Báo cáo khoa học: H1-Protein Crosslinking by Transglutaminases docx

Szondy Department of Biochemistry and Molecular Biology, University of Debrecen, Debrecen, Hungary.. E-mail: fesus@indi.dote.hu Transglutaminase 2 TG2, a multifunctional enzyme with Ca2+

H1-Protein Crosslinking by Transglutaminases

H1–001

Introduction to the chemistry and biology of

transglutaminases (TGs)

L Lorand

Cell and Molecular Biology, Northwestern University Feinberg

Medical School, Chicago, IL USA

E-mail: l-lorand@northwestern.edu

Eukaryotic TGs evolved from the papains, but utilizing a Trp/

Cys/His/Asp catalytic tetrad instead of Gln/Cys/His/Asp for

acy-lation-deacylation Because saturable binding of specific amine

substrates, aminolysis is preferred over hydrolysis in deacylation

Transamidation leads to protein remodelling of the gamma

amides of select Gln residues by crosslinking to epsilon amines of

select Lys’s or by incorporation of small primary amines Such

post-translational modifications have important biological

conse-quences Thrombin/Ca2+-activated plasma Factor XIII, a

mem-ber of the TG family, is essential for stabilizing blood clots; it

can also promote angiogenesis Another form of the factor

acti-vated by Ca2+ alone, crosslinks the angiotensin receptor and

causes adhesion of monocytes, thereby initiating atherosclerosis

in hypertension In platelets, it catalyzes protein crosslinking and

incorporation of serotonin into G proteins to promote release of

procoagulants Other TGs are essential for forming the cornified

envelope of skin; stabilize dermo-epidermal junctions; mediate

cell-matrix interactions and aid mineralization; bind and

hydro-lyze GTP TGs are involved in autoimmune, inflammatory and

degenerative conditions, and apoptosis, while diverse hereditary

diseases are caused by the deficiency of a TG (Nature Rev Mol

Cell Biol2003; 4: 140–156) TGs are important catalysts for the

synthesis of novel biocompatible polymers, also for incorporation

of a variety of bioactive substances, including growth factors

H1–002

Allosteric regulation of the transamidase

activity of transglutaminase 2 by guanine

nucleotide

G E Begg1, S E Iismaa1, L Carrington2, S Holman1,

A Husain1,3and R M Graham1,4

1Victor Chang Cardiac Research Institute, Sydney, NSW

Australia,2School of Mol and Microbial Sci., University of

Queensland, Brisbane, QLD Australia,3Physiology Department,

University of Alabama, Birmingham, AL, USA,4University of

NSW, Sydney, NSW Australia

E-mail: b.graham@victorchang.unsw.edu.au

Transglutaminase 2 (TG2) is a multifunctional protein, with

actions including receptor signalling, extracellular matrix

assem-bly and apoptosis It has two enzyme activities: ted signalling that requires GTP binding, and calcium-activatedtransamidation (TGase activity) that is inhibited by GTP Theseactivities are associated with distinct conformations induced bythe allosteric regulators, calcium and GTP The recently reportedcrystal structure of human TG2 bound to GDP identified a novelguanine nucleotide-binding site comprised of residues in the firstbeta barrel and core domains [Liu, S., Cerione R.A., & Clardy J.Proc Natl Acad Soc USA 2002; 99, 2743–2747] Here we haveinvestigated the allosteric mechanism by which GTP inhibitsTG2 TGase activity We have shown that of the proposed GTPbinding-site residues, S171, F174 and R579 are indeed critical forGTP binding, while R476 and R478 are individually less import-ant We demonstrate using sedimentation velocity analysis that inthe absence of GTP, TG2 undergoes dynamic sampling of expan-ded and compact conformations, and that GTP-binding shiftsthe equilibrium to a compact conformational substate Thisallows a hydrogen bond to form between the active site cysteine

receptor-stimula-in the core domareceptor-stimula-in, and a tyrosreceptor-stimula-ine residue receptor-stimula-in the first beta-barreldomain We show that this Cys/Tyr bond not only stabilizes thecompact conformation, but is critical for inhibition of TGaseactivity by GTP Furthermore, we show that R579 is a conform-ational switch residue that, in the absence of GTP, destabilizesthe compact conformation Our findings support a unique modelfor allosteric regulation of TG2, in which GTP not only masksthe destabilizing R579 residue, but also links the core and firstbeta-barrel domains, thereby stabilizing the compact conforma-tion, facilitating C277/Y516 H-bond formation, and inhibitingsubstrate access to the active site

H1–003 Chemistry and Biology of Human Transglutaminase 2: its role in celiac sprue and other diseases

C KhoslaDepartments of Chemistry, Chemical Engineering, andBiochemistry, Stanford University, Stanford, CA, USA

E-mail: khosla@stanford.eduTissue transglutaminase (a.k.a transglutaminase 2; TG2) cata-lyzes cross-linking between selected glutamine c-carboxamidegroups and lysine e-amino groups in proteins Although thechemistry of this enzyme is well understood, its multi-faceted role

in mammalian biology is only just beginning to be appreciated.TG2 also has considerable medicinal significance, as it is believed

to play a crucial role in the pathogenesis of diverse human ders, including celiac sprue, neurological disorders and certain

disor-types of cancers We have taken a chemical approach toward

understanding the biology and pharmacology of human TG2

Our studies focus on the design, evaluation and application of

new substrates and inhibitors of this remarkable enzyme Of

fore-most interest is the role of this enzyme in celiac sprue

pathogene-sis Our recent findings on this subject will be presented

H1–004

Transglutaminase-mediated immobilization of

growth factors within fibrin for tissue healing

J A Hubbell

Integrative Biosciences Institute, Ecole Polytechnique Fe´de´rale de

Lausanne, Lausanne, Switzerland E-mail: jeffrey.hubbell@epfl.ch

Growth factor-based therapies have held much promise in tissue

repair, but the clinical results of such therapies have not met with

initial expectations One limitation may be in the mode of

presen-tation of the factors: in nature, most growth factors are presented

bound to extracellular matrix components, and moreover

signal-ling involves ligation of both the growth factor receptors and also

the extracellular matrix adhesion protein receptors In contrast to

this, most growth factors have been explored in therapy as soluble

proteins, substantially unrelated to their natural mode and contact

of presentation in development and repair To begin to address

the disconnect between the natural function and therapeutic

pres-entation of growth factors, our group has explored the design and

production of growth factors, as well as related adhesion

mole-cules, capable of binding clinically useful cell invasion matrices

We have focused on fibrin as such as matrix material Growth

fac-tor variants have been designed as fusion proteins of a

fibrin-bind-ing immobilization site, a proteolytic cleavage site, and the growth

factor of interest As a fibrin-binding domain, we have selected a

factor XIIIa substrate from the N-terminus of alpha 2 plasmin

inhibitor, and we have demonstrated that fusion proteins

contain-ing this domain are effectively and nearly quantitatively coupled

into fibrin during coagulation Chronic dermal wound may related

to an angiogenic deficit in the skin, and for this reason we have

addressed the function of several candidate angiogenic growth

fac-tors Vascular endothelial growth factor A 121 (VEGF-A121) was

expressed as such a fusion protein, with the transglutaminase

sub-strate (TG) at the N-terminus, an adjacent plasmin-sensitive

clea-vage site (pl), and a C-terminal growth factor domain In vivo, this

growth factor bound within fibrin was demonstrated to induce

angiogenesis that was more localized, more intense, and most

importantly more morphologically normal than the free growth

factor For example, in the chicken chorioallantoic membrane,

blood vessels were well formed, with a clearly demarcated basal

lamella and associated pericytes, whereas free VEGF-A121

induced angiogenesis without these morphological features

Sim-ilar work platelet-derived growth factor variants is under way

H1–005

Transglutaminase 2 in the balance of cell

survival and death

L Fe´su¨s and Z Szondy

Department of Biochemistry and Molecular Biology, University of

Debrecen, Debrecen, Hungary E-mail: fesus@indi.dote.hu

Transglutaminase 2 (TG2), a multifunctional enzyme with Ca2+

-dependent protein crosslinking activity and GTP dependent G

protein functions, is often upregulated in cells undergoing

apop-tosis Studies from several laboratories have revealed that in

cul-tured cells TG2 may exert both pro- and anti-apoptotic effects

depending upon the type of cell, the kind of death stimuli, the

intracellular localization of the enzyme and which of its activities

is switched on The majority of data support the notion that

transamidation by TG2 can both facilitate and inhibit apoptosis,

while the GTP-bound form of the enzyme generally protects cellsagainst death Recently described biochemical properties of TG2,including its protein disulphide isomerase and protein kinaseactivities, also may contribute to either survival or death of cells

In vivostudies using TG2 knock out mice confirm the Janus faceaction of TG2 in the molecular pathways of the apoptotic pro-gram They have revealed an additional role of TG2: the preven-tion of inflammation, tissue injury and autoimmunity once theapoptosis has already been initiated This function of TG2 is par-tially achieved by being expressed also in macrophages engulfingand digesting apoptotic cells TG2 mediated processes participate

in the communication between the phagocytic and the apoptoticcells facilitating both death and clearance of dying cells

H1–006 Characterization of transglutaminase and its role in pancreatic b-cell function and survival

S Dookie1, N G Morgan2, M Griffin3and E A M Verderio1

1

School of Biomedical and Natural Sciences, The NottinghamTrent University, Nottingham, UK,2Peninsula Medical School,Plymouth, UK,3Aston University, Birmingham, UK

E-mail: shakthi.dookie@ntu.ac.ukTransglutaminases (TGs) are Ca2+-dependent enzymes that cata-lyze the formation of protein crosslinks via intermolecular iso-peptide bonds Previous work has shown that pancreatic isletsexpress functional tissue-type TG (tTG) and recent work under-taken in tTG-deficient mice points to a possible role for tTG inthe development of type-II diabetes Although these data implythat TGs are required for the maintenance of adequate pancre-atic b-cell function, this has never been characterized at themolecular level Moreover, the recently described survival rolefor matrix-associated tTG (Verderio et al, JBC, 2003) has notbeen investigated in islets Here we show, for the first time, thatthe predominant form of TG expressed in both a clonal rat insu-linoma b-cell line (BRIN-BD11) and pancreatic islets from differ-ent species is a novel 55 kDa TG protein which may be involved

in stimulus-secretion coupling due to increased enzyme activityupon glucose stimulation Furthermore, this 55 kDa TG proteinwas not a proteolytic fragment of tTG since its expression wasunchanged following inhibition of matrix-associated proteases Apossible protective role for TGs in conditions resembling the dia-betic milieu has also been investigated in vitro, by inducingconditions of glucotoxicity, oxidative stress and lipotoxicity Apre-conditioned matrix secreted by urinary bladder carcinomacells (5637), known to support pancreatic b-cell survival, appeared

to improve cell spreading and inhibit caspase-3 activity of clonalb-cells when supplemented with exogenous purified tTG In con-clusion, these findings suggest the predominant expression andfunction of a novel TG in pancreatic islets but also provides evi-dence for a survival role of matrix tTG, which may be deposited

by different cell types surrounding b-cells in vivo

H1–007P Tissue transglutaminase promotes survival of differentiated SH-SY5Y human neuroblastoma cells following exposure to MPP+

K Beck1, L A De Girolamo1, M Griffin2, A Hargreaves1and

E E Billett1

1

Interdisciplinary Biomedical Research Centre, School of cal and Natural Sciences, Nottingham Trent University, Notting-ham, UK,2School of Life and Health Sciences, Aston University,Birmingham, UK E-mail: ellen.billett@ntu.ac.uk

Biomedi-Tissue transglutaminase (tTG) is a multi-functional, Ca2+

dependent enzyme, which modifies proteins by transamidation of

specific polypeptide bound glutamate residues, resulting in

pro-tein crosslinking or incorporation of polyamines into substrates

The role of tTG in cell survival and death pathways remains

elu-sive, but it has been implicated in the pathology of several

neuro-degenerative diseases MPTP via its active metabolite MPP+

induces symptomatic, biochemical and pathological features of

Parkinson’s disease in primates, including man The role of tTG

was investigated in pre-differentiated SH-SY5Y cells exposed to

cytotoxic concentrations of MPP+(1 and 5 mm) for 24 h

West-ern blot analysis revealed that MPP+ treatment significantly

reduced tTG protein levels On the other hand, MPP+ dose

dependently increased TG transamidating activity, measured by

incorporation of a polyamine substrate (EZ-link-5-[biotinamido]

pentylamine) into proteins, detected using a Neutravidin, HRP

conjugated secondary antibody and ECL In the presence of a

competitive TG inhibitor (putrescine) and a specific, irreversible

inhibitor of internal tTG, (R283), MPP+-mediated TG activity

was reduced, suggesting it be, in part mediated by tTG activity

To investigate the role of tTG in MPP+-mediated toxicity, the

effects of tTG inhibitors on the viability of cells treated with

MPP+ were assessed using the MTT reduction assay Results

demonstrated that tTG inhibition exacerbated MPP+ toxicity,

suggesting that tTG promotes survival in this toxicity paradigm

Given that tTG catalysed crosslinks have been found co-localized

with alpha-synuclein in Lewy bodies of Parkinsonian brains,

fur-ther research into the role of tTG in MPP+-mediated toxicity is

Apoptosis Research Group, Biochemistry and Molecular Biology,

University of Debrecen, Debrecen, Hungary,2Retroviral

Biochem-istry Goup, BiochemBiochem-istry and Molecular Biology, University of

Debrecen, Debrecen, Hungary,3Apoptosis Research Group,

Biochemistry and Molecular Biology, University of Debrecen,

Debrecen, Hungary E-mail: cseva@indi.biochem.dote.hu

Transglutaminases catalyze the Ca2+-dependent formation of

e-amino-c-glutamil isopeptide bonds between the c-carboxamide

groups of glutamine residues and e-amino groups of lysines or

primary amines The protein substrates of transglutaminases are

numerous, ranging from proteins of the immune system to

pro-teins bearing gene regulatory functions So far the studies of

the primary amino acid sequence for known Gln substrate

pro-teins have not revealed a consensus sequence for modification

by transglutaminase We have constructed a database of

transgl-utaminase substrate proteins (http://www.biochem.dote.hu/

TRANSDAB) which contains structural information about

sub-strates of six different transglutaminase types Using a computer

program, we have analyzed the 3D structure of substrate

pro-teins, examining the atoms present in a 1.5 nm radius sphere

around the C(delta) of each Gln residue The first detailed

study was carried out for tissue transglutaminase (TG2) using

crystal structure data of thirteen substrate proteins where the

modified Gln residues are known We also have calculated the

radial distribution function (RDF) for pairs of Gln C(delta)

and any other type of atom The RDF values showed

differ-ences for substrate and for non-substrate glutamines These

results indicate that the steric and/or electrostatic environment

might be different around the substrate and the non-substrate

Gln residues of TG2 determining which Gln residue is

recog-nized by the enzyme

H1–009P Human cyclo-statherin: specific cyclization of salivary statherin by transglutaminase as a potential early event involved in the formation

of oral protein pellicle

T Cabras1, R Inzitari2, C Fanali2, C Olmi2, M Patamia3,

M T Sanna1, E Pisano1, B Giardina2,3,4, M Castagnola2,3,4and I Messana1

1

Department of Sciences Applied to Biosystems, CagliariUniversity, Cagliari, Italy,2Institute of Biochemistry and ClinicalBiochemistry, Catholic University, Rome, Italy,3Institute for theChemistry of Molecular Recognition, National Research Council(CNR), Rome, Italy,4International Scientific Institute Paolo VI,Catholic University, Rome, Italy E-mail: tcabras@unica.itStatherin is a phospho-peptide of 43 amino acid residues pecu-liar of human saliva with unusual characteristics, such as highcontent of Tyr, Pro and Gln and a high degree of structuralasymmetry Recently, a significant low level of statherin in sal-iva of subjects affected by pre-cancerous and cancerous lesions

of the oral cavity, but not in those affected by tumours of ary glands, was demonstrated [1] The pivotal multifunctionalrole of statherin in the oral cavity could be connected to itsinvolvement in the formation, through protein crosslinking, of aprotein network with proline-rich proteins and histatins Thisoral protein pellicle could interact with the oral epithelial-cellplasma membrane, contributing to mucosal epithelial flexibilityand turnover [2] The enzyme responsible for the covalent cros-slink formation is an oral transglutaminase (TGA) highly pre-sent in oral epithelial cells [3] In the present study thereactivity of statherin in the presence of TGA from human oralepithelial cells was investigated by RP-HPLC ESI-ion trap MS.The mass of the reaction product (5363 vs 5380 amu) evidencedthat the early event of the homotypic TGA reaction is the spe-cific formation of a cyclo-statherin The RP-HPLC ESI-ion trap

saliv-MS analysis of the proteolytic digest of cyclo-statherin byproteinase K allowed establishing that the intra-molecular cros-slinking involves Lys-6 and Gln-22 Interestingly, a SIM strat-egy performed on different human salivary samples evidenced insome subjects the presence of cyclo-statherin, even though at aconcentration near to the limit of detection of the analyticalmethod (about 0.1 lm/l)

References

1 Contucci et al., Oral Diseases 2005; 11: 95–99

2 Bradway et al., Biochem J 1989; 261: 887–896

3 Bradway et al., Biochem J 1992; 284: 557–564

H1–010P Neural transglutaminase as a potential target for organophosphate toxicity

R Howden1, P L R Bonner1, M Griffin2and

A J Hargreaves1

1Interdisciplinary Biomedical Research Centre, School of cal and Natural Sciences, Nottingham Trent University, Notting-ham, Nottinghamshire, UK,2School of Life and Health Science,Aston University, Birmingham, West Midlands, UK

Biomedi-E-mail: alan.hargreaves@ntu.ac.ukTransglutaminases (TGases) are a group of calcium-dependentenzymes that are responsible for the posttranslational modifica-tion of proteins by the transamidation of polypeptide boundglutamines The reaction occurs either through the formation ofprotein crosslinks or the incorporation of polyamines into sub-strate proteins Of particular interest is the fact that modified

TGase activity is known to be involved in several

neurodegener-ative conditions in mammals Since chemically induced

neuropa-thies, such as organophosphate toxicity, share a number of

common features with these disorders, it was of interest to

determine whether TGase might also be affected by exposure to

organophosphates Treatment of differentiating N2a cells with

the organophosphate phenylsaligenin phosphate (PSP) is known

to inhibit the formation of axon-like processes TGase assays [1]

were carried out on extracts of N2a mouse neuroblastoma cells

induced to differentiate for 24 h in the presence of 3 lm PSP,

using biotin labelled peptide substrates Both extracts were

shown to prefer substrate peptides enriched in glutamine

resi-dues Extracts from PSP treated cells showed a significant

increase in TGase activity compared with non-PSP treated

con-trols A similar result was obtained in porcine brain cytosol

extracts treated with 3 lm PSP compared to controls Our

find-ings suggest that PSP exposure is able to induce a significant

increase in TGase activity in mouse neuroblastoma cells and

mammalian brain extracts, and is consistent with the possibility

that modified TGase activity may be a key molecular event

fol-lowing exposure to PSP

Reference

1 Trigwell, S.M., Lynch, P.T., Griffin, M., Hargreaves, A J and

Bonner P.L.R Analytical Biochemistry 2004; 330: 164–166

H1–011P

Extracellular matrix changes induced by

transglutaminase 2 block angiogenesis and

tumor growth

R A Jones1, P Kotsakis1, T S Johnson2, D Chau1,

E Verderio1, S Ali1and M Griffin3

1

School of Biomedical and Natural Sciences, Nottingham Trent

University, Nottingham, UK,2Sheffield Kidney Institute, Northern

General Hospital, Sheffield, UK,3School of Life and Health

Sciences, Aston University, Birmingham, UK

E-mail: richard.jones2@ntu.ac.uk

Destabilization of the extracellular matrix (ECM) is known to

be important in both tumor invasion and angiogenesis Here we

describe that by promoting ECM accumulation, the protein

crosslinking enzyme transglutaminase 2 (TG2) can serve as a

potent inhibitor of angiogenesis in vitro and tumor growth

in vivo Administration of active TG2 to in vitro angiogenesis

assays suppressed capillary tube formation without causing

cel-lular toxicity, whilst inhibition of endogenous enzyme activity

had no effect on angiogenesis The applied TG2 led to the

accumulation of a complex ECM that surrounded capillary

tubes, resulting in the blockade of capillary growth and the

dis-appearance of endothelial cells from vessels TG2-induced

mat-rix accumulation was accompanied by a decreased rate of ECM

turnover and an increased resistance to matrix

metalloprotein-ase-1 Addition of TG2 to purified collagen I in vitro led to an

accelerated rate of collagen fibril formation, which was

depend-ent on its protein crosslinking activity In vivo, mice bearing

CT26 colon carcinoma tumors demonstrated a reduction in

tumor growth, prolonged survival, and in some cases tumor

regression following intratumor injection of TG2

Immunohisto-chemical and bioImmunohisto-chemical analysis of TG2-treated tumors

revealed fibrotic-like tissue rich in the applied TG2, collagen,

TG2-generated a˚(a˜-glutamyl)lysine isopeptide crosslink, and a

lack of organized vasculature TG2-mediated modulation of the

ECM within a tumor to inhibit angiogenesis and promote

tumor fibrosis may provide a new approach to solid tumor

therapy

H1–012P Coeliac autoantibodies can enhance transamidating and inhibit GTPase activity of tissue transglutaminase

R Kira´ly1, Z Vecsei1, T Deme´nyi1, I R Korponay-Szabo´2and

L Fe´su¨s1

1

Department of Biochemistry and Molecular Biology, Medical andHealth Science Center, University of Debrecen, Debrecen,Hungary,2Heim Pa´l Children’s Hospital, Budapest, Hungary.E-mail: kiralyr@indi.biochem.dote.hu

Enzyme functions of tissue transglutaminase (TG2) and theirmodification by anti-TG2 autoantibodies may play a role in mani-festations of coeliac disease, but the effects observed in previousstudies have been controversial We aimed to evaluate the effect

of coeliac autoantibodies on transamidation, deamidation andGTPase reactions catalyzed by TG2 by a systematic biochemicalapproach and in relation to observed clinical presentation type.Effect of antibodies from coeliac patients representing four groups

on the activity of human recombinant TG2 was studied usingamine-incorporation into solid and immobilized casein and a kin-etic assay GTPase activity was determined by charcoal method.Coeliac patient antibodies did not have significant inhibitory effect

on transamidation/deamidation activity In contrast, globulins from patients with severe malabsorption enhanced thereaction velocity to 105.4–242.2% (mean: 189.5%) compared withcontrol antibodies This activating effect was dose-dependent, andmost pronounced with immobilized glutamine-acceptor substrates,and correlated inversely with the basal specific activity of theenzyme Immunoglobulins purified from the same patients after agluten-free diet did not affect transamidating activity significantly.GTPase activity of TG2 decreased to 67.0–73.4% in the presence

immuno-of antibodies from all coeliac groups In conclusion, an early andsevere malabsorptive clinical manifestation of celiac disease isassociated with the presence of autoantibody population thatenhances transamidation activity of TG2

H1–013P Calreticulin-bound transglutaminase 2 is poorly accessible to celiac autoantibodies

I Korponay-Szabo´1,2, T Halttunen2, K Laurila2, I Dahlbom3,

J B Kova´cs4and M Ma¨ki2

1Dept of Paediatrics, Univ of Debrecen, Debrecen, Hungary,

2Paediatric Research Centre, Univ of Tampere, Tampere, Finland,

3Research Unit, Pharmacia Diagnostics, Uppsala, Sweden,4Dept

of Gastroenterology-Nephrology, Heim Pa´l Children´s Hospital,Budapest, Hungary E-mail: ilma.korponay-szabo@uta.fiBackground and aims: Tissue transglutaminase (TG2) is amajor autoantigen in celiac disease, an autoimmune disorderinduced by the ingestion of gliadin-containing cereal foods Cal-reticulin (CRT), another putative celiac autoantigen, acts as aregulatory subunit for TG2 during receptor signaling CRT hassome homology with gliadin and belongs to molecular chaper-ones We investigated whether CRT is able to interact directlywith TG2 also in vitro and whether this interaction influencesautoantigenic properties of TG2 or of both

Methods: Purified CRT (Sigma) was coated to ELISA plates as

a sole antigen or as a capture compound for TG2 (Sigma) toestablish a sandwich ELISA with a joint CRT-TG2 antigen.Anti-TG2 monoclonal antibodies (MAb) (n = 4) and serum sam-ples of untreated patients with proven gluten-enteropathy(n = 62) were tested with these antigens and with directly coatedTG2 antigen as control Antibody binding to tissue sections rich

in TG2 was also investigated by immunofluorescence

Results: CRT bound TG2 in a dose-dependent manner in thepresence of Ca++ Both free and CRT-bound TG2 were well

recognized by the MAb ETG 2742, therefore the reactivities of

other antibodies were expressed as percentages (AU) of this

stand-ard The MAb CUB7402 specific for the linear epitope 447–478 of

TG2 did not bind to the CRT-bound TG2 (2.9 AU vs 108.3 AU

for the free TG2), the other MAbs gave intermediate results None

of the MAbs while 19 of 62 patient samples reacted with CRT

alone, which correlated with the high positivity for gliadin

anti-bodies The mean reactivity of patient samples with the joint

CRT-TG2 was 19.0 ± 21.5% of that obtained with the free TG2

antigen after background values with CRT had been subtracted

MAb CUB7402 was not able to bind to intracellular TG2

co-localized with CRT in immunofluorescent studies on tissues, while

MAb ETG 2742 showed a partial co-localization Gliadin

inter-fered with the CRT-TG2 complexing in a dose-dependent manner

Conclusions: Binding of CRT to TG2 involves the 447–478

ami-noacids of TG2 and results in the masking of antigenic epitopes

relevant for celiac autoantibodies Improper chaperone function

or displacement of CRT by gliadin might contribute to the

induc-tion of TG2 autoantibodies in celiac disease

H1–014P

Proteolytic processing and secretion of Factor

XIII in articular cartilage

C M Gohr and A K Rosenthal

Department of Rheumatology, Medical College of Wisconsin,

Milwaukee, WI, USA E-mail: akrose@mcw.edu

Transglutaminase enzymes (Tgases) have recently been implicated

in the pathologic matrix mineralization that occurs in articular

cartilage in advanced osteoarthritis Surprisingly, plasma

transgl-utaminase (factor XIII) is present in articular cartilage in addition

to the ubiquitous enzyme, tissue (type II) Tgase We hypothesize

that extracellular factor XIII plays a key role in pathologic matrix

calcification in osteoarthritic articular cartilage, yet little is known

about mechanisms of activation and secretion of cartilage factor

XIII We sought to examine processes of proteolytic activation

and extracellular secretion for cartilage factor XII

Immunohisto-chemistry demonstrated high levels of extracellular factor XIII

concentrated in areas of matrix mineralization in human

osteoar-thritic cartilage Factor XIII was present in extracellular matrix in

cultured chondrocytes and in association with articular cartilage

matrix vesicles These membrane-bound, chondrocyte-derived

ves-icles play a key role in matrix mineralization in cartilage Like

fac-tor XIII, chondrocyte Tgase activity was stimulated by calpain

and furin as well as thrombin and trypsin To determine potential

mechanisms of factor XIII secretion, chondrocytes were exposed

to brefeldin, an inhibitor of classical protein secretion, or

methyl-amine, an inhibitor of non-classical protein secretion Brefeldin

caused no change in extracellular matrix factor XIII levels In

con-trast, methylamine dramatically reduced quantities of factor XIII

in chondrocyte matrix These findings support an important role

for factor XIII in promoting matrix mineralization in articular

cartilage Understanding the processing and secretion of factor

XIII in articular cartilage may lead to the development of novel

therapies for osteoarthritis

H1–015P

An attempt to identify the active center for

protein disulfide isomerase reaction in

tissue-type transglutaminase

Y Michiyama, M Kikuchi, G Hasegawa and Y Saito

Department of Biological Sciences, Graduate School of Bioscience

and Biotechnology, Tokyo Institute of Technology, Yokohama,

Japan E-mail: ysaito@bio.titech.ac.jp

We have found that tissue-type transglutaminase (tTG), also

called as TGc, TGase2, and Gah, has the activity of protein

disulfide isomerase (PDI) We have shown that tTG convertscompletely reduced/denatured inactive RNase A molecule to thenative active enzyme It was substantially inhibited by the simul-taneous presence of other potential substrate proteins such ascompletely reduced BSA, but not by native BSA This activitywas especially high in the presence of GSSG, but not GSH Theaddition of GSH to the reaction mixture in the presence ofGSSG at a fixed concentration up to at least two hundred timesdid not very substantially inhibit the PDI activity It is possiblethat tTG can exert PDI activity in fairly reducing environmentlike cytosol, where most of tTG is found It is quite obvious fromthe following observations that PDI activity of tTG is catalyzed

by domain different from that used for the transglutaminasereaction Although the alkylation of Cys residues in tTG com-pletely abolished the transglutaminase activity as was expected, itdid not affect the PDI activity at all This PDI activity did notrequire the presence of Ca2+ It was not inhibited by nucleotidesincluding GTP at all unlike the other activity of tTG Our resultsindicated that a disulfide bond(s) might serve as the active site

We have been able to show the presence of the complex made upwith the enzyme and the substrate via mixed-disulfide bond(s)

We have started the investigation to elucidate the active sitedisulfide bond For this matter we synthesized a novel convenientmodification reagent for -SH groups We have not completed theelucidation of the active site, but yet I believe we are very close

to the end

H1–016P Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas-mediated cell death

Z Sarang1, P Molna´r2, T Ne´meth2, S Gomba2, T Kardon3,

G Melino4, S Cotecchia5, L Fe´su¨s1and Z Szondy1

TG2 is a protein cross-linking enzyme known to be expressed byhepatocytes, and to be induced during the in vivo hepatic apopto-sis program TG2 is also a G protein that mediates intracellularsignaling by the alpha-1b-adrenergic receptor (AR) in liver cells.Fas/Fas ligand interaction plays a crucial role in various liver dis-eases, and administration of agonistic anti-Fas antibodies to micecauses both disseminated endothelial cell apoptosis and fulminanthepatic failure Here we report that an intraperitoneal dose ofanti-Fas antibodies, which is sublethal for wild-type mice, killsall the TG2 knock-outs within 20 h While TG2-/-thymocytes die

in the presence of anti-Fas antibodies at the same rate as wildtypes, TG2-/-hepatocytes have increased sensitivity towards anti-Fas treatment both in vivo and in vitro, with no change in theircell surface expression of Fas or the levels of FLIPL, but adecrease in the Bcl-xL expression, and appearance of apoptoticcells with unusual morphology The same dose of anti-Fas anti-bodies in wild-type mice induced mostly endothelial cell apopto-sis and consequent necrosis in hepatocytes Administration ofchloroethylclonidine, an AR antagonist, to wild-type mice resul-ted in down-regulation of Bcl-xL in the liver, and sensitizedhepatocytes for Fas-mediated apoptosis Accordingly, mice defici-ent in AR expression were also more sensitive to Fas-inducedkilling, their liver was more sensitive to Fas-mediated apoptosis,and expressed decreased levels of Bcl-xL In conclusion, our data

demonstrate that the loss of TG2 sensitizes hepatocytes for

Fas-mediated apoptosis, and this is related to an impaired AR

signa-ling that regulates the levels of Bcl-xL

T034191, T034 918, ETT 48/2000, ETT 04/605-00 and CT-2002-02017

QLK3-H2–Oxidation of Proteins

H2-001

Oxidation of proteins: an overview

A Benedetti

Dipartimento di Fisiopatologia, Medicina Sperimentale e Sanita

Pubblica, Universita di Siena, Siena, Italy

E-mail: benedetti@unisi.it

Chemically speaking, protein oxidation reactions may involve a

variety of reactions including SH oxidation, hydroxylation of

cer-tain amino acids, and a number of modifications of proteins,

which are directly or indirectly mediated by free radicals In the

endoplasmic reticulum, oxidation of -SH groups is a key event in

the folding of newly synthesized peptides This involves luminal

oxidases/isomerases (such as PDI and Ero1) and requires an

oxi-dative local environment, which, in turn, is maintained by the

interplay between such enzymes and membrane specific

transport-ers Hydroxylation of amino acid residues such as prolyl, lysyl

and asparaginyl is a process relevant not only for the

post-tran-scriptional modification of collagen and other proteins in the

endoplasmic reticulum, but also, in the cytosol, for the regulation

of hypoxia-inducible transcription factors, which are main actors

in certain pathophysiological cellular responses to oxygen A

num-ber of free radical-mediated alterations of cell proteins, often

non-enzymatic, have been described in the last decades They include

not only direct oxidation of amino acid residues and of the

pro-tein backbone, but also the indirect damage of propro-teins by lipid

peroxidation products and glycation reactions A huge number of

studies reports on the involvement of free radical-induced protein

damage in a variety of biopathological conditions (e.g ageing,

neurodegenerative diseases, atherosclerosis, diabetes), nonetheless,

the true role for this event remains to be clarified

H2-002

Redox control in the endoplasmic reticulum.

R Sitia

Laboratory of Transport and Secretion of Protein, DiBiT,

Universita` Vita-Salute, Milan, Italy E-mail: sitia.roberto@hsr.it

Many secretory proteins contain disulfide bonds that are essential

for folding and function The process of achieving the correct

ter-tiary structure and tying it with disulfide bonds occurs in the ER,

under the assistance of specialized protein machineries that

con-trol the conformity of the result and finally decide the fate of the

newly formed protein (quality control) The main oxidative

fold-ing pathway requires the transfer of oxidative equivalents to

newly synthesized cargo proteins via protein disulfide isomerase

(PDI), which in turn is oxidized by Ero1 proteins (Ero1a and

Ero1b in mammals, the latter being induced during ER stress)

However, oxidation must be limited, as some reduced PDI is

necessary for disulfide isomerization and reduction from

termin-ally misfolded proteins prior to their dislocation to the cytosol

for proteasomal degradation How is Ero1 activity regulated? We

tackled this question following different lines of investigation In

a search for proteins capable of interacting with Ero1, we

identi-fied ERp44, a novel multifunctional ER folding assistant In

par-allel, the characterization of a wide panel of Ero1a mutants

revealed interesting features in intra-molecular electron transfer

We also demonstrated that import of cytosolic GSH is necessary

to balance the oxidative power of Ero1 Ero1a over-expressionincreases the GSH content in HeLa cells, implying the existence

of inter-compartmental regulatory pathways that control redoxsignaling and homeostasis Our results shed some light on themolecular networks involved

H2-003 Endoplasmic reticulum lumen: a Janus-faced compartment

G Ba´nhegyiEndoplasmic Reticulum Research Group, Department of MedicalChemistry, Molecular Biology and Pathobiochemistry, SemmelweisUniversity, Budapest, Hungary E-mail: banhegyi@puskin.sote.huThe oxidizing redox environment in the endoplasmic reticulum(ER) lumen is both a pre-requisite and a consequence of the oxi-dative folding of secretory and membrane proteins Luminal thi-ols (including protein cysteines and glutathione) are present inmore oxidized state than the cytosolic ones While the ratiobetween reduced and oxidized glutathione is 100:1 in the cytosol,this value is around 1–2:1 in the ER lumen The redox potentialscalculated from these values are -0.24 and -0.18 V, respectively.Generalizing the observations regarding the redox state of thiolsfound in the ER of cells engaged in protein secretion, now it issupposed that the redox conditions in the ER lumen are oxid-izing However, several intraluminal reactions have been des-cribed, which require reducing equivalents Such reactionsinvolve the isomerization of disulfide bonds, the steps of the vita-min K cycle, the biotransformation of several xenogenousketones and aldehydes and the local activation of cortisone tocortisol This circumstance suggests that more redox systems exist

in the ER lumen Since their redox state must be different, it canalso be suggested that these systems are functionally uncoupled.While molecular oxygen as the ultimate electron acceptor can beused for the direct generation of oxidizing power in the lumen,reducing equivalents need to be transported from the cytosol.The lecture will focus on ER transporters and intraluminal mech-anisms responsible for the support of luminal reducing reactions

H2-004 Cellular oxygen sensing and the regulation of HIF by protein hydroxylation

P J RatcliffeHenry Wellcome Building for Molecular Physiology, Oxford, OxonUnited Kingdom E-mail: pjr@well.ox.ac.uk

Hypoxia inducible factor (HIF) is an alpha/beta heterodimerictranscriptional complex that plays a key role in directing cellularresponses to hypoxia Recent studies have defined novel oxygensensitive signal pathways that regulate the activity of HIF bypost-translational hydroxylation at specific residues within thealpha-subunits HIF prolyl hydroxylation governs proteolyticregulation of HIF, whereas HIF asparaginyl hydroxylationmodulates interaction with transcriptional co-activators Thesehydroxylations are catalyzed by a set of non-haem Fe(II) 2-oxo-glutarate (2OG) dependent dioxygenases During catalysis, the

splitting of molecular oxygen is coupled to the hydroxylation of

HIF and the oxidative decarboxylation of 2-oxoglutarate to give

succinate and CO2 Hydroxylation at two prolyl residues within

the central ‘‘degradation domain’’ of HIF-alpha increases the

affinity for the von Hippel-Lindau (pVHL) E3 ligase complex by

at least three orders of magnitude, thus directing HIF-alpha

polypeptides for proteolytic destruction by the

ubiquitin/protea-some pathway Since the HIF hydroxylases have an absolute

requirement for molecular oxygen this process is suppressed in

hypoxia allowing the HIF-alpha to escape destruction and

acti-vate transcription Co-substrate and co-factor requirements for

Fe (II), ascorbate and the Krebs cycle intermediate (2OG) and

inducible changes in the cellular abundance of three closely

rela-ted HIF prolyl hydroxylases (PHD1–3) provide additional

inter-faces with cellular oxygen status that may be important in

regulating the oxygen sensitive signal

H2-005

Prolyl 4-hydroxylases, key enzymes in the

synthesis of collagens and the response of

cells to hypoxia

J Myllyharju

Collagen Research Unit, Department of Medical Biochemistry and

Molecular Biology, University of Oulu, Oulu, Finland

E-mail: johanna.myllyharju@oulu.fi

Collagen prolyl 4-hydroxylases (C-P4Hs) are essential enzymes in

collagen synthesis Three vertebrate C-P4H isoenzymes are

currently known We have recently produced knock-out mice

for C-P4Hs I and II The C-P4H-I-/-mice died at E10.5, while the

C-P4H-II-/-mice have no obvious phenotypic abnormalities The

C-P4H-I+/); C-P4H-II-/-double mutants, i.e mice with no C-P4H-II

activity and a decreased C-P4H-I activity, are likewise viable but

smaller than the wild-type mice Detailed structure of C-P4H is

currently unknown, but we have determined the domain structure

of its catalytic a subunit, identified catalytically critical residues in

it and solved the structure of its peptide-substrate-binding domain

It was found to belong to tetratricopeptide repeat domains and

possess a groove lined by tyrosines, which were shown to be critical

for the binding of collagen-like peptide substrates The response of

cells to hypoxia is chiefly mediated by the hypoxia inducible

tran-scription factor HIF that upregulates the expression of (e.g.)

ery-thropoietin, VEGF and glycolytic enzymes in hypoxic cells A

novel family of P4Hs and a novel asparaginyl hydroxylase (FIH)

have recently been found to play a key role in the adaptation of

cells to hypoxia by regulating the stability and activity of HIF We

have expressed the three human HIF-P4Hs and FIH as

recombin-ant proteins and characterized their catalytic properties The Km

values of the HIF-P4Hs for O2were slightly above the

concentra-tion of dissolved O2in air, thus indicating that they are very

effect-ive oxygen sensors The Kmof FIH was distinctly lower, indicating

that a larger decrease in O2concentration is needed for a significant

decrease in FIH activity We have also analyzed inhibition

proper-ties of the HIF hydroxylases Furthermore, our studies on the

sub-strate specificity of the HIF-P4H isoenzymes and FIH indicate that

they hydroxylate the three HIF isoforms with varying efficiencies

H2-006

Detection of protein and lipid oxidation in

living cells using fluorescent probes

D Sakharov and K W Wirtz

Bijvoet Institute, Department of Biochemistry of Lipids, Utrecht

University, Utrecht, The Netherlands

E-mail: k.w.a.wirtz@chem.uu.nl

A novel strategy will be presented for the visualization of

ROS-induced protein oxidation in living cells using acetyl-tyramine

fluorescein (acetylTyrFluo) This membrane-permeable probelabels intracellular proteins, which become oxidized at tyrosineresidues under conditions of oxidative stress in a reaction similar

to o,o-dityrosine formation The fluorescein-labelled proteins can

be visualized after gel electrophoresis and subsequent Westernblotting using the anti-fluorescein antibody Identification oflabelled proteins were based on mass spectrometry Exposure ofhuman fibroblasts to hydrogen peroxide (H2O2) resulted in thespecific labelling of endoplasmic reticulum resident proteins.Photodynamic treatment (PDT) of these cells, loaded with thephotosensitizer Hypocrellin A as to generate singlet oxygen resul-ted in the oxidation of a different set proteins Under the condi-tions, where the extent of protein oxidation was comparable,PDT caused massive apoptosis, whereas hydrogen peroxide treat-ment had no effect on cell survival This suggests that the oxida-tive stress generated by PDT with Hypocrellin A activatesapoptotic pathways, which are insensitive to hydrogen peroxidetreatment By using a ratiometric oxidation-sensitive probe,C11-BODIPY581/591 (C11-BO), which reports on lipid peroxida-tion, oxidative stress was visualized by subjecting cells to PDTloaded with a phthalocyanine photosensitizer Pc4 Interestingly,the photosensitizer showed a broad localization in the cytoplasm,whereas the oxidative stress was mostly limited to vesicular peri-nuclear organelles, most likely lysosomes As a result of PDTlysosomes disappeared, indicating that lysosomes were damaged

We propose that the massive apoptosis observed is associatedwith the damage to the lysosomes

References

1 Van der Vlies D et al Biochem J 2002; 366: 825–830

2 Avram D et al Proteomics 2004; 4: 2397–2407

3 Sakharov DV et al Eur J Biochem 2003; 270: 4859–4865

4 Sakharov DV et al FEBS Lett 2005 In press

H2-007P Reactivity and functional significance of cysteine residues of mammalian dipeptidyl peptidases III

M Abramic1, U Imaga1, M Osmak2, L E`Iicin-Ain2,

B Vukelic1, K Vlahovicec3and L Dolovcak1,4

1

Laboratory of Cellular Biochemistry, Department of OrganicChemistry and Biochemistry, Rudger Boskovic Institute, Zagreb,CROATIA,2Department of Molecular Medicine, Rudger BoskovicInstitute, Zagreb, CROATIA,3Protein Structure and Bioinformat-ics Group, International Centre for Genetic Engineering and Bio-technology, Trieste, ITALY,4Department of Molecular Biology,University of Zagreb, Zagreb, CROATIA

E-mail: abramic@irb.hrDipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopepti-dase involved in the intracellular protein catabolism of eukaryo-tes Although inhibition by thiol reagents is a general feature ofDPP III originating from various species, the function of activityimportant sulfhydryl groups is still inadequately understood Thepresent study of the reactivity of these groups was undertaken inorder to clarify their biological significance The inactivation kin-etics of human and rat DPP III by sulfhydryl reagent p-hydroxy-mercuribenzoate (pHMB) was monitored by determination of theenzyme’s residual activity with fluorimetric detection Inactivation

of this human enzyme exhibited pseudo-first-order kinetics, gesting that all reactive SH-groups have equivalent reactivity,and the second-order rate constant was calculated to be 3520M-1min-1 Rat DPP III was hyperreactive to pHMB and showedbiphasic kinetics indicating two classes of reactive SH-groups.The second-order rate constants of 3540 M-1s-1 for slower react-ing sulfhydryl, and 21,855 M-1s-1, for faster reacting sulfhydrylwere obtained from slopes of linear plots of pseudo-first-order

sug-constants versus reagent concentration Peptide substrates

protec-ted both mammalian DPPs III from inactivation by pHMB

Phy-siological concentrations of biological thiols and H2O2

inactivated the rat DPP III Human enzyme was resistant to

H2O2attack and less affected by reduced glutathione than the rat

homologue A significantly lower DPP III level, determined by

activity measurement and Western blotting, was found in the

cyt-osols of highly oxygenated rat tissues These results provide

kin-etic evidence that cysteine residues are involved in substrate

binding of mammalian DPPs III

H2-008P

Thiol oxidase activity of bovine lens aldose

reductase modified by copper ion

A Del Corso, M Cappiello, G M De Donatis, F Gorini and

U Mura

Laboratory of Biochemistry, Department of Physiology and

Biochemistry, University of Pisa, Pisa, Italy

E-mail: delcorso@dfb.unipi.it

Aldose reductase (ALR2), the first enzyme of the polyol

path-way, catalyzes the NADPH-dependent reduction of aldoses and

aldehydes Besides its damaging role during diabetes, linked to

sorbitol production, ALR2 appears to be involved in the

detoxifi-cation of harmful lipid peroxidation products Despite being not

a metal binding protein, aldose reductase is extremely sensitive to

Cu(II) The bovine lens enzyme is readily inactivated by

stoichio-metric concentrations of the metal through an oxygen

independ-ent modification process The modified enzyme (Cu-ALR2)

retains two copper ions and carries a disulfide bond between

Cys298 and Cys303 Both copper ions present on the enzyme

molecule retain their ability to catalyzes oxidation of thiol

com-pounds When thiols are incubated in the presence of Cu-ALR2,

a loss of reduced thiols is observed in a time dependent manner

The rate of thiol oxidation is proportional to the concentration

of Cu-ALR2 and depends on the nature of the thiol used and on

pH Among physiological thiols, while GSH and homocysteine

appear to be quite insensitive to oxidation, Cys, cysteamine and

Cys-Gly are more prone to oxidation, as it occurs when free

cop-per is used as catalyst The metal ions bound to the enzyme

appear as effective as free copper in catalyzing the oxidation of

different thiols The only exception appears to be the case of

Cys-Gly, whose susceptibility to oxidation increases, when

Cu-ALR2 is used as catalyst instead of free copper Copper

che-lation by ALR2 should represent a potentially damaging event

contributing to the oxidative damage induced by the metal

H2-009P

A radish peroxidase intrinsically stable

towards hydrogen peroxide

P Gil1, C Ferreira-Batista2, R Vazquez-Duhalt1and

B Valderrama1

1

Department of Cellular Engineering and Biocatalysis, Institute of

Biotechnology, National University of Mexico, Cuernavaca,

More-los Mexico,2Department of Molecular Medicine and Bioprocesses,

Institute of Biotechnology, National University of Mexico,

Cuerna-vaca, Morelos Mexico E-mail: gil@ibt.unam.mx

Peroxidases are ubiquitous enzymes that catalyze a variety of

oxygen-transfer reactions and are thus potentially useful for

mul-tiple applications However, peroxidases are unstable and are

readily inactivated by their substrate, hydrogen peroxide Here

we report the identification, biochemical characterization and

clo-ning of the gene encoding a novel hydrogen peroxide-tolerant

peroxidase isoenzyme isolated from roots of radish (Raphanus

sativus) and named Zo peroxidase, after the greek word meaning

permanence Furthermore, we show that the tolerance of Zo oxidase towards hydrogen peroxide is an intrinsic property of theenzyme, not due to the presence of catalase activity

per-H2-010P Thionylation of PKC by reactive sulfur species: disulfide S-monoxide and disulfide S-dioxide

F L Huang and K P HuangEndocrinology and Reproduction Research Branch, NICHD, NIH,Bethesda, Maryland United States of America

E-mail: huangk@mail.nih.govDisulfide S-monoxide (DSMO) and -dioxide (DSDO) are power-ful signal molecules during oxidative stress These disulfide S-oxi-des (DSOs) can be generated by oxidation of thiols (glutathione(GSH), captopril (CPSH)) with H2O2 in the presence of iron ormethyltrioxorhenium and the formation of DSOs was moreeffective with free thiols than with their corresponding disulfides,suggesting that sulfenic and sulfinic acids are the intermediatesfor the formation of these compounds Both DSMO and DSDOwere purified by HPLC and identified by mass spectrometry andthey were highly reactive toward thiol to form mixed disulfide.The reaction rate of DSDO was at least an order of magnitudefaster than that of DSMO when tested by reaction with 5-merca-pto-2-nitro benzoate Both DSOs caused dose-dependent inacti-vation of PKC, whose glutathionylation was detected by Westernblot with a specific antibody The IC50 of GS-DSDO-mediatedinactivation of PKC (50 ll) was ~ 10-fold less than that ofGS-DSMO and mechanistically, the former mainly causedthionylation and the latter both thionylation and polymerization

of PKC DSOs derived from CPSH, CP-DSMO and CP-DSDOwere less potent as PKC inhibitors than their correspondingderivatives from GSH, suggesting that the individual DSOs havetheir distinct reactivities in spite of possessing similar functionalgroups Inactivation of PKC by DSOs could be reversed by DTT

at low levels of thionylation but became irreversibly inactivatedafter extensive thionylation These results suggest that DSOsderived from a variety of thiols constitute a repertoire of signa-ling molecules with distinctive roles in regulating oxidant-medi-ated cellular responses

H2-011P Changes in activity levels of zinc- and non-zinc enzymes in rat brain stem and salivary glands with zinc meal

S Hayashi1, Y Kojima2and S Fujiwara1

1

Department of Biochemistry, University of Kyushu DentalCollege, Kitakyushu, Fukuoka Japan ,2Department of PediatricDentistry, University of Kyushu Dental College, Kitakyushu,Fukuoka Japan,3Department of Biochemistry, University ofKyushu Dental College, Kitakyushu, Fukuoka Japan

E-mail: haya-sue@mail.kyu-dent.ac.jpD-aspartate oxidase (D-AspO) is a flavoprotein that catalyzesoxidative deamination of D-aspartate to oxaloacetate, H2O2 and

NH3 D-AspO has been found in many mammal tissues, showingthat D-AspO differs from D-amino acid oxidase (DAO) and islocated in the peroxisomes of mammal liver and kidneys In ourprevious studies, we used rats to study the effects of low intakes

of zinc and calcium on the growth and development in tibiametaphysis However, we have no report on the enzyme forcofactor zinc in rats salivary glands and brain stem We aimed toproduce and eliminate hydrogen peroxide Twenty, three week-old male rats were divided into four groups and given a diet con-taining different levels of zinc: normal, deficient, low and high,

respectively for six weeks Using the methods of sucrose density

gradient centrifugation, we examined the activities of alkaline

phosphatase and copper, zinc superoxide dismutase (Cu,

Zn-SOD) for cofactor zinc enzyme and manganese superoxide

dismutase (Mn-SOD), D-AspO and DAO for non-zinc enzyme to

assess zinc nutrition in the brain stem and salivary glands The

results indicated that, the activities of Cu, Zn-SOD in the salivary

glands were higher than those other tissues, and the highest in

the sublingual gland, and significantly decreased in efficient zinc

group The activity of Cu, Zn-SOD in the brain stem slightly

increased in high Zn and Zn deficient diet compared with control

diet These results suggest that the activity of Cu, Zn-SOD is

sen-sitive to Zn supplementation and deprivation in the sublingual

gland and parotid gland In the brain stem, the activity of DAO

slightly increased in Zn deficient diet and D-AspO increased in

high Zn diet In sublingual glands, activity of DAO decreased

in high Zn and low Zn diet The activity of D-AspO increased in

sublingual glands and parotid glands in high Zn diet and

decreased in sublingual glands and submandibular glands in Zn

deficient diet Implications of the present studies for hydrogen

peroxide metabolism were stated above and compared with Zn

metabolism These studies will be discussed, including new

evi-dence that aging or death is going through the process of a loss

of the capacity to erase active oxygen from the peroxisomes and

that life span extension mechanism is depend on the acquisition

of the capacity

H2-012P

Effect of sodium hypochlorite on isolated

human erythrocyte membranes

I V Halets and V M Mazhul

1

Laboratory of Proteomics, Institute of Biophysics and Cellular

Engineering, National Academy of Sciences, Minsk, Belarus

E-mail: inessahalets@mail.ru

Hypochlorous acid (HOCl) is a powerful oxidant generated in

neutrophils and eosinophils This oxidative agent plays an

important role in membrane components damage under

inflam-matory conditions The aim of our study was to investigate the

effect of sodium hypochlorite (NaOCl) on slow (millisecond)

internal dynamics (ID) of proteins of erythrocyte membranes

and accumulation of lipid peroxidation products in them Room

temperature tryptophan phosphorescence (RTTP) analysis had

been used as a monitor of protein ID The lipid peroxidation

was studied by determining the amount of thiobarbituric acid

reactive (TBAR) products, mainly MDA The estimation of

total membrane SH-groups was realized using the Ellman

rea-gent It has been found that the dependence of both fast (x1)

and long (x2) lifetime components of RTTP of erythrocyte

membranes on oxidative reagent concentration in the range

0.25–50 mM is given by the curve with peak at 2.7 mM The

considerable increasing of lifetime components of RTTP at

con-centrations below of 2.7 mM oxidative reagent correlates with

the reducing of total amount membrane SH-groups The

treat-ment of isolated erythrocyte membranes with higher

concentra-tions of NaOCl (4.5–50 mM) results in a reduction of x1 and

x2 of RTTP At that in aforementioned range of NaOCl

con-centrations characterized by the increasing of x1 and x2 of

RTTP the level of lipid peroxidation products remains constant

Thus our studies reveals that NaOCl treatment of erythrocyte

membranes results in protein modification related to the

oxida-tion of SH-groups, restricoxida-tion of membrane protein structure

ID in concentration range 0.25–2.7 mM of NaOCl and

accumu-lation of lipid peroxidation products at more higher

concentra-tions (9–50 mM) of this oxidative reagent

Lipoprotein(a) is one of the plasma lipoprotein that is composed

of an LDL core and glycoprotein apo(a) Lp(a) is present inhuman plasma in four heterogenous subspecies (Lp(a)Lys-,Lp(a)Lys+1, Lp(a)Lys+2, Lp(a)Lys+3) This subclassification ismade according to their ability to bind to lysine sepharose Patho-genicity of Lp(a) as a risk factor for cardiovascular disease, maydepend on its lysine binding site [LBS] activity, which imparts aunique function to Lp(a), including a potential to inhibit fibrinoly-sis Previous studies have shown that Lp(a)Lys+, Lp(a)Lys+2and Lp(a)Lys+3 have athero-thrombosis properties Several evi-dences indicate that serum lipoproteins are sensitive to copper-induced oxidation In this study the effect of copper ion on lysinebinding site activity of Lp(a) was investigated For this purposefour subspecies of Lp(a) were isolated using affinity chromatogra-phy on lysine sepharose from pooled human serum Serum lipidswere oxidized in the presence of 15, 30, 50, 75 and 100 m ofCuCl2 Lipid oxidation was measured as conjugated diene forma-tion determined by spectrophotometric method at 245 nm Fourspecies of Lp(a) in the oxidized serum was isolated The resultsshowed a concentration-dependent increase in all the Lp(a)Lys+.These data suggest that copper ion induce a chemical modification

to lipoprotein(a) that lead to an increase in LBS activity Lp(a)and thus can promote athero-thrombosis

H2-014P Relationships between ascorbic acid, hydroxyproline and collagen, of wound healing in diabetic rats

M Khaksari Haddad1, A R Rezaizadeh2, M Mardani3,

G R Asadi Karam4and M Mahmoodi4

1Department of Physiology, Kerman Medical University, Kerman,Iran,2Department of Anatomy, Rafsanjan University of MedicalSciences, Rafsanjan, Iran,3Department of Anatomy, Isfahan Univer-sity of Medical Sciences, Isfahan, Iran,4Department of Biochemis-try, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.E-mail: khaksar38@yahoo.co.uk

It was reported that increased oxidative stress in diabetes mellitusassociated with decrease in collagen synthesis In this presentstudy, the role of ascorbic acid on hydroxyproline and collagen

in diabetic rats is reported This study was performed on 160male rats Diabetes was induced by injection of streptozotocin[STZ, S.C , 55 mg/kg].Animals were divided into four groups,and then rats were remained diabetic for 8 weeks Control group(I), received the vitamin c deficient diet Prophylaxia group (II),received 250 mg/lit vitamin c, one month before induction of dia-betes Treatment group (III), received vitamin c after induction

of diabetes Combination group (IV), received the vitamin c onemonth before diabetic induction, until wound healing Full thick-ness incision induced on back skin of rat, 8 weeks after diabeticinduction Content of collagen and biochemical measurement ofhydroxyproline at days 1, 3, 7, 11.15, and 20 after operation weremeasured The results showed, the mean of collagen content andhydroxyproline in group I was more than all examined groups,

at days of 11, 15 and 20, in addition, in group III this indicationwas less than group IV during all days This data suggest that,vitamin c supplementation in chronic diabetic rats impaired

wound healing, and decreased collagen and hydroxyproline

con-tent, while used as prophylaxis or combination

H2-015P

Oxidative modulation of macromolecular

interactions within the cell surface-activated

kinin-generating system

A Kozik, M Rapala-Kozik, U Blaszczyk and I Guevara-Lora

Department of Analytical Biochemistry, Faculty of Biotechnology,

Jagiellonian University, Krakow, Poland

E-mail: akozik@mol.uj.edu.pl

Bradykinin and related peptides, collectively known as kinins,

regulate many physiological processes and are involved in

virtu-ally all inflammatory responses Kinins are excised from

precur-sor proteins, kininogens, due to the proteolytic action of

kallikreins The plasma kinin-generating system, called the

con-tact system, includes a high molecular mass form of kininogen

(HMWK), prekallikrein (PK) and factor XII, and is activated on

negatively-charged cell surfaces In this work, we tested a

hypo-thesis that during inflammatory episodes the macromolecular

interactions within the contact system may be modified due to

the action of reactive oxygen species Therefore, we oxidized

sol-uble components of this system, HMWK, PK and factor XII,

and studied their assembly and activation on the surfaces of

var-ious cells (neutrophils, macrophages etc.) For protein oxidation,

we used the neutrophil myeloperoxidase-hydrogen

peroxide-chloride system The interactions between soluble components of

the contact system was studied by high-performance gel filtration,

a microplate-binding assay, time-resolved fluorescence

polariza-tion measurements and steady-state fluorescence energy transfer

measurements A destructive effect of oxidation on the complex

formation was observed for any pair of interacting proteins

Especially, the oxidized HMWK completely lost the ability to

bind PK, apparently due to the modification of methionine and

tryptophan residues at the PK-binding site Hence, the oxidized

HMWK when bound to the cell surface could not be effectively

processed+ after subsequent attachment of PK and factor XII

Hence, the kinin production was quenched This may happen in

real inflammatory foci unless some other proteinases take over

the kallikrein role

H2-016P

Evaluation of antioxidant activity of tomato

treated with an ethylene inhibitor

M A Murcia, M Martı´nez-Tome´ and A M Jime´nez-Monreal

Department of Food Science and Technology, Faculty of

Veterinary, Universidad de Murcia, Murcia, Spain

E-mail: mamurcia@um.es

The majority of antioxidants have a phenolic structure and

var-ious phenolic compounds with antioxidative activity and present

in the various plant tissues Tomato contains several antioxidants

that could be important in the diet; they are the main foodstuffs

contributing to the dietary intake of lycopene Lycopene is able

to act as an antioxidant and scavenger of free radicals, being

more effective than carotenes Prolonging the shelf life of fresh

vegetables is a very important concern for both consumers and

food packers Factors affecting the surface colour are related

mainly to packaging methods, light exposure, time/temperature

regime during storage and the presence of oxidants and

antioxi-dants 1-Methyl cyclopropene is an ethylene inhibitor, which is

used as a delayer of senescence of the tomato and we have

assayed the antioxidant properties of tomato treated with this

ethylene inhibitor, by using the deoxyribose assay All tomatoes

analyzed (treated or not with the ethylene inhibitor) exhibited

better antioxidant activities than some common food additivesanalyzed such as BHA and BHT Results demonstrated that thetreatment of tomatoes with the ethylene inhibitor did not signifi-cantly affect to their antioxidant activity

H2-017P L-Glutamine effect on liver citrulline level

J I Nikolic1, D T Sokolovic1and B J Djindjic2

1Department of Biochemistry, University of Nis, Nis, Serbia andMontenegro,2Department of Pathological Physiology, University

of Nis, Nis, Serbia and Montenegro

E-mail: nikdan@bankerinter.netTrauma, systemic inflammation, sepsis or toxins can modulatetissue functions Glutamine has been shown to have beneficialeffects in sepsis, trauma, infections, chemotherapy etc The aim

of this study was to investigate the possible beneficial effect ofL-glutamine on acute hepatotoxicity induced by mercury chloride(HgCl2) To study possible role of nitric oxide in mechanismstoxicity of mercury chloride we have study interaction betweenL-Glutamine and nitric oxide synthesis in tissue metabolism.Male Spraque-Dawley rats weighing about 200 g were used inthe experiment Acute toxicity was induced by i.p administration

of mercury chloride in a dose of 3 mg/kg One group of animalswas pre-treated with L-glutamine (200 mg/kg) 1 h before mercurychloride administration Control group of animals was treatedwith equal volume of saline The animals were killed 24 h afterHgCl2administration The level of citrulline, by product in nitricoxide synthesis, was measured by diacetylmonoxime reaction.Results of the study show that acute toxicity of mercury chlorideleads to elevation of tissue level of citrulline compared to controlgroup (P < 0.01) Administration of glutamine to control ratsdecreases slowly citrulline level compared to control, while pre-treatment of animals with glutamine before mercury chlorideadministration leads to the most greater decreases in citrullinelevel (P < 0.01) Heaving in mind that overproduction of nitricoxide has cytotoxic effects administration of L-glutamine may bebeneficial in mercury toxicity through decreasing nitric oxidelevel

H2-018P Peptides are formed when RNAase is treated with Fenton reagent in the presence of copper (II)

V R Oso´rio e Castro1and G B Domont2

1Laboratory of Biochemistry, Department of Biosciences,Politechnic Institute of Castelo Branco, Castelo Branco, BeiraInterior Portugal,2Laboratory of Biochemistry, Department ofBiochemistry, Federal University of Rio Janeiro, Rio de Janeiro,Rio de Janeiro Brazil E-mail: valdemar@esa.ipcb.pt

The inactivation of RNAase by Fenton reaction, within certainconditions, shows that after 30 min of reaction 22% of the ori-ginal activity is lost Two active fractions are separated, by filtra-tion of the reaction mixture through Sephadex G-75 columns,suggesting that the smaller active fraction is formed as the result

of Fenton reaction However, the inactivation increases in thepresence of copper (II) For example, with 4.4 x 10–4 Cu++ itreaches 92% after 1 min and 98% after 30 min of reaction,showing three fractions (F1Cu, F2Cu, F3Cu) by Sephadex G-75gel filtration Electrophoretic methods reveal that RNAase, modi-fied by free oxygen radicals, from Fenton reactive, has an elec-trophoretic mobility lower than the control, suggesting a decrease

of positive charges on its surface On the other hand, they alsoindicate peptides in fractions F1Cu and F2Cu Particularly F2Cuhave, at least, seven peptides, well separated by high voltage

paper electrophoresis In a control experiment with H2O2 and

copper (II) enzymatic activity also decreases, but no peptides are

formed The composition of two of those peptides were

deter-mined by an Aminoacid Analyser, to gain a preliminary idea

about the peptide bonds, which are lysed in the presence of

cop-per (II) The reactivity of Cu++to RNAase histidine residues, as

well as its effect in Fenton mixture, should be taken into account

to explain the observed results

H2-019P

Effect of Metroxylon sago polyphenol feeding

on the free radical scavenging enzymes in

hamster tissue

R Perumal, D Laura and H Melissa

BioMedicine & Therapeutics, School of Medicine, University

Malaysia Sabah, Kota Kinabalu, Sabah Malaysia

E-mail: perumal@ums.edu.my

The aqueous extract of Metroxylon sago (SAE), containing

poly-phenols, was investigated for its antioxidant as well as free

rad-ical- scavenging activities, using various chemical as well as

enzymatic assays The toxicity of the extract was evaluated by

the brine shrimp test and was found to be non-toxic The extract

was fractionated into eighteen fractions by column

chromatogra-phy on Sephadex LH20 Further purification was achieved by

C18 reversed phase High Performance Liquid Chromatography

Dietary studies were carried out using the Syrian hamsters,

Mesocricetus auratus The hamsters were divided into three

diet-ary groups, with the first being fed on a basal diet, as negative

controls, the second being fed on a diet supplemented with

butyl-ated hydroxy toluene (BHT), as positive control and the third

group, the experimental, being fed on a diet supplemented with

the sago aqueous extract (SAE), for a period of eight weeks

con-tinuously At the end of each week, for the first four weeks and

at the end of the eighth week, the hamsters were sacrificed in

groups of three from each dietary group and the tissues from

brain, lungs, liver and kidneys assessed for the free

radical-scav-enging enzymes, superoxide dismutase (SOD), catalase (CA) and

glutathione peroxidase (GPX) All the three enzymes assayed

showed that SAE was a more potent antioxidant than BHT The

polyphenolic compounds from the aqueous extract of Metroxylon

sago were shown to effectively lower the endogenous levels of

free radicals in all these hamster tissues thereby reducing the

activities of all the free radical-scavenging enzymes assayed

H2-020P

Investigations of the COX-2, CYP4F8 and

microsomal PGE synthase complex in human

seminal vesicles and in epidermis

K Stark1, H To¨rma¨2and E Oliw1

1Oliw Laboratory, Department of Pharmaceutical Biosciences,

Uppsala University, Uppsala, Sweden,2Dermatology, Department

of Medical Sciences, Uppsala University, Uppsala, Sweden

E-mail: Katarina.Stark@farmbio.uu.se

Cytochrome P450s are known to metabolize both endogenous

compounds like steroids, fatty acids, and bile acids, as well as

environmental pollutants and drugs CYP4 members oxidize fatty

acids, prostaglandins and leukotrienes CYP4F8 is a member of

the CYP4F subfamily and was originally cloned from human

seminal vesicle, and is known to metabolize PGH to

19-hydroxy-PGE compounds CYP4F8 is located in a cluster on chromosome

19p13.1–2 together with the other CYP4F subfamily members

One of the other CYP4F-members, CYP4F3, is known to be

expressed in two isoforms exhibiting different tissue distributions,

and catalytical properties Prostaglandins and their 19-hydroxymetabolites were initially discovered in human seminal fluid andare mainly formed by biosynthesis in seminal vesicles, presuma-bly in series by cyclooxygenase type 2 (COX-2), cytochromeP-450 4F8 (CYP4F8) and microsomal prostaglandin E synthase-1(mPGES-1) Quantitation by real-time PCR suggested that PGHsynthase-2, CYP4F8, and microsomal PGE synthase-1 wereco-localized and abundant and correlated in seminal vesicles ofseven patients (Stark et al 2004) COX-2 is known to be upregulated by inflammatory stimuli in many tissues, often togetherwith mPGES-1 COX-2 occurs along with CYP4F8 in non-lesion-

al epidermis, and the latter is strongly up regulated in psoriaticlesions (Stark et al 2003) We have also in co-operation investi-gated the co-localization and mRNA expression of COX-2,CYP4F8, and mPGES-1 using immunofluorescence and real-time PCR CYP4F8 and COX-2 are known to be present inhuman epidermis and CYP4F8 has been found to be stronglyupregulated in psoriatic lesions of psoriasis Treatment with poly-unsaturated fatty acids have been found to modify the inflamma-tory responses in psoriasis and we are presently in progressinvestigating if CYP4F8 may metabolize other fatty acids thenArachidonic acid In the genital organs CYP4F8 and 19-hydrox-y-prostaglandins are believed to have an anti-inflammatory func-tion and our over all hypothesis is that CYP4F8 in epidermismay have a similar function

Acknowledgments: This work was supported by det Medicin (03X-06523), Edvard Welanders Stiftelse and Finsenstiftensen

Vetenskapsra˚-H2-021P Implication of oxidative stress in protein degradation, acetylcholinesterase, (Na+, K+)- ATPase and Mg2+-ATPase activities in rat brain after forced swimming

T Tsakiris1, P Angelogianni1, C Tesseromatis2, S Tsakiris1and

C Tsopanakis1

1Laboratory of Experimental Physiology, Medical School,University of Athens, Athens, Greece,2Laboratory of ExperimentalPharmacology, Medical School, University of Athens, Athens,Greece E-mail: ctsop@cc.uoa.gr

The aim of this study was to investigate, whether exercise stress[short (2 h) or prolonged (5 h) forced swimming in rats] couldmodulate brain total antioxidant status (TAS), tissue protein con-centration and the activities of acetylcholinesterase (AChE),(Na+, K+)-ATPase and Mg2+-ATPase Protein concentration,TAS and enzyme activities in homogenized rat brain were deter-mined spectrophotometrically Protein concentration wasdecreased by 15% (P < 0.01) and by 30% (P < 0.001) after 2 hand 5 h of forced swimming, respectively TAS was decreased by20–25% after 2 h or 5 h of exercise AChE was inhibited by 30%(P < 0.001) and 45% (P < 0.001) after 2 h and 5 h of forcedswimming, respectively In contrast, (Na+, K+)-ATPase and

Mg2+-ATPase were stimulated by 80% (P < 0.001) and 40%(P < 0.001), respectively, after 2 h of swimming and by 100%(P < 0.001) and 60% (P < 0.001), respectively, after 5 h ofexercise Control values in non-treated rats were unaltered(P < 0.05) In conclusion, short or prolonged forced swimminginduces oxidative stress in rats, probably resulting in a reduction

in brain protein concentration and AChE activity In addition, a(Na+, K+)-ATPase and Mg2+-ATPase activation was observedunder the above mentioned experimental conditions This stresscondition may modulate brain intracellular Mg2+ concentration,neural excitability, metabolic energy production and neurotrans-mission

Low total antioxidant status is implicated with

degradation of erythrocyte membrane protein

content in patients with classical

galactosaemia

S Tsakiris1, H Michelakakis2, T Tsakiris1and K H Schulpis2

1

Laboratory of Experimental Physiology, Department of Medical

School, University of Athens, Athens, Greece,2Aghia Sophia’

Chil-dren´s Hospital, Institute of Child Health, Athens, Greece

E-mail: stsakir@cc.uoa.gr

Objective: Classical galactosaemia is characterized by high levels

of galactose-1-phosphate (Gal-1-P), galactose and galactitol The

aim of this study was to evaluate the erythrocyte membrane

pro-tein content (PC) of the galactosaemic patients and to correlate it

with Gal-1-P concentration in blood and their total antioxidant

status (TAS)

Patients and Methods: Nine patients (n = 9) originally on

‘‘loose diet’’ (group B) were requested to follow their diet strictly

(group A) Twelve healthy children were the controls (group C)

PC was determined by Lowry method TAS and Gal-1-P in

blood were determined spectrophotometrically In the in vitro

study, erythrocyte membranes from controls were pre-incubated

with Gal-1-P (300 lm)

Results: TAS and PC were significantly (p < 0.001) reduced

(0.89 ± 0.02 mmol/l, 36.8 ± 2.0 g/l, respectively) in group B as

51.5 ± 3.1 g/l, respectively) and controls (1.65 ± 0.12 mmol/l,

64.0 ± 3.5 g/l, respectively) Gal-1-P levels in group B was

signif-icantly higher than those in group A and controls Negative

correlation coefficients were found between PC and TAS with

Gal-1-P concentrations

Conclusions: High blood Gal-1-P concentrations resulted in low

TAS and PC The oxidative stress, induced in patients with

clas-sical galactosaemia, may break some structural proteins of their

erythrocyte membranes

H2-023P

Protection by 6-amino nicotinamide against

oxidative stress in cardiac cells

J P Hofgaard, K S Sigurdardottir and M Treiman

Department of Physiology, University of Copenhagen, Copenhagen,

Denmark E-mail: M.Treiman@mfi.ku.dk

We used a cell model of heart reperfusion injury employing

H2O2 to impart an oxidative stress in H9C2(2–1) cells and

neo-natal cardiac myocytes (CMC) In this model, pre-incubation

for 6 to 23 h with 6-aminonicotinamide (6AN) strongly

attenu-ated cell death after H2O2 application Survival was determined

for 2 h after the completion of H2O2exposure, and was

increased from about 12–16% to 57–75% in H9C2 cells, and

from 10% to 48% in CMC, dependent on the specific 6AN

treatment protocol 25 lM 6AN was sufficient for maximal

pro-tection when present for 23 h prior to 4 h in basal medium,

fol-lowed by 1 h of H2O2 stress This 6AN-mediated protective

effect was associated with a modest increase (by up to 55%) of

the cytosolic free Ca2+, and a decrease of total cell GSH down

to 56% This protective effect was blocked by inhibition of

ryanodine receptors, as well as by inhibition of Ca2+ent isoforms of PKC The longest of the 6AN treatments tested(18 or 23 h) induced the Unfolded Protein Response (UPR) inH9C2 cells UPR is an adaptative, transcriptional upregulation

-independ-of S/ER-resident chaperones caused by a disturbance -independ-of theS/ER lumenal environment, and is highly conserved in eukary-otic cells Although in some systems UPR has been shown toexert an antioxidative protection, this appeared not to be thecase in the present work, as the protective effect of 6AN wasonly slightly affected by a complete inhibition of protein synthe-sis, which did abolish UPR We propose that 6AN induced anoxidative shift in the cytosol redox status owing to an impairedglutathione regeneration via the oxidative pentose pathway,leading to an increased Ca2+ leak through ryanodine receptors

to the cytosol This leak in turn appeared to trigger a state of

an enhanced antioxidative resistance, perhaps similar to the lier described ‘‘Ca2+ pre-conditioning’’ of cardiomyocytes

ear-H2-024P Heroin abuse caused oxidative stress and oxidative damages of protein

Q Zheng1, B Xu1, G Li1, X Sun1and Z Wang1

1Ocean School, Yantai University, Yantai, Shandong PR China,

2Pharmacy School, Yantai University, Yantai, Shandong PRChina E-mail: zqsyt@sohu.com

Background/Aims: Chronic opiate intoxication has been shown

to cause various pathologic changes in the liver almost in 100%

of cases Our aim is to study if the mechanism of oxidative stressinvolved in hepatotoxicity induced by heroin

Methods: A dose-increasing method was used to develop theheroin-dependent model in mice The content of malondialdehyde(MDA) was measured by thiobarbituric acid (TBA) method, pro-tein carbonyl by DNPH reagent, reduced glutathione (GSH) byDTNB method and percentage of cell with damaged DNA bycomet method, and the activity of alanine aminotransferase(ALT) by commercial kit in hepatocytes of heroin administeredmice Coefficient of linear pair wise regression was used to evalu-ate the correlation between oxidative damage and hepatotoxicityinduced by heroin

Results: The oxidative damaged products, MDA, protein bonyl and the percentage of cells with damaged DNA increased

car-in hepatocytes of herocar-in admcar-inistered mice, while the ant enzyme and substances, activities of superoxide dismutase(SOD), catalase (CAT), and glutathione peroxidase (GPx) aswell as the content of GSH decreased, on the other hand, thehepatotoxic index, activity of ALT in serum of heroin adminis-tered mice elevated remarkably Positive correlations betweenALT activity and MDA content (r = 0.5442, n = 15), and car-bonyl group (r = 0.86, n = 15) in the mice liver, while the neg-ative correlation between ALT activity in serum and GSHcontent in hepatocytes (r = –0.6648, n = 15) were found How-ever, there is no close correlation between ALT activity andDNA damage

antioxid-Conclusions: Increase of oxidative damaged DNA, protein andlipid with decrease of antioxidant activities implied that therewas a seriously oxidative stress in mice liver induced by heroin.Oxidative stress may cause hepatotoxicity

H3–Protein Targets in Oxidative Stress

H3-001

Reactive oxygen species signaling in the

hippocampus

E Klann

Departments of Molecular Physiology & Biophysics and

Neuroscience, Baylor College of Medicine, Houston, TX, USA

E-mail: eklann@bcm.tmc.edu

Reactive oxygen species (ROS) have been studied intensely in the

central nervous system with respect to their role in oxidative

stress associated with excitotoxicity and neurodegeneration

How-ever, ROS also have been shown by a number of laboratories to

play a critical role as signaling molecules during synaptic

plasti-city and memory formation We recently have shown that a

func-tional NADPH oxidase is present in hippocampal neurons

(Tejada-Simon et al Mol Cell Neurosci, in press) and that

super-oxide produced via NADPH oxidase is required for NMDA

receptor-dependent activation of extracellular signal-regulated

kinase (ERK) in the mouse hippocampus (Kishida et al J

Neuro-chem, in press) Because activation of ERK is required for

hippo-campal synaptic plasticity and memory, we initiated studies to

determine whether NADPH oxidase also is required for these

hippocampal functions Using pharmacological and genetic

approaches, we have found that NADPH oxidase is necessary for

hippocampal synaptic plasticity and memory We are currently

conducting studies to identify protein targets that are modified

by ROS produced via NADPH oxidase during synaptic

plasti-city In addition to its role in normal neuronal function, NADPH

oxidase also has been implicated in neurodegenerative diseases

such as Alzheimer’s disease Consistent with this notion, we have

found that amyloid beta peptide activates ERK via NADPH

oxidase in the hippocampus In addition, we have found that the

activation of NADPH oxidase and ERK by amyloid beta peptide

requires alpha7 nicotinic acetylcholine receptors Taken together,

our data suggest that ROS produced via NADPH oxidase are

involved in both physiological and pathophysiological processes

Department of Pharmacology, University of Pittsburgh, Pittsburgh,

PA, USA E-mail: iannmda@pitt.edu

Mitochondria are believed to be one of the major sources of

oxidant signals within cells Alterations in mitochondrial oxidant

signals may be responsible for a number of pathological states

where cell death is triggered by oxidative stress However, it is

often not easy to establish the link between oxidants and their

targets Our recent studies have focused on zinc as an

endog-enous toxin in central neurons Normally, free zinc

concentra-tions are very low as the result of high affinity binding of zinc by

proteins such as metallothionein Exposing proteins to oxidant

sources results in the oxidation of protein sulfhydryls, and this

results in the release of zinc from the proteins into the

intracellu-lar milieu While there are several potential sources of oxidants

that can cause zinc release, we have recently found that

activa-tion of glutamate receptors causes intracellular zinc release, and

that this probably arises from the stimulation of oxidant

produc-tion by mitochondria We have also shown that zinc can

modu-late several different mitochondrial functions In particular, zinc

can inhibit complex I of the electron transport chain, and therebystimulate oxidant generation In this way, zinc provides an exam-ple of an important neurotoxin that is mobilized as the result ofthe modulation of mitochondrial oxidant signals, and which canalso modulate mitochondrial itself, possibly in a feed-forwardloop This kind of oxidant signaling could prove to be of greatimportance in neurodegenerative disease

H3-003 Functional consequences of protein tyrosine nitration and oxidation

H IschiropoulosStokes Research Institute and Department of Biochemistry andBiophysics, Children’s Hospital of Philadelphia, and The University

of Pennsylvania, Philadelphia, PA, USA

E-mail: ischirop@mail.med.upenn.edu

A better understanding for the role of oxidants in human ease may be derived by the identification of specific protein tar-gets that are post-translationally modified by oxidants and byexamining the effect of these selective modifications upon pro-tein function Tyrosine nitration is now recognized as a covalentprotein modification derived from the reaction of proteins withnitrating agents Biological protein nitration appears to be arather selective process; not all tyrosine residues in proteins andnot all proteins are nitrated in human and model systems ofdisease Published studies and work in progress has revealedthat the presences of specific tyrosine residues in local environ-ments of proteins are the primary determinants of the site ofnitration Proteomic analysis, specific monoclonal antibodiesand immunoprecipitation methodologies have identified a num-ber of proteins that are nitrated under physiological conditionsand in human pathologies Protein tyrosine nitration does notalways modify protein function, despite alterations in secondarystructure, and quite often the coexistence of other amino acidoxidation precludes from attributing changes in function totyrosine nitration To gain a better understanding of the biolo-gical consequences of protein tyrosine nitration we investigatedthe role of this modification on the induction of inappropriateprotein aggregation The data indicated that protein tyrosinenitration selectively even at low yield may facilitate the inappro-priate aggregation of proteins implying a role of this modifica-tion in the formation of a-synuclein hallmark lesions in humanneurodegenerative diseases such as Parkinson’s disease and forthe association between nitrative stress and risk for coronaryartery disease will be discussed

dis-H3-004

H2O2signaling through cysteine modifcations

L Monceau1, S Fourquet1, A Sekowska-Danchin1, F Tacnet1,

G Rousselet1, E Hidalgo2and M B Toledano1 1

Laboratoire Stress Oxydants et Cancer, Service De BiologieMole´culaire Syste´mique, Commissariar a l’Energie Atomique,Gif-sur-Yvette, France,2Cell Signalling Unit, Departament deCie`ncies Experimentals i de la Salut, Universitat Pompeu Fabra,Barcelona, Spain E-mail: toledano@dsvidf.cea.fr

Microbial H2O2 sensors are master regulators of cellular H2O2

homeostasis They are activated by oxidation when intracellularlevels of H2O2 increase, and they set the expression of oxidant-scavengers in response to this increase Such regulation, meant

to prevent oxidative stress-induced cellular damage, is essentialfor aerobic life and has the hallmarks of a homeostatic control

In mammals, regulators activated by reversible oxidation with

mechanisms similar to those of microbial H2O2 sensors are

being described However their physiological function is not to

regulate H2O2 homeostasis Rather they respond to the second

messenger H2O2 and regulate specific cellular pathways The

yeast S cerevisiae Orp1-Yap1 and S pombe Tpx1-Pap1

two-component H2O2 sensors share an overall common mechanism

of activation involving reversible disulfide bond formation

H2O2-induced oxidation of the Yap1 and Pap1 transcription

factors is not direct involving the thiol-based peroxidases Orp1

(GPx-like enzyme) and Tpx1 (a peroxiredoxin family member)

respectively that act as the pathway sensors and redox

transduc-ers However, Pap1 but not Yap1 activation is restricted within

a narrow range of H2O2 concentration We show that Pap1

restricted activity is the consequence of Tpx1 oxidation to an

inactive cysteine-sulfinic acid form, eventually reversed by

ATP-dependent reduction by sulfiredoxin Conservation of these

mechanisms in higher eukaryotes will be addressed These

mech-anisms illustrate the built-in high specificity of cysteine-based

H2O2 redox signaling and suggest the existence of specific

path-ways of cysteine oxidation by H2O2

H3-005

Calcium release mediated by redox activation

of ryanodine receptors induces CREB and ERK

phosphorylation in N2a cells and hippocampal

neurons

M A Carrsaco1, U Kemmerling1,2, P Mun˜oz3and C Hidalgo1

1

FONDAP CEMC, Facultad de Medicina, ICBM, Universidad de

Chile, Santiago, RM Chile,2FONDAP CEMC, Facultad de

Cien-cias de la Salud, Universidad de Talca, Talca, VI Regio´n Chile,

3

FONDAP CEMC, Facultad de Ciencias, Universidad de Chile,

Santiago, RM Chile E-mail: chidalgo@med.uchile.cl

Neuronal calcium entry signals can be amplified and propagated

via calcium-induced calcium release from intracellular stores In

particular, ryanodine receptor (RyR) mediated calcium release

might play a role in the generation of calcium signals that

sti-mulate neuronal gene expression RyR are highly susceptible to

redox modifications; oxidation/nitrosylation of critical cysteine

residues enhances RyR activity whereas their conversion to the

free sulfhydryl state has the opposite effect We have

investi-gated whether RyR channels activated by oxidation promote

the sequential activation of the calcium-dependent

transcrip-tional regulators ERKs and CREB, which is required for long

lasting synaptic plasticity in the hippocampus To this end, we

have investigated the effects of hydrogen peroxide (0.2 mm) on

the generation of calcium release signals and the

phosphoryla-tion of ERK/CREB in hippocampal cells in primary culture

and in the neuronal cell line N2a Confocal imaging of cells

revealed that hydrogen peroxide induced calcium release signals

in primary hippocampal cells, which were not observed in cells

preincubated with 0.05 mm ryanodine to block selectively

RyR-mediated calcium release Similar results were obtained in N2a

cells Hydrogen peroxide also induced transient CREB and

ERKs phosphorylation in both N2a and hippocampal neurons;

these effects were significantly inhibited by ryanodine Nitric

oxide donors produced the same effects as hydrogen peroxide

when added to N2a cells These results suggest that redox

acti-vation of RyR-mediated calcium release might enhance the

ERK/CREB-controlled calcium-dependent gene expression in

the hippocampus that is required for enduring synaptic

plasti-city

Acknowledgments: This work was supported by FONDAP

CEMC 15010006 and FONDECYT 1030988

H3-006 ROS and PKC delta: a bidirectional intracellular talk able to decide cellular fate

B Marengo1, C De Ciucis1, L Raffaghello2, V Pistoia2,

D Cottalasso1, M A Pronzato1, U M Marinari1and

C Domenicotti1

1

Department of Experimental Medicine, University of Genoa,Genoa, Italy,2Laboratory of Oncology, Gaslini Hospital, Genoa,Italy E-mail: cdomeni@medicina.unige.it

The by-product of biological processes is represented by reactiveoxygen species (ROS) which have high chemical reactivity andcan damage a variety of biomolecules, such as lipids, proteins,carbohydrates and nucleic acids Recent studies imply that ROSare not only destructive elements for cells, but also they areessential for cell biology and physiology; in fact they play a crit-ical role in differentiation, proliferation and apoptosis acting as

‘‘second messengers’’ able to regulate sulphydryl groups in ling molecules Among these signal proteins, a crucial role in cellresponses is played by protein kinase C (PKC), a family of isoen-zymes also implicated in tumorigenesis Oxidative regulation ofdelta and epsilon PKCs has been demonstrated to be important

signa-in the development of cancer preventive or therapeutic agents.Neuroblastoma is characterised by the production of oxygenintermediates and L-buthionine-S,R-sulfoximine (BSO), a gluta-thione (GSH)-depleting agent commonly used in its clinical treat-ment, exploits this biological peculiarity to induce cell death Inour study, we have found that the production of ROS induced

by BSO is able to trigger mitochondrial pathway of apoptosis inneuroblastoma cells with MYCN amplification This process ismediated by the oxidative activation of the pro-apoptotic PKCdelta which might be involved also in the production of ROSamplifying the apoptotic cascade Future studies will beaddressed to alter the expression of PKC isoenzymes in order toinduce cancer cell death; this offers new strategies for increasingthe efficacy of chemiotherapy in neuroblastoma clinical treat-ment

Acknowledgments: This work was supported by grants fromGenoa University (ex 60%) and from FIRB 2001 (Prof G Poli)

H3-007P Alteration of oxidant-prooxidant status of rats’ blood at experimental gastric ulceration

P A Anatolievich1, P L Anatolievna2, T T Nikolaevna3and

L E Anatolievna4

1

Pathophysiology, Academy of Medical Sciences of Ukraine, tute of Gastroenterology, Dnepropetrovsk, Ukraine,2Biochemistry,Academy of Medical Sciences of Ukraine, Institute of Gastroenter-ology, Dnepropetrovsk, Ukraine,3Pathophysiology, Academy ofMedical Sciences of Ukraine, Institute of Gastroenterology,Dnepropetrovsk, Ukraine,4Biochemistry, Ukrainian StateScientific Research Institute of Medical and Social Problems ofDisability, Dnepropetrovsk, Ukraine E-mail: patphys@ua.fmInvestigation of oxidant-prooxidant status of rats’ blood byintroduction of medical bile in the stomach resulted in intensifica-tion of free radical process in the blood of the animals So thelevel of the thiobarbituric acid (TBA) active products in plasma

Insti-of blood increased on 94.6% Observable phenomena are fied after the rats’ immobilization and subjection to cold(4 ± 1
C) stress for 1 h Similar tendencies were valid for theerythrocytes considering as a model of general cells reactions.Decrease of antioxidant status was simultaneously observed,activity of catalase, SOD, glutathione peroxidase, glutathionereductase and a level of restored glutathione were reduced.Administration of l-arginin-l-glutamat (20 mg/100 g) resulted in

intensi-stabilization of studed parameters Level TBA active products

was increased in comparison with the control parameters on

52% Thus we assume, that l-arginin-l-glutamat carries out not

only hepatoprotection function But it also stabilizes general

anti-oxidant status of rats at investigated pathology

H3-008P

Possible protective effect of kolaviron

(a garcinia kola seed extract) on carbon

tetrachloride-induced erythrocyte damage in

rats

O A Adaramoye1and O Akinloye2

1Laboratory of Drug Metabolism, Department of Biochemistry,

University of Ibadan, Ibadan, Oyo Nigeria,2Laboratory of

Metabolic Research, Department of Chemical Pathology, Ladoke

Akintola University of Technology, Osbongo, Osun Nigeria

E-mail: aoadaramoye@yahoo.com

The possible protective effect of kolaviron (KV) (a Garcinia

kola seed extract) on rat erythrocytes following simultaneous

administration of kolaviron (100 mg/kg body weight/day) with

carbon tetrachloride (CCl4) (1.2 g/kg body weight/day) by

sep-arate intraperitoneal injections was investigated KV, a

biflavo-noids fraction of the defatted alcoholic extract of Garcinia kola

seed, inhibits the accumulation of lipid peroxidation products in

erythrocytes A significant reduction (P < 0.05) by about 34%

of lipid peroxidation products was observed in erythrocytes of

rats treated simultaneously with CCl4 and KV when compared

to CCl4- only treated rats Similarly, the significant increase

(P < 0.05) in membrane cholesterol levels observed in CCl4

-treated rats was significantly decreased (P < 0.05) in rats

trea-ted simultaneously with CCl4 and KV Therefore, there was no

significant difference (P < 0.05) in cholesterol–phospholipids

molar ratio (C/P) of rats treated with CCl4+ KV, and

con-trols Thus, KV normalizes the CCl4-induced change in

erythro-cyte membrane lipid composition In addition, KV antagonizes

the effect of CCl4 on the activity of the membrane bound

enzyme, Ca2+-ATpase These results suggest that kolaviron

pro-tects erythrocyte membranes from free radical attack, on both

lipids and proteins

H3-009P

The effects of 5-fluorouracil on proliferative

capacity of enterocytes in experimental

animals

K Bajin-Katic1, K Stankov1, Z Kovacevic2, I Milenkovic1and

V Mijatovic1

1

Department of Biochemistry, University of Novi Sad, Novi Sad,

Serbia and Montenegro,2Serbian Academy for Sciences and Arts,

Branch in Novi Sad, Novi Sad, Serbia and Montenegro

E-mail: stankovkarmen@yahoo.com

The cytotoxic effects of antimetabolites chemotherapy are based

on their role as substrates from the same transport processes and

enzymes involved in anabolism and catabolism as the natural

sub-strates As the uracil analog, 5-fluorouracil (5-FU), may be

util-ized by several metabolic routes The main goal of our study was

to analyze the dose-dependent antiproliferative effects of 5-FU on

intestinal mucosa and enterotoxic potential of 5-FU in

experimen-tal animals Our results of enterotoxicity induced by

intraperito-nealy administered 5-FU showed statistically significant decrease

of DNA content in small intestine samples of experimental

ani-mals, as well as significant pathohystological changes, indicating

the diagnosis of intestinal mucosa inflammation, without

signifi-cant changes in total protein content

H3-010P Protection against radicals formed from xenobiotics: a novel role for carbohydrate moieties in glycoproteins?

V Martinek1, J Sklenar1,2, M Sulc1,2, K Bezouska1,2, E Frei3and M Stiborova1

1

Faculty of Science, Department of Biochemistry, Charles sity Prague, Prague, Czech Republic,2Institute of Microbiology,Academy of Sciences, Prague, Czech Republic,3Division ofMolecular Toxicology, German Cancer Research Center,Heidelberg, Germany E-mail: bezouska@biomed.cas.czGlycosylation is one of the most important post-translationalmodification of protein [1] It has been suggested previously thatcarbohydrate moieties may be involved in glycoprotein folding,protection against proteolysis, and participate in specific recogni-tion functions [2] We observed that radicals generated from twoxenobiotics, 1-phenylazo-2-hydroxynaphthalene (Sudan 1) andellipticine, by horseradish peroxidase readily attach to non-gly-cosylated proteins such as HSA, while glycosylated proteins such

Univer-as the peroxidUniver-ase itself were not modified Sudan I is a liver andurinary bladder carcinogen in mammals known to be metaboli-cally activated by both cytochromes P450 and peroxidases toreactive species that bind covalently to nucleic acids and proteins[3] Ellipticine is an alkaloid with antineoplastic and anti-HIVactivities that is oxidized by peroxidases generating radicals parti-cipating in its pharmacological efficiency [4] We established aremarkable correlation between the resistance of glycoproteins(asialofetuin, fetuin, ovomucoid, Tamm-Horsfall glycoproteinand alpha1-acid glycoprotein) towards modification by the aboveradicals, and the degree of glycosylation Comparison of theseactivities using RNase A (nonglycosylated) and RNase B (glycos-ylated) supported our findings on two proteins having an identi-cal protein moiety and differing only in glycosylation Ourfindings indicate a novel role for protein glycosylation, namelythe protection of protein against the attack by radicals generatedfrom a one-electron oxidation of the xenobiotics

Acknowledgments: This work was supported by Ministry ofEducation of Czech Republic (MSM 0021620808), by the Institu-tional Research Concept (AVOZ5020963), and by the GrantAgency of the Academy of Sciences of Czech Republic(A5020403)

References

1 Kornfeld S J Clin Invest 1998; 101: 1293

2 Gagneux P, Varki A Glycobiology 1999; 9: 747

3 Stiborova M, Martinek V, Rydlova H, Hodek P, Schmeiser

HH, Frei E Cancer Res 2000; 62: 5678

4 Frei E, Borek-Dohalska L, Wiessler M, Stiborova M Proc

Am Assoc Cancer Res2001; 42: 252

H3-011P Studies on the selenoproteome in the respiratory tract: subcellular distribution of selenium and selenium-containing proteins in the lung and trachea of the rat

K Bukalis, A Kyriakopoulos, D Alber and D BehneDepartment of Molecular Trace Element Research in the LifeSciences, Berlin, Germany E-mail: k.bukalis@hmi.deThe lung and trachea are constantly exposed to many gases andparticular matter present in the atmosphere They thereforerequire a specific defence system against oxidants and free radi-cals The trace element selenium seems to play an importantrole in this defence system In the form of selenocysteine it is

an essential component of glutathione peroxidase, an enzymeinvolved in the detoxification of peroxides In addition, several

other selenoproteins are known which may likewise be of

signi-ficance in the antioxidant defence system of the respiratory

tract It is therefore of great interest to analyze the

selenium-containing proteins present in the lung and trachea and to

obtain information on their subcellular distribution In these

studies element analytical methods and radiotracer techniques

have been combined with biochemical separation procedures

Subcellular separation of the lung and trachea tissues has been

achieved by differential ultracentrifugation Instrumental

neut-ron activation analysis has been used to determine the

concen-trations of total selenium in the whole organ and its subcellular

fractions The selenium-containing proteins in these

compart-ments have been investigated by labeling of rats in vivo with

Se-75, gel electrophoretic separation of the proteins and

autora-diographic detection of the tracer Our results show that

selen-ium is distributed inhomogeneously among the cellular

compartments of the rat lung and trachea After combination

of the tracer techniques with gel electrophoresis about 24

Se-containing proteins could be distinguished The molecular

masses of the subunits were in the range 10–30 and 50–80 kDa,

with pI value between 3 and 10

H3-012P

Protective effects of different vitamin E doses

on cisplatin-induced oxidative damage to renal

tissue in rats

M O Dillioglugil1, H M Kir1, M D Gulkac2, A O Kanli2,

S Kuskay1, H K Ozdogan1and C Eraldemir1

1Department of Medical Biochemistry, Kocaeli University, Faculty

of Medicine, Kocaeli, Turkey,2Department of Medical Biology,

Kocaeli University, Faculty of Medicine, Kocaeli, Turkey

E-mail: mozden@superonline.com

Cisplatin (CP) is one of the most potent antitumor drugs, but

it is able to generate reactive oxgen species In this study, we

aimed the effect of 100, 200 and 400 mg/kg bw doses vitamin

E (VE) administration on malondialdehyde (MDA), nitric

oxide (NO), glutathione (GSH) and superoxide dismutase

(SOD) in kidney of CP-induced toxicity in rats Thirty male

Wistar rats were used Control group received olive oil CP

groups, 5 mg/kg bw CP intraperitoneally administrated Groups

of VE100 + CP, VE200 + CP, VE400 + CP received VE once

a day for 2 days by gavage before CP injection Kidney tissues

MDA, GSH, NO levels and SOD activities were determined to

designate the oxidant–antioxidant balance In the VE200 + CP

and VE400 + CP groups when compared with the CP alone

group, kidney MDA and NO levels were found to be

signifi-cantly lower (MDA: P < 0.05 and P < 0.0001; NO: P < 0.05

and P < 0.01, respectively), whereas there were significant

increase GSH levels and SOD activities (GSH: P < 0.05 and

P< 0.01; SOD: P < 0.01 and P < 0.0001, respectively) In

the VE100 + CP and VE200 + CP groups, MDA and NO

levels were found to be significantly higher (MDA: P < 0.01

and P < 0.05; NO: P < 0.0001 and P < 0.01, respectively),

but GSH levels and SOD activities were significantly lower

(GSH: P< 0.01 and P< 0.01; SOD: P< 0.01 and

P< 0.05, respectively) than the control group Among the

VE100 + CP, VE200 + CP, and VE400 + CP groups, MDA

and NO levels decreased (P < 0.05; P < 0.05, respectively)

and GSH levels and SOD activities increased (P < 0.01;

P< 0.05, respectively) significantly as the amounts of VE

increased In conclusion, that the toxic effect of CP is

some-how minimized by a compensatory mechanism involving VE

via induction of antioxidant enzyme activities under received

intraperitoneal injection of CP

H3-013P Post-translation modification of alpha- synuclein by oxidative stress in differentiated human neuroblastoma cells

B Dozza and P StrocchiDepartment of Pharmacology, University of Bologna, Bologna,Italy E-mail: bdozza@biocfarm.unibo.it

Alpha-synuclein is known to be a brain pre-synaptic protein and

to be a structural component of the filaments in Lewy bodies inParkinson’s disease brains and other neurodegenerative disorders.Alpha-synuclein has been found to be phosphorylated on serine

129 in Lewy bodies The phospho-serine 129 alpha-synuclein motes fibril formation in vitro, suggesting the importance ofphosphorylation of the filamentous protein in the pathogenesis ofParkinson’s disease

pro-Recent findings of abnormal protein folding, coupled with tive stress, provide scientific rationale for studies designed to char-acterize the effect of oxidative stress on alpha-synuclein post-translation modifications We report here that oxidative stress,induced by the pro-oxidant pair iron-ascorbate, modulates thephosphorylation state of alpha-synuclein as detected by Westernblot analysis using specific phospho-antibodies and modified theintracellular distribution of this protein in differentiated SH-SY5Ycells These results demonstrated that oxidative stress inducedpost-translation modification of the alpha-synuclein protein sup-porting the notion of the role of oxidative stress in neurodegenera-tive disease characterized by alpha-synuclein positive lesions.Acknowledgments: This work was supported by the University

oxida-of Bologna, Funds for Selected Research Topics

H3-014P Increased cardiac activity enhances RyR2 S-glutathionylation

P Donoso1,2, G Sa´nchez1,2, Z Pedrozo1, R Domenech1and

C Hidalgo1,2

1Instituto de Ciencias Biome´dicas, Universidad de Chile, Santiago,Chile,2Calcium Signaling Group, FONDAP CEMC, Santiago,Chile E-mail: pdonoso@med.uchile.cl

Reactive oxygen species (ROS) have been involved in the ment of ischemic preconditioning in the heart but the source ofROS and their mechanisms of action are uncertain Short episodes

develop-of tachycardia or exercise can also induce preconditioning, andboth maneuvers increase the activity of ryanodine receptors (RyR)/calcium release channels in sarcoplasmic reticulum (SR) fractionsisolated from dog heart Since RyRs are redox sensitive and theiractivity increases by oxidation, we investigated whether theNAD(P)H oxidase, a ROS producing enzyme, played a role in theincrease in cardiac RyR2 activity produced by tachycardia or exer-cise We found that tachycardia, as well as exercise, increased theassociation of rac1 and of the NADPH oxidase cytosolic regulatorysubunit p47phox to the isolated cardiac SR fraction Both tachy-cardia and exercise increased twofold the production of superoxideanion and hydrogen peroxide by this isolated SR membrane frac-tion, and increased the endogenous level of RyR2 S-glutathionyla-tion by 70% In vitro activation of the NAD(P)H oxidase present

in this fraction produced a similar increase in RyR2 lation, with a concomitant increase in RyR2 activity These resultssuggest that the increased NAD(P)H oxidase activity induced bytachycardia and exercise is responsible for the increase in RyR2redox modifications (S-glutathionylation) over the endogenous lev-els This mechanism may be one of the primary factors that causethe higher RyR2 activity found in these conditions

S-glutathiony-Acknowledgments: This work was supported by Fondecytgrants 1030446, 1030449 and FONDAP 15010006

The activity of 20S proteasome from the yeast

S cerevisiae is modulated by redox

mechanisms including S-thionylation and

reduction by glutaredoxin2 and cytosolic

thioredoxins

M Demasi1, G M Silva2, F P Cavalher2, J A Ba´rcena3,

C R A Bertoncini4and L E S Netto2

1

Instituto Butantan, Sao Paulo, SP Brazil,2Universidade de Sa˜o

Paulo, Sao Paulo, SP Brazil,3Universidad de Co´rdoba, Co´rdoba,

Spain,4Universidade Federal de Sa˜o Paulo, Sao Paulo, SP Brazil

E-mail: marimasi@butantan.gov.br

We have previously reported that the yeast 20S proteasome is

modified by redox mechanisms such as the oxidation of its Cys

residues (Cys-SH) to Cys-sulfenic acid (Cys-SOH) followed by

S-glutathionylation (Cys-S-SG) As observed, such modifications

implied on an important reduction of mainly the proteasomal

chymotrypsin-like activity Since we hypothesize that the

modula-tion of proteasomal activity, by means of such modificamodula-tions,

might play a role upon intracellular signaling against oxidative

stress, we have investigated potential intracellular mechanisms

able to recover proteasomal activity On that, studies conducted

thus far have shown that ascorbate is able to partly reduce the

oxidized 20S proteasome-Cys-SOH residues, concomitantly to

partial rescue of chymotrypsin-like activity Moreover,

recombin-ant glutaredoxin2 and cytosolic thioredoxin isoforms (Trx1 and

Trx2) released glutathione from S-glutathionylated 20S

protea-some core which correlated with the recover of proteolytic

activ-ity To know whether 20S proteasome hydrolytic activity is redox

modified in vivo, we performed studies by comparing cells grown

to stationary phase into glycerol- and glucose-containing media in

order to compare two distinct intracellular redox conditions

Results obtained showed a positive correlation among 20S

protea-somal activity, glutaredoxin2 expression and reductive

intracellu-lar capability accessed by the determination of GSH/GSSG ratio

We hypothesize that 20S proteasomal activity is dependent on

either enzymatic or non-enzymatic intracellular reductive systems

H3-016P

Suppression of chemokine and inflammatory

cytokine expression by gallotannin in A549

cells is not related to inhibition of

poly(ADP-ribose) glycohydrolase

K Erdelyi1, E Bakondi1, C Szabo2, P Gerely1and L Virag1

1

Department of Medical Chemistry, University of Debrecen,

Med-ical and Health Science Center, Debrecen, Hungary,2Inotek

Phar-maceuticals, Beverly, MA, USA E-mail: erdelyika@freemail.hu

Oxidative stress is known to stimulate poly(ADP)-ribosylation, a

post-tranlational modification carried out by the concerted action

of poly(ADP)-ribose polymerases (PARP) and

poly(ADP)-ribose-glycohydrolase (PARG) Poly(ADP-ribosyl)ation has been

impli-cated in the transcriptional regulation of inflammatory mediators

By utilizing a chemokine-focused thematic macroarray and

RT-PCR, we found that immune-stimulated A549 type II lung

epithelial cells express many chemokines and inflammatory

cytok-ines Pre-treatment of cells with the PARG inhibitor gallotannin

(GT) abolished the expression of most chemokines/cytokines

whereas the potent PARP inhibitor PJ34 only had marginal

effect Activation of AP-1 and NF-kappaB, transcription factors

playing crucial role in the signal transduction of

TNFalpha/IL-1beta, was determined with EMSA In the presence of GT we

observed marked decrease in the DNA binding activities of both

transcription factors In the case of NF-kappaB GT inhibited the

nuclear translocation of the transcription factor due inhibition ofphosphorylation of the inhibitor of NF-kappaB (I-kappaB).Regarding the AP-1 signaling pathway, GT induced a maximalphosphorylation of JNK and c-jun and a moderate phosphoryla-tion of p38 and ATF-2 in the absence of cytokines Cytokine-induced p38 and ATF-2 phosphorylation, however, was blocked

by GT TNFalpha/IL-1beta did not cause poly(ADP-ribose)accumulation either in the absence or in the presence of GT indi-cating that the effects of GT are not related to PARG inhibition

We propose that gallotannin inhibits an early step of theNF-kappaB pathway (proximal to IkappaB phosphorylation)and inhibits the p38-ATF-2 pathway and/or the DNA binding ofAP-1 despite of activation of the JNK-c-jun pathway

H3-017P Protective effect of green tea on erythrocyte membrane of different age rats intoxicated with ethanol

Z A Figaszewski1, I Dobrzyn˜ska1, B Szachowicz-Petelska1,

J Ostrowska2and E Skrzydlewska2 1

Laboratory of Electrochemistry, Department of Biology andChemistry, University of Białystok, Bialstok, Poland,2Department

of Analytical Chemistry, Medical Academy of Białystok,Białystok, Poland E-mail: elchem@uwb.edu.pl

It is known that ageing is characterized by changes in cell lism resulting e.g in modification of the structure and function ofcell membrane components being mainly the consequences ofreactive oxygen species action These disturbances are alsoenhanced by different xenobiotics e.g ethanol Therefore the aim

metabo-of this paper has been to examine green tea influence on total oxidant status and on composition and electric charge of erythro-cyte membrane phospholipids in ethanol intixicated rats ofvarious ages Antioxidant abilities of erythrocytes were estimated

anti-by measuring total antioxidant status (TAS) Qualitative andquantitative composition of phospholipids in the membrane wasdetermined by HPLC, while the extent of lipid peroxidation wasdetermined by spectrophotometric measurement of the level oflipid peroxidation products as thiobarbituric reactive substances(TBARS) Electrophoresis was used to determine the surfacecharge density of the rat erythrocyte membrane It was shown thatthe process of ageing was accompanied by a decrease in TAS and

in the total amount of phospholipids as well as by increase in lipidperoxidation and surface charge density of erythrocyte membrane.However ethanol administration caused decrease in TAS andincrease in the amount of all phospholipids and in the level oflipid peroxidation products Ethanol as well significantly enhancedchanges in surface charge density of erythrocyte membrane Theingestion of green tea partially prevented decrease in erythrocytesantioxidant abilities observed during aging and ethanol intoxicat-ion Moreover long-term drinking of green tea protects the struc-ture of the erythrocytes membrane disturbed during aging processand/or chronic ethanol intoxication

H3-018P Significant role of cytoprotective kinase signaling mechanisms in the protective effect

of PARP inhibitors in a murine septic shock model

F Gallyas Jr, B Veres, B Radnai, E Hocsak and B SumegiDepartments of Biochemistry and Medical Chemistry, University

of Pecs, Pe´cs, Hungary E-mail: ferenc.gallyas@aok.pte.huActivation of poly(ADP-ribose) polymerase (PARP), a highcopy-number nuclear enzyme, was reported to have a significant

role in many pathological conditions Since PARP-1 knock-out

mice proved to be resistant against septic shock We studied the

effect of some structurally different PARP inhibitors in LPS

induced septic shock in mice, as well as the underlying kinase

signaling pathways We found that the PARP inhibitors (i)

signi-ficantly improved the survival rate among the animals, (ii)

attenuated the pathological changes in morphology of liver,

kid-ney, lung and intestines, (iii) decreased the formation of

pro-inflammatory cytokines and the (iv) activation of nuclear factor

jB Based upon our results on the kinase cascades, it seems that

activation of the cytoprotective PI3-kinase/Akt pathway was

strongly involved in the protective effect of the PARP inhibitors

All these results suggest that non-toxic PARP inhibitors may

have therapeutic values in alleviating the pathomechanisms of

septic shock

H3-019P

Inhibition of matrix metalloproteinases mimics

the cardioprotective effect of preconditioning

in normal and hyperlipidemic rats

Z Giricz1, M M Lalu2, C Csonka1, P Bencsik1, R Schulz2,3

and P Ferdinandy1,4

1Cardiovascular Research Group, Department of Biochemistry,

University of Szeged, Szeged, Hungary,2Cardiovascular Research

Group, Department of Pharmacology, University of Alberta,

Edmonton, Alberta Canada,3Cardiovascular Research Group,

Department of Pediatrics, University of Alberta, Edmonton,

Alberta Canada,4PharmaHungary 2000 Co., Szeged, Hungary

E-mail: macsek@biochem.szote.u-szeged.hu

Background: The cardioprotective effect of preconditioning is

attenuated in hyperlipidemia, however, the underlying

mecha-nisms are unknown We have previously reported that

precondi-tioning decreased the activation and release of matrix

metalloproteinase-2 (MMP-2) in ischemic-reperfused hearts from

normolipidemic rats Here we investigated whether

hyperlipide-mia interferes with the cardioprotective effect of preconditioning

through the modulation of MMP-2

Methods and results: Hearts isolated from male Wistar rats

fed 2% cholesterol-enriched or control chow for 9 weeks were

subjected to a preconditioning protocol (three intermittent

peri-ods of ischemia/reperfusion of 5 min duration each) or a

time-matched non-preconditioning protocol This was followed by a

test ischemia/reperfusion (30 min ischemia and 120 min

reper-fusion) in both groups Preconditioning decreased infarct size

in the control but not the cholesterol-fed group

Cardioprotec-tion in the precondiCardioprotec-tioned control group but not in

choles-terol-fed rats was associated with a 18 ± 3% (P < 0.05)

inhibition of test ischemia/reperfusion-induced MMP-2

activa-tion and release Myocardial protein levels of tissue inhibitors

of MMPs (TIMP-2 and TIMP-4) were not changed in either

group A reduction of infarct size in non-preconditioned hearts

could be produced in the cholesterol-fed group with the MMPs

inhibitor ilomastat (0.25 lm), a concentration producing

MMP-2 inhibition comparable to the effect of preconditioning in the

control group

Conclusions: We conclude that: (i) hyperlipidemia blocks

pre-conditioning-induced cardioprotection, (ii) hyperlipidemia

abol-ishes the preconditioning-induced inhibition of cardiac MMP-2

activation and release, (iii) preconditioning-induced inhibition of

MMP-2 is not mediated by TIMPs, and (iv) cardioprotection

could be produced in hyperlipidemic rats through

pharmacologi-cal inhibition of MMPs

H3-020P

An oxygen consumption enzyme involved in bacterial stress response when grown on non-ionic surfactants

G C Hung, C H Chen, G L Guo and S L HuangMicrobial Proteomics Laboratory, Department of Life Science,National Central University, Jhongli City, Taiwan PR China.E-mail: slhuang@cc.ncu.edu.tw

The octylphenol polyethoxylates (OPEOn) are recalcitrant ionic surfactants The release of OPEOn has resulted in concerndue to their metabolites in the environment showing estrogenicactivity However, little is known about the degradation path-ways, microorganisms and enzymes involved in the degradation ofOPEOn A Gram-negative rod, Pseudomonas nitroreducens TX1,was shown to be able to grow on 0.05–20% (v/v) of OPEOn assole carbon source An OPEOn-dependent oxygen consumptionactivity was observed to be induced in strain TX1 when grownsolely on OPEOn An oxygen consumption enzyme was isolated

non-by column chromatography from crude extract of the bacterium.The purified enzyme was identified as dihydrolipoamide dehydrog-enase by peptide sequences from mass spectrometry It was shown

to uptake oxygen in the presence of excess of NAD(P)H andmetals In addition, the functional proteomic study also foundthat this enzyme was up-regulated for 3–5-fold in OPEOn-grownproteome compared to succinate-grown one by 2D-PAGE Thegene of dihydrolipoamide dehydrogenase and its promoter in

P nitroreducensTX1 were also cloned and sequenced The aminoacid sequence of dihydrolipoamide dehydrogenase showed a highidentity (94%) to those from P aeruginosa PAO1 and P fluores-cens The sequence motifs in the promoter of dihydrolipoamidedehydrogenase gene showed the binding sites for several regulatorproteins involved in many stress responses such as heat shock,oxidative, alcohol and osmotic stresses This is the first report onsuch a novel function of dihydrolipoamide dehydrogenase, whichmay be involved in the bacterial stress response to surfactant

H3-021P

In vitro effects of iron overload on toxicological parameters in isolated hepatocytes obtain from adult rats

A A Harandi1, A Allameh1and P J O’Brien1

1Department of Biochemistry, Ahvaz Medical Science University,Ahwaz, Ahvaz Iran,2Dr Allameh Laboratory, Department ofBiochemistry, University of Trabiat Modares, Tehran, Iran,

3

Dr O’Brien Laboratory, Department of Pharmacy, University ofToronto, Toronto, Ontario Canada

E-mail: a.aminiharandi.utoronto.caIron is toxic to the liver as well as to other organs and tissues ofthe body This toxicity is most likely related to enhancement ofoxidative stress and free radical reaction stimulated by Iron Majorpathophysiological consequences of iron on the liver are the devel-opment of hepatic fibrosis and cirrhosis, porphyries cutanea tardaand development of hepatocellular carcinoma Suspension ofhepatocytes was prepared by in situ perfusion of rat liver using col-lagenase and then toxicity of iron was determined Because toxicity

of iron is related to free radicals that generated by Fenton tion, lipid peroxidation, reactive oxygen species in (ROS) andglutathione as an effective factor to follow up oxidative stress weredetermined Decreased glutathione (>90%) as index for oxidativestress and increase of ROS in incubated isolated hepatocytes byiron show that toxicity of iron is related to oxidative stress thatgenerated by iron overload Lipid peroxidation of hepatocytes byfree radicals that produce in Fenton reaction was responsible forhepatocytes damage and finally caused cell death

Chemistry Laboratory, Department of Material Engineering,

Atilim University, Ankara, Turkey,2Biochemistry Laboratory,

Department of Chemistry, Middle East Technical University,

Ankara, Turkey,3Biochemistry Laboratory, Department of

Biolo-gical Sciences, Middle East Technical University, Ankara, Turkey

E-mail: bisgor@atilim.edu.tr

Living organisms are continuously exposed to herbicides, insect

pathogens or non-nutritional foreign chemical species that are

harmful to their survival These xenobiotics induce Phase II

detoxification enzymes, namely glutathione S-transferases (GSTs),

which protect animals and their cells against toxic and neoplastic

effects of reactive oxygen metabolites as well GSTs function by

conjugating the reactive molecules to glutathione (GSH) In

mammals, at least seven classes of soluble GST isozymes, namely

alpha, mu, pi, theta, sigma and zeta have been identified GST

activity has been detected in a wide variety of species including

mammals, birds, insects, plants, fishes and microorganisms In

this study, bovine liver cytosolic GSTs are screened by using

sev-eral purification steps including DEAE-cellulose,

S-hexylglutathi-one agarose, and dye binding orange A column chromatography

Affinity matrix bound and unbound GST isozymes were

charac-terized by their activities against CDNB, 1-NBC and 1-MS The

bovine liver cytosol contains GST activity towards CDNB

(180 unit/mg), 1-MS (25.6 unit/mg) and 4-NBC (6.5 unit/mg) As

the 0–1 m NaCl gradient eluates from DEAE-Cellulose column

are collected and applied to affinity column, the flowthru

frac-tions exhibit GST activity against 1-MS (27 unit/mg), while GSTs

activity towards CDNB (4791 unit/mg) retains on the column,

and eluted by 15 mm glutathione The GSTs in the flowthru

frac-tions (Theta isozymes) are separated on orange A column and

characterized by their activity towards 1-MS (98 unit/mg) and

4-NBC (55 unit/mg) separately The diversity of GST isozymes are

confirmed by positive staining with primary antibodies against

alpha, mu and pi isozymes in affinity bound fractions and with

theta antibodies in flowthru fractions

H3-023P

Influence of metal-organic substances on

activity of antioxidant enzymes

I D Leus1, E D Zabitskaya1, N I Shtemenko1and

A V Shtemenko2

1

Laboratory of Biochemistry, Department of Biophysics and

Biochemistry, Dnepropetrovsk National University,

Dnepropetrovsk, Ukraine Ukraine,2Laboratory of Synthesis of

Metal-organic Substances, Department of Inorganic Chemistry,

Ukranian State University of Chemical Technology,

Dnepropetrovsk, Ukraine Ukraine E-mail: ashtem@a-teleport.com

Activity of catalase (C) and superoxide dismutase (SOD) were

measured in plasma and red blood cells (RBC) of healthy and

Guerin’s carcinoma-bearing Wistar rats under influence of

cis-platine (CP) and cluster rhenium compounds with organic

lig-ands (CROL) introduction These two enzymes are known to be

sensitive as to development of tumor as to introduction of any

substance with redox properties We found the significantly

reduced values of C and SOD activity in blood of tumor-bearing

animals and SOD (in four times) in healthy animals after CP

introduction as a result of a depression of defensive ability of

organism Introduction of CP (strong tumor-reducing agent) to

tumor-bearing animals led to practically close to control

mean-ings of the activity of C and reduced (on 25%) SOD CROL

(weak tumor-reducing agents) have not changed the activity ofboth enzymes in healthy animals that confirmed low toxicity ofthese substances Being introduced to tumor-bearing animalsCROL showed themselves as strong inhibitors of C (on 40–50%)and SOD in plasma but positive modulators of SOD in RBC.Especially interesting fact is that influence of CROL on RBCSOD is strongly dependent from the structure of radical in themolecule of CROL Possible mechanism of these cell-specificactions of CROL is speculated

H3-024P Cambialistic-superoxide dismutase from diatom: cloning, expression, and enzyme stability

C.-T Lin1, Z.-X Huang1and R.-H Juang2

to coordinate the single manganese ion were conserved in allreported Mn-SOD sequences This cDNA was introduced in anexpression vector, pET-20b(+) and transformed into E coli Theexpressed SOD protein was then purified by a His-tag column.The half-life of deactivation was 14.7 min, and its thermal inacti-vation rate constant Kd was 3.21· 10–2/min at 65
C heating.The enzyme was inhibited either in acidic pH (below 5.0) or inthe presence of imidazole (above 1.6 m), and had only moderateeffect under SDS (above 4%), whereas it was not affected under

an alkaline pH (above 9.0) The atomic absorption spectrometricassay showed that 0.3 atom of iron/manganese (2:1) was present

in each subunit of SOD, reconstituted activity suggesting thatdiatom SOD was cambialistic -SOD The finding of this SODcDNA could be used for a reference in comparing the differencesamong marine phytoplankton species and as a probe to detectthe transcription level of this enzyme, which can be applied incosmetics for skin protection caused by oxygen-containing freeradicals

H3-025P Zinc, copper, selenium and activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in blood of pregnant women after vitamin-mineral supplementation

T Laskowska-Klita1, M Chelchowska1and P Kubik2

1

Department of Biochemistry, Institute of Mother and Child,Warsaw, Poland,2Department of Obstetrics and Gynecology,Institute of Mother and Child, Warsaw, Poland

E-mail: lipidy@imid.med.plThe aim of the present work was to study the effect of vitamin-mineral supplementation (15 mg of zinc, 2 mg of copper and

20 lg of selenium daily) on activities of superoxide dismutase(SOD) and glutathione peroxidase (GPx) in blood of pregnantwomen Zinc, copper and selenium as cofactors for theseenzymes, were also determined (by ICP MS method) Activities

of SOD during pregnancy were similar in supplemented and

unsupplemented women In both groups on the course of

preg-nancy increasing concentration of Cu and decreasing that of Zn

was observed Activity of GPx in supplemented women was in

the same range at the I trimester (44.7 U/gHb) and in the III

(41.44 U/gHb) and was accompanied by unchangeable

concentra-tion of selenium (75–71 lg/l) In unsupplemented group

glutathi-one peroxidase activity decreased by 16% to 38.4 U/gHb at term

of delivery The difference was statistically significant (P < 0.01)

In this group amount of selenium in serum fall from 75 lg/l

(I trimester) to 67 lg/l (at delivery) was observed Above results

indicated that vitamin-mineral supplementation (Vibovitmama,

Polfa-Kutno, Poland) may improved antioxidant defence and

should be recommended during pregnancy without the risk of

overdosing especially with reference to selenium

H3-026P

Thioredoxin system in HeLa cells upon

oxidative challenge

S M L Lam and K S Lee

Department of Applied Biology and Chemical Technology,

The Hong Kong Polytechnic University, Hong Kong, PR China

E-mail: 03900867r@polyu.edu.hk

Thioredoxin system, comprising that of the enzyme thioredoxin

reductase (TR), thioredoxin (Trx) and NADPH is well known

for its protective function against oxidative stress Recent

find-ings suggested that TR could be expressed at elevated levels

under oxidative stress and the process could be transcription

fac-tors mediated Nevertheless, the direct relationship between

thi-oredoxin system and ROS is still not well understood In this

study, the level and activity of TR and Trx were studied in HeLa

cells challenged with H2O2and prior treatment of cyclohexamide

Cyclohexamide treated cells were found significantly more

sensi-tive to the cytotoxic effect of H2O2, indicating that de novo

pro-tein synthesis is essential for cell survival under oxidative stress

However we also observed increased basal activity of the whole

thioredoxin system in cells challenged with low levels of H2O2

and with and without the prior addition of cyclohexamide Our

results suggested that both de novo protein synthesis and possibly

a direct activation of the thioredoxin system may be involved in

response to oxidative stress

Acknowledgments: Levina Suk-mi Lam is a recipient of MPhil

studentship of The Hong Kong Polytechnic University The

study is also supported by The Hong Kong Polytechnic

Univer-sity Internal Grant No A-PD96

H3-027P

Free radical activity in obese patients with

type 2 diabetes mellitus

M Mohora, B Virgolici, F Paveliu, I Stoian and D Lixandru

Laboratory of Biochemistry, Department of Biochemistry,

University of Medicine ’Carol Davila’, Bucharest, Romania

E-mail: mohoramaria@yahoo.com

Oxidative stress plays critical roles in the pathogenesis of

var-ious diseases The aim of the present study was to investigate

the relationship between oxidative stress, measured by plasma

malondialdehyde (MDA) and plasma antioxidant defences,

measured by total antioxidant capacity expressed as Trolox

equivalent (TAC-TE), reduced glutathione (G-SH) in whole

blood, protein thiols in plasma and erythrocyte superoxide

dismutase activity in obese patients with type-2 diabetes

melli-tus, clinically free of complications Twenty obese patients with

type-2 diabetes mellitus were examined as well as twenty healthy

controls (matched for age and sex against the obese diabetic

49 vs 390.01 ± 71 U SOD/g Hb in the controls, P < 0.05) Therewere no significant differences in the concentration of plasma thiols(7.37 ± 0.4 vs 7.63 ± 0.4 nmol/g protein in the controls) andplasma total antioxidant capacity (1.58 ± 0.1 vs 1.55 ± 0.09 mMtrolox in the controls) measuring the combined free radicals scav-enging ability of non-enzymatic antioxidants This reduction insuperoxide dismutase activity and the imbalance in the redox status

of the plasma demonstrated is consistent with an increase in freeradical activity in obese patients with type 2 diabetes

H3-028P The level of GSH and antioxidant enzymes activity: GPx and Cu/Zn SOD in patients with pancreatitis

H Milnerowicz1, M Jablonowska1and S Milnerowicz2

1Departament of Biomedical and Environmental Analyses, law University of Medicine, Wroclaw, Poland,2Department andClinic of Gastointestinal and General Surgery, Wroclaw University

Wroc-of Medicine, Wroclaw, Poland E-mail: hm@tox.am.wroc.plIntroduction: The imbalance of reactive oxigen species (ROS)generating and ROS scavengic processes is thought to lead to thedamage of pancreatic cells that initiate autodigestion of the entireorgan Antioxidative enzymes play an important role in the regu-lation of ROS action The aim of this study was to determinedserum activity of Cu/Zn SOD and plasma activity of GPx, andalso the blood level of GSH

Methods: The activity of Cu/Zn SOD (Superoxide DismutaseAssay-IBL, Germany) and GPx (Glutathione Peroxidase Colo-rim/Kinetic Assay-IBL, Germany), and the blood GSH level weremeasured in: 35 patients with chronic pancreatitis (CP), 20patients with chronically exacerbated pancreatitis (CEP), 15patients with acute pancreatitis (AP), 22 normal healthy subject.Results: In all examined groups of patients a decrease in GSH

(448.0 ± 82.7 nmol/ml), furthermore, the highest decrease wasnoticed among patients with AP (135.8 ± 46.5 nmol/ml) Addi-tionally, together with decrease in GSH level, the highest increase

(66.9 ± 17.9 nmol/min/ml) in comparison with healthy subjects(41.6 ± 3.8 nmol/min/ml) However, the highest activity in Cu/

Zn SOD was detected in patients with CEP (3.9 ± 1.4 U/ml)and CP (2.6 ± 0.6 U/ml) in comparison with control group(1.7 ± 0.3 U/ml)

Conclusion: The study suggests that in progress of AP theGSH/GPx system plays the role of the first line of defenceagainst ROS, however in CP and its exacerbation (CEP) the Cu/

Zn SOD overtakes such function

H3-029P Reversion of the hydrogen peroxide-induced modification of eukaryotic initiation factor 4F activity by N-acetyl-cysteine involves distinct signaling pathways

A O’Loghlen, M I Perez-Morgado, M Salinas and

M E MartinLaboratory of Biochemistry, Department of Research, HospitalRamon y Cajal, Madrid, Spain E-mail: ana.o.loghlen@hrc.esDuring the oxidative stress generated by hydrogen peroxide(H2O2) in nerve growth factor (NGF)-differentiated PC12 cells,