Báo cáo khoa học: V1–Varia V1-001P Hemolysis of human red blood cells by riboflavin-Cu(II) system: enhancement by azide. ppt

Rahmati2 1 Molecular Biology, Biochemistry, Tarbiat Modarres University, Tehran, Tehran Iran,2Molecular Biology, Biochemistry, Science and Research Unit- Islamic Azad University, Tehran,

V1-001P

Hemolysis of human red blood cells by

riboflavin-Cu(II) system: enhancement by

azide.

I Ali1and I Naseem2

1

Faculty of Pharmacy, Applied Science university, Amman,

Amman Jordan,2Department of Biochemistry, Faculty of Life

Sciences, Aligarh Muslim University, Aligarh, UP India

E-mail: iyad74@yahoo.com

Photoactivated riboflavin in the presence of Cu(II) generates

reactive oxygen species (ROS) which can hemolyse human red

blood cells (RBC) In the present work we examined the effect

of sodium azide (NaN3) on RBC in the presence of riboflavin

and Cu(II) The addition of NaN3 to the riboflavin-Cu(II)

sys-tem enhanced K+ loss and hemolysis The extent of K+ loss

and hemolysis were time and concentration dependent

Batho-cuproine, a Cu(I)-sequestering agent inhibited the hemolysis

completely Among various free radical scavengers used to

iden-tify the major ROS involved in the reaction, thiourea was

found to be the most effective scavenger Thiourea caused

almost 85% inhibition of hemolysis suggesting that •OH is the

major ROS involved in the reaction Using spectral studies and

other observations, we propose that when NaN3 is added to

riboflavin-Cu(II) system, it inhibits the photodegradation of

riboflavin resulting in increased•OH generation Also, the

possi-bility of azide radical formation and its involvement in the

reac-tion could not be ruled out

V1-002P Involvement of thromboxane A2 synthase in the dexamethasone altered response to electrical field stimulation in mesenteric arteries from spontaneously hypertensive rats

R M Aras, M Ferrer and G Balfago´nDepartamento de Fisiologı´a, Universidad Auto´noma de Madrid,Madrid, Spain E-mail: arasmadrid@wanadoo.es

The aim of this study was to analyze whether dexamethasone altersthe vasoconstrictor response induced by electrical field stimulation(EFS) in endothelium denuded mesenteric arteries from spontane-ously hypertensive rats (SHR) For this purpose, superior mesen-teric arteries from male SHR rats were used to analyze whetherdexamethasone alters: (i) the vasomotor response to EFS, (ii) thevasoconstrictor response to exogenous noradrenaline (NA), and(iii) the EFS-induced release of [3H]-NA Preincubation with dexa-methasone for 5 h decreased the contractile response induced byEFS but did not modify the contraction induced by exogenous

NA The EFS-induced [3H]-NA release was increased by methasone preincubation The thromboxane A2synthase inhibitor,furegrelate, reversed the decreased response to EFS induced bydexamethasone without modifying the unaltered response to NA.These results show that preincubation with dexamethasone for 5 hincreases the NA release induced by EFS, and indicate decreasedthromboxane A2synthase activity under these conditions.Acknowledgment: This work was supported by grants fromFIS (PI020335 and C03/01) and DGCYT (BFI2001–1324)

Effects of Nat5 aminotermional

acetyltransferase inhibition on HeLa cells

A Ametzazurra, E Larrea, M P Civeira, J Prieto and

R Aldabe

Gene Therapy and Hepatology, FIMA, Pamplona, Navarra Spain

E-mail: raldabe@unav.es

Alpha aminoterminal acetyltransferases (NAT) acts

cotranslation-ally on the vast majority of eukaryotic proteins In yeast there are

at least three NATs: NatA, NatB and NatC being Nat3p one

sub-unit of NATB We have focused on analyzing the effects induced

by the inhibition of Nat5, human homologue of Nat3p, in HeLa

cells using siRNAs We have observed that there is no induction of

apoptosis in the cells that have Nat5 downregulated but there is a

clear growth arrest of the HeLa cells that posses Nat5 inhibited

that directly correlates with the degree of Nat5 inhibition observed

in the cells The cell cycle disruption induced by the

downregula-tion of Nat5 expression is induced by the activadownregula-tion of p53 that

induces an increase of p21 mRNA and protein presents in the cell

V1-004P

Plants as alternative systems for recombinant

protein production: Medicago truncatula as an

emerging production system

R Abranches1, S Marcel2, E Arcalis2, P Fevereiro1and

Plants are emerging as a promising alternative to conventional

platforms for the large-scale production of recombinant proteins

This field of research, known as molecular farming, is developing

rapidly and several plant-derived recombinant proteins are already

in advanced clinical trials However, the full potential of molecular

farming can only be realized if we gain a fundamental

understand-ing of biological processes regulatunderstand-ing the production and

accumu-lation of functional recombinant proteins in plants Recent studies

indicate that species- and tissue-specific factors as well as plant

physiology can have a significant impact on the amount and

qual-ity of the recombinant product More detailed comparative studies

are needed for each product, including the analysis of expression

levels, biochemical properties, in vitro activity and subcellular

localization Here we include the first results from an extensive

comparative study in which the highly-glycosylated enzyme

phy-tase (from the fungus Aspergillus niger) was produced in different

plant species (including tobacco, rice and the model legume

Medi-cago truncatula) Special emphasis is placed on M truncatula,

whose leaves accumulated the highest levels of active phytase We

discuss the potential of this species as a novel production host

V1-005P

Role of plasma bilirubin and superoxide

dismutase in erythrocytes in elevation of total

antioxidant activity of plasma in growing rats

intoxicated with acetaminophen.

A Allameh1, A Dadkhah1, F Fatemi1, Z I Islamifar1and

M Rahmati2

1

Molecular Biology, Biochemistry, Tarbiat Modarres University,

Tehran, Tehran Iran,2Molecular Biology, Biochemistry, Science

and Research Unit- Islamic Azad University, Tehran, Tehran Iran

E-mail: allameha@modares.ac.ir

Ferric reducing ability of plasma (FRAP) represents the total

anti-oxidant capacity (TAC) of plasma that encompasses different

enzy-matic and non-enzyenzy-matic antioxidant factors Recently wereported that TAC of plasma, determined as FRAP is remarkablyinduced in developing rats pretreated with high dose vitamin K1 ormenadione Increased FRAP was inversely related to formation oflipid peroxidation products in plasma and erythrocytes In the pre-sent study, the role of selected antioxidant factors in the drug-rela-ted changes in FRAP was examined Unlike adults, in growing ratstreated with a sub-lethal dose of acetaminophen (450 mg/kg b.w)there was about 4–5 fold increase in FRAP Under these circum-stances, no significant changes recorded in total protein and uricacid levels in plasma as well as catalase activity in erythrocytes Incontrast, increased FRAP value was associated with plasma biliru-bin as well as erythrocyte superoxide dismutase (SOD) activities.Kinetic studies showed that FRAP is elevated during 1–4 h afteracetaminophen treatment Increased FRAP occurred simulta-neously with a surge in total bilirubin 6 h after acetaminophenadministration However, elevation in SOD in erythrocytesoccurred with a delay (12 h after drug administration) Theseresults suggest that in immature rats, plasma bilirubin and eryth-rocyte SOD play a distinct role in elevation of total antioxidantcapacity of plasma leading to suppression in the level of lipid per-oxidation products in plasma

V1-006P Functional analysis of peptides from the skin extracts of the North African frog Rana saharica

F Abassi1, M Amich2, P Nicolas2and K Hani1

1Laboratoire de Biochimie, Departement de Biochimie, Faculte´ deMedecine de Sousse, Sousse, Sousse Tunisia,2Laboratoire deBioactivation des Peptides, Institut Jacques Monod, Universite dePierre et Marie Curie, Paris, Paris France

E-mail: feten20022002@yahoo.frDuring the course of evolution, nature has developed a vastnumber of peptides in all living and past species that display anexceeding diversity of structure and biological effects, such ashormonal and enzyme-controlling activity, communicationbetween cells, and participation in host defense These peptidesexhibit broad-spectrum activity against a wide range of microor-ganisms including Gram-positive and Gram-negative bacteria,protozoa, yeast, fungi and viruses A few peptides have alsobeen found to be active to sperm and tumor cells These bacte-ricidal, fungicidal, virucidal and tumoricidal properties and thefact that they have potential to overcome bacterial resistancemake these peptides promising candidates for therapeutic drugs.They serve as research tools and have potential as diagnosticbiomarkers and for the development of peptide and peptidomet-

ic drugs In the present study, we have used, for the first time,the ‘‘Tunisian frog’’ as a model to isolate, characterize andstudy the pharmacological properties of new structures, synthes-ized by the skin of tunisian frogs from the genus Rana Thesepeptides can be used as phylogenetic markers and as probes inthe experiments of homologous cloning, to reveal the presence

of new peptides with therapeutic interest and to assess the value

of amphibian antimicrobial peptides as taxonomic and genetic markers A primary purification of the skin of the frogRana saharicawas done using Sep-Pak C18 cartridges; the frac-tions obtained were tested for their ability to inhibit growth ofpathogenic microorganisms Many fractions inhibited thegrowth of reference strains of Gram+ bacterium, Gram– bac-terium and fungi strains

National Research Center for Genetic Engineering &

Biotechno-logy, Tehran, Iran,2M Department Faculty of Science, Tehran

University, Tehran, Iran,3Khatam University, Tehran, Iran,

4

Digest Diseases Research Center, Tehran University of Medical

sciences, Tehran, Iran E-mail: fereshteh_mail

Background: One reason for the persistence of H pylori in the

human stomach might be a stage in the life cycle of H pylori in

which the bacterium establishes as an intracellular symbiont in

yeast In the previous study H pylori-specific genes, universal

prokaryotic 16S rDNA and vacA(s1/s2) were detected in yeasts

DNA In this study cagA was targetted

Methods: DNA was extracted from 18 yeasts in which the

pres-ence of bacterium-like bodies was confirmed by light microscopy

H pylori-specific cagA gene was targeted by designing specific

primers PCR conditions were optimized and the amplified

prod-ucts were analyzed on agarose gel H pylori and S cerevisiae

were used as controls The fragments were cloned for sequencing,

then compared with data in Gene bank

Results: cagA was amplified from eight of 18 yeasts isolates and

H pylori, as a 814bp product, when primer pair No.1 was

recrui-ted When seminested-PCR was performed on all of the 18 PCR

products of the first reaction cagA gene was amplified, as 316bp

band, from 15 samples and H pylori Such product was not

amplified from S cerevisiae The PCR product of cagA in

H pyloriand yeast DNA were sequenced and showed more than

98% homology to the cagA gene of H pylori

Discussion: One critical measurement to counteract H pylori is

to block the routes of its transmission Universal prokaryotic

16S rDNA, vacA, and cagA genes were detected in oral yeasts,

it is concluded that yeasts are important in transmission of

H pylori

V1-008P

Circadian rhythmicity of rPER1 and rPER2

proteins in the rat peripheral tissues.

Z Bendova´, K Laurinova´ and A Sumova´

Department of Neurohumoral Regulations, Institute of Physiology,

Academy of Sciences, Prague, the Czech Republic

E-mail: bendova@biomed.cas.cz

In living organisms, there exists many physiologic and behavioural

processes that oscillate in time with circadian period In mammals,

these oscillations are controlled by a circadian clock located in the

suprachiasmatic nuclei (SCN) of the hypothalamus SCN

clock-work mechanism is based on the transcriptional and translational

positive and negative feedback loops of clock genes and their

cor-responding proteins These include the PAS (Per-ARNT-Sim)

pro-teins PER1 and PER2 Several studies revealed that circadian

rhythms in the expression of these proteins in the SCN were

delayed by about 6 h relative to the mRNA Recently,

compo-nents of the SCN clockwork system has been found also in

mul-tiple peripheral tissues of mammals Several analysis show the

circadian rhythmicity in clock genes expression in lung, liver,

heart and others but the results are discussed mostly in the context

of organization of circadian oscillators within the body In our

study, we present the circadian profiles of rPER1 and rPER2

pro-teins in lung, liver, heart and kidney in rats maintained under LD

12:12 as well as under long (LD 16 : 8) and short (LD 8:16)

pho-toperiod as they were analyzed by Western blots Our data

indi-cate a tissue specific pattern of both rPER proteins expression and

the results will be communicated in context of molecular work mechanism in peripheral tissue compared to the SCN.Acknowledgment: This work was supported by Grant Agency

clock-of the Czech Republic, Grant No: 309/02/D093

V1-009P Reconstitution of human hypoxia inducible factor 1 (HIF-1) in yeast cells: a simple in vivo system to identify hif-1 inhibitors

G G Braliou, E Venieris and G SimosLaboratory of Biochemistry, School of Medicine, University ofThessaly, Larissa, Greece E-mail: gbraliou@tee.gr

Hypoxia inducible factor 1 (HIF-1) is a transcription factor lating a number of genes involved in oxygen and energy homeos-tasis Furthermore, HIF-1 is highly involved in the pathology ofvarious diseases including tumors, angiogenesis-related disordersand preeclamsia Thus, inhibitors of HIF-1 activity are of highinterest as potential pharmaceutical or anti-cancer agents HIF-1

regu-is a heterodimer consregu-isting of the hypoxia-regulated subunitHIF-1alpha and the constitutively expressed subunit HIF-1beta(ARNT) To be functional, the two subunits of HIF-1 have totranslocate inside the nucleus, dimerize and bind to DNAsequences called Hypoxia Response Elements (HREs), locatedwithin promoter or enhancer regions of target genes We show inthis work, that these processes can also efficiently occur in

S cerevisiae cells, which have been genetically modified toco-express the two human HIF-1 subunits and a suitable reportergene Therefore, HIF-1 can exert its transcriptional activity inyeast, a finding that has important implications for its function

in human cells In addition, this simple and genetically tractable

in vivosystem can be used for identifying other proteins, peptides

or chemical compounds that directly affect the activity of HIF-1.The potential of this system is currently being investigated

V1-010P Tyrosine diazoquinone (quinone diazide) and other diazo compounds are the result of NO-dependent protein modifications

N Beda and A NedospasovInstitute of Molecular Genetics, Moscow, Russian Federation.E-mail: beda@img.ras.ru

Nitric oxide (NO) and reactive NO-derived species (RNS) can duce various NO-dependent modifications (NODM) of proteins.The essential characteristics of NODM are their relative independ-ence of enzymes, the involvement of very small ions and molecules(NO, NO+, NO-, NO2etc.) and the possibility of autocatalytic for-mation of RNS, e.g NO-oxidation products, within protein mole-cules Micellar catalysis of NO oxidation can maintainuninterrupted fluxes of NO and oxygen to the interior of the pro-tein molecule, formation of RNS inside the molecule, and transfer

pro-of the reaction products out pro-of the molecule Tyrosine and phan residues are the main targets for nitration in proteins How-ever, the biochemistry of consequent conversions of nitratedmolecules is studied insufficiently The possibility of nitrotyrosineresidues reduction in proteins to amino derivatives has been reli-ably demonstrated Here we show that aminotyrosine residues inproteins can be converted by NODM into tyrosine quinone diazide(Tyr-QD) form, which is in a pH-dependent equilibrium withdiazo-, and diazoate forms At physiological pH values these diazoforms are highly reactive and can react with some amino acidsand bases of nucleic acids They can be reduced to native tyrosine

fi Tyr-H + N2In cyclicmetabolic paths, stationary concentrations of each product of a

cycle depend on their formation/decomposition rates ratio

There-fore, an increase in the observed concentrations of individual

prod-ucts, for instance, nitrotyrosine or quinone diazide forms of a

particular protein, might be a consequence of both their synthesis

acceleration and decomposition deceleration Hundreds of proteins

are known to undergo nitration and nitrosation in vivo Their diazo

forms might be likewise participants of cyclic NO-dependent

con-versions of these proteins Their distribution and functional

signifi-cance remain to be elucidated

V1-011P

Glycan differences between healthy and

pancreatic adenocarcinoma serum

ribonuclease 1.

S Barrabe´s1, G Tabare´s1, E Fort2, P M Rudd3, P Rosa1and

D L Rafael1

1

Biochemistry of Cancer, Biochemistry and Molecular Biology

Department, University of Girona, Girona, Spain,2Digestive

Department, Hospital Universitari Dr J Trueta, Girona, Spain,

3

Glycobiology Institute, Departament of Biochemistry, University

of Oxford, Oxford, UK E-mail: silvia.barrabes@udg.es

Pancreatic adenocarcinoma has a poor prognosis because of its

high death : incidence ratio, close to 1 Its diagnosis is still

diffi-cult due to its low symptomatology and the absence of a specific

marker One of the general features of tumour cells consists of

changes in their cell surface, which can be also reflected in the

gly-cosylation pattern of their secreted glycoproteins Ribonuclease 1

is a glycoprotein expressed mainly by the pancreas and it is also

found in serum RNase 1 from human healthy pancreas presents

a different glycosylation pattern when secreted by

adenocarcino-ma cell lines Capan-1 and MDA-Panc-3 One of the most

signifi-cative differences is the presence of sialic acid on RNase 1 from

tumour cells In order to elucidate whether similar differences in

the glycosylation pattern can be found between serum RNase 1

from healthy and pancreatic cancer patients, two-dimensional

electrophoresis studies were performed Three healthy patients

sera and five pancreatic adenocarcinoma patients sera samples

were pretreated by two different chromatographic methods in

order to enrich RNase ratio, and analyzed by two-dimensional

electrophoresis Some differences due to changes in pI were

attrib-uted to different glycosylation patterns In order to get a further

insight in these differences, a sandwich Glycosylation

Immuno-Sorbent Assay (GISA), determining the sialylation potential by

Sialyl-Transferase activity, was carried out With the aim to fully

characterize the glycan structure of healthy and pancreatic

adeno-carcinoma serum RNase 1, glycan sequencing is in progress The

results presented show differences in glycosylation structures of

serum RNase 1, which indicate a possible way for characterize

pancreatic adenocarcinoma and could be useful to improve the

pancreatic cancer diagnostic

V1-012P

Yellow head virus infection induces a

modulation of cytoskeletal-related proteins in

hemocyte from black tiger shrimp

A Bourchookarn, P Chongsatja and C Krittanai

Institute of Molecular Biology and Genetics, Mahidol University,

Nakhon Pathom, Thailand E-mail: mrchookarn@yahoo.com

Yellow head virus (YHV), a rod shaped, single-stranded RNA

virus, is one of the most severe infectious agents to black tiger

shrimp (Penaeus monodon) Rather than lymphoid organ and gills,

the hemocytes were previously reported to be infecting by YHV

Light microscopy of hemocytes from forty-eight hours

post-infec-tion has shown a clear morphological change Proteomic profiles

of hemocytes from YHV-infected shrimps by two-dimensional gelelectrophoresis were elucidated showing up and down regulatedproteins These proteins were in gel digested and analyzed by massspectrometry Some of the down-regulated proteins were identified

as tropomyosin and actin A vast modulation of tropomyosin andactin could clearly explain the transformation of hemocytes mor-phology observed in an infected shrimp The possible relation ofthe cytoskeletal-related proteins in mechanism of YHV infectionwas also investigated and discussed

V1-013P Identification of a novel mitochondrial protein that is essential for peroxisomal membrane synthesis in the yeast Hansenula polymorpha

E Bener Aksam1, R Pries1, S Kohlwein2and I J van der Klei1

1

Department of Eukaryotic Microbiology, Groningen BiomolecularSciences and Biotechnology Institute (GBB), University ofGroningen, Haren, The Netherlands,2SFB Biomembrane ResearchCenter, Institute of Molecular Biosciences, University of Graz,Graz, Austria E-mail: e.bener@biol.rug.nl

Peroxisomes are important subcellular organelles, which canfunction in a variety of metabolic processes, dependent onenvironmental conditions and the organism in which they occur.Yeasts are attractive model organisms to study peroxisome bio-genesis and function In recent years, vast studies have aimed atthe elucidation of the mechanisms involved in these processes.Various genes (PEX genes) have been identified which are essen-tial for peroxisome biogenesis Here we report on the isolation of

a mitochondrial protein that functions in the formation/stability

of the peroxisomal membrane This protein of yet unknown tion has been identified within a novel collection of conditionaltemperature sensitive (ts) mutants of the yeast Hansenula poly-morpha, affected in the utilization of methanol at restrictive tem-peratures This growth defect appeared to be associated with thedestabilization of peroxisomes in the cells due to the disintegra-tion of the peroxisomal membrane Total peroxisome desintegra-tion occurred in a time interval of 2-4 h after the shift of cellsfrom permissive (37
C) to restrictive (44
C) temperature Themutant gene was sequenced and appeared to contain a pointmutation that changed D407 into N The D407-N mutation wassubsequently introduced into a WT H polymorpha strain to con-firm the mutant phenotype Fusion of the gene to GFP localizedthe protein to mitochondria Hence, this protein represents thefirst known mitochondrial protein involved in peroxisome mem-brane assembly Mass spec lipid analysis was performed to ana-lyze the phospholipid components affected in the mutant strain

func-V1-014P Phthalocyanine conjugates of oligonucleotides

as new reagents for sensitized and catalytic modification of DNA and DNA-binding proteins

A A ChernonosovLaboratory of Investigation of Biopolymer’s Modification, Institute

of Chemical Biology and Fundamental Medicine, Novosibirsk,Russian Federation E-mail: sandy@niboch.nsc.ru

Phthalocyanines (Ptc) is now of interest as a drugs for namic therapy of malignant tumors Their therapeutic effect isbased on the ability to sensitize the generation of singlet molecu-lar oxygen1O2under irradiation Besides, some metal-Ptc can cat-alyze in dark conditions the formation of other reactive oxygenspecies-•O2, H2O2,•OH These properties of Ptc make them veryattractive as a reactive group to be linked to antisense oligonucle-

photody-otides for specific DNA chemical modification We have

devel-oped the solid-phase method of synthesis of

deoxyribooligonucle-otide conjugates carrying Al(III)–, Zn(II)–, and

Co(II)-tetracarboxy-Ptcs groups The yields of conjugates were 30–40%

Thermodynamics of interaction of Ptc-oligonucleotide conjugates

with ss- and dsDNA were measured by UV melting method and

CD spectroscopy The results demonstrated the stabilizing effect

of Ptc moiety on the formation of both duplexes and triplexes

DNA Co(II)-Ptc-oligonucleotide conjugate was found to result in

modification of target DNA in duplexes and triplexes in the

pres-ence of molecular oxygen/reducing agent or hydrogen peroxide

The Al(III)- and Zn(II)-Ptc conjugates caused DNA modification

under irradiation with Hg-lamp or He/Ne laser lights at 340 and

633 nm, respectively The damages in DNA were revealed by the

treatment of products with piperidine or

8-oxoguanine-DNA-gly-cosylase from E coli The interactions of Ptc-oligonucleotide

con-jugates with proteins of blood plasma were studied

Acknowledgment: This work was supported by grants from the

Russian Foundation for Basic Research (05-04-48447) and

Minis-try of Education and Sciences of Russia

V1-015P

Bile acids in bile and serum of a mouse model

of alpha-methylacyl-CoA racemase-deficiency

K Savolainen1, T J Kotti1, W Schmitz2, T I Savolainen1,

J K Hiltunen1and E Conzelmann2

1Department of Biochemistry, University of Oulu, Oulu, Finland,

2Physiologic Chenistry II, Biozentrum, University of Wuerzburg,

Wuerzburg, Germany E-mail: conz@biozentrum.uni-wuerzburg.de

required for the degradation of branched-chain fatty acids and is

also involved in the synthesis of bile acids from cholesterol The

inherited deficiency of the enzyme in humans causes adult-onset

sensory and motor neuropathy To study the physiological role of

the enzyme, an AMACR-deficient mouse strain was generated Bile

acid composition of bile and serum from knockout animals and

from their normal litter-mates was quantitatively analyzed by

FT-ICR mass spectrometry The extremely high resolution of this

method allowed the simultaneous determination of all relevant

compounds without prior chromatographic separation In the bile

of enzyme-deficient animals, total concentration of mature (C24)

bile acids was reduced to approx half the normal content, while the

C27 precur-sors, which are barely detectable in normal controls,

made up for most of the difference Similar changes were found in

serum and in liver tissue The presence of significant amounts of

normal bile acids, in spite of the block of an essential step, indicates

the operation of an alternative pathway, with lower capacity

V1-016P

Src family kinases regulates the activation of

phospholipase D2 and degranulation in RBL

2H3 cells

W S Choi1, Y M Kim2, E Her1, H Y Lee3, J W Han4,

H W Lee4, T Hiragun5and M A Beaven5

1Laboratory of Immunology, College of Medicine, Konkuk

Univer-sity, Chungju, South Korea,2Department of General Education,

Duksung Women’s University, Seoul, South Korea,3College of

Medicine, Konyang University, Nonsan, South Korea,4College of

Pharmacy, Sungkyunkwan University, Suwon, South Korea,

5

Laboratory of Molecular Immunology, NHLBI, National

Institutes of Health, Bethesda, MD 20892 USA

E-mail: wahnchoi@kku.ac.kr

We reported previously that both phospholipase D1 (PLD1)

and PLD2 regulate degranulation in RBL 2H3 mast cells

However, the activation mechanism for PLD2 is still unclear

As reported here, PLD2 but not PLD1 is phosphorylatedthrough the Src kinases, Fyn and Fgr, and this phosphoryla-tion appears to regulate PLD2 activation For example, onlyPLD2 was tyrosine phosphorylated in antigen-stimulated mastcells This phosphorylation was blocked by an Src kinaseinhibitor or by small interfering RNAs directed against Fynand Fgr and was enhanced by overexpression of Fyn and Fgrbut not by other Src kinases The phosphorylation and activity

of PLD2 were further enhanced by the tyrosine phosphataseinhibitor, Na(3)VO(4) Mutation of PLD2 at tyrosines 11, 14,

165, or 470 partially impaired, and mutation of all tyrosinesblocked, PLD2 phosphorylation and activation, although two

of these mutations were detrimental to PLD2 function PLD2phosphorylation preceded degranulation, both events wereequally sensitive to inhibition of Src kinase activity, and bothwere enhanced by coexpression of PLD2 and the Src kinases.The findings provide the first description of a mechanism foractivation of PLD2 in a physiological setting and of a role forFgr in Fc epsilon RI-mediated signaling

V1-017P

A systems biology approach of metabolomics applied to design new targets in cancer therapy

M Cascante, A Ramos, P Viza´n, P de Atauri, J Centelles,

L Boros, S Mazurek, W Frederiks, P Lee and J BorenBiochemistry and Molecular Biology, Barcelona, Barcelona,Catalunya Spain E-mail: martacascante@ub.edu

In the last few years, science has concentrated their efforts incharacterizing the molecular elements that conform cells Sev-eral techniques as DNA sequencing, expression arrays, andproteomic and metabolomic experiments have provided us alarge amount of new information that cannot be easily inter-preted Moreover, the integration of all these information in invivo models is likely to be the most interesting tool to under-stand and to complete an overview picture of the cellular pro-cesses We must take into account, that metabolic profile is inmost cases the end point of the signalling events, where chan-ges caused by diseases like cancer may be reflected Therefore,using bioinformatics – specially a tool as Metabolic ControlAnalysis- we are able to identify the main steps that control ametabolic pathway after the integration of all the experimentaldata These control points may be used as new therapeuticaltargets In this way, we are working in new antitumoral treat-ments based on the inhibition of the synthesis of DNA andRNA because of their importance for cell proliferation Wehave focused on ribose-5-phosphate synthesis, which is a com-ponent of nucleotides First of all, we characterized the meta-bolic pathways (utilizing gas chromatography coupled to massspectrometry) implied in glucose metabolism and ribose synthe-sis This was followed by the integration of the obtained data

in mathematical models using MCA, which led us to identifythe main enzymes controlling ribose-5-P synthesis: transketo-lase and glucose-6-phosphate dehydrogenase Finally, we valid-ated the obtained targets using specific inhibitors and then, westudied the effects produced in cell proliferation as well as inthe proteomic profile Consequences of the utilization of theseinhibitors on protein-protein interactions and on supramolec-ular organization of the tumor metabolism have also beenstudied

Grape seed procyanidins extract and

oleoyl-estrone improve hepatic antioxidant enzyme

systems in obese Zucker rats

V M Castrillejo1, M Romero2, M Esteve2, A Arde´vol1,

C Blade´1, G Pujadas1, J Ferna´ndez-Larrea1, M Blay1,

L Arola1and M J Salvado´1

1

Biochemistry and Biotechnology Department, University Rovira i

Virgili, Tarragona, Spain,2Nutrition and Bromathology

Department, University of Barcelona, Barcelona, Spain

E-mail: vanessa.castrillejo@ estudiants.urv.es

Recent studies have revealed that pathologies, such as obesity

and other metabolic disorders are associated with an imbalance

between the rate of free radicals and Reactive Oxygen Species

(ROS) production and that of their degradation, known as

tive stress Using the obese Zucker rat as animal model of

oxida-tive stress, we assayed the effect of procyanidins (GSPE), the

most common flavonoids present in red wine, and oleoyl-estrone

(OE), a natural hormone with slimming effects, on the hepatic

antioxidant enzyme systems Antioxidant activities of

Cop-per,Zinc-superoxide dismutase (Cu,Zn-SOD), catalase (CAT),

glutathione peroxidase (GPx), glutathione reductase (GR) and

glutathione S-transferase (GST) were assayed by

spectrophotom-etry Glutathione (GSH) content was also determinated

spectro-photometrically in order to test the cellular redox status When

we compared obese Zucker rats versus lean Zucker rats, the

hep-atic antioxidant activities of Cu, Zn-SOD, GPx, and GR in obese

rats were significantly increased while GSH content was

decreased, indicating an imbalance in the redox homeostasis In

obese rats, treatment with GSPE and GSPE plus OE resulted in

a decrease of antioxidant enzyme activities and in an

enhance-ment of glutathione levels OE treatenhance-ment decreased GPx activity

and also enhanced glutathione levels So, all treatments recovered

the redox homeostasis of obese rats GSPE acts as a free radical

and ROS scavenger while OE, due to its slimming effects and

mobilization of internal fat stores, seems to reduce the organic

hydroperoxides formation As conclusion, we showed that GSPE

and OE treatments improve the oxidative stress condition in

obese Zucker rats by different ways and at different levels

V1-019P

Elucidation of the biosynthetic pathway

of glucosylglycerate Characterization of

key-enzymes

J C Costa1, N Empadinhas2and M da Costa2

1Microbiologia, Zoologia e Centro de Neurocieˆncias e Biologia

Experimental, Coimbra, Coimbra, Portugal,2Microbiologia,

Bioquı´mica e Centro de Neurocieˆncias e Biologia Experimental,

Coimbra, Coimbra, Portugal E-mail: jcosta@cnc.uc.pt

Glucosylglycerate (GG) is a structural analogue of

mannosylgly-cerate (MG), a compatible solute that occurs predominantly in

thermophilic or hyperthermophilic prokaryotes GG has been

detected in low amounts in the Methanohalophilus sp FDF1 [1],

in E chrysanthemi Strain 3937 [2] and in a H elongate Strain

CHR63 [3] We explored public databases for genes similar to

those involved in the synthesis of MG in most prokaryotes The

ongoing genome sequence of Methanococcoides burtonii, a

cold-adapted archaeon, closely related to the GG-accumulating

Methanohalophilus sp FDF1, revealed a sequence with high

homology to MG phosphatase [4] The analysis of the gene

immediately upstream revealed conserved motifs with

glucosyl-transferases Altogether, these data raised the hypothesis that this

glucosyltransferase gene could encode the enzyme responsible for

the synthesis of GG The incubation of the cell-free extract

carry-ing the recombinant glucosyltransferase with GDP-glucose and3-phosphoglycerate catalyzed the formation of a compound thatwas dephosphorylated to GG by alkaline phosphatase Theseobservations allowed us to elucidate the biochemical pathwayleading to the synthesis of GG in M burtonii, while proceeds intwo steps through a phosphorylated intermediate and by the con-secutive action of a glucosyl-3-phosphoglycerate synthase (GpgS)and a glucosyl-3-phosphoglycerate phosphatase (GpgP) The cat-alytic properties of the two enzymes are presented These resultsrepresent an important step towards the elucidation of the realphysiological role of GG synthesis

P Clerc, X Leverve and E FontaineLaboratoire de Bioe´nerge´tique Fondamentale et Applique´e,University of Grenoble, Grenoble, France

E-mail: pascaline.clerc@ujf-grenoble.frThe yield of the oxidative phosphorylation (P/O = JATP/JO2)can be modified both at short and long term Short-term modifi-cations include activation of UCPs or exogenous uncouplers,which decrease P/O Long-term modifications can be achieved bynutritional modifications or by changing thyroid status, whichdecrease (hyperthyroidism, polyunsaturated fatty acids deficiency)

or increase (chronic ethanol intoxication, hypothyroidism) P/O

It has been recently reported that P/O inversely correlates withcytochrome c oxidase content in rat liver mitochondria Becausenitric oxide (NO) inhibits cytochrome c oxidase, we have studiedthe effect of NO on P/O We found that a mild inhibition of therespiratory chain (20% inhibition of basal respiratory rate) usingeither NO donor DPTA-NONOate or cyanide led to a decrease

in the maximal ATP production but to an increase in the P/O.Such a mild respiratory chain inhibition did not affect membranepotential in the presence of oligomycin, but decreased the mem-brane potential for a given respiratory rate in phosphorylatingconditions Therefore, the oxygen consumption required for agiven membrane potential was always lower when cytochromeoxidase was partly inhibited We propose that NO is an endog-enous messenger that may short term regulate the oxidative phos-phorylation efficiency

V1-021P Expression of hepatic transcription factors is modulated by dietary procyanidins.

J Ferna´ndez-Larrea, J M del Bas, A Arde`vol, M Blay,

G Pujadas, M J Salvado´, L Arola and M C Blade´

Biochemistry and Biotechnology department, University Rovira iVirgili, Tarragona, Spain

E-mail: josepmaria.delbas@estudiants.urv.esDietary procyanidins, a group of polyphenolic compounds, haveshown neuroprotective, cardioprotective and chemoprotectiveactions Although these beneficial properties were attributedfirstly to its antioxidant capabilities, some studies have revealedtheir aptitude as signalling molecules, able to interact with intra-

cellular signalling pathways and thus modulating gene expression.

To study the effect of procyanidins in the gene expression of liver

transcription factors and related cofactors, we treated healthy

Wistar rats with an oral, high and non-toxic dose of grape seed

procyanidins extract (GSPE) Microarray hybridization

tech-niques were used to study the changes induced by GSPE on the

liver gene expression profile 5 h after treatment We found

signifi-cant upregulation of small heterodimer partner (SHP), Max

interacting protein 1 (Mxi1), zinc finger protein 354A (Znf354a),

Spalt-3, Spi-B, forkhead box E1 (Foxe1) and regenerating liver

inhibitory factor-1 (RL-IF-1) in the GSPE group versus the

con-trol group On the other hand, the expressions of TRIP-Br2,

He-patocyte nuclear factor 3 beta (HNF3b), High mobility group

box 1 (Hmgb1) and hematopoietically expressed homeobox

(Hhex) were downregulated in the GSPE treated rats These

find-ings reinforce the results found by our and other groups, which

show that procyanidins are able to regulate gene expression

Moreover, as transcription factors are key regulators of a wide

variety of pathways, the regulation of the expression of these

proteins can clarify the different actions that procyanidins exert

on diverse processes as lipidic and glucose metabolisms or

apop-tosis

V1-022P

Transcriptional repression of human CYP17 by

transforming growth factor beta is mediated

by Smad signalling pathway

N Derebecka-Holysz, T P Lehmann, M Holysz and

W H Trzeciak

Department of Biochemistry and Molecular Biology, University of

Medical Sciences, Poznan, Poland

E-mail: derebeckanatalia@o2.pl

Cytochrome P450c17, encoded by CYP17 gene, is a component

of an enzyme complex that catalyses a key reaction in the

biosyn-thesis of glucocorticoids and androgens in the adrenal cortex

CYP17 expression is stimulated at the transcriptional level by

ACTH acting via cAMP/PKA signalling pathway Both basal

and cAMP-induced levels of CYP17 mRNA are decreased by

transforming growth factor beta (TGF-b), however the

mechan-ism of TGF-b action on CYP17 expression remains unknown In

the present study we have examined this mechanism in human

adrenocortical cells H295R Using quantitative PCR we

demon-strated that CYP17 mRNA level was decreased in H295R treated

with both TGF-b and transcription blocker, actinomycin D in

comparison with cells treated with TGF-b or actinomycin D

alone Moreover, protein synthesis blocker, cycloheximide

increased basal level of CYP17 expression and reduced the

inhi-bitory effect of TGF-b Inhibitor of histone deacetylase,

tric-hostatin A partially abolished stimulatory effect of forskolin

on CYP17 expression but did not affect CYP17 inhibition by

TGF-b In transient transfection experiments with plasmid

con-taining -57 bp CYP17 promoter fragment and luciferase reporter

gene we have demonstrated that transcriptional activity of this

region was significantly reduced by TGF-b Moreover,

over-expression of Smad7 evoked decrease of inhibitory effect of

TGF-b on both -57 bp CYP17 promoter activity and endogenous

CYP17 mRNA level We conclude that CYP17 repression by

TGF-b is the multi-level process On the one hand, the repression

of transcription is histone deacetylase-independent and involves

type I TGF-b receptor On the other hand, experiments with

actinomycin D imply that CYP17 mRNA level could also be

decreased by TGF-b post-transcriptionally

V1-023P Recombinational telomere elongation in telomerase deletion mutant of Kluveromyces lactis

B Debelec1, G Kantarci1, W McRae2, M J McEachern2and

Z Topcu1

1

Department of Pharmaceutical Biotechnology, University of Ege,Faculty of Pharmacy, Izmir, Turkey,2Department of Genetics,University of Georgia, Fred Davison Life Sciences Building,Athens, Georgia USA E-mail: bilged79@hotmail.comEukaryotic cells lacking telomerase are sometimes capable ofmaintaining their telomeres via a telomerase-independent mech-anism, which is shown to be recombination-dependent (Topcu et

al, 2005) In this study we addressed the question whether a gle telomere could be used to elongate the other telomeres Wetransformed telomerase-deficient K lactis cells with a singlemutant telomeric repeat bearing URA3 as marker gene that givesrise an AccII recognition site for analyses of telomeric lengthregulation Transformed colonies were followed by scoring theirgrowth phenotypes from 4 to 0.5; from normal to senescing phe-notype, under microscope with serial passaging Upon six succes-sive passaging, the colonies were collected for DNA isolation toestimate their average telomeric lengths using Southern blot andcompared to wild type K lactis Our results showed that thetransformed K lactis cells manifested a varying cell growth capa-bility One third of the colonies reached near-to-senescing statusand around 50% of these cells recovered their growth phenotypefollowing an initial growth decline while the remaining kept theirinitial phenotypes The deviation among Acc mutants emphasizesthe significance of recombinational telomere elongation, an issue

sin-to relate with Alternative Lengthening of Telomeres (ALT),manifested in some human cancers

Acknowledgment: This study was supported by Turkish tific Research Council (Grant number SBAG 2791)

Scien-V1-024P Protein expression profile of Mesocestoides corti larva submitted to metabolic depression

A Esteves, L Canclini and R EhrlichLaboratory of Biochemistry, Department of Cellular andMolecular Biology, Faculty of Sciences, University of the Republic,Montevideo, Uruguay E-mail: aesteves@fcien.edu.uy

Mesocestoides – corti – Cestoda: Cyclophyllidea – despite notbeing of sanitarian interest, it presents important properties as amodel organism The easy manipulation in the laboratory makes

of it the selected model to begin the studies of functional ics in cestodes Metabolic rate depression constitutes an import-ant survival strategy for many animal species submitted tovariable conditions The central aim of this work is the study ofmetabolic pathways of parasite platyhelminths submitted tohypometabolic conditions The molecular comprehension ofmetabolism in these parasites is scarce; it could be an importantsource of key molecules with putative use as drug targets or vac-cines We focused our attention to the implementation of largerange of differential expression techniques to evidentiate changes

genom-in the expression profile as a result of the different stimuliresponse Previously we standardize the metabolic labelling of thelarva stage of M.– corti – (tetrathyridia) and the bidimensionalelectrophoretic analysis Tetrathyridia were aseptically extractedfrom intraperitoneally infected mice, cultured by different times,and submitted to nutritional starvation, thermal and pH stressÆExpression profile of each conditions was analyzed by 2D elec-trophoresis or by auto–radiography Since protein phosphoryla-tion plays a central role in biochemical regulation of metabolic

rate depression, protein phosphorylation was also analyzed in

each situation In a next step, in order to identify the genes

responsible of the observed expression profile changes subtraction

libraries will be performed

Acknowledgment: This work has been supported by

Department of Biochemistry, Dept of Biochemistry, Uppsala

University, Uppsala, Sweden,2Department of Comparativ

Bioscienses, University of Wisconsin, Madison, Wisconsin USA

E-mail: Birgitta.Eklund@biokemi.uu.se

The thiopurine derivatives trans-acetylvinylthioguanine (tAVTG)

and cis-acetylvinylthiopurine (cAVTP) are glutathione-activated

prodrugs of 6-thioguanine (6-TG) and 6-mercaptopurine have

(6-MP) The activation has been investigated kinetically with

human glutathione transferases (GSTs) and glutathione (GSH)

GSTs have broad substrate specificities and are involved in several

cellular processes, but their main function is to detoxify xenobiotic

molecules by conjugating them with GSH In contrast, the

ration-ale behind the prodrugs studied is toxification The design of the

prodrugs make them more tissue specific chemotherapeutic agents,

since they will preferentially exert their cytotoxicity in tumour cells

that have increased levels of GSH The reactions between GSH

and the prodrugs are presumed to be GST-catalyzed, but activities

of human GSTs have not previously been determined Cytosolic

human GSTs investigated were obtained by heterologous

expres-sion in Escherichia coli and included members of the Alpha class:

A1-1, A2-2, A3-3, A4-4; the Mu class: M1-1, M2-2, M3-3; Omega

class: O1-1; Pi class: P1-1; and Theta class: T1-1 The results

estab-lish that all of the Alpha and the Muclass GSTs catalyze the

reac-tion between GSH and the thiopurine produgs With tAVTG

GST A4-4 and GST M1-1 are the most active enzymes With

cAVTP GST M1-1 and GST M2-2 have the highest specific

activ-ities with all Alpha class members have specific activactiv-ities lower by

an order of magnitude GST A4-4, which has the highest activity

with tAVTG, has markedly lower activity with cAVTP

V1-026P

Synthesis of phosphorodithoate compounds

and study of their inhibitory effects on

acetylcholinesterase (AChE) enzyme

S Emadi, B Kaboudin and D Elhamifar

Organic Lab 2, Department of Chemistry, Institute for Advanced

Studies in Basic Sciences, Zanjan, Iran E-mail: emadi@iasbs.ac.ir

Phosphorodithioates are among thiolic derivatives of phosphoric

acid which show different degrees of toxicity and have found many

application in pharmacology and pesticide research Their toxicity

is primarily due to binding to the enzyme acetylcholinesterase

which play pivotal role in neurotransmission process In this study

the anticholinesterase activities of five synthesized compounds were

investigated on acetylcholinesterase from electric eel, human

eryth-rocytes by the spectrophotometric method of Ellman and his

coworkers This study showed that pre-incubation of the enzymes

with the synthesized compounds caused AChEs to show lower

activities (10–30% lower than control sample) in compare with the

enzyme mixture containing no synthetic phosphorodithioate

com-pounds The work is in progress, and with special attention to the

mode of interaction of these compounds with AChE, we are at the

process of examining other synthesized phosphorodithioate

com-pounds with presumably greater inhibitory effects

V1-027P Stannous chloride induces alterations

in enzyme activities, lipid peroxidation and histopathology in male rabbit: antioxidant role

40 mg AA/kg BW; 20 mg SnCl2/kg BW (1/500 LD50); 20 mgSnCl2 plus 40 mg AA/kg BW Rabbits were orally administeredtheir respective doses every other day for 12 weeks Liver andKidney specimens were processed for histopathologic studies.Results obtained showed that SnCl2 significantly (P < 0.05)induced free radicals in rabbit liver, testes, kidney, lung, brainand heart While, the activity of glutathione S-transferase (GST)and the levels of sulfhydryl groups (SH-groups) were decreased(P < 0.05) in all tested organs except brain and heart Aspartateaminotransferase (AST) activity was increased (P < 0.05) in liverand decreased in testes, but alanine aminotransferase (ALT) didnot change The activities of alkaline phosphatase (AlP) and acidphosphatase (AcP) were decreased (P < 0.05) in liver, testes,kidney and lung Also, the activity of acetylcholinesterase(AChE) was significantly decreased in brain and plasma of rab-bits treated with SnCl2 compared to control group Histopatho-logic studies showed marked changes in hepatocytes as well asproliferation of duct epithelium, dilatation and congestion ofblood vessels as well as mononuclear inflammatory infiltrate Thekidneys were also severely affected by SnCl2the Bowman’s spacewas increased, with infiltration of renal parenchyma by mononu-clear inflammatory infiltrate and changes in cells lining convolu-ted tubule Ascorbic acid alone significantly decreased the levels

of free radicals, and increased the activity of GST and the levels

of SH groups in tested organs except brain and heart While, therest of the tested parameters were not affected Results showedthat AA alleviated the harmful effects of SnCl2 This was provedhistpathologically by the great improvement in liver and kidneyhistology were hepatocytes retained normal architecture withmild dilatation and congestion of blood vessels Bowman’s space

of kidneys were almost normal, with normal lining of proximaland distal convoluted tubules In conclusion AA could be effect-ive in the protection against stannous chloride toxicity

V1-028P Evaluation of optimal conditions for arginase activity in streptozotocin-induced diabetic rats

M Ery´ty´r1, E Erc¸el2, S Yilmaz1and S T Ozan1

tigated and compared The physicochemical and kinetic

proper-ties of liver arginase in diabetic and control rats were very

sim-ilar, those of kidney arginase were significantly different It was

found that pre-incubation temperature (68
C), pre-incubation

period (20 min), optimal pH (10.1) of liver arginase and Km(3.2)

to its substrate, l-arginine, did not change in diabetic and

non-diabetic rats As a consequence of diabetes, the optimum Mn2+

concentration for liver arginase only changed from 1 to 2 mm

Although pre-incubation temperature and period for activation

of kidney arginase in control rats was unnecessary, they were

found to be 56
C and 12 min in diabetic rats pH profile of

argi-nase in kidney of diabetic rats was different than that of control

rats The Km value (6.7) of arginase for l-arginine in kidney is

unchanged in diabetes, whereas a marked decrease in Vmax was

found Optimum Mn2+concentration (2 mm) for kidney arginase

unchanged in diabetes The activity of arginase in liver of

dia-betic animals was higher 1.5- to 1.7-fold than that of controls

Diabetes caused about 53% decrease of arginase activity in

kid-ney of female rats, 26% in that of male These findings may

sug-gest the idea that encoded arginases by separate gene loci may be

differently affected by pathological and hormonal status

V1-029P

Characterization of Pur3, a monophosphatase

from the puromycin biosynthetic pathway of

Streptomyces alboniger

P Barrado, M B Sa´nchez, A Jime´nez and M Ferna´ndez Lobato

Centro de Biologı´a Molecular, Universidad Auto´noma of Madrid,

Madrid, Spain E-mail: mfernandez@cbm.uam.es

Pur3 is the product of a gene (pur3) from the puromycin

biosyn-thetic pur cluster of Streptomyces alboniger A comparative

ana-lysis of its deduced amino acid sequence with those from data

banks indicated that Pur3 is a phosphatase enzyme These

comparisons detected in Pur3 a Mg++binding motif and other

motifs that are supposed to be implicated in the catalytic activity

of a variety of phosphatases A structural modeling of Pur3,

based on the SWISS-MODEL program, indicated that Pur3

con-tains these motifs located in a central core of 155 amino acids In

contrast to other phosphtases, the G201, A226, G227 and G228

residues of Pur3 are located opposite to the active center at the

external face of the molecule These findings might suggest an

implication in regulatory function(s) The pur3 gene was

expressed in Escherichia coli as a recombinant protein fused to a

polyhistidine tag and then was purified to apparent homogeneity

by filtration through a TALON niquel column In accordance to

the above referred to high similarity this recombinant protein

showed monophosphatase activity It was able to

dephosphory-late a wide variety of substrates amongst which lowest Kmvalues

were obtained for the putative intermediates of the puromycin

biosynthesis pathway 3’-amino-3´-dAMP (Km= 1.37 mm) and

puromycin aminonucleoside (Km= 1.40 mm) The identification

of this activity has allowed a revision of the previously proposed

puromycin biosynthetic pathway

V1-030P

Application of dotblotting for detecting the

expression of p5cs gene in transgenic olive

plantlets

M Farzaneh, F Rastegar Jazi and N Motamed

Biochemistry, Department of Biology, University of Tehran,

Tehran, Enghelab, Iran E-mail: mfarzaneh57@yahoo.com

Olive is an ever-green tree of the Oleaceae family and of the

genus Olea, grown under a wide range of climates and soils all

over the world in Iran, 50% of all growing areas encountered byhigh-salt concentration conditions and drought Water deficitcaused by drought and high salinity has been an important factorlimiting crop productivity To cope with these environmentalstresses, application of molecular plant breeding is necessary Inresponse to osmotic stress elicited by condition of high salt, theexpression of P5CS gene altered in olive plants for this, con-structs (S,E,X) were prepared by introducing P5CS sequence in

to PBI121 Constructs cloned in binary vector were transformed

to the disarmed Agrobactrium tumefaciens Olive embryo weretransformed with A tumefaciens harboring the various gene con-structs and salt-tolerance plants were regenerated in media con-taining adequate concentration of salt The total protein wasextracted from fresh leaves of both control and transformedregen To probe the effect of p5cs overexpression on salinitystress tolerance, poly clonal antibody developed in rabbit againstp5cs protein The result of immunological methods for specificdetection of p5cs gene, strongly indicated P5CS up-regulation instressed transformed plants vs non-transformed plants, eratedplants

V1-031P Repression of SOX6 transcriptional activity by SUMO modification

R Fernandez-Lloris1,2, N Osses1, E Jaffray2, L Shen2,

O A Vaughan2, D Girdwood2, R Bartrons1, J L Rosa1,

R T Hay2and F Ventura1

14171, Ciencies Fisiologiques II, Universitat de Barcelona, let De Llobregat, Spain,22.08, Biomolecular Sciences, University

Hospita-of St Andrews, St Andrews, UK E-mail: rakylloris@yahoo.esSOX6, a member of the SOX family of Sry-type HMG transcrip-tion factors, plays key functions in several developmental proces-ses, including neurogenesis, sex determination and skeletonformation In this report, we show that SOX6 is covalently modi-fied in vitro and in vivo by SUMO1, 2 and 3 on two consensussites (IK364NE and VK377DE) Mutation of both lysines toarginine abolished SOX6 sumoylation and increased SOX6 tran-scriptional activity as well as SOX6/SOX9 synergistic activation

of the Col2a1 enhancer SUMO dependent repression of SOX6transcription was demonstrated by Uubc9 overexpressionwhereas siRNA to Ubc9, cotransfection of a catalytically inactiveUBC9 or a SUMO specific protease increased SOX6 transcrip-tional activity Immunofluorescence analysis showed a predomin-ant diffused nuclear localization of SOX6 when expressed alone.Coexpression of SOX6 with SUMO1 and/or SUMO2 results inthe appearance of SOX6 in a punctate nuclear pattern that colo-calized with PML PML body localization of SOX6 was abol-ished by mutations in SOX6 sumoylation sites Thus, SUMOmodification of SOX6 alters its subnuclear localization and leads

to transcriptional repression

V1-032P Usage of the mannose-binding protein (lectin) Con A and the fucose-binding lectins UEA-I and PA-IIL for the study of glycoproteins from human milks and other body fluids

N Gilboa-Garber, B Lerrer and E Lesman-MovshovichFaculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.E-mail: garben@mail.biu.ac.il

Human milk, saliva, and seminal fluid are rich in glycoconjugatesthat act as decoys to which pathogens are attracted instead of bind-ing to the host cells for infection initiation Such compounds func-tion in the front line against infections in adults, and are especially

important for the newborn protection The fucose-binding Ulex

europaeus lectin (UEA-I), which is selective for the H type 2

tope, detects only the glycans of the ’secretors’ who contain H

epi-tope in their secretions, The ‘‘non-secretor’’ body fluids, that do

not express the same H epitope, contain other fucosylated

glyco-forms, which are not detected by UEA-I but might be detected by

other fucose-specific lectins The present communication describes

the usage of fucose-binding lectin of Pseudomonas aeruginosa

(PA-IIL), which may contribute to this pathogen adhesion, as a

probe for fucosylated anti-adhesive glycans in the human body

fluids PA-IIL was found to display highest sensitivity to milk

glycans, followed by those of seminal fluids and salivas, while

UEA-I was only inhibited by ‘‘secretor’’ body fluids, most strongly

by seminal fluids, followed by milks and salivas Conclusions:

Body fluids are rich in fucosylated glycans, detectable by UEA-I

and the P aeruginosa lectin PA-IIL These lectins are useful as

probes for identification of glycoprotein glycans that might

effi-ciently block fucose-dependent adhesion of that bacterium and of

other pathogens

Acknowledgments: These results are parts of the PhD theses

of B.L and E.L.-M

V1-033P

NMR Conformational studies of the interaction

between the V3 loop of HIV-1 coat

glycoprotein gp120 and chemokine receptor

CCR5, at peptide level

P A Galanakis1, N Kandias1, G A Spyroulias1,

M Sioumpara2, D Morikis3, A Rizos4and E Krambovitis2

1

Department of Pharmacy, University of Patras, Patras, Greece,

2

Department of Applied Immunology & Biochemistry, Institute of

Molecular Biology & Biotechnology, FORTH, Heraklion, Greece,

3Department of Chemical and Environmental Engineering,

Univer-sity of California at Riverside, Riverside, CA, USA,4Department

of Chemistry, University of Crete, Heraklion, Greece

E-mail: pgalanak@upatras.gr

Recent convincing evidence indicates that the majority of the

cells that die due to HIV-1 are not actually infected by the virus

Instead, HIV-1 or its components lead these cells to programmed

cell death by altering their physiological function (Zafiropoulos

A et al BBRC 2001; 284: 875–879] after the activation of

apop-totic mechanisms Ionic interactions between the variable V3

domain of the HIV-1 coat glycoprotein gp120 and the amino

ter-minal of the chemokine receptor CCR5 (Baritaki S et al BBRC

2002; 298: 574–580] play a prominent role in this process

Stand-ard multidimensional and multinuclear NMR spectroscopy was

applied to probe the structural and physicochemical determinants

of three representative peptides from V3 domain of the HIV-1

and a 22-residue peptide, representing the amino terminal of the

chemokine receptor CCR5, in their free or interacting state

Titration of CCR5 peptide with V3-peptides was performed in

NMR tube, at 286K 1D 1H NMR spectra and 1H-15N HSQC

were recorded after each addition of V3 peptides Analysis of the

NOESY maps, acquired for free and interacting peptides at

278K, suggests that the free CCR5 construct is structured, giving

rise to numerous NOEs Data analysis of HSQC and NOESY

spectra verifies the interaction of the V3-CCR5 peptide constructs

and suggests a remarkable role for the (i) 7-residue CCR5

N-ter-minal domain and particularly for Tyr3, which is the first among

the four N-terminal tyrosines of CCR5, and (ii) for the

7/9-resi-due V3 N-terminal peptide fragment, which especially in V3 LAI

peptide is rich in basic residues

V1-034P Construction of a double copy (DC) retroviral vector for efficient expression of small hairpin RNAs (shRNAs) in avian cells

P Gooz and S HoffmanDepartment of Medicine, Rheu, Medical University of SouthCarolina, Charleston, SC, USA E-mail: goozp@musc.eduRNA interference (RNAi) has recently become a powerful toolfor studying gene functions in vitro and in vivo The human H1promoter is widely used for expression of shRNAs within thecells These shRNAs are processed into double stranded siRNAmolecules that lead to specific degradation of their target mRNAs

We wanted to validate the use of the H1 promoter in avian tems, which are useful models for studying angiogenesis andembryonic development We directly compared the green fluores-cence protein (GFP) silencing efficiency of the human H1 promo-ter in the quail QCE-6 and the human HEK 293 cell lines, whichwere previously transfected with GFP We found that in the aviancell line the silencing effect developed slower (6 days vs 2 days)and the silencing efficiency was lower (~60% vs >95%), com-pared to the 293 cells On Northern blot we were not able todetect the shRNAs transcribed from the H1 promoter in theQCE-6 cells while the 293 cells produced high amount of the fullyprocessed siRNA product Thus we hypothesized that the humanH1 promoter cannot drive sufficiently high level transcription ofshRNA products in avian cells To increase the level of transcripts

sys-in QCE-6 cells beside the ssys-ingle copy (SC) vector we constructed adouble copy (DC) retroviral vector by cloning the human H1 pro-moter into the U3 region of the 3’LTR of the pLEGFP-N1 retro-viral vector (Clontech, Palo Alto) This vector expresses eGFPthat we used to assess infection efficiency and to obtain pure pop-ulations of cells that express the shRNA by Fluorescence Activa-ted Cell Sorting Then we designed several shRNAs targeting the

75 kDa gelatinase that is expressed endogenously by the QCE-6cells and cloned them into both SC and DC vectors Northernblot analysis showed a significant increase in the expression ofsiRNA molecules when the cells were infected with the DC retro-virus as compared to the single copy vector Moreover one ofthese constructs very effectively (>98%) inhibited the expression

of 75 kDa gelatinase when expressed as a DC design The mum inhibition developed about 5 days after infection and thesilencing effect was stable for more then 10 passages (~2 month)

maxi-of the cells We concluded, that we constructed a retroviral vectorthat can be used for highly efficient gene silencing in avian cells

V1-035P Changes in antioxidant enzyme activities, glutathione and lipid peroxidation levels in rat erythrocytes with age

S Gumuslu and O OzturkDepartment of Biochemistry, Akdeniz University, Faculty ofMedicine, Antalya, Turkey E-mail: sgumuslu@akdeniz.edu.tr

We have studied the activities of enzymes [glucose-6-phosphatedehydrogenase (G-6-PD), copper,zinc-superoxide dismutase(Cu,Zn-SOD), catalase (CAT), selenium-dependent glutathioneperoxidase (Se-GSH-Px) and glutathione-S-transferase (GST)],and the levels of reduced glutathione (GSH), oxidized glutathione(GSSG) and thiobarbituric acid-reactive substances (TBARS) inrat erythrocytes and estimated the ratio of GSH/GSSG and theredox index Male Wistar rats at ages of 1, 6 and 12 months wereused The activities of G-6-PD and Cu,Zn-SOD, the levels ofGSSG and TBARS were increased, while the activity of Se-GSH-

Px and the level of GSH were decreased with age GSH/GSSGratio was significantly decreased with age We found a positive

correlation between age and G-6-PD (r = 0.476, P < 0.01),

Cu,Zn-SOD (r = 0.291, P < 0.01), CAT (r = 0.254, P < 0.01)

and GST activities (r = 0.250, P< 0.05), and GSSG

(r = 0.708, P < 0.05) and TBARS levels (r = 0.802, P < 0.01),

whereas the correlation between age and Se-GSH-Px activity

(r = –0.376, P < 0.05), GSH level (r = –0.603, P < 0.01) and

GSH/GSSG ratio (r = –0.685, P < 0.05) were negative

V1-036P

DNA-binding and ATPase activity of truncated

and mutant forms of the AtoC, response

regulator of the AtoS-AtoC two-component

system

A Grigoroudis1, E Lioliou1, M Matta2, C Panagiotidis2and

D Kyriakidis1

1

Laboratory of Biochemistry, Department of Chemistry, Aristotle

University of Thessaloniki, Thessaloniki, Greece,2Laboratory of

Pharmacognosy/Pharmacology, 2 Department of Pharmacy,

Aristotle University of Thessaloniki, Thessaloniki, Greece

E-mail: asteris1@chem.auth.gr

The cloning of the E coli atoC structural gene was first reported

by Canellakis et al (1993) as the gene encoding the ornithine

decarboxylase antizyme The gene product was found to share

significant homology with bacterial transcription activators of the

two-component regulatory system family; these systems consist of

a ‘‘sensor’’ kinase and a response regulator Later evidence

indi-cated that the E coli ornithine decarboxylase antizyme and the

response regulator of the AtoS-AtoC/Az two-component system

are one The gene next to atoC, atoS, encodes the sensor kinase

that modulates the AtoC/Az activity by phosphorylation, thus

positively regulating the expression of genes of the atoDAEB

operon, encoding enzymes involved in short-chain fatty acids

metabolism Mutations in the putative phosphorylation sites

D55G and H73L were introduced, as well as the double mutant

in these residues Furthermore, a PCR-amplified part of the atoC

gene encoding the C-terminal of the protein (amino acids 140–

461) lacking the phosphorylation domain, has been sub-cloned

The His-tagged forms of the truncated AtoC/Az protein as well

as the three AtoC mutant forms, were overexpressed and purified

in order to determine their properties as transcriptional activators

as well as the effect of their differences with the wild-type form

on the transcription of the regulated genes Measurement of the

ATP-ase activity of the truncated and mutant forms revealed

some interesting variations on levels comparing to the native

AtoC, depending on various conditions of DNA-binding and

phosphorylation by putative donors All forms were shown to

bind at different extension on specific pallindromic sequences of

the atoDAEB operon’s promoter, sharing a characteristic

prop-erty of the sigma 54 activators’ family

V1-037P

Structural studies of Group A Neisseria

meningitidis strains L10, L11, L12

lipooligosaccharides

M Mieszała1, C Jones2, H J Jennings3and A Gamian1

1Medical Microbiology, Immunology Infectious Diseases, Inst

Immunology Exp Therapy, Wroclaw, Poland,2National Institute

for Biological Standards and Control, Blanche Lane, South

Mimms, Herts UK,3National Research Council, Institute for

Bio-logical Sciences, Ottawa, Ontario Canada,4Medical Microbiology,

Immunology Infectious Diseases, Inst Immunology Exp Therapy,

Wroclaw, Poland E-mail: gamian@immuno.iitd.pan.wroc.pl

Neisseria meningitidisis a bacterial colonist of the upper

respirat-ory tract and is known to cause localized diseases such as

con-junctivitis, otitis media, pneumonia, pericarditis and arthritis aswell as severe bacteraemic disease, particularly acute meningitisand bacteremia, sometimes with septic shock [1] Neisseria menin-gitidisbelonging to groups A, B and C cause by far the largestnumber of cases, accounting for about 90% of all cases Whereasserogroups B and C strains generally cause sporadic cases andsmall outbreaks, serogroup A strains are still leading cause of epi-demics [2] These large epidemics of serogroup A meningococcalmeningitis are still prevailing (every 6–10 years) in the People’sRepublic of China and Sub-Saharan zone of Africa so-called the

‘‘meningitis belt’’ [3] Meningococcal lipooligosaccharide (LOS) isthe major glycolipid molecule present in outer membrane and isresponsible for endotoxic and immunostimulating activities ofbacteria Based on LOS structure N meningitidis can be serologi-cally divided into 12 immunotypes (L1–L112) Immunotypes L8,L9, L10, L11, L12 are found within group A meningococci, but

of these L10, L11 and L12 are prevalent and uniquely associatedwith this serogroup [4] However, as far little is known about thestructure of lipopolysaccharides of these strains The present stud-ies demonstrate the results of our structural study on group Ameningococcal strains LOS L10, L11 and L12 Chemical specificdegradations, analytical determinations, ESI and MALDI massspectrometry as well as NMR spectroscopic analysis of carbohy-drate part of these lipopolysaccharides revealed the structural het-erogeneity in non-carbohydrate substituents, but in case of L10also of sugar backbone The nature of the heterogeneity relies in agreat extent on the O-acetyl, phosphate and glycine substituents,which participate in specific epitopes formation

References

1 Bannister B (ed Duerden B.I.), J Med Microbiol 1988; 26: 61–187

2 Peltola H Rev Infect Dis 1983; 5: 71–91

3 Salih MAM, Danielsson D, Backman A, Caugant DA, man M, Olcen PJ Clin Microbiol 1990; 28(8): 1711–1719

Acht-4 Verheul AFM, Snippe H, Poolman JT, Microbiol Rev 1993;57: 34–49

V1-038P Proteom differences in canola plants subjected

to cold stress, cold shock and cold acclimation

R Trischuk1, X Liu1, V Ruddat2, J Lorch2and L Gusta1

in turgor occurs as a result of a decreased hydraulic conductivity

n the roots and stomatal shock (remain open) caused by rapidcooling of the plant Wilting of this nature causes increases in thelevel of abscisic acid (ABA) within the plant that causes a globalhormonal state of disarray Photoinhibition results in the produc-tion of reactive oxygen species (ROS), causing an oxidative stresswithin the chloroplast due to reduction in photosynthesis Not all

of the genes and proteins identified under these conditions may

be associated with LT survival and cold acclimation In an

attempt to separate LT and acclimation associated proteins out

from those associated with chilling injury we have conducted an

in depth proteomic analysis of winter Brassica napus exposed to a

variety of short and long term LT treatments Proteoms of the

dif-ferentially LT treated plants were compared utilizing conventional

two dimensional polyacrylamide gel electrophoresis (2D-PAGE)

and two dimensional Differential Gel Expression (2D-DIGE),

which is capable of measuring minor changes in accumulation

patterns due to the inclusion of an internal standard on

multi-plexed gels Results reveal that a different proteom exists in plants

exposed to a LT shock compared to that of an acclimated plant

Proteins present in a LT shocked plant are indicative of growth

under low temperature conditions (e.g chilling injury) and have

little to do with survival at sub zero temperatures

Van ’t Hoff Laboratory, Chemistry Department, University of

Utrecht, Utrecht, The Netherlands

E-mail: joanke.graveland@nizo.nl

Nanotubes are formed by self-assembly of partially hydrolysed

alpha-lactalbumin, a milk protein There are several promising

applications of these alpha-lactalbumin tubes, in food, pharmacy

and nanotechnology We studied the mechanism of self-assembly,

the structure and the properties of the nanotubes Limited

pro-teolysis of the alpha-lactalbumin (by a serine protease) makes the

molecule prone to self-assembly In the presence of Ca2+tubular

structures are formed Other divalent ions like Mn2+ and Zn2+

can also induce tubular self-assembly, while Mg2+ leads to

ran-dom aggregation Light scattering showed that the self-assembly

is reversible, which is of relevance for controlled release

applica-tions On the other hand, we could also make stable tubes by

cross linking, which would be a requisite for several other

appli-cations From AFM and SAXS measurements, we obtained

val-ues for the outer diameter: 22 nm; and the inner diameter: 7 nm

AFM revealed the helical structure of the tube wall; it is a

left-handed helix By performing nano indentations with AFM we

determined mechanical properties of the tubes The tubes were

shown to be relatively resilient upon small deformations; the

elas-tic modulus is of the order of MPa

V1-040P

Antimicrobial activity, structure and

mechanism relationship of antimicrobial

peptides, HP (2–20), derived from N-terminus

of Helicobacter pylori Ribosomal Protein L1

(RPL1)

Y Park, S.-C Park, J.-Y Kim, M H Kim, S O Shin,

S J Kang, C.-Y Cheong and K.-S Hahm

Research Center for Proteineous Materials, Chosun University,

Gwangju, South Korea E-mail: kshahm@chosun.ac.kr

HP (2–20) (AKKVFKRLEKLFSKIQNDK) is the antimicrobial

sequence derived from N-terminus of Helicobacter pylori

Ribo-somal Protein L1 (RPL1) HP (2–20) was used as model peptides

for designing and developing novel synthetic antimicrobial

pep-tides Analogues including truncated or cyclic-peptides of HP

(2–20) peptide were designed and synthesized and their structure–

activity and mechanism relationships were investigated by

analyz-ing antibacterial, antifungal, and/or antitumor activities and the

secondary and tertiary structures utilizing CD and NMR spectra

The results showed the importance of hydrophobicity, helicity, and some amino acid residues for the antimicrobialactivity In addition, the mechanism of these antimicrobialpeptides was studied by employing fluorescence activated flowcytometry, confocal laser scanning microscopy, and various artifi-cial liposomes The present data suggest that the peptides mayexert their antimicrobial activities through the pore formation ordetergent-like disruption of cell membranes From these studies,several novel synthetic antimicrobial peptides having improvedantibiotic activity with no hemolytic activity were identified asnew model peptides for commercial development

alpha-V1-041P Overall nitrile pathway in the industrial Pseudomonas chlororaphis B23

Y Hashimoto, H Hosaka, K.-I Oinuma, M Goda,

H Higashibata and M KobayashiGraduate School of Life and Environmental Sciences,The University of Tsukuba, Tsukuba, Ibaraki Japan

E-mail: yh@agbi.tsukuba.ac.jpObjectives: We have extensively studied the biological metabo-lism of toxic compounds, nitriles [1] The microbial degradation

of nitriles proceeds through two enzymatic pathways: nitrilasecatalyzes the direct cleavage of nitriles to yield acids and ammo-nia; and nitrile hydratase (NHase) catalyzes the hydration of nit-riles to amides, which are subsequently hydrolyzed to acids andammonia by amidase NHase of P chlororaphis B23, which waspreviously utilized as a catalyst for acrylamide manufacture, isnow used for the production of 5-cyanovaleramide, a herbicideintermediate We have already cloned the NHase gene, and dis-covered the existence of a gene cluster containing the aldoximedehydratase, NHase and amidase genes The downstream regionwas analyzed to clarify an overview of the gene cluster

Results: Two open reading frames (nhpS and acsA) were fied immediately downstream of the NHase gene cluster Theamino acid sequence deduced from acsA shows similarity to that

identi-of acyl-CoA synthetase Purified AcsA was shown to be a chain acyl-CoA synthetase, according to the catalytic efficiencies(kcat/Km) for various acids The substrate specificity of AcsA wassimilar to those of aldoxime dehydratase, NHase, and amidase.Pseudomonas chlororaphisB23 grew when cultured in a mediumcontaining butyraldoxime as the sole carbon and nitrogen source.The activities of aldoxime dehydratase, NHase, and amidasewere detected together with that of acyl-CoA synthetase underthe culture conditions used Together with the adjacent locations

short-of these genes, these findings suggest that the four enzymes aresequentially correlated with one another in vivo to utilize butyral-doxime as a carbon and nitrogen source We here propose anoverall ‘‘nitrile pathway’’ (aldoxime fi nitrile fi amide fiacid acyl-CoA) comprising these enzymes

Reference

1 Nature Biotechnol, 1998; 16: 733–736

V1-042P Induction of nitric oxide synthase (NOS) II in macrophages by viral vaccine results in a cytotoxic effect on COS 7 cell line

A Hraba´k1, T Bajor2, I Csuka1and L K Csata´ry3

beneficial anti-tumor effects in people suffering from various

tum-ors without causing serious side effects in treated patients Rat

peritoneal macrophages were elicited by an NDV vaccine in vivo

The number of macrophages increased in a dose-dependent

man-ner The production of nitrite (end product of NO) by isolated

macrophages also increased This effect may be due to the

enhanced expression of the inducible NOS II also dependent on

vaccine doses COS 7 cell line was used as target cell to test the

cytotoxic effect of NO COS 7 cells were killed by NO as tested by

the addition of NO-donors Similar cytotoxicity was observed

when COS 7 cells were co-cultured with macrophages and this

effect was reduced by NOS inhibitors The cause of the induction

of NOS II is not clear: the levels of some cytokines as

interferon-gamma and TNF-alfa were not changed in cell cultures, while the

amount of IL-1beta increased significantly Phagocytic capacity of

elicited macrophages was also enhanced Our results strongly

suggest that the oncolytic property of NDV vaccines may partly

be due to the induction of NOS II in macrophages These

obser-vations support the role of NO in the anti-tumor responses in

mammalian organisms

Acknowledgments: This work was supported by the grants of

OTKA 43075, ETT 213/2003 and by the UCRI

V1-043P

Transition state analog induced structural

change of pigeon liver malic enzyme

C Hui-Chuan1and C Gu-Gang2

1Department of Biochemistry, Avademic Sinica, Taipei, Taiwan

ROC,2Laboratory of Chang Gu-Gang, Department of Life

Science, National Yang-Ming University, Taipei, Taiwan ROC

E-mail: chceve@pchome.com.tw

Pigeon liver malic enzyme catalyzes a divalent metal ion (Mn2+

or Mg2+)-dependent reversible oxidative decarboxylation of

l-malate to yield CO2 and pyruvate with a concomitant

constructed an N-terminal His-tagged malic enzyme and purified

the enzyme by affinity chromatography We got a single band in

SDS-PAGE with molecular mass of 65 kDa, which is also

posit-ive on Western blot hybridization with anti-His-tag antibody A

single band was also shown in the native-PAGE gel, which can

be stained for enzyme activity The kinetic parameters of the

tagged-enzyme were the same as untagged, 106.8/s for kcat and

0.15 ± 0.06 mm, 2.9 ± 0.5 lm and 9.4 ± 0.7 lm for the Kmof

malate, Mn2+, and NADP+, respectively A slow binding

phe-nomenon was observed for oxalate (transition state analog)

0.0069 ± 0.0005 and 0.0029 ± 0.0003/s, respectively The

bind-ing constant for oxalate (Ki) was found to be 35.1 ± 9.5 lm

This result showed that oxalate induces a conformational change

of the enzyme form the ground state to an activated transition

state, however, since oxalate is not a true transition state

interme-diate, it is catalytically incompetent and the enzyme activity is

inhibited This inhibition could be reversed by the true substrate

malate In accordance with the native-PAGE, AUC provides an

excellent tool to explore the quaternary structure of protein The

tagged malic enzyme exists as tetramer in solution with

sedimen-tation coefficient in 10 S The sedimensedimen-tation coefficient but not

the molar mass was changed if the tagged enzyme was incubated

with high salt concentration If the tagged enzyme was incubated

under acidic or basic conditions, the tetramer was dissociated to

monomer These results indicated that the major interfacial

inter-actions of malic enzyme included charge-charge interaction or

hydrogen bonding

V1-044P Utilization of regulatory expression mechanisms of nitrile-degrading enzymes in Streptomyces

S Herai, Y Hashimoto, H Kabumoto, H Higashibata and

M KobayashiInstitute of Applied Biochemistry and Graduate School of Life andEnvironmental Sciences, The University of Tsukuba, Tsukuba,Ibaraki Japan E-mail: s-herai@bsys.tsukuba.ac.jp

The microbial degradation of nitriles proceeds through two matic pathways: nitrile hydratase (NHase) catalyzes hydration ofnitrile to amide, whereas nitrilase catalyzes hydrolysis of nitrile

enzy-to acid and ammonia Both nitrile-converting enzymes havepotential abilities from the application standpoints; particularly,NHase in an actinomycete Rhodococcus rhodochrous J1 is usedfor the industrial production of acrylamide and nicotinamide [1].NHase is inducibly overexpressed approximately 50% of all sol-uble protein in the J1 strain cultured in the presence of urea TheJ1 strain also contains nitrilase, which is inducibly formed up to35% of all soluble protein by the addition of isovaleronitrile orepsilon-caprolactam to the culture medium Based on the experi-mental data of SDS-PAGE and Northern blot analyses, we clar-ified the transcriptional regulation mechanisms of the bothNHase gene and nitrilase gene The NHase expression mechan-ism requires both NhhC and NhhD as the transcriptional regula-tor proteins, whereas, the nitrilase expression is regulated by theonly one positive regulator protein, NitR These strong inductionmechanisms are expected to be applicable for constructing regula-tory expression systems for actinomytcetes, especially Streptomy-ces, which is the most important antibiotics-producing genus Weinvestigated whether the both Rhodococcus regulatory systemsfunction in Streptomyces Both expression mechanisms func-tioned even in Streptomyces; nitrilase and NHase expression pat-terns were inducible and constitutive, respectively Very recently,

we succeeded in construction of a novel hyper-inducible sion vector for Streptomyces based on the nitrilase mechanism.Development of NHase-based constitutive gene expression system

expres-is also in progress

Reference

1 Nature Biotechnol 1998; 16: 733–736

V1-045P Deduced enzymatic function of the proteins encoded by the biosynthetic (ata) gene cluster

of the nucleoside antibiotic A201A

I Saugar, E Sanz, B Molloy, A Ceballos,

M Ferna´ndez Lobato and A Jime´nezCBMSO, Auto´noma Madrid, Madrid, Spain

E-mail: ajimenez@cbm.uam.esA201A is an aminonucleoside antibiotic that is produced byStreptomyces capreolus Its biosynthetic (ata) gene cluster (34 kb)was inserted in the chromosome of Streptomyces lividans Thisbacterium was shown to produce A201A A total of 28 ORFswere detected Comparison of their deduced peptide sequencesand complementation of a variety of heterologous mutants per-mitted both to assign possible functions to all of them and todepict an A201A biosynthetic pathway The Ata P3 (phospha-tase), 5 (methyltransferase), 4 (aminotransferase), 10 (NAD-dependent dehydrogenase) and 7 (pyrophosphatase) wouldsynthesize the aminonucleoside moiety of A201A from ATP

AtaPks1 (acyltransferase, AT), AtaPks2 (acyl carrier protein,

ACP), Pks3 (keto synthase, KS), PKS4 (control lengh factor,

CLF), Ata4 (ketoreductase,KT) and Ata2 (dehydratase) would

be implicated in the biosynthesis of polyketide moiety

(a-methyl-p-coumaric acid) The joining of the carboxyl group of this

moiety to the amino group of the aminonucleoside one would be

achieved by a non-ribosomal peptide bond synthesis Ata15

(amide bond formation), 18 (adenylating enzyme with an

AMP-binding site) and 19 (it contains a 4’-phosphopantetein AMP-binding

motive; ACP) could act as a multimodular synthase An

unsatur-ated hexofuranose moiety could be synthesized from d-rhamnose

by oxidoreductases Ata7, 10 and 14 Ata5, a possible

UDP-glyco-syltransferase, could link it to the polyketide moiety The

d-rhamnose moiety of A201A could be synthesized from

d-rhamnose by Ata12 (NDP-hexose 4, 6 dehydratase) and Ata17

(NDP-hexose ketoreductase) Ata13 (glycosyltransferase) could

link it to that hexofuranose Methylation of these two sugars

could be performed by the SAM-dependent Ata6, 8 and 11

Laboratory of Dr Ettrich, Institute of Physical Biology of USB

and Institute of Landscape Ecology of AS CR, Nove Hrady, Czech

Republic,2Laboratory of Dr Bickle, Department of Microbiology,

Biozentrum,University of Basel, Basel, Switzerland,3Department

of Molecular Cancer Research, University of Zurich, Zu¨rich,

Switzerland E-mail: jindrova@greentech.cz

Although the DNA-cleavage mechanism of Type I

restriction-modification enzymes has been extensively studied, the mode of

how these enzymes introduce DNA double-strand breaks still

remains elusive In this work, DNA ends produced by EcoKI,

EcoAI and EcoR124I, members of the Type IA, IB and IC

famil-ies, respectively, have been characterized by cloning and

sequen-cing of restriction products from reactions with a plasmid DNA

substrate containing a single recognition site for each enzyme

We show that all three enzymes cut this DNA randomly with no

preference for a particular base composition surrounding the

cleavage site, producing both 5’- and 3’-overhangs of varying

lengths EcoAI preferentially generated 3’-overhangs of 2–3

nucleotides, whereas EcoKI and EcoR124I displayed some

pref-erence for formation of 5’-overhangs in a length of about 6–7

and 3–5 nucleotides, respectively A mutant EcoAI endonuclease

assembled from wild-type and nuclease-deficient restriction

sub-units generated a high proportion of nicked circular DNA,

whereas the wild-type enzyme catalyzed efficient cleavage of both

DNA strands We conclude that Type I restriction enzymes

require two restriction subunits to introduce DNA double-strand

breaks, each providing one catalytic center for phosphodiester

bond hydrolysis Possible models for DNA cleavage are further

studied and discussed

Acknowledgments: This research was supported by the

Minis-try of Education, Youth and Sports of the Czech Republic

(MSM6007665808) and by the Academy of Sciences of the Czech

Republic (Institutional research concept AVOZ60870520)

V1-047P Mouse brain serine racemase, novel approaches to activity determination

J Jiraskova1,2, K Strisovsky1, D Koval1, C Barinka1and

J Konvalinka1,2

1

Department of Biochemistry, Institute of Organic Chemistry andBiochemistry, Academy of Sciences of the Czech Republic, Prague,Czech Republic,2Department of Biochemistry, Charles University,Faculty of Natural Sciences, Prague, Czech Republic

E-mail: jjiraskova@yahoo.co.ukSerine racemase is a novel enzyme catalyzing reversible conver-sion between l- and d-serine d-serine has been previously identi-fied in the mammalian brain where it serves as ‘‘co-agonist’’ ofthe ‘‘glycine binding site’’ of the N-methyl-d-aspartate-receptors(NMDA) NMDA receptor overactivation has been implicated in

a number of pathological conditions (such as brain stroke, betic neuropathy or Alzheimer disease) and serine racemase rep-resents thus potentially interesting target for therapy Serineracemase is a pyridoxal-5’-phosphate (PLP)-dependent enzymethat shares some properties of PLP-binding enzymes, like cata-lysis of side reactions and low substrate specificity Some of theproperties of serine racemase are unique among the PLP-bindingenzymes, for example activation by ATP-Mg2+complex Newsubstrates, inhibitors and activators have been identified andstudied New activity determination approaches were introducedusing HPLC or capillary electrophoresis (CE) based methods.Direct metabolic studies in mouse serine racemase transfectedhuman embryonal kidney cells (HEK 293) have been performed

dia-In order to enable structure analysis of the important enzyme,screens for crystallization conditions have been performed to pro-duce diffracting crystal of mouse serine racemase

V1-048P The effect of antracyclines on histone proteins

in active and inactive chromatin

M Jafari1,2and A Rabbani1

11Institute of Biochemistry and Biophysics, University of Tehran,Tehran, Iran,2Department of Biochemistry, Faculty of Medicine,Baqiyatallah University, Tehran, Iran E-mail: jafari@bmsu.ac.irDaunomycin and adriamycin are the most effective anthracyclineantibiotics, which are widely used in treatment of variety of can-cers The main target of these drugs is DNA in which inhibitDNA replication and transcription In chromatin, the binding ofhistones and non-histone to DNA, producing nucleosomalstructure probably influence the binding accessibility of DNA todrugs The present study represents an attempt to investigate theeffects of these drugs on histone proteins of active and inactivechromatin Therefore, calf thymus chromatin was fractionatedinto active (S1 and S2) and inactive (P2) chromatin by micrococ-cal nuclease digestion Then, the interaction of these drugs withthese fractions was investigated employing absorption anddifference UV.Vis spectroscopy and SDS gel electrophoreses Theresults show that with increasing drugs concentration, theamount of released proteins in the S2 and P2 decreased, while inS1 did not change Also, the tendency of adriamycin was morethan daunomycin The results suggest that as drug concentration

is increased in the mixture, core histones are aggregated and cipitate with the chromatin The extent of condensation in P2 ishigher when compared to the S2 fraction

Alteration of lymphocyte subsets and MHC

class II expressed cells after chronic methanol

exposure

N P Jeya Parthasarathy, R Srikumar and R S Sheela Devi

Laboratory of Neuroimmunomodulation, Department of

Physiol-ogy, University of Madras, Chennai, Tamilnadu, India

E-mail: njeyaparthasarathy@yahoo.co.in

Severe exposure to methyl alcohol leads to liver damage,

blind-ness, and finally death The adverse effects of methanol toxicity

receive attention only when severe signs and symptoms of mass

methanol intoxication sets in or after death Its action on the

immune system remains unexplored In this study, attempts

have been made to explore the effects of chronic methanol

explore (2.37 g/kg body weight in saline/day IP) to male wistar

albino rats for 30 days Along with control and immunized

con-trol animals group The results were analyzed by one way

ana-lysis of variance (ANOVA) followed by Tukeys multiple

comparison There was a significant decrease (P < 0.05) in the

thymocytes count when compared to the control group and no

significant alteration was observed in the thymus immunized

groups compared to the control However no significant

altera-tion in the splenocytes count was observed The chronic

metha-nol exposure as well as in immunized chronic methametha-nol

exposure showed a significant increase (P < 0.05) in the

leuko-cyte migration inhibition (LMI index) compared to their

respec-tive controls Antibody titer was found to be significantly

decreased (P < 0.05) in immunized chronic methanol exposed

group compared to the control immunized animals These

results indicate that chronic exposure of methanol at this

dosage can affect thymus cells, cell mediated immunity and

humoral immunity Lymphocyte subsets and MHC class II

expressed cells and macrophages count results will be showed in

time of presentation

V1-050P

An unusual RNA–protein interaction leading to

tRNA import into yeast mitochondria: in vitro

and in vivo studies

P Kamenski1, N Entelis1, I A Krasheninnikov2, R P Martin1

and I Tarassov1

1

FRE 2375 of the CNRS, IPCB, Strasbourg, France,2Department

of Molecular Biology, Faculty of Biology, Lomonosov Moscow

State University, Moscow, Russian Federation

E-mail: p-kamensky@mail.ru

Only one tRNA is imported into yeast mitochondria from the

cytoplasm This tRNA is one of the two yeast lysine cytosolic

tRNAs One of the tRNA import factors was identified as the

precursor of mitochondrial lysyl-tRNA synthetase (preMSK1p)

It is transported into mitochondrial matrix due to its

N-ter-minal presequence Inside the mitochondria, its mature form

(MSK1p) aminoacylates the mitochondrial lysine tRNA but not

the imported one Interaction with preMSK1p is absolutely

necessary for tRNA import In order to understand which parts

of preMK1p are important for tRNA mitochondrial targeting,

we performed an alignment of all known lysyl-tRNA synthetases

and found six unique motifs in preMK1p molecule We

constructed several mutant preMSK1p versions with the

dele-tion of one or several unique motifs and measured tRNA

import efficiency in vivo Unexpectedly, in almost all the mutant

strains import efficiency was even higher than in wild type

yeast From our previous studies, it was known that preMSK1p

N-terminal domain was able to direct tRNA import into

isolated yeast mitochondria In this work, we constructed fourshorter versions of preMSK1p N-terminal domain, each of themhas a native mitochondrial N-terminal pre-sequence and lacksseveral structural elements on the C-terminus Unexpectedly,truncated versions were shown to direct import but to looseimport directing specificity, permitting import of tRNAs thatare normally not imported The mechanisms of this phenom-enon are discussed

Acknowledgments: This study was supported by RFBR andPICS (CNRS)

V1-051P Development of antiviral drugs targeting the flaviviruses fusion mechanism

T Kampmann1, R Yennamalli4, M Sto¨ermer3, B Kobe2and

P Young1

1

Department of Microbiology and Parasitology, University ofQueensland, Brisbane, Queensland, Australia,2Department ofBiochemistry and Molecular Biology, University of Queensland,Brisbane, Queensland, Australia,3Institute for MolecularBioscience, University of Queensland, Brisbane, Queensland,Australia,4Bioinformatics Center, School of InformationTechnology, Jawaharlal Nehru University, New Delhi, India.E-mail: s4021216@student.uq.edu.au

We have successfully identified and tested small molecules whichinhibit the dengue virus fusion mechanism, a process essential forviral infection of cells The compounds were identified by in silicodocking of a compound library against the dengue virus envelope(E) protein We hypothesize that these compounds inhibit thetransition of the E protein from its metastable pre-fusion form toits stable post-fusion form We tested the compounds in plaqueand MTT assays and identified one compound with an IC50 of

1 lm with no mammalian cytotoxicity This lead compound isnow undergoing further studies for the development of a denguevirus fusion inhibitor Furthermore, the high conservation of Eprotein structural elements amongst the flaviviruses suggests thatthis compound may also serve as a lead for the development offusion inhibitors against other members of the flavivirus family,for example the West Nile fever and Japanese encephalitis vir-uses Flaviviruses, in particular, dengue virus are a major publichealth problem in tropical and subtropical areas of the world

Up too 100 million people are infected annually and the dence of these diseases is increasing over the last two decades.Infection with dengue virus can result in acute and potentiallyfatal haemorrhagic fever Currently there are no therapeuticagents available for treating flavivirus infections The identifica-tion of our lead compound is a first step toward their develop-ment

inci-V1-052P Mandelate pathway regulation

in Pseudomonas putida ATCC 12633

G L Kenyon, M M Kneen and M J McLeishMedicinal Chemistry, University of Michigan, Ann Arbor, MI,USA E-mail: gkenyon@umich.edu

The mandelate pathway in Pseudomonas putida ATCC 12633consists of four enzymes that facilitate the conversion of (R,S)-mandelate to benzoate, thereby permitting growth on mandelate

as a sole carbon source A fifth enzyme, mandelamide hydrolase(MAH), appears also to be associated with the pathway Whilethe structure and function of the enzymes of the mandelate path-way have been studied, the regulatory mechanisms of the path-way are not understood The genes encoding the four mandelate