarriages after informed consent, between 13th and 28th week

of gestation The mtDNA amount and gene expression levelswere analyzed by the real-time PCR method using SybrGreen I

In both fetal liver and muscle tissues, mtDNA content andTFAM 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 wasincreasing only in fetal liver tissue (r = 0.54; P < 0.01) NRF1was unchanged in both fetal tissues and PGC1 was slightly risingbetween 13th and 28th week of gestation in liver tissue(r = 0.42; P < 0.05) Our results showed that POLG expressionvaries between two different tissues during gestation and proba-bly is not associated with mtDNA content as tightly as TFAMexpression The increase of PGC1 transcript level is statisticallysignificant 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 toits specific sites on TFAM promoter In conclusion, this studydescribed mainly the increasing trends of gene expression in thepathway leading from expression of PGC1 to mtDNA contentchanges in fetal liver and muscle tissues during second trimester

of gestation According to our results, we suppose that the chondrial proliferation is growing up between 13th and 28thweek of gestation and therewithal it is the first hallmark of pre-pare for postnatal adaptation on transcriptional level

mito-Acknowledgement: This work was supported by grantGAUK25755707, IGAMZ-NR9410,GACR305/08/H037

P1–77 DNA methylation in LDL receptor and Apolipoprotein B100 genes promoter in patients with hypercholesterolemia

M Piechota1, A Banaszewska1, E Guzniczak2, R Link2,

G Rosinski1, T Siminiak2and R Plewa1

1

Department of Animal Physiology and Development, Adam iewicz University, Poznan, POLAND,2Cardiac and RehabilitationHospital Kowanowko, Poznan University of Medical Sciences,Poznan, POLAND

Mick-The DNA methylation is an epigenetic modification present inProkaryoticand Eukaryotic genomes The CpG alterations lead

to selective utilization of gene information and are responsiblefor tissue specific gene expression, embryonic development, geno-mic imprinting and X-chromosome inactivation Hypermethyla-tion of promoters is usually associated with silencing of the geneexpression Disorders of the methylation pattern may contribute

to autoimmune disease development, cancer as well as metabolicand 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 ofLDLRor/and ApoB100 can influence on decrease the synthesis

of proteins and the accumulation LDL-cholesterol in the wallsand advance atherosclerosis disease The purpose of the studywas to evaluate LDLR and ApoB100 expression level and themethylation status in patients with hypercholesterolemia Theinvestigated group consisted of 25 patients with no mutation inLDLRexons Expression of LDLR was significantly lower andaverage 24–45% then in the healthy individuals The analysis ofCpG 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

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 withlack 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 HovorunDepartment of Molecular and Quantum Biophysics, Institute ofMolecular Biology and Genetics NAS of Ukraine, Kiev, UKRAINEMultiple steps in replication cycle of human immunodeficiencyvirus type 1 (HIV-1) causing AIDS illness are directed by controlelements located both in untranslated and translated regions ofHIV-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 itenables to use phylogenetic approach to study mechanisms ofvirus 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) Atpresent database contains secondary structures predicted by themfold program (Zuker, 2003) of the region encompassing DIS,

SD and Psi hairpins of 5¢-UTR for about 1300 HIV-1 isolatesselected from NCBI (GenBank) The database has been devel-oped to assist in analyzing of frequencies of different variants ofcontrol elements, influence of mutations on the secondary struc-ture, dependencies of secondary structures of control elements onsubtype and geographical region, evolution of control elements

In particular we found tolerant and intolerant combinations ofbase changes in DIS, SD and Psi hairpins We present theschemes of hypothetical transitions between all frequent variants

of DIS, SD and Psi hairpins via single base change The authorsbelieve CESSHIV-1 database to be a useful guide for a widerange 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 RubioAndalusian Center for Molecular Biology and RegenerativeMedicine (CABIMER), Senalizacio´n Celular 4, Sevilla, SPAIN14-3-3 is a ubiquitous protein that in many cases had beeninvolved in antiapoptotic signals in cells, promoting cell survival

by association with proapoptotic proteins Here, a proteomic andbiochemical analysis was used to identify apoptotic-related 14-3-3e-binding proteins aimed to have a comprehensive knowledge ofsurvival functions of 14-3-3e isoform in cells Tandem affinitypurification, LC-MS/MS analysis and biochemical validationwere combined to characterize 46 proteins associated with 14-3-3e which binding status is regulated by C2-ceramide-inducedapoptosis in HeLa cells Among this pool of proteins, 16 of themwere directly implicated in the apoptotic process comprising pro-teins related with regulation of intermediate filament integrityand 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 inearly stages of apoptosis as DNA protein kinase (DNA-PKcs).The functional diversity of theses identified proteins suggests that14-3-3e regulates the apoptotic process through new mechanisms

in addition to others previously characterized The majority of

these proteins contained one or several

phosphorylation-depen-dent 14-3-3-binding sites indicating a potential direct interaction

with 14-3-3e Accordingly of survival function of 14-3-3e isoform,

a weak ectopic 14-3-3e expression delay cell death and

knock-down of this isoform sensitized cells to C2-ceramide induced

apoptosis Finally, these biochemical and functional analysis

show 14-3-3e isoform as a survival factor during

C2-ceramide-induced apoptosis and characterized novel C2-ceramide regulated

14-3-3e interacting proteins related with processes that control

life or death in Hela cells

P1–82

Computational analysis of miRNA targets and

CpG islands in human genes

E Rodriguez, A Ferre´, M Gonzalez-Porta, M A Montero,

E Olle´, E Daura, C Rojas, M Mulero, M Cabre´,

J L Paternain and A Romeu

Biochemistry and Biotechnology, Rovira i Virgili University,

Tarragona, SPAIN

MicroRNAs (miRNAs) are small noncoding RNAs

(single-stranded RNA molecules of about 21–23 nucleotides in length)

that regulate gene expression by targeting messenger RNA

(mRNA) transcripts CpG islands are genomic regions that

con-tain a high frequency of CG dinucleotides Both, CpG islands

and miRNA are involved in the epigenetic landscape, i.e.,

heri-table changes in gene expression caused by mechanisms other

than changes in the underlying DNA sequence CpG islands are

DNA sequence features which typically occur at or near the

transcription start site of genes, particularly housekeeping genes;

and cytosines in such an arrangement tend to be methylated

However, miRNAs, are epigenetic factors acting at

post-tran-scriptional level, through their targets in mRNAs The aim of

this work is to explore the possible relationship between

miR-NA targets and CpG islands in human genes Genomic data

have been retrieved from NCBI the current Homo sapiens Build

36.3 We have used the MySQL relational database management

system, MySQL and Perl scripts First, taking into account all

human genes, the overlapping percentage of gene sequence

(including the flanking regions) by CpG islands were evaluate

The plot CpG-gene interactions vs number of miRNA targets,

clearly shows an inverse correlation In order to identify

whether this behaviour plays a significant role in genes which

are under an epigenetic regulation, we have analysed the

inci-dence of CpG islands and miRNA targets in hypomethylated

and hypermethylated in specific genes of colon cancer; and in a

set of human experimentally identified imprinted genes Project

AGL2007-65678/ALI

P1–83

DNA damage induces a post-translational

modification of the mismatch protein hMLH1

F Romeo, N Ahmad, M Di Sanzo, D Scumaci,

M Saccomanno, G Cuda, M C Faniello, B Quaresima and

F S Costanzo

Experimental and Clinical Medicine, ‘Magna Graecia’ University,

Catanzaro, ITALY

DNA mismatch repair (MMR) system contributes to the

mainte-nance of the genomic stability in both prokaryotes and

eukary-otes, through the correction of replication errors, the suppression

of recombination between non identical, but homologous

sequences, and the activation of cell cycle arrest and apoptosis in

response to DNA damage Moreover, the inactivation of the

MMR pathway is linked to cancer predisposition Recent

evidence supports a potential role of MMR system in signallingthe DNA damage response, in particular, has been observed theinduction of hMLH1 in response to several DNA-damagingagents, including cisplatin, adriamycin and MNNG Further-more, the stabilization of the remaining hMutL (hPMS1 andhPMS2) proteins and their nuclear compartmentalization inresponse to DNA damage requires hMLH1 Here, we examinedthe induction of hMLH1 protein in response to adryamicin-induced DNA damage We have used the MCF7 cells, treatedand untreated with adriamycin, and performed Western blot andRT-PCR assays We found that hMLH1 and BRCA1 (molecularpartner of hMLH1) were activated by adryamicin treatment intime-dependent manner On the basis of these results specificshRNA for BRCA1 was used for silencing endogenous BRCA1

in MCF7 cells We found that hMLH1 stabilization is mediated

by BRCA1 in response to DNA damage To further uncover apotential link between hMLH1/BRCA1, we looked for post-translational modifications of hMLH1, like Ser/Thr phosphoryla-tion Using immunoprecipitation and 2D gel approaches wefound a post-translational modification of hMLH1 Moreover,the post-translational modifications of hMLH1 protein is cur-rently under investigation by LC/MS-MS approaches

P1–84 Functional genomic resolution of pharmacogenetic nexus between metabolic syndrome and morphogenesis

M Krupkova1, M Janku1, L Sedova1, L Kazdova2, F Liska1,

D Krenova1, V Kren1and O Seda3

1First Faculty of Medicine Charles University, Institute of Biologyand Medical Genetics, Prague, CZECH REPUBLIC,2Institutefor Clinical and Experimental Medicine, Department ofMetabolism and Diabetes, Prague, CZECH REPUBLIC,

3CHUM, Research Centre, Montre´al, QC, CANADA

We tested the hypothesis of common genetic factors forming anexus between morphogenesis and metabolic syndrome We utilizedmetabolic and transcriptomic profiling of a unique set of ratmodels with previously ascertained disparity in both limb devel-opment and metabolic syndrome features Adult male rats(n = 12/strain) of spontaneously hypertensive rat (SHR) andcongenic SHR.PD-(D8Rat42-D8Arb23)/Cub (SHR-Lx) strains,differing in 1.4 Mb region of chromosome 8, were fed a highsucrose diet (HSD) for 2 weeks and subsequently treated with

RA (15 mg/kg) for 16 days, while still on HSD We contrastedmetabolic(insulin sensitivity, adipokines, free fatty acids, triacyl-glycerol and cholesterol in 20 lipoprotein fractions) and transcrip-tomic(Affymetrix Rat Exon 1.0 ST, liver) profiles between SHRand SHR-Lx under conditions of standard, HSD andHSD + RA administration We observed noticeable distinction

in effect of RA between SHR and SHR-Lx strains SHR-Lxreacted with significant impairment of glucose tolerance and lessfavorable distribution of cholesterol and triaylglycrols into thelipoprotein fractions compared to SHR Significant interactionsbetween strain and diet/RA factors were found for free fatty acidand insulin levels Transcriptomic data corroborated the meta-bolic profile as they revealed a concerted shift in distinct path-ways between the strains in response to RA We demonstratedinteraction of retinoic acid with a 14- gene region of rat chromo-some 8, affecting concurrently the features of metabolic syndromeand, as previously shown, the limb morphogenesis.Our results sup-port the notion of interconnection of morphogenetic and metabolic

processes, assumed in human syndromes, e.g., Bardet-Biedl or

Smith-Lemli-Opitz

P1–85

Expression and turnover of small RNAs in

Salmonella typhimurium

I J Silva, S C Viegas and C M Arraiano

ITQB, Control of Gene Expression Lab, Lisboa, PORTUGAL

Eukaryotic and prokaryotic cells contain a wealth of small RNAs

(sRNAs) with determinant roles in the post-transcriptional

con-trol of gene expression The mechanisms by which sRNAs

modu-late gene expression are diverse and they can be differently

classified according to their mode of action In pathogenic

bacte-ria, a large number of sRNAs coordinate adaptation to stress

and expression of virulence genes We have analysed the

expres-sion profiles of sRNAs, conserved among many enterobacteria,

in different growing conditions This has given us valuable

infor-mation about the conditions of expression of these sRNAs in

Salmonella, which are probably related to their function and

tar-gets in this bacterium Namely, the higher levels of these sRNAs

in growth conditions under which the expression of the

Salmo-nellaPathogenicity Island (SPIs) genes is induced may indicate a

relation with virulence functions To understand the regulation

by sRNAs it is fundamental to study their turnover since this

may impact on the regulatory capacity of a sRNA The specific

ribonucleases (RNases) involved in the degradation of these

sRNAs should be one of the main factors responsible for these

differences We have constructed Salmonella typhimurium

mutants for several ribonucleases or Poly(A) Polymerase I The

processing and stability of the four sRNAs was then analyzed in

the RNase mutant strains constructed and cleavage sites were

mapped in order to understand the mechanism of decay In this

work we have identified some of the main RNases involved in

the post-transcriptional control of each of these sRNAs Our

glo-bal results give important information about the expression,

pro-cessing and turnover of these sRNAs, bringing insights into the

mechanisms of sRNA function in Salmonella

P1–86

Analysis of the p53 temperature-dependent

mutants in H1299 cell line

J Slovackova1, D Grochova1, J Navratilova2and J Smardova1

1Department of Pathology, University Hospital and Faculty of

Medicine Masaryk University, Brno, CZECH REPUBLIC,

2Faculty of Science Masaryk University, Institute of Experimental

Biology, Brno, CZECH REPUBLIC

The p53 tumor suppressor plays an important role in preventing

cancer development by regulating cell cycle, apoptosis and

main-tenance of genome integrity Missense mutations in the p53 gene

are often associated with cancerogenesis They can alter

expres-sion profile of the p53-target genes thus resulting in different

bio-logical outcomes Using functional assay in yeast, we have

collected 23 temperature-dependent (td) mutants of the p53 gene

The transactivation abilities of these p53 mutants are partially or

fully restored at decreased or increased temperature In this

study, we investigated relationship between type of mutation and

ability to transactivate the p53 target genes (p21, bax, RGC) in

the p53-null human H1299 cell line transiently transfected with

p53 td mutants Then, we compared the p21, bax and MDM2

gene expression in H1299 cells permanently transfected with the

p53 td mutants by real-time PCR In order to pursue temperature

sensitivity we have carried out both assays at 32C and 37C We

found that p53 td mutants exhibit very diverse transactivation

activity and discriminative character (i.e., different affinity to ious responsive elements of the p53 target genes) In some cases,p53 tdmutants showed even higher ability to transactivate sometarget genes than wt p53 We conclude that even a single aminoacid exchange in the p53 protein can lead to diverse transactiva-tion spectra, which in turn could cause diverse biologicalresponses Better understanding of various functional variants ofp53 can be important for development of new anticancer thera-pies

var-Acknowledgement: This work was supported by grants No.NR/9305-3 of IGA MZ CR and MSM 0021622415

P1–87 New specific molecular targets for radiochemotherapy in colorectal cancer

K Snipstad1, C G Fenton1, J Kjaeve2, E Anderssen3and

The preferred treatment for patients with advanced colorectalcancer is preoperative radiochemotherapy The early side effects

of this treatment have been considered to be acceptable The aim

of the study was to identify the effect on gene expression intumour and normal rectal tissue samples before and after preop-erative radiochemotherapy For that purpose, tissue samplesfrom twenty patients with operable rectal adenocarinomas wereused for a whole genome-based gene expression analysis, usingmicroarray technology A factorial experimental design allowed

us to look solely at the radiation effect on tumours This resulted

in 4496 differentially expressed genes in tumour tissue with

P< 0.05 In addition to known markers for radiochemotherapy,

a Gene Set Enrichment Analysis (GSEA) showed that the effect

on tumour tissue is specific and mostly associated with cell sion and leukocyte transendothelial migration (TEM) pathways,represented by 618 genes Only 61 genes were differentiallyexpressed (P < 0.05) in normal tissue, none of which areinvolved in pathways described for tumour tissue We concludethat radiochemotherapy mainly and specifically affect tumour tis-sue, and that the effect on normal tissue is not only limited, butalso different from tumour tissue The profound change of celladhesion molecule expression in tumour tissue could eitherdecrease the tumours invasive potential, or it could increase therisk of metastasis Further characterization of genes involved incell adhesion and leukocyte TEM may give new insights into themolecular responses to radiochemotherapy, and open the possi-bility for design of new tools for colorectal cancer therapy

adhe-P1–88 The ge´nolevures consortium: evolution of protoploid saccharomycetaceae genomes

G The Ge´nolevures ConsortiumInstitut de Botanique, GDR Ge´nolevures, Strasbourg, FRANCEThe Ge´nolevures Consortium (http://www.genolevures.org/) isdeeply involved in the comparative genomics of Hemiascomycet-ous yeasts An exploration of 13 hemiascomycete genomesrevealed their broad evolutionary range (Souciet et al., 2009) butafter complete sequencing of the genomes of a number of species(Dujon et al., 2004) it appears that the Hemiascomycetous yeastsencompass three major internal subdivisions In a recent compar-ative genomic analysis (Souciet et al., 2009) we concentrate on

five species, we establishe the complete and fully annotated

sequences of three new species, of Saccharomycetacea

(Zygosac-charomyces rouxii, Sac(Zygosac-charomyces kluyveri and Kluyveromyces

thermotolerans) that we call « protoploid » because they diverge

from the S cerevisiae lineage prior to its whole genome

duplica-tion The main points of the work are:

1 Three fully sequenced and manually annotated genomes of

yeasts, selected according to previous phylogeny as part of the

less extensively documented sections of Saccharomycetaceae,

des-ignated ‘protoploid’ to distinguish them from the extensively

studied species derived from whole genome duplication Yeast

species of various habitats and origins

2 Analysis of genome content and composition, including genes

for non-coding RNA Discovery of an unexpected compositional

heterogeneity Discovery of a novel class transposable element

3 Classification of entire proteomes into protein families et

char-acterization of a core protein repertoire for the

Saccharomyceta-ceaeyeasts

4 Recognition and analysis of ancestral duplications and

numer-ous paralogs among the set of protoploid species Analysis of

conserved and species-specific paralogs, either dispersed or

form-ing tandem gene arrays

5 A new method to define gene orthologs by synteny

Multidi-mensional sequence comparisons of corresponding orthologous

proteins, revealing long evolutionary distances

6 Conservation of synteny, and first analysis of relationship

between sequence divergence and loss of synteny in yeasts

Reconstruction of an ancestral genome

7 General discussion on the common characteristics of

Sacchar-omycetaceae and the evolutionary variations along each special

lineage

References:

1 Souciet et al., FEBS Lett 2000; 487: 3–12

2 Dujon et al., Nature 2004; 430: 35–44

3 Souciet et al., Genome Research 2009

Faculty of Medicine, Histology and Embryology, Balikesir,

TURKEY,2Faculty of Science and Art, Biology, Balikesir,

TURKEY

Cell-cell and cell-matrix interactions play role not only in

physio-logical processes like embryogenesis and development of the

organism but also in pathological situations like tumour

metasta-sis and AIDS One factor, which controls these interactions, is

proteinases ADAMTS (a disintegrin and metalloproteinase with

thrombospondin motifs) proteins, firstly described in 1997, are

the newest members of the proteinase family and up-to-date 19

different ADAMTS proteins have been determined There are

several studies about the expression of the different ADAMTS

proteins in different tissues and cell lines But, there is no

infor-mation about the expression of these proteins in hepatocellular

carcinoma cell lines The aim of this study was to determine the

expression of ADAMTS-1, -2, -3 and -8 proteins in a human

hepatocellular carcinoma cell line, namely Hep3B cells

ADAM-TS-1 and -8 were included to the study because they have

anti-angiogenesis effect and ADAMTS-2 and -3 were included to the

study because they function in collagen synthesis and liver

cirrho-sis may provide background for hepatocellular carcinoma Total

cellular RNA was prepared from Hep3B cells and subjected to

RT-PCR using ADAMTS-1, -2, -3 -8 and GAPDH specific

prim-ers There was ADAMTS-1 and ADAMTS-2 expression in

Hep3B cells However, neither ADAMTS-3 nor ADAMTS-8expression was detected

P1–90 Ubiquinone biosynthesis in Saccharomyces cerevisiae: Molecular organization of O-methylase Coq3p depends on Abc1p/Coq8p

A Tauche, U Krause-Buchholz and G Ro¨ delInstitute of Genetics, Dresden University of Technology, Dresden,GERMANY

The redox active lipid coenzyme Q (Q) functions as an electroncarrier in the mitochondrial respiratory chain Q biosynthesis inSaccharomyces cerevisiaerequires at least nine proteins (Coq1p–Coq9p), including Coq8p, which contains motifs found ineukaryotic-type protein kinases Absence of Coq8p results in lack

of Q and concomitant accumulation of the intermediate HHB.Here we show Coq8p is a soluble homomeric protein of the mito-chondrial matrix or associated with the inner mitochondrialmembrane on the matrix side As revealed by 2D-Blue Native/SDS-PAGE analysis, Coq8p is not integral part of a Coq3p,Coq5p and Coq9p containing 1.3 MDa Q-biosynthesis complex,but – together with Coq10p – mainly associated with a complex

of about 500 kDa, and only weakly or transiently associated withthe 1.3 MDa complex Deletion of COQ8 results in the release ofCoq3p, but not of Coq9p from the 1.3 MDa Q-biosynthesis com-plex This effect cannot be restored by Q6supplementation Wedemonstrate further that the 1.3 MDa Q-biosynthesis complex isnot associated with the complex III and IV supracomplexes ofthe respiratory chain 2D-Isoelectric Focusing data indicate theCoq8p kinase activity, with Coq3p as a target We propose amodel for the role of Coq8p in the Q-biosynthetic complex

P1–91 Detection of enteroviruses and hepatitis A virus RNA in milk by reverse transcriptase- polymerase chain reaction

G Terzi1, H Albayrak2, B Siriken1, O Cadirci1and Z Yazici3

1Ondokuz Mayis University Veterinary Faculty, Food Hygiene andTechnology, Samsun, TURKEY,2Samsun Veterinary Control andDirectory of Research Institute, Virology, Samsun, TURKEY,

A new amino acid sequence variant in

NADPH-cytochrome P450 oxidoreductase found in a

healthy control

M Tomkova1, C C Marohnic2, K M McCammon2,

B S Masters2and P Martasek1

1Department of Paediatrics and Adolescent Medicine, Charles

University in Prague First Faculty of Medicine, Prague, CZECH

REPUBLIC,2Department of Biochemistry, University of Texas

Health Science Center at San Antonio, San Antonio, TX, USA

Microsomal cytochrome P450 enzymes are involved in metabolism

of drugs and xenobiotics and in catalysis of steroid biosynthesis A

single protein, NADPH-cytochrome P450 oxidoreductase

(CY-POR), supplies electrons to all microsomal P450s CYPOR is a

flavoprotein, containing both FAD and FMN, that uses NADPH

as electron donor Mutations in the POR gene (encoding CYPOR)

have been associated with an autosomal recessive disorder called

POR deficiency To identify CYPOR amino acid sequence

varia-tions in the Czech population we sequenced POR in 37 healthy

controls We have identified a new CYPOR amino acid sequence

variant P384L, which is a one-base substitution (c1151C>T) on

one allele in exon 10 Wild type (WT) CYPOR and P384L variant

were bacterially expressed and purified Flavin analysis

indicated-that the protein:FAD:FMN ratio of P384L was the same as WT

(1 : 1 : 1) CYPOR function was quantified by measuring both

NADPH-cytochrome c reduction and CYP1A2 catalyzed

ethoxy-resorufin-O-dealkylase (EROD) activity The P384L variant

retained 3 60% of WT turnover in the cytochrome c reductase

assay with no effect on KmNADPH= 1 lM A similar result was

obtained from the EROD assay, with 3 70% of WT activity

observed for P384L According to the location of the P384 residue

in the CYPOR connecting domain, it is surprising that activity was

not more severely affected by P384L substitution It is not

surpris-ing, however, that a minor loss of function from a single allele had

no apparent affect on the health of the individual

Acknowledgement: Supported by grants GAUK 252000

100007 and NIH grant GM081568

P1–93

Analysis of genetic variability of Salmonella

enterica strains based on prophage genes and

variable number tandem repeats

L Tothova, H Drahovska, T Szemes, H Al-Alami,

D Brotanova, A Soltysova and J Turna

Department of Molecular Biology, Comenius University,

Bratislava, SLOVAK REPUBLIC

Salmonella entericarepresents a leading cause of food born

poison-ing universally in developed and developpoison-ing countries Rapid and

reliable identification is a key step in the tracking and management

of infection outbreaks Conventional phenotype based methods,

which are in use currently, are increasingly showing their limits

when compared to modern molecular methods based on PCR,

fragment analysis or sequencing analysis In our study, we

ana-lyzed a set of salmonella strains belonging to 21 different serotypes

using three methods – MLVA (Multi Locus VNTR Analysis),

PCR phage typing and sequencing based phage typing, which we

selected for typing of serovar Typhimurium in our previous study

We revised our previously used VNTR markers, excluding those

lacking variability or presence of markers, and used a set of seven

markers in a multiplex fragment analysis assay We observed high

polymorphism in selected markers with the total number of 36

dif-ferent MLVA profiles Inter-serovar variability was detected in

strains of serovars Anatum, Bredeney, Derby, Dublin, Enteritidis,

Havana, Indiana, Saintpaul, Senftenberg and Typhimurium Onthe other hand, four strains of S Agona and four strains of S Wor-thingtonshared the same profile Selected genes from salmonellabacteriophages P22, ST104, Gifsy-1, Gifsy-2, Fels-1, Fels-2 andSopEFi were detected in strains by PCR based phage typingapproach We were able to observe that presence of phage genesand their combinations were mostly serotype specific, despite beingable to observe some intra-serotype variability Taking the PCRphage typing to the level of nucleotide sequence, applying sequenc-ing analysis, we observed further improvement in discriminationpower of PCR based phage typing

P1–94 Analysis of the tellurite resistance determinant present in uropathogenic Escherichia coli

L Valkovicova, S Vavrova, J S Aradska and J TurnaDepartment of Molecular Biology, Comenius University,Bratislava, SLOVAK REPUBLIC

Tellurium compounds have a long history as antimicrobial andtherapeutic agents Prior to the development and marketing oftoday’s antibiotics, they were used to treat conditions such as lep-rosy, tuberculosis, dermatitis, cystitis and eye infections As a con-sequence, many Gram-positive and Gram-negative bacteriadeveloped resistance to potassium tellurite Despite the rare occur-rence of tellurite in nature, the determinant of tellurite resistanceencoded by ter genes has been found in relatively wide spectrum ofpathogenic microorganisms, including the emergent haemorrhagicstrain Escherichia coli O157:H7 We have studied tellurite resis-tance determinant located on the previously described pTE53 plas-mid which was originally isolated from uropathogenic strain E coliKL53 The real time PCR analysis has determined that the ter-ZABCDEgenes are represented by a single RNA transcript, form-ing a single transcriptional unit that is expressed from a singlepromoter – predicted also by large scale bioinformatics analysis

We functionally confirmed the existence of one common functionpromoter for terZABCDE genes by cloning probable promoterregion, also called PPRR (the potential promoter rich region) intothe vector pBAD-GFPuv in the fusion with reporter gfpUV gene.The functional fusion of proposed promoter region with gfpUVresulted in the cells expressing GFP, which was observed with fluo-rimetry assay The terW gene codifies a protein of 155 amino acidsand recently, our research group demonstrated its DNA bindingactivity In this work, the terW-derived protein containing a N-ter-minal His-tag was overexpressed, purified and used for in vitroDNA-binding reactions By method EMSA, it was found thatTerW binds specifically to DNA containing the potential promoterregion of terZABCDE EMSA is one of the most widely usedmethods to explore interactions between DNA and proteins Ami-noacid sequence comparison and specific bioinformatic predictionsuggest that TerW is related to the other DNA-binding proteinswith transcriptional regulator function However the mechanism oftellurite resistance still remains unclear, but with the promisingresults given above we hope to uncover its secret soon

P1–95 Molecular modelling study of PrnB, the major proline transporter of Aspergillus nidulans

Y Vangelatos1, M Billini2, D Vlachakis1and

V Sophianopoulou1

1

NCSR ‘Demokritos’, Biology, Athens, GREECE,2JGI,Production Genomics Facility, San Francisco, CA, USAPrnB, the L-proline transporter of Aspergillus nidulans, belongs

to the YAT family, one of the ten members of the Amino acid

Polyamine Organocation transporter superfamily In silico

analy-sis and limited biochemical evidence suggest that YAT

transport-ers comprise 12 transmembrane segments connected with

relatively short hydrophilic loops However, little is known on

the structure-function relationships in !AT transporters We have

made use of the A nidulans PrnB transporter to address

struc-ture-function relationships by selecting, constructing and

analy-sing several prnB mutations (Tavoularis et al., 2003; Vangelatos

& Sophianopoulou, unpublished data) Most isolated missense

mutations affecting PrnB function map in the borders of

cyto-plasmic loops with transmembrane domain In a recent study,

PrnB structure-function relationships were addressed by

employ-ing a Cys-scannemploy-ing approach, based on the engineeremploy-ing of a

func-tional Cys-less PrnB genetic background (Kafasla et al., 2007)

The 3D structures of only few Secondary transporters have been

solved to date After obtaining them from the Protein DataBank,

the structure of PrnB was established using a novel,

hydropho-bicity-based, approach to conventional homology modelling

tech-niques More than one templates were chosen and structurally

combined together to produce a reliable alignment Loop

search-ing and buildsearch-ing was done by Modeller, followed by Gromacs

molecular dynamics simulations, using explicit water molecules in

an NVT periodic box The viability of the PrnB homology model

was evaluated by in silico scoring functions, such as Procheck

and Verify3D Currently exhaustive molecular dynamics

simula-tions of PrnB are carried out within the membrane bilayer to

evaluate binding of a series of substrates

ITQB, Control of Gene Expression lab, Lisboa, PORTUGAL

Eukaryotic and prokaryotic cells contain a wealth of small RNAs

(sRNAs) with determinant roles in the post-transcriptional

con-trol of gene expression In pathogenic bacteria, a large number of

sRNAs coordinate adaptation to stress and expression of

viru-lence genes MicA is a small RNA conserved among many

en-terobacteria Its main targets in Salmonella are the outer

membrane protein ompA and LamB maltoporine We have

anal-ysed the expression profile of MicAin this bacterium under

differ-ent growing conditions To understand the regulation by sRNAs

it is also fundamental to study their turnover since this may

impact on the regulatory capacity of a sRNA We have

con-structed Salmonella typhimurium mutants for several of the main

ribonucleases and Poly(A) Polymerase I The processing and

sta-bility of MicA sRNA was analyzed in these mutants PNPase

and RNase E mutations were shown to strongly influence its

sta-bility However, a mutation in the endoribonuclease III was one

that mostly affected MicA decay We have purified several of

these main ribonucleases of Salmonella and we are performing

in vitroactivity assays to clarify its role on MicA processing and

decay We are also analysing the stability of MicA in the absence

of its mRNA target(s) to investigate whether there is a coupled

MicA-target regulation Taken together, the results of this study

provide initial insight into the mechanisms of sRNA decay in

Salmonella, and indicate specific contributions of the RNA decay

machinery components to the turnover of individual sRNAs

P1–97 Shear flow enhances s-nitrosylation of proteins in endothelial cells

L WangAcademia Sinica, Institute of Biomedical Sciences, Taipei,TAIWAN

Endothelial cells (ECs) constantly exposed to blood flow increasenitric oxide (NO) production via the activation of eNOS NO-mediated S-nitrosylation is recently identified as an importantposttranslational modification that may alter signaling and/orproteins’ function In the present study, a CyDye switch methodwas utilized to examine the S-nitrosylated proteins in humanumbilical vein ECs exposed to flow by a flow chamber setup.After flow, free thiols in proteins were blocked by alkylation andthe S-NO bond was reduced by ascorbate followed by a CyDye(Cy3 or Cy5) labeling to this reduced cysteine in proteins col-lected from respective static- or shear flow- treated ECs TheCyDye-labeled proteins were mixed and subjected to two-dimen-tional electrophoresis The enhanced S-nitrosylation of proteinsafter flow treatment was reflected as an increase of Cy5-labellingproteins More than one hundred S-nitrosoproteins were detected

in static and shear-treated ECs Among those, 12 major proteinswere shown to have significantly increases in S-nitrosylationunder shear flow ECs pretreated with an eNOS inhibitor (L-NAME) followed by flow treatment decreased the flow-inducedS-nitrosylation Those S-nitrosylated proteins were heterogeneousorigin The S-nitrosylated residue in tropomyosin and vimentinwere identified and localized in the hydrophobic motif Similar S-nitrosylations were shown in ECs treated with a NO donor(SNAP) Our results demonstrate that the S-nitrosylation ofendothelial proteins could be detected by this CyDye switchmethod coupled with 2-D DIGE Shear flow to ECs increases S-nitrosylation in endothelial proteins This eNOS-dependent S-nit-rosylation of proteins may be essential for the adaptation/remod-eling in ECs under flow

P1–98 Significance of nucleotide changes in I subunit

of cytochrome oxidase gene in Polish pyrethroid-resistant populations of Melighetes aeneus

P Wieczorek1, K Nowaczyk1, P Wegorek2and

A Obrepalska-Steplowska1

1Institute of Plant Protection, Interdepartmental Laboratory ofMolecular Biology, Poznan, POLAND,2Department of Zoology,Institute of Plant Protection, Poznan, POLAND

Pollen beetle (Melighetes aeneus F.) is a common pest of oilseedrape cultivation It could cause great damage amounting to 80%

of rape harvest by feeding onpollen flowers Therefore, ate prevention steps have been undertaken tocontrol occurrence

appropri-of this insect Pyrethroids are insecticides commonly usedtorestrict insect pests Many populations of pollen beetle developedresistance towards the pyrethrin analogues Resistance might be-caused by: mutations in the pyrethroid-binding site in voltage-sensitive sodium channel (knockdown resistant) or other genesinvoled in detoxication, for example esterases or oxidases, or bychanges in their gene expression level In our study we haveinvestigated significance of mutational changes within partial

sequence of cytochrome oxidase subunit I (mtCOI) gene of Polish

populations of pyretroid resistant pollen beetles insects Total

DNA was isolatedfrom thirteen Polish populations of M aeneus

followed by PCR reaction with primers amplifying mtCOI gene

sequence Afterwards, SSCP analysis (single strand conformation

polimorphism) was performed followed by DNA sequencing To

assess whether analyzed samples were in homo- or heterozygotic

state HRM (high resolution melting analysis) was performed

The studied DNA sequences were analyzed with BioEdit,

GENE-DOC and BLAST software Then analysis of secondary structure

of the mtCOI protein was done with psipred, jnet and sam and

profsec software The comparison between nucleotide sequences

of mtCOI with those available in GeneBank (UK populations)

has shown several point changes present within considered gene

fragment Single G849>A849 substitution was readily detected

with HRM, but it does not change amino acid Other mutations

found, changed amino acids sequence, however bioinformatical

modelling has localised them on the ends of helises, outside of

the core of the enzyme Therefore, we suppose that all changes

observed might occur due to geographical isolation of examined

populations of M aeneus and are not significant to enzymatic

abilities nor structure of the cytochrome oxidase, especially that

we could not refer it to population completly sensitive to

pyreth-roids, because such population was not identified in Poland

Therefore we suggest that different detoxification mechanisms,

for example increased level of esterases or oxidases might be

asociated with pyrethroid resistance in Polish strains of pollen

beetle

P1–99

Identification of human blood group A/A-Leb/y

and B/B-Leb/y glycotopes co-expressed from

two distinct ovarian cyst glycoproteins

A M Wu1, S Y Yu2, Z Yang3and K H Khoo4

1

College of Medicine, Chang-Gung University, Tao Yuan,

TAIWAN,2Institute of Biochemical Sciences, National Taiwan

University, Taipei, TAIWAN,3Institute of Molecular and Cellular

Biology, Chang Gung University, Tao-yuan, TAIWAN,4Academia

Sinica, Institute of Biological Chemistry, Taipei, TAIWAN

Although the individual human blood group A and B

determi-nants are well defined, their co-expression pattern on a particular

glycan carrier in individuals of blood group AB status has not

been delineated To address this issue, complex O-glycans were

isolated from two distinct sources of human ovarian cyst

glyco-proteins (HOC 89 and Cyst 19) and profiled by advanced mass

spectrometry analyses, in conjunction with defining their binding

characteristics against a panel of lectins and monoclonal

antibod-ies The major O-glycans of HOC 89 were found to correspond

to sialyl Tn, mono- and disialyl Tstructures, whereas those of

Cyst 19 were apparently much less sialylated, extended to larger

sizes, and overall more heterogeneous A minimal structure that

carries both A and B determinants on the same molecule was

identified, in which the A epitope is attached directly to the core

GalNAc, whereas the B epitope is preferentially located on the

six arm of a core two structure Both arms can be further

extended with internal fucosylation that appears to be restricted

to those non-sialylated chains already carrying the terminal ABH

determinants, thus giving rise to rather prominent A/B-Leb/y

gly-cotopes on larger O-glycans

P1–100 Degeneration of lipocalin-type prostaglandin D synthase in cerebrospinal fluids from patients with rheumatoid arthritis

Y Yamamoto1, H Sato2, S Fukasaku3, K Takeuchi4,

A Okazaki4, K NIshimura4, M Izeki4and E Inada4

1Tokyo Metropolitan Institute for Medical Science, Cancer peutics, Tokyo, JAPAN,2Tokyo Metropolitan Cancer and Infec-tious Diseases Center Komagome Hospital, Anesthesiology, Tokyo,JAPAN,3Juntendo University School of Medicine, Orthopedics,Tokyo, JAPAN,4Juntendo University School of Medicine, Anes-thesiology, Tokyo, JAPAN

Thera-Introduction: The cause of rheumatoid arthritis (RA) pathology

is still not fully known We studied disease-specific proteins inthe cerebrospinal fluid (CSF) of patients with RA by proteomicanalysis L-PGDS, a major component in the central nervous sys-tem, is a dual functional protein acting as a PGD2-synthesizingenzyme and a transporter for lipophilic ligands Enzymatic kinet-ics was analyzed by measuring the level of PGD2, and the role ofL-PGDS in RA is discussed

Methods: Proteins in CSF were assayed by isoelectric focusingand SDS-polyacrylamide gel electrophoresis Protein spots wereanalyzed and identified by mass spectrometry for expression-changed spots PGD2and 15-deoxy-D12, 14-PGJ2(15d-PGJ2) lev-els were measured

Results and Discussion: Differential analysis of proteins inCSF of RA and controls by two-dimensional gel electrophoresisrevealed several expression-changed proteins, among which theexpression of lipocalin-type prostaglandin D synthase (L-PGDS)

in CSF of RA was 1.5-fold stronger than that in controls Wefocused on L-PGDS, a pain-regulatory protein, and despite anapparent increase in its expression, the level of PGD2in RA sam-ples was significantly lower than in controls The level of 15d-PGJ2, a non-enzymatic degraded product of PGD2, was similarlylow The decrease of these products was evaluated by examiningthe reactivity of CSF proteins and the anti-L-PGDS antibody,which showed two primary bands: one of an L-PGDS monomerand one with a larger molecular weight These results suggestthat L-PGDS in RA patients may associate with other compo-nents and that the conformational change might be due to adecrease in L-PGDS activity

P1–101 Effects of resveratrol on oxidant/antioxdant status and sirtuin 1 and Matrix

metalloproteinase-9 mRNA expression in streptozotocin-induced diabetic rat hearts

A S Yar1, E Alp1, D Sahin2, N Altan2and S Menevse1

1Gazi University Faculty of Medicine, Medical Biology andGenetics, Ankara, TURKEY,2Gazi University Faculty ofMedicine, Medical Biochemistry, Ankara, TURKEYDiabetic cardiomyopathy is related directly to hyperglycemia thatincreases oxidative stress inducing cardiac damage in animals andhumans with diabetes Sirtuin 1 (SIRT1) has been implicated inmodulating transcriptional silencing and cell survival Resveratrol(RSV), exhibits potent antioxidative and anti-inflammatoryeffects, has potent pharmacological agonist of SIRT1 activity.The reduction of SIRT1 activity because of high glucose is corre-

lated to activation of Matrix metalloproteinase-9 (MMP-9)

tran-scription MMP-9, belong to a large MMP family capable of

degrading the constituents of extracellular matrix, is decreased by

hyperglycemia The present study was designed to clarify the

effect of RSV treatment on diabetic cardiomyopathy and to

determine the MMP-9 and SIRT1 mRNA expression and ferrous

oxidation xylenol orange (FOX), malondialdehite (MDA) levels

and superoxide dismutase (SOD) activity in diabetic rat hearts

Three months-old, 44 Wistar albino male rats were used for the

study Rats were divided into six groups After the induction of

Streptozotocin (55 mg/kg), every day 10 mg/kg RSV was

admin-istered intraperitoneally for 4 weeks In this study, SIRT1

mRNA levels decreased in diabetic group (P < 0.05) SIRT1

mRNA levels increased in RSV treated diabetic group when

com-pared to the diabetic group (P < 0.05) On the other hand,

RSV was no significant effects on MMP-9 mRNA expression

among groups (P > 0.05) SOD activity decreased in RSV

trea-ted sodium sitrat buffer (sham) control and RSV treatrea-ted diabetic

group (P < 0.05) In conclusion, higher doses or different

peri-ods of RSV treatment may alter antioxidant capacity Activation

of SIRT1 by RSV may prevent proinflammatory phenotypic

alterations in heart tissues

P1–102

Molecular cloning of a wheat a-amylase

inhibitor and its functional studies

S Yasothornsrikul1and G R Reeck2

1Biochemistry, Naresuan University, Phitsanulok, THAILAND,

2Biochemistry, Kansas State University, Manhattan, NY, USA

The kernels of wheat contain a number of proteins which can

inhibit a-amylases of various origins These protein inhibitors

can be classified into subfamilies and families on the basis of

their amino acid sequence similarity The wheat 0.28 a-amylase

inhibitor can inhibit Tenebrio molitor a-amylase but has very

little effect on human salivary a-amylase We have isolated a

cDNA clone, W22.1, encoding a wheat a-amylase inhibitor and

investigated its inhibitory function towards a-amylases from

various sources The cDNA clone consists of 435 nucleotides

flanked by a 33 nucleotide 5¢-untranslated sequence and a

3¢-non coding sequence of 126 nucleotides One putative

polyaden-ylation signal is located 47 base pairs upstream from the

poly-adenylation site The longest open reading frame translates into

145 amino acid residues, with 30 residues of a signal peptide

The deduced amino acid sequence shows that it belongs to the

wheat 0.28 a-amylase inhibitor family We expressed and

puri-fied the mature protein from clone W22.1 in E coli, and its

inhibitory activity against a-amylases from Sitophilus oryzae,

Tribolium casteneum, and Tenebrio molitor was analyzed

Results reveal that the E coli-expressed protein from the W22.1

clone can inhibit a-amylase activities of SOA1, SOA2, TCA1,

20 ± 7%, and 73 ± 5%, respectively These data suggest

speci-ficity and selectivity of this a-amylase inhibitor towards

a-amy-lases from Sitophilus oryzae Such characteristic of this

inhibitor could be further used as a model to study structure/

function relationships toward specificity and selectivity of other

a-amylase inhibitors of the same family

P1–103 Using RNAi-based high-throughput screen to identify novel signaling pathways regulating oral cancer cells proliferation

M H Yeh1, P Y Lee2, Y L Chen3, W L Tsai3, T C Tsai4,

M H Tsai5and M C Kao6

1National Defense Medical Center, Graduate Institute of lifesciences, Taipei, TAIWAN,2Graduate Institute of Basic MedicalScience, China Medical University, Taichung, TAIWAN,

3Department of Medical Laboratory Science and Biotechnology,China Medical University, Taichung, TAIWAN,4Department ofMicrobiology and Immunology, National Chiayi University,Chiayi, TAIWAN,5Department of Otolaryngology, China MedicalUniversity Hospital, Taichung, TAIWAN,6Department ofBiological Science and Technology, China Medical University,Taichung, TAIWAN

Oral carcinoma is a worldwide healthy issue and the first leadingcause of head and neck cancer death in Taiwan Moreover, oralcancer patients often present symptoms at a late stage and show

a high recurrence rate after treatment Therefore, to identifynovel biomarkers for early diagnosis or clinical oral caner ther-apy is an urgent topic In this research we employed the ‘anti-ki-nome’ and ‘anti-phosphatome’ RNAi subsets (including 737kinases, 209 phosphatases and 30 genes with dual function) fromthe RNAi consortium (TRC), to high-throughput screen and sys-temically identify the novel regulators of oral cancer cells Thescreen results showed two groups of genes: growth inhibitiongroup (GIG) contained 51 genes revealing more than 90%growth inhibition; and growth promotion group (GPG) con-tained 42 genes revealing more than one fold of growth promo-tion from human oral cancer HSC-3 cells These two groups ofgenes were submitted to MetaCore analysis, resulting in fourand three major putative molecular pathways in GIG & GPGrespectively They are TGF-beta receptor type II, FLT3, IPP-1,and EGFR pathways in GIG, and ErbB2/ErbB4-IGF-1 receptor,Insulin receptor and p57 pathways in GPG These novel regula-tors may have potential as biomarkers for early diagnosis of theoral cancer Moreover, these regulators will also be used aspotential targets for anti-cancer drug design and help to develop

a novel method to combat with oral cancer

P1–104 Identification of GPI-anchored proteins using position specific score matrix

Y Ikeda1, O Oura1, T Sasaki1, Y Yabuki2and M Ikeda3

1Department of Electronics and Bioinformatics, Meiji University,Kawasaki, JAPAN,2Information and Mathematical ScienceLaboratory Inc., Life Science Group, Tokyo, JAPAN,3Consoli-dated Research Institute for Advanced Science and Medical Care,Waseda University, Tokyo, JAPAN

Glycosylphosphatidylinositol (GPI) is one of the most importantpost-translational modifications, which can bind soluble polypep-tides to plasma membrane In this regard, the development ofcomputational methodology to predict GPI modification fromprotein sequences is highly awaited GPI-anchored protein hassignal peptides in the N-terminal region because the GPI anchor

is modified in the endoplasmic reticulum The propeptide of anchored protein, which is a polypeptide positioned behind theGPI-anchored position (omega-site), is removed The GPI-anchored protein has the propeptide consists of hydrophobic resi-dues and small amino acid residues exist near the omega-site Aprediction method based on the characteristics of C-terminalamino acids has been reported by Eisenhaber et al (JMB, 1999)

Here we describe a new method for the discrimination of

GPI-anchored proteins, which uses the position specific score matrix

(PSSM) combined with the hydropathy profile First, the

sequences of mammalian GPI-anchored proteins (411 sequences

from SWISS-PROT ver 54.0) were scanned in terms of average

hydrophobicity, and non-GPI-anchored proteins having similar

hydropathy profiles to GPI-anchored proteins were used as

nega-tive control (368 sequences) Sequences were aligned according to

residue size in the C-terminal region, and position-specific amino

acid propensity depending on alignment position was analyzed

for both GPI-anchored proteins and negative controls ThePSSM was devised using each amino acid propensity and thematching score was estimated for each dataset The thresholdwas set from the score distribution The discrimination accuracybetween GPI-anchored proteins and negative controls was higherthan 90%, evaluated on the basis of the cross-validation test.Reference:

1 Eisenhaber B, Bork P & Eisenhaber F Prediction of potentialGPI-modification sites in proprotein sequences J Mol Biol1999; 292: 741–758

P2 Protein Structure and Interactions

P2–1

Investigation of blip – beta-lactamase binding

in-vivo

A E Akkaya1, A Hortacsu1, E O Olmez1and B S Akbulut2

1Department of Chemical Engineering, Bogazici University,

Istan-bul, TURKEY,2Department of Bioengineering, Marmara

University, Istanbul, TURKEY

The widespread use of beta-lactam antibiotics has caused bacteria

to acquire resistance to these antibiotics which is a major health

problem The use of beta-lactamase inhibitors is one of the

strat-egies to activate the therapeutic value of beta-lactam

antibiot-ics.Recent studies show that beta-lactamase inhibitor protein

(BLIP) from Streptomyces clavuligerus has inhibitory properties

against class A beta-lactamases such as TEM-1 In this work,

experimental studies were carried out to investigate in-vivo

beta-lactamase inhibition by BLIP For this purpose, BLIP was cloned

into expression vector pET-26b(+) and expressed as a his-tagged

periplasmic proteinin E coli BL21 (DE3) cells for binding and

inhibition studies In order to investigate in-vivo beta-lactamase

inhibition by BLIP in the periplasm, pUC18 plasmid, carrying

the gene for R-TEM-1 beta-lactamase was transformed into the

E coli BL21 (DE3) cells harboring recombinant pET-26b(+)

vector BLIP expression was under the control of IPTG

induc-tion whereas beta-lactamase expression was constitutive

Beta-lac-tamase inhibition was monitored by analyzing the growth profile

of the recombinant cells Furthermore osmotic shock fluid from

these cells was analyzed on SDS-PAGE for the complex

forma-tion between BLIP and beta-lactamase

P2–2

The electron transfer complex between D.

gigas Superoxide Reductase and Rubredoxin

R Almeida, S Pauleta, I Moura and J J G Moura

REQUIMTE, Quimica, Caparica, PORTUGAL

Superoxide reductase (SOR) is a 29 kDa homodimeric

metallopro-tein present in the cytoplasm of Desulfovibrio (D.) gigas cells This

protein allows this anaerobic organism to survive under mild

expo-sure to aerobic atmospheres, by converting the radical anion

superoxide to hydrogen peroxide without formation of molecular

dioxygen The D gigas protein is a class II SOR, thus it contains

only one iron center per monomer: a Fe atom bound to four

histid-inyl residues’ side chains in the equatorial plane, with a fifth ligand

provided by a cysteinyl sulfur in the axial plane (1) Several

attempts have been made to identify and characterize the

electron-transfer (ET) partners of SOR (2) Rubredoxin (Rd) is proposed to

be one such partner This small (6 kDa) monomeric metalloprotein

is capable, among other things, of accepting electrons from

NADPH:Rubredoxin Oxidoreductase and transferring them to the

terminal oxidase of the oxygen detoxification pathway,

Rubredox-in:Oxygen Oxidoreductase Its metal center is very similar to center

I of class I SORs.ET has been demonstrated to occur between

SOR and Rd in vitro (2) In this work, we provide evidence for the

formation of the ET complex via 2D NMR titration This

experi-ment showed that the complex is on fast exchange in the NMR

time scale We were also able to determine the Rd aminoacid

resi-dues affected by the formation of the complex Furthermore, we

estimate a value for the dissociation constant (Kd) consistent with

the formation of a low-affinity, high-turnover complex

Acknowledgements: Fundacio para a Ciencia e Tecnologia –

MCTES (PhD grant SFRH/BD/25342/2005 and project POCI/

QUI/57741/2004); MARIE CURIE HOST FELLOWSHIPS

FOR EARLY STAGE RESEARCH TRAINING (1/4/04–31/5/08) n MEST-CT-2004-504391, ‘NMR in Inorganic StructuralBiology’; Financial support by the Access to Research Infrastruc-tures activity in the 6th Framework Program of the EC (Contract

#RII3-026145, EU-NMR) for conducting the research at CERM

is gratefully acknowledged

References:

1 Pereira AS, Tavares P, Folgosa F, Almeida RM, Moura I &Moura JJG Eur J Inorg Chem 2007; 18: 2569–2581

2 Auche´re F, Pauleta SR, Tavares P, Moura I & Moura JJG

J Biol Inorg Chem.2006; 11(4): 433–444

a copper-containing enzyme which catalyzes the hydroxylation ofmonofenols to o-diphenols and the oxidation of o-diphenols too-quinones by using molecular oxygen It is responsible for enzy-matic browning reaction occurring during the handling, storageand processing of fruits and vegetables The present work sought

to carry out a screening of PPO activity in four different Turkey(northwestern Turkey) plants, goosefoot plant (Chenopodium ant-helminthicum), common mallow (Malva sylvestris), dandelion (Ta-raxacum officinale) and cress (Lepidium sativum) consumed asfood and also used for medicinal purposes by local populations.The all plants were homogenized in 0.1 M sodium phosphatebuffer pH 7.0 with 0.5% PVP, 2% ascorbic acid and 5% Triton-X100 at 4C The extract was filtered through 4 layers of cheesecloth and centrifuged at 10 000 g for 15 min at 4C PPO activitywas determined with catechol as a substrate by measuring theincrease in absorbance at 420 nm The enzyme from each plantwas characterized for thermal stability, pH and kinetic with dif-ferent catechol concentrations at proper pH’s The results showedthat dandelion and goosefoot plant PPO have 11.74 and11.41 mM Kmvalues for catechol at pH 6.5 and 7.5 However,common mallow and cress have 23.5 and 20.99 mM Km valuesfor catechol at pH 7.0 and 5.0 have 23.5 and 20.99 mM Kmval-ues for catechol at pH 7.0 and 5.0 Dandelion and goosefootplant PPO are more active and efficient to catechol substrate atdifferent pH’s and 35C Thus, different plants have differentenzyme activities at the same conditions

P2–4 Lipocalin-type prostaglandin D synthase: a transporter of prostaglandin D2 and scavenger

of prostaglandin D2-degraded products

K Aritake1, M Sakata1, S Shimamoto2, T Tsurumura1and

Y Urade1

1Osaka Bioscience Institute, Molecular Behavioral Biology, Suita,JAPAN,2Pharmaceutical Science, Osaka University, Suita,JAPAN

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) isresponsible for the production of PGD2, a potent endogenoussleep substance, and is secreted into the cerebrospinal fluid (CSF)

as beta-trace, a major protein of human CSF L-PGDS is amember of the lipocalin gene family, which consists of smallsecretory transporter proteins of various lipophilic ligands such

as retinoic acid, retinal, biliverdin, bilirubin, gangliosides with

high affinities PGD2 is chemically unstable in aqueous solution

and non-enzymatically dehydrated to produce the J series of

PGs, such as PGJ2,D12-PGJ2, and 15-deoxy-D12,14

-PGJ2in vitro

However, the stability and metabolism of PGD2 in vivo remain

to be clarified In this study, we characterized the binding affinity

of L-PGDS to PGD2and its degraded products In the presence

of L-PGDS but not albumin, we could detect only PGD2 in

aqueous solution without any detection of neither metabolites

nor catabolites over 6 h Surface plasmon resonance analysis

revealed that PGD2bound to L-PGDS in a

concentration-depen-dent manner with high affinities of Kd = 20 nM, and its binding

was reversibly in the short duration On the other hand, the

degraded products, 15-deoxy-D12,14-PGJ2-binding to L-PGDS

was irreversible Using MALDI-TOF mass spectroscopy, we

showed that the binding of PGD2 to L-PGDS was covalent and

irreversible 15-deoxy-D12,14

-PGJ2 dose-dependently inactivatedL-PGDS enzyme activities We identified that Cys65 is the resi-

dues for the PGD2-binding, proven by peptidase-digestion and

site-directed mutagenesis We therefore propose that PGD2is

sta-bilized via binding to L-PGDS providing a stable transport

towards its receptors Moreover, L-PGDS consecutively acts as

scavenger for J-type PGs through covalent binding providing a

balance in the biological PGD2system

P2–5

Binding cooperativity and allosterism make

liver fatty acid binding proteins ideal

chaperones of lipids and lipid-functionalized

drugs

M Assfalg, M D’Onofrio, M Pedo’, S Zanzoni, C Cogliati,

M Guariento and H Molinari

Dipartimento di Biotecnologie, Universita’ degli Studi di Verona,

Verona, ITALY

Lipids are vital components of many biological processes and

crucial in the pathogenesis of numerous common diseases, but

the specific mechanisms coupling intracellular lipids to biological

targets and signalling pathways are not well understood (1)

Intracellular lipid chaperones known as fatty acid binding

pro-teins (FABPs) coordinate lipid responses in cells and are strongly

linked to metabolic and inflammatory pathways FABPs display

a wide range of sequence diversity, but share a common

struc-tural fold Among these proteins, human liver FABP is

increas-ingly attracting the interest due to its capability of regulating not

only fatty acids but also bile acids pathways This protein, which

is the most abundant cytosolic protein in hepatocytes, is

proba-bly the most versatile chaperone in terms of its ligand repertoire

As a consequence of its abundance, it was suggested that

L-FAB-Ps may be involved not only in the transport of endogenous

lip-ids, but also of exogenous lipophilic drugs Our group is

focussing its research on the elucidation of the complex binding

mechanisms of FABPs (2) Here we present recent results on the

structural and the dynamic characterization of protein-ligand

ad-ducts, obtained primarily by Nuclear Magnetic Resonance

(NMR) spectroscopy We describe measurements of translational

diffusion and site-specific protein mobility that have highlighted

functionally relevant protein regions These data, performed both

on L-FABP and on model proteins, have been combined with

calorimetry and mass spectrometry data: the emerging picture is

consistent with the occurrence of strong cooperative binding to

multiple sites, originated by protein allosterism (3) Furthermore,

we show very recent NMR data referred to experiments

per-formed on FABPs in solutions containing phospholipids vesicles

and in living cells The obtained results suggest that these

pro-teins can act as molecular switches allosterically activated by lipid

molecules and/or membranes in the regulation of lipid trafficking

As an application of the acquired knowledge, we show the acterization of the interaction of FABPs with lipid-functionalizedgadolinium chelates to be used as potential hepatospecific con-trast agents for MRI (4)

char-References:

1 Furuhashi M & Hotamisligil GS Fatty acid-binding proteins:role in metabolic diseases and potential as drug targets NatRev Drug Discov 2008;7(6): 489–503 Review

2 Eliseo T, Ragona L, Catalano M, Assfalg M, Paci M, Zetta L,Molinari H & Cicero DO Structural and dynamic determi-nants of ligand binding in the ternary complex of chicken liverbile acid binding protein with two bile salts revealed by NMR.Biochemistry2007;46(44):12557–12567

3 Pedo` M, D’Onofrio M, Ferranti P, Molinari H & Assfalg M.Towards the elucidation of molecular determinants of coop-erativity in the liver bile acid binding protein (Submitted)

4 Tomaselli S, Zanzoni S, Ragona L, Gianolio E, Aime S,Assfalg M & Molinari H Solution structure of the supramo-lecular adduct between a liver cytosolic bile acid bindingprotein and a bile acid-based gadolinium(III)-chelate, a poten-tial hepatospecific magnetic resonance imaging contrast agent

J Med Chem 2008;51(21):6782–6792

P2–6 Dysferlin interacts with alpha-tubulin in skeletal muscle

B A Azakir, C Therrien, S Di Fulvio and M SinnreichMontreal Neurological institute, Neurology, Montre´al, QC,CANADA

The pathogenic basis for many muscular dystrophies is theinability of the muscle cell to maintain its sarcolemmal integrity,due to defective or deficient membrane associated structural pro-teins or components of the membrane repair machinery Animportant protein implicated in surface membrane repair in mus-cle is dysferlin Mutations in dysferlin are a frequent cause forthe recessively inherited limb girdle muscular dystrophies(LGMD), defining the common subtype of LGMD2B In addi-tion, dysferlin mutations also cause Miyoshi Myopathy (MM)and distal anterior compartment myopathy, both distal forms ofmuscular dystrophy To better understand the cellular function

of normal dysferlin, we set out to identify novel protein bindingpartners Using affinity purification followed by liquid chroma-tography/mass spectrometry, we have identified a number of pro-teins in skeletal muscle, which showed association with dysferlin.Importantly, we were able to identify the few proteins that werepreviously shown to interact with dysferlin by co-immunoprecipi-tation The newly identified proteins fall into categories of surfacemembrane proteins, proteins involved in cellular trafficking and

in protein degradation We have identified alpha-tubulin as adysferlin binding partner and confirmed this interaction throughco-immunoprecipitation assays and co-localization studies Wefound the interaction to be calcium dependant and propose a rolefor alpha-tubulin in dysferlin trafficking after sarcolemmal injury

P2–7 Structural studies on the catalytic subunit of the oxaloacetate decarboxylase Na+pump

M Balsera, R M Buey, F W Winkler and X D LiPaul Scherrer Institute, Structural Biology, Villigen,SWITZERLAND

The oxaloacetate decarboxylase Na+ pump (OAD) is a functional enzyme that converts the free energy of a specific deca-

multi-rboxlylation reaction into an electrochemical gradient of Na+

ions across the membrane This membrane protein complex is

functional and essential in the anaerobic citrate fermentation

pathway of some bacteria It is composed of three subunits: a

bi-modular soluble alpha subunit that is posttranslationally

biotiny-lated and contains a metal ion in its catalytic center where the

decarboxylation of oxaloacetate to pyruvate occurs, a bitopic

gamma subunit that binds tightly to the alpha enzyme and

local-izes it to the membrane, and a multispan transmembrane beta

subunit that constitutes the Na+channel and whose function is

fully dependent on the transfer of the carboxyl group from the

decarboxylation reaction on the alpha subunit Large structural

arrangements have been proposed for the alpha subunit in order

to promote the carboxy-transfer reaction from its active site to

the beta subunit niche through biotin With the aim of

under-standing the molecular mechanisms underlying OAD, we have

carried out solution structural analyses by small-angle X-ray

scat-tering in combination with homology modelling on the enzymatic

subunit Our results let us hypothesize about the conformational

rearrangements associated with the carboxy-biotin transfer

mech-anism within the complex

P2–8

Saturation transfer difference NMR study on

the specific binding of sinomenine by human

serum albumin

G Bazylak1, C Ludwig2and U L Gunther2

1

Department of Pharmaco-Bromatology & Molecular Nutrition,

Faculty of Pharmacy Collegium Medicum Nicolaus Copernicus

University, Bydgoszcz, POLAND,2HWB NMR, CR UK Institute

for Cancer Studies University of Birmingham, Edbagston

Birmingham, UK

Sinomenine (SN), a natural dextrorotatory morphinan analog, is

alkaloid isolated from the Chinese medicinal plant Sinomenium

acutum It has immunomodulating and anti-inflammatory

acitivi-ties and is used in traditional Chinese arthritic and

anti-rheumatic therapies The in vitro human serum albumin (HSA)

binding rate of SN was estimated as 63% on the introductory

results of equilibrium dialysis assay Thus interaction process

occurring at the protein-solvent interface for SN-HSA system

was investigated by NMR using the saturation transfer difference

(STD) technique which is valid in the lM–mM range of the

ligand-protein binding constants Similar procedure was used for

the common tussive alkaloid codeine and non-steroidal

anti-inflammatory drug naproxen, thus the comparison of the binding

ability of SN and codeine towards two various forms of HSA

have been made In contrast to naproxen, exhibiting high binding

affinity to the both fatty-acid-loaded-HSA and

fatty-acid-free-HSA, we observed that SN binds exclusively to the second

men-tioned form of HSA, what is contrary to codeine which indicated

no binding affinity to these both forms of HSA The location of

specific SN binding site(s) of fatty-acid-free-HSA have been

pro-posed to explain these STD-NMR results, thus enabling in vitro

prediction of HSA binding ability and evaluate pharmacokinetic

properties of SN and co-administered NSAIDs during

pharmaco-logical treatment of rheumatoid arthritic subjects

Acknowledgement: Supported by the grant from EU NMR

project – Contract No RII3-026145

P2–9 Inhibition of A277V mutant of human butyrylcholinesterase by malachite green

K Biberoglu1, O Tacal1and H Akbulut2

1Department of Biochemistry, Hacettepe University School ofPharmacy, Ankara, TURKEY,2Department of Medical Oncology,Ankara University School of Medicine, Ankara, TURKEYHuman butyrylcholinesterase (BChE) is a serine hydrolase which

is toxicologically and pharmacologically important in scavengingand detoxifying cocain, organophosphorus pesticides, carbamatepesticides and chemical warfare agents In this study, we testedthe role of Ala277 in inhibition of BChE by malachite green Themutation A277V was introduced into human BChE by PCR-mediated site-directed mutagenesis The presence of A277V muta-tion was verified by automated sequencing Recombinant wild-type and A277V mutant of human BChE were transientlyexpressed in human embryonic kidney cells and partially purified

by procainamide-Sepharose affinity chromatography For kineticstudies, BChE activity was assayed spectrophotometrically at25C, in 50 mM MOPS buffer (pH 8), containing 0.05–0.4 mMbutyrylthiocholine as substrate, and 0–3 lM dye Malachite greenacted as linear mixed inhibitiors of mutant enzyme and wild typeBChE Based on the rapid equilibrium inhibitory model, Kival-ues were 0.166 mM for A277V mutant enzyme and 0.044 mMfor recombinant wild type BChE In conclusion, malachite greenwas found four-fold less effective as an inhibitor of A277Vmutant, pointing Ala 277 may be important in the binding ofmalachite green to BChE

Acknowledgements: This study was supported by a grant(SBAG-3677) from Scientific and Technical Research Council ofTurkey

P2–10 The study of multimerization leading to the assembly of immature particles of Mason– Pfizer monkey virus and Human

immunodeficiency virus

K Bohmova, R Hadravova, J Stokrova, I Pichova and

M RumlovaGilead Sciences & IOCB Research Centre Institute of OrganicChemistry and Biochemistry Academy of Sciences of the CzechRepublic, Prague, Czech Republic, Viral and microbial proteins,Prague, CZECH REPUBLIC

The basic interactions leading to the assembly of immature viral particles are mediated by protein-protein and protein-nucleicacids contacts Protein-protein interactions are mediated mainly

retro-by capsid protein (CA) molecules whereas nucleocapsid protein(NC) is responsible for the binding of RNA During the assem-bly, two Cys-His boxes of NC that are highly conserved amongretroviruses specifically incorporate retroviral genomic RNA.However, the role of NC in the process of immature capsidassembly remains unclear Studies from several laboratories sug-gest that the NC might function as a dimerization domain, as areplacement of NC protein by heterologous dimerization domainallows the polymerization of Gag and virus-like particles assem-bly To study the multimerization effect on the assembly ofimmature particles, the nucleocapsid proteins from both, Mason-Pfizer monkey virus (M-PMV) and Human immunodeficiencyvirus (HIV-1), were replaced by heterologous dimerization andtrimerization leucine zipper domains from GCN4 and the dimer-ization domain from CREB Using a bacterial expression/assem-bly system, in vitro and in vivo assembly assays was found that

replacement of entire M-PMV NC and HIV-1 NC domains with

both, GCN4 and CREB multimerization domains, completely

abrogated the assembly The efficient assembly of M-PMV and

HIV-1 particles was observed only in the presence of basic

resi-dues of either N-terminal part of NC or CREB, suggesting the

importance of these residues for an initiation of virus-like particle

assembly The impact of these replacements on the immature

par-ticles assembly will be also studied in vivo in 293 cells

P2–11

Role of the protein S1 in translation initiation

and ribonuclease RegB activation; an NMR and

SAXS analysis

F Bontems1, C Sizun1, J B Cre´chet2, J Perez3, M Uzan4,

P Aliprandi1, P Giraud1, F Mareuil1and S Caputo1

1Institut de Chimie des Substances Naturelles, RMN structurale,

Gif-sur-Yvette, FRANCE,2Ecole Polytechnique, RMN structurale,

Palaiseau, FRANCE,3Synchrotron Soleil, SWING beam line,

Gif-sur-Yvette, FRANCE,4Institut Jacques Monod, dynamique du

ge´nome, Paris, FRANCE

S1 is the largest protein of the Escherichia coli ribosome It is

strictly required to the correct recognition of the translation

initi-ation codon of most, if not all, messenger RNAs by the

ribo-somal 30S subunit It is also used by several bacteriophages at

their own benefit It is one of the subunits of the phage Qb

repli-case It also enhances the reaction rate of the phage T4

endoribo-nuclease RegB, which inactivates the phage early mRNAs when

their translation is no more required by cleaving them in the

mid-dle of their Shine-Dalgarno sequence We are interested in

ana-lyzing and comparing S1 mode of action in these different

functions By combining biochemical, NMR and SAXS

tech-niques, we identified a functional fragment of the protein formed

of three domains and analyzed its structure and its interactions

with different RNAs corresponding to different functions We

also undertook to compare the interactions of S1 with the

ribo-somal 30S subunit and with the Qb replicase All our results

sug-gest that the different S1 functions are associated to an unique

mechanism We also undertook to revisit the relationships

between the forms of the S1 protein found in the gram-negative

and the gram-positive bacteria

P2–12

Partial purification, characterization, molecular

cloning, recombinant expression and

immunocytochemical localization of a

beta-1,3-glucanase from the midgut of

Spodoptera frugiperda larvae

I Bragatto1, F Ariel Genta2, W Ribeiro Terra1and C Ferreira1

1University of Sao Paulo Institute of Chemistry, Biochemistry, Sao

Paulo, BRAZIL,2Instituto Oswaldo Cruz FIOCRUZ Brazil,

Dbbm, Sao Paulo, BRAZIL

b-1,3-glucanases hydrolyze b-1,3-glucans from fungal or plant cell

walls and are little known in insects b-1,3-glucanase activity is

similar along the midgut of S frugiperda The purified enzyme

(SLAM) has a molecular mass of 37 kDa and optimum pH of

9.0 Using laminarin, the enzyme has kcat= 8.72 s-1,

Km= 0.14 ± 0.004% (w/v) SLAM does not signicantly form

small products, hence being an endo-b-1,3-glucanase, with

multi-ple attack degree of 0.4 A cDNA that codes for a protein similar

to those in family 16 glucanase was cloned from a midgut cDNA

library The protein produced in Escherichia coli BL21-DE3 cells

using a pET28-A expression system, although inactive, was used

to raise polyclonal antibodies in rabbits The antiserum nizes the major b-1,3-glucanase from midgut and by immunocy-tolocalization showed that the enzyme is present in midgutcolumnar cells, associated to the cell glycocalyx and in largesecretory vesicles from posterior midgut This distribution agreeswith that of tissue b-1,3-glucanase activity SLAM produced inPichia pastoris GS115 cells using a pPIC9K expression systemhas a Km of 0.13 ± 0.08% (w/v) and optimum pH of 9.0 withlaminarin as substrate, similar to the parameters previouslyobtained for purified enzyme from the midgut The action ofSLAM against several substrates showed that laminarin is thebest substrate The straight substrate specificity of the enzymemay be due to an adaptation to hydrolyse fungal polysaccharidesand the enzyme may help in preventing midgut infections byfungi

recog-P2–13 Kv1.2-based model of the calcium channel is consistent with published data obtained by substituted cysteine accessibility method

I Bruhova and B ZhorovBiochemistry and Biomedical Sciences, McMaster University,Hamilton ON, CANADA

Calcium channels are important drug targets, but their lution structures are unavailable Zhen et al (2005) used thesubstituted cysteine accessibility method to identify pore-liningresidues in CaV2.1 and concluded that their results are inconsis-tent with published sequence alignments between Ca2+ and K+

high-reso-channels This conclusion casts doubts on homology models of

Ca2+ channels Here we modeled MTSET-substituted Cysmutants (MTSETC) of CaV2.1 using the alignment reported forthe Kv1.2-based model of CaV1.2 (Tikhonov & Zhorov, 2008).Energetically favorable orientations ofMTSETC were predicted byMonte-Carlo minimizations In our models, the inner-helix resi-dues in positions i15, i18, i19, and i22 face the pore In agree-ment with this, MTSET blocks CaV2.1 with Cys substitutions inpositions i15 and i19 of all four domains In our models, Ci22ofany domain is surrounded by large hydrophobic residues, whichwould prevent reaction with MTSET This explains why MTSETdoes not block channels with Ci22in any domain According tocalculations,MTSETC2i18and MTSETC4i18 occlude the inner pore,whereasMTSETC1i18andMTSETC3i18protrude into domain inter-faces This explains why MTSET blocks C2i18and C4i18, but not

C1i18 or C3i18 Intriguingly, MTSET blocks the outer-helixmutants C2o10and C4o10, but not C3o10 These observations alsoagree with our models, in whichMTSETC2o10 and MTSETC4o10

extend toward the inner pore, whereas access ofMTSETC3o10 tothe inner pore is prevented by F3i18 Our calculations validate thereported alignment between Ca2+and K+channels and suggestsimilar dispositions of transmembrane helices in these channels.Supported by CIHR

P2–14 CV-IIL lectin: an ideal helper for understanding mutation-caused binding preference changes

in the PA-IIL lectin family

L Brunova1, J Adam1, M Pokorna1and M Wimmerova2

1

National Centre for Biomolecular Research, Masaryk University,Brno, CZECH REPUBLIC,2National Centre for BiomolecularResearch and Department of Biochemistry, Masaryk University,Brno, CZECH REPUBLIC

Lectins are sugar-binding proteins that play important role in ognition processes Bacterial lectins could be crucial agents in the

rec-process of colonization and in the bacterial virulence by binding to

specific saccharide moieties at host cells´ surface Understanding

the nature of the interaction can be helpful in designing

anti-adhe-sion therapy CV-IIL is a lectin from human opportunistic

patho-gen Chromobacterium violaceum and belongs to PA-IIL lectin

family, which includes structurally similar lectins from several

other pathogenic bacteria The interaction between CV-IIL and

the saccharide is mediated by two calcium ions in the binding site

The specificity of the interaction is directed by the composition of

the binding site, namely a triad of amino acids called

‘specificity-binding loop’ in the positions 22-23-24 Opposed to other members

of the PA-IIL family that prefer either mannose or fucose as a

binding partner, CV-IIL lectin could recognize both sugars with

high affinity and specificity (1) This feature makes CV-IIL an ideal

candidate for investigating the change of sugar preference in either

direction upon amino acid mutations Specific mutations in

bind-ing site were prepared by in vitro mutagenesis method Surface

plasmon resonance was used to characterize their binding

prop-erty The in silico mutagenesis method (complemented with

molec-ular docking) offers additional insight into the structural reasoning

behind the sugar preference

Acknowledgements: This work is supported by Ministry of

Education of the Czech Republic (MSM0021622413, LC06030)

Reference:

1 Pokorna´, M et al., Unusual Entropy-Driven Affinity of

Chro-mobacterium violaceumlectin CV-IIL toward Fucose and

Man-nose Biochemistry 2006; 45(24): 7501–7510

P2–15

Interaction of bovine serum

albumin-polyelectrolyte followed by a novel polarity

sensor

Y Budama Battal1, Z Mustafaeva1, S Ercelen2and

A P Demchenko3

1Bioengineering Department, Yildiz Technical University, Istanbul,

TURKEY,2Tubitak Marmara Research Center, Genetic

Engineer-ing and Biotechnology Institute, Kocaeli, TURKEY,3A.V Palladin

Institute of Biochemistry, Kiev, UKRAINE

Introduction: Serum albumin is the most abundant of all

pro-teins in blood plasma of many species, where it reaches a

concen-tration of about 40 mg/ml Biological and pharmacokinetic

functions of albumin are very important It is the major

trans-porter in the blood of non-esterified fatty acids as well as of

many other low-polar metabolites and drugs In our previous

studies, the complex formation of Bovine Serum Albumin (BSA)

with anionic polyelectrolyte (polyacrylic acid) in aqueous solution

was examined using the turbidimetric titration, HPLC and

fluo-rescence methods It is revealed that the character of the

interac-tions and solubility of the polycomplex particles depends on the

protein/polyelectrolyte ratios and the pH of solution

Methods: The polycomplexes have been examined using

UV-VIS Spectrophotometer, Steady-State Flourescence Spectrometer

Two seperate methods were used to determine the conformation

of BSA in BSA-PAA complexes by adding prob, which is the

new ratiometric 3-hydroxychromone derivative

2-(6-diet-hylaminobenzo[b]furan-2-yl)-3-hydroxychromone (FA) displays a

dramatic solvent-dependent transformation of fluorescence

spec-tra in the range of low-polar envoriment In the first method,

BSA-PAA complexes were formed and examined under different

ratios and pH values by adding equal amount of FA probe In

the second method, different ratios of polyelectrolyte solutions

were added to BSA-FA complexes and examined under different

pH values

Results and Conclusions: The change in the fluorescence erties of FA bound to the hidrofobic moiety of BSA upon thebinding of PAA let us think about the change in conformation

prop-of BSA Experiment will continue to examine this effect at ent pH values

differ-P2–16 Investigation of in-vivo beta-lactamase inhibition by beta-lactamase-inhibitor-protein based peptides

N Budeyri1, E O Olmez2and B S Akbulut3

1

Bioengineering, Marmara University, Istanbul, TURKEY,

2Chemical Engineering, Bogazici University, Istanbul, TURKEY,

3Biongineering, Marmara University, Istanbul, TURKEYBeta-Lactamase-Inhibitor-Protein (BLIP) is an effective inhibitor

of class A beta-lactamases such as TEM-1, SHV-1 and SME-1that render bacteria resistant to beta-lactam antibiotics Due tothe resistance of beta-lactamase developed against small organicinhibitors, beta-lactamase inhibition by BLIP is an interestingresearch field for peptide based inhibitor development In-vitrostudies have shown that small peptides derived from BLIP havethe potential to be used as inhibitors (1,2) However there has yetbeen no report on the in-vivo behavior of these possible therapeu-tic agents against beta-lactamase inhibition It is well documentedthat large biotinylated peptides can readily be transported intogram (-) bacteria such as E coli (3) In this study, this tool hasbeen used to investigate the in-vivo inhibition potential of two N-terminal biotinylated BLIP based peptides of different lengths[Biotin-NH2-AAGDYY-COOH (residues 46-51BLIP) and Biotin-

30-49BLIP)] Escherichia coli K12 strain harboring the pUC18 mid that carries the gene of RTEM-1 beta-lactamase, was usedfor periplasmic beta-lactamase production Following a predeter-mined time of incubation of the cells in the presence and absence

plas-of the biotinylated peptides, samples were taken to count colonyforming units In addition to this, osmotic shock fluids from bothcultures were assayed for beta-lactamase activity using CENTA

as the substrate The results obtained were used to evaluate tiveness of this method for peptide transport and potential of thetwo peptides for in-vivo beta-lactamase inhibition

effec-References:

1 Rudgers et al., Protein Eng 2001; 14(7) 487–492

2 Rudgers et al., Antimicrob Agents Chemother 2001; 45(12)3279–3286

3 Walker et al., Appl Environ Microbiol 2005; 71(4) 1850–1855

P2–17 Allozymic variations in the genus dryomys (rodentia: gliridae) distributed in Turkey

S Bulut1, N Yigit2, E Colak2, R Colak2, P Cam3and

F Saygili2

1Faculty of Science-Art, Biology, Corum, TURKEY,2Faculty ofScience, Biology, Ankara, TURKEY,3Faculty of Science-Art,Biology, Sinop, TURKEY

Allozymic variations were analyzed in two species representingthree populations of Dryomys nitedula from Central Anatolia,Turkish Thrace and the Black Sea region of Turkey and onepopulation of Dryomys laniger, is endemic mammal species forTurkey, from the southern part of Turkey The genetic analysiswas carried out on 36 specimens on 17 enzyme systems Nine oftwenty loci (a-Gpdh, Me, Idh-1, G6pdh, Sod, Ca-2, Acon, Mpi,Pgm) were found to be polymorphic in D nitedula populations

and five polymorphic loci (Idh-1, Sod, Fum, Pgm ve Mpi) in

D laniger Ldh and Gpi loci were fixed in the different alleles

between D nitedula and D laniger The percentage of

polymor-phic loci varies from 25 to 40 in D nitedula populations and is

25 in D laniger population The mean value of the fixation index

was FST = 0.13 indicating moderate/high genetic differences

between the subpopulations of D nitedula Nei’s measure of

genetic distance varied from D = 0.006 to D = 0.030 between

the subpopulations of D nitedula and the highest D value

appeared between the Black Sea subpopulation of D nitedula

and D laniger (D = 0.187)

P2–18

Tracking of single A-beta oligomers on the

plasmamembrane reveals an heterogeneous

dynamic behaviour

M Calamai1, M Zanni2, F Chiti3and F Pavone1

1Lens – European Laboratory for Non-linear Spectroscopy,

University of Florence, Sesto Fiorentino (Florence), ITALY,

2Department of Chemistry, University of Wisconsin, Madison, WI,

USA,3Department of Biochemistry, University of Florence,

Florence, ITALY

Alzheimer’s disease (AD) is a neurodegenerative pathology

char-acterized by the extracellular deposition in the brain of fibrillar

aggregates consisting predominantly of A-beta peptide Recent

evidence points at early prefibrillar A-beta oligomers, rather than

mature amyloid fibrils, as main cause for neurotoxicity A-beta

oligomers interact with the cellular membrane, but the specific

manner through which they bind and cause neuronal dysfunction

is still a matter of debate Distinct results indicate that oligomers

may insert non-specifically into the lipid bilayer, or bind to

spe-cific targets, such as excitatory post-synaptic locations and

gan-gliosides characteristic of lipid rafts In general, most of these

studies have been focused on the averaged features of an

ensem-ble of molecules Here, we have been aensem-ble to successfully monitor

the mobility of single A-beta oligomers on the plasmamembrane

of living NRK fibroblasts Preformed oligomers were incubated

with cells and subsequently labelled with monoclonal primary

antibodies and secondary Fab fragments coupled to small (10–

30 nm) and extremely photostable fluorescent probes called

‘quantum dots’ (QDs) Single QDs bound to the oligomers were

then tracked The analysis of the trajectories shows that the

olig-omers display an heterogeneous dynamic behaviour Some

oligo-mers laterally diffuse on the membrane following a free

Brownian motion, while others show a highly confined mobility

The latter result, although preliminary, suggests a potential

inter-action of the oligomers with molecules linked to the cytoskeleton

The full characterization of the membrane dynamics of A-beta

oligomers will enable a better understanding of AD and thus

contribute to the development of new therapies

P2–19

AFM and SAXS Structural Analises of two

Centrins from Fungi Blastocladiella emersonii

A I Camargo1, P C Camargo2, L Barbosa3, R Itri3and

L Beltramini1

1

Instituto de Fisica de Sao Carlos, Biophysics, Universidade de Sao

Paulo, Sao Carlos, BRAZIL,2Physics, Universidade Federal do

Parana, Curitiba, BRAZIL,3Biophysics, Universidade de Sao

Paulo, Sao Paulo, BRAZIL

Centrins are EF-Hand calcium binding protein required for

duplication of centrioles It may also play a role in severing of

microtubules by causing calcium-mediated contraction, usually

located at microtubule-organizing centers The majority of trin in the cell is non-centrosomal, whose function is not yetclear Two centrins, BeCen1 and BeCen3, from the aquatic fungiBlastocladiella emersonii were overexpressed, purified and charac-terized by CD, AFM and SAXS Both proteins showed CDspectra compatible with alpha helix content and exhibited a self-assembly capacity depending on Calcium presence and tempera-ture The transitional temperature (Tm) of 42C for BeCen1 and49C for BeCen3 had an increase of 5C in Calcium presence.After denaturation process (90C), only BeCen1 showed a refoldcapacity (submitted manuscript) Recent results showed that bothcentrins are in soluble form below 35C (with or without Cal-cium) and above this temperature, the formation of insolubleorganized filaments happens in calcium presence The drasticconformational changes as a function of temperature in the Cal-cium presence (from monomeric, oligomeric and filaments forms)were identified by AFM and SAXS AFM images of BeCen1 andBeCen3, deposited on mica slice, showed organized filaments atdifferent temperatures Measurements of SAXS showed the oligo-merization process, starting from monomers until trimmersbetween 20 and 35C, above these temperatures the oligomersare formed, in the absence and presence of Calcium ions.Acknowledgement: Supported by Brazilian agencies: FAPESP,CNPq, CAPES

cen-P2–20 The allosteric modulation of heme-serum albumin

M Coletta1,2, A Bocedi3, C Ciaccio1, A Di Masi4,

F P Nicoletti5, G Fanali6, G De Sanctis7, G Smulevich5,

M Fasano6and P Ascenzi4

1

Department of Experimental Medicine and Biochemical Sciences,University of Roma Tor Vergata, Roma, ITALY,2InteruniversityConsortium for the Research on Chemistry of Metals in BiologicalSystems, Bari, ITALY,3Department of Molecular Genetics andMicrobiology, Duke University, Durham, NC, USA,4Department

of Biology, University of Roma Tre and National Institute forInfectious Diseases I.R.C.C.S ‘Lazzaro Spallanzani’, Roma,ITALY,5Department of Chemistry and Interuniversity Consortiumfor Science and Technology of Materials, University of Firenze,Sesto Fiorentino (FI), ITALY,6Department of Structural andFunctional Biology and Center of Neuroscience, University ofInsubria, Busto Arsizio (VA), ITALY,7Department of Molecular,Cellular and Animal Biology, University of Camerino, Camerino(MC), ITALY

Human serum albumin (HSA) is the most abundant protein ent in the bloodstream (reaching a plasmatic concentration of0.7 mM) and it is characterized by an extraordinary ligand bind-ing capacity for several natural compounds, such as metabolites,drugs and hormones It displays seven binding sites for fattyacids, one of which shows a very high tendency to bind heme.Heme-HSA, is able to bind gaseous ligands, such as O2 and CO,

pres-in the Fe (II) form and oxygen and nitrogen radicals, such asperoxynitrite, in the Fe (III) form Furthermore, this binding pro-cess can be modulated by the allosteric interaction between theheme site and the six additional binding sites Therefore, theoccupancy of one of these sites can alter the reactivity of theheme We have investigated in details the allosteric effect induced

by two drugs (namely warfarin and ibuprofen) on the reactiveproperties of heme-HSA toward CO in the Fe (II) form (by war-farin) and toward peroxynitrite in the Fe (III) form (by ibupro-fen) This study allowed us to characterize most of the kineticparameters for the binding of both heme ligands to heme-HSAand of drugs to their relative sites These measurements have

been carried out as a function of drugs concentration, rendering

possible a quantitative description of the energetics associated to

the allosteric interactions Parameters obtained from this study

turn out to be important for the knowledge of reciprocal

interac-tions between different molecules bound to HSA, unraveling the

possibility of interference between different ligands present in the

bloodstream

P2–21

Human scFv derived from a naive library that

target gp41 are strong candidates to inhibit

HIV-1 fusion process

A Couto and J Goncalves

Instituto de Medicina Molecular, URIA-CPM, Lisboa,

PORTUGAL

The fusion process of the Human Immunodeficiency Virus type 1

(HIV-1), is mediated by the gp120 surface protein and the gp41

transmembrane protein The gp120-CD4 interaction initiates this

process, and induces conformational changes in gp41 that result

in an active conformation of the fusion peptide (fusogenic

confor-mation) promoting the fusion of the cellular and viral membranes

Since the fusion process depends on the conformational changes

of the gp41 ectodomain, by interfering with this process, it is

pos-sible to block viral entry into the cell Thus the aim of this study

is to select single chain antibodies (scFv) against epitopes present

in the gp41 ectodomain critical to gp41 conformational changes,

with the purpose of blocking HIV-1 fusion, and consequently

inhibiting HIV-1 infectivity In this work we have selected by

phage display technology, single chain antibodies (scFv) against

the gp41 ectodomain These scFv were selected from a human

scFv phage library obtained from HIV non-immunized donors

The inhibition of HIV-1infectivity is being tested in vitro using

scFv that presented good expression levels and a strong binding

to gp41 The scFv molecules that present higher inhibition of

HIV-1 infectivity, revealed a high prevalence of Ser and Tyr in

the CDR1 and CDR3 particularly in the light chain These scFv

were subjected to affinity maturation strategies restricted to

ran-domization of only four amino acids, Ser, Tyr, Ala and Asp

These results suggest that this scFv anti-gp41 represent a

promis-ing alternative in the development of new fusion inhibitors

P2–22

Biological cost of the 16S rRNA modification

conferred by aminoglycoside resistance

methyltransferase Sgm

S Cubrilo, F Babic and G M Vlahovicek

Faculty of Pharmacy and Biochemistry, Department of

Biochemis-try and Molecular Biology, University of Zagreb, Zagreb,

CROATIA

Sgm methyltransferase from actinomycete Micromonospora

zion-ensisis a member of the Arm family of enzymes that confer high

level resistance to aminoglycoside antibiotics Their mode of

action is modification of the A-site of the ribosome by

methyla-tion of the nucleotide G1405 at the posimethyla-tion N-7 Structure and

function of the A-site of the ribosome is highly conserved

throughout all three kingdoms of life and so are the nucleotide

sequence and posttranscriptional modification content of the 16S

rRNA contained within Nucleotide G1405 is located in the

upper part of helix 44 in 16S rRNA, in the decoding center of

the ribosome In the vicinity of G1405, there are several

nucleo-tides modified by housekeeping methyltransferases, such as

m4Cm1402, m5C1407 and m3U1498 Here we propose that the

addition of another methyl group in this highly evolutionary served region in the ribosome could have its biological cost Weinvestigated how the presence of the Sgm enzyme affects theexponential growth of E coli cells in various media and tempera-ture conditions and the ability of bacteria to compete with thecells not expressing the enzyme To see if m7G1405 methylationinfluences the ribosome assembly, we compared ribosomal pro-files of cells carrying Sgm methyltransferase with the wild typecells, both in the presence and the absence of kanamycin or gen-tamicin Our results revealed that E coli cells expressing Sgmenzyme grow slightly slower compared with the non expressingcells Moreover, cells without Sgm outcompeted Sgm expressingcells when grown together, suggesting that the presence of theSgm enzyme is advantageous only in the presence of antibiotics.Since the ribosome assembly was not influenced by the presence

con-of Sgm enzyme, we propose that the biological cost con-of m7G1405methylation in the ribosomal A-site could be caused by the hin-drance of the protein synthesis process

P2–23 Structural and functional features of post- translationally modified cytochrome c, a bifunctional protein

I Diaz-Moreno1, J M Garcia-Heredia1, A Diaz-Quintana1,

P Nieto2, M Orzaez3, E Perez-Paya3and M A De la Rosa1

c oxidase (1) In addition to its well-established role in energymetabolism, regulation of programmed cell death (PCD) hasemerged as a second major function of Cc Early events in PCDinvolve the release of Cc from mitochondria to the cytoplasm so

as to trigger the assembly of the apoptosome by interacting withthe Apoptotic protein-activating factor (Apaf-1) and to activatethe cysteine-aspartic acid proteases (caspases) that finally leads tocell death (2) The function of Cc in cell signalling seems to beregulated by post-translational modification of the hemeprotein.Actually, one of the most common effects of the so-called RNOS(Reactive Nitrogen and Oxygen Species), which are being pro-duced in mitochondria, is protein nitration (3), with respiratory

Cc – in particular, its five tyrosine residues – being an ing target for the RNOS (4) To determine the specific effect oftyrosine nitration on the structure-function relations of human

outstand-Cc, we have produced a set of monotyrosine Cc mutants – inwhich all the tyrosine residues but one are replaced by phenylala-nines (5) Using both the nitrated and non-nitrated species of

WT and mutant Cc, a biophysical approach has been performed

by combining NMR and CD techniques with theoretical MD culations In addition, the impairment of procaspase-9 activationdepending on Cc and Apaf-1 interaction has been investigated as

cal-a function of specific nitrcal-ation of Cc tyrosines

References:

1 Maneg et al., Biochim Biophys Acta 2004; 1655: 274–281

2 Kim et al., Proc Natl Acad Sci USA 2005; 102: 16545–17550

3 Aulak et al., Am J Physiol Heart Circ Physiol 2004; 286:H30–H38

4 Batthya´ny et al., Biochemistry 2005; 44: 8038–8046

5 Rodrı´guez-Rolda´n et al., Biochemistry 2008; 47: 12371–12379