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

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:
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 Biol 2003; 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. Begg
1
, S. E. Iismaa
1
, L. Carrington
2
, S. Holman
1
,
A. Husain
1,3
and R. M. Graham
1,4
1
Victor Chang Cardiac Research Institute, Sydney, NSW
Australia,
2
School of Mol. and Microbial Sci., University of
Queensland, Brisbane, QLD Australia,
3
Physiology Department,

University of Alabama, Birmingham, AL, USA,
4
University of
NSW, Sydney, NSW Australia.
E-mail:
Transglutaminase 2 (TG2) is a multifunctional protein, with
actions including receptor signalling, extracellular matrix assem-
bly and apoptosis. It has two enzyme activities: receptor-stimula-
ted signalling that requires GTP binding, and calcium-activated
transamidation (TGase activity) that is inhibited by GTP. These
activities are associated with distinct conformations induced by
the allosteric regulators, calcium and GTP. The recently reported
crystal structure of human TG2 bound to GDP identified a novel
guanine nucleotide-binding site comprised of residues in the first
beta barrel and core domains [Liu, S., Cerione R.A., & Clardy J.
Proc Natl Acad Soc USA 2002; 99, 2743–2747]. Here we have
investigated the allosteric mechanism by which GTP inhibits
TG2 TGase activity. We have shown that of the proposed GTP
binding-site residues, S171, F174 and R579 are indeed critical for
GTP binding, while R476 and R478 are individually less import-
ant. We demonstrate using sedimentation velocity analysis that in
the absence of GTP, TG2 undergoes dynamic sampling of expan-
ded and compact conformations, and that GTP-binding shifts
the equilibrium to a compact conformational substate. This
allows a hydrogen bond to form between the active site cysteine
in the core domain, and a tyrosine residue in the first beta-barrel
domain. We show that this Cys/Tyr bond not only stabilizes the
compact conformation, but is critical for inhibition of TGase
activity by GTP. Furthermore, we show that R579 is a conform-
ational switch residue that, in the absence of GTP, destabilizes

the compact conformation. Our findings support a unique model
for allosteric regulation of TG2, in which GTP not only masks
the destabilizing R579 residue, but also links the core and first
beta-barrel domains, thereby stabilizing the compact conforma-
tion, facilitating C277/Y516 H-bond formation, and inhibiting
substrate access to the active site.
H1–003
Chemistry and Biology of Human
Transglutaminase 2: its role in celiac sprue
and other diseases
C. Khosla
Departments of Chemistry, Chemical Engineering, and
Biochemistry, Stanford University, Stanford, CA, USA.
E-mail:
Tissue transglutaminase (a.k.a. transglutaminase 2; TG2) cata-
lyzes cross-linking between selected glutamine c-carboxamide
groups and lysine e-amino groups in proteins. Although the
chemistry 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 disor-
ders, including celiac sprue, neurological disorders and certain
Abstracts
408
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:
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 cells
against death. Recently described biochemical properties of TG2,
including its protein disulphide isomerase and protein kinase
activities, also may contribute to either survival or death of cells.
In vivo studies using TG2 knock out mice confirm the Janus face
action 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 the
apoptosis has already been initiated. This function of TG2 is par-
tially achieved by being expressed also in macrophages engulfing
and digesting apoptotic cells. TG2 mediated processes participate
in the communication between the phagocytic and the apoptotic
cells facilitating both death and clearance of dying cells.
H1–006
Characterization of transglutaminase and its
role in pancreatic b-cell function and survival
S. Dookie
1
, N. G. Morgan
2
, M. Griffin
3
and E. A. M. Verderio

1
1
School of Biomedical and Natural Sciences, The Nottingham
Trent University, Nottingham, UK,
2
Peninsula Medical School,
Plymouth, UK,
3
Aston University, Birmingham, UK.
E-mail:
Transglutaminases (TGs) are Ca
2+
-dependent enzymes that cata-
lyze the formation of protein crosslinks via intermolecular iso-
peptide bonds. Previous work has shown that pancreatic islets
express functional tissue-type TG (tTG) and recent work under-
taken in tTG-deficient mice points to a possible role for tTG in
the development of type-II diabetes. Although these data imply
that TGs are required for the maintenance of adequate pancre-
atic b-cell function, this has never been characterized at the
molecular level. Moreover, the recently described survival role
for matrix-associated tTG (Verderio et al, JBC, 2003) has not
been investigated in islets. Here we show, for the first time, that
the 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 activity
upon glucose stimulation. Furthermore, this 55 kDa TG protein
was not a proteolytic fragment of tTG since its expression was
unchanged following inhibition of matrix-associated proteases. A

possible protective role for TGs in conditions resembling the dia-
betic milieu has also been investigated in vitro, by inducing
conditions of glucotoxicity, oxidative stress and lipotoxicity. A
pre-conditioned matrix secreted by urinary bladder carcinoma
cells (5637), known to support pancreatic b-cell survival, appeared
to improve cell spreading and inhibit caspase-3 activity of clonal
b-cells when supplemented with exogenous purified tTG. In con-
clusion, these findings suggest the predominant expression and
function 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. Beck
1
, L. A. De Girolamo
1
, M. Griffin
2
, A. Hargreaves
1
and
E. E. Billett
1
1
Interdisciplinary Biomedical Research Centre, School of Biomedi-
cal and Natural Sciences, Nottingham Trent University, Notting-

ham, UK,
2
School of Life and Health Sciences, Aston University,
Birmingham, UK. E-mail:
Tissue transglutaminase (tTG) is a multi-functional, Ca
2+
dependent enzyme, which modifies proteins by transamidation of
Abstracts
409
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
warranted.
H1–008P
An in silico study of substrate preference for
transglutaminase 2
E
´
. Cso
˜

sz
1
, P. Bagossi
1
and L. Fe
´
su
¨
s
1
1
Apoptosis Research Group, Biochemistry and Molecular Biology,
University of Debrecen, Debrecen, Hungary,
2
Retroviral Biochem-
istry Goup, Biochemistry and Molecular Biology, University of
Debrecen, Debrecen, Hungary,
3
Apoptosis Research Group,
Biochemistry and Molecular Biology, University of Debrecen,
Debrecen, Hungary. E-mail:
Transglutaminases catalyze the Ca
2+
-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 ( />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. Cabras
1
, R. Inzitari
2
, C. Fanali
2
, C. Olmi
2

, M. Patamia
3
,
M. T. Sanna
1
, E. Pisano
1
, B. Giardina
2,3,4
, M. Castagnola
2,3,4
and I. Messana
1
1
Department of Sciences Applied to Biosystems, Cagliari
University, Cagliari, Italy,
2
Institute of Biochemistry and Clinical
Biochemistry, Catholic University, Rome, Italy,
3
Institute for the
Chemistry of Molecular Recognition, National Research Council
(CNR), Rome, Italy,
4
International Scientific Institute Paolo VI,
Catholic University, Rome, Italy. E-mail:
Statherin is a phospho-peptide of 43 amino acid residues pecu-
liar of human saliva with unusual characteristics, such as high
content of Tyr, Pro and Gln and a high degree of structural
asymmetry. 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 saliv-
ary glands, was demonstrated [1]. The pivotal multifunctional
role of statherin in the oral cavity could be connected to its
involvement in the formation, through protein crosslinking, of a
protein network with proline-rich proteins and histatins. This
oral protein pellicle could interact with the oral epithelial-cell
plasma membrane, contributing to mucosal epithelial flexibility
and 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 the
reactivity of statherin in the presence of TGA from human oral
epithelial cells was investigated by RP-HPLC ESI-ion trap MS.
The mass of the reaction product (5363 vs. 5380 amu) evidenced
that the early event of the homotypic TGA reaction is the spe-
cific formation of a cyclo-statherin. The RP-HPLC ESI-ion trap
MS analysis of the proteolytic digest of cyclo-statherin by
proteinase 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 in
some subjects the presence of cyclo-statherin, even though at a
concentration near to the limit of detection of the analytical
method (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. Howden
1
, P. L. R. Bonner
1
, M. Griffin
2
and
A. J. Hargreaves
1
1
Interdisciplinary Biomedical Research Centre, School of Biomedi-
cal and Natural Sciences, Nottingham Trent University, Notting-
ham, Nottinghamshire, UK,
2
School of Life and Health Science,
Aston University, Birmingham, West Midlands, UK.
E-mail:
Transglutaminases (TGases) are a group of calcium-dependent
enzymes that are responsible for the posttranslational modifica-
tion of proteins by the transamidation of polypeptide bound
glutamines. The reaction occurs either through the formation of
protein crosslinks or the incorporation of polyamines into sub-
strate proteins. Of particular interest is the fact that modified
Abstracts
410
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. Jones
1
, P. Kotsakis
1
, T. S. Johnson
2
, D. Chau

1
,
E. Verderio
1
, S. Ali
1
and M. Griffin
3
1
School of Biomedical and Natural Sciences, Nottingham Trent
University, Nottingham, UK,
2
Sheffield Kidney Institute, Northern
General Hospital, Sheffield, UK,
3
School of Life and Health
Sciences, Aston University, Birmingham, UK.
E-mail:
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 biochemical 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
´
ly
1
, Z. Vecsei
1
, T. Deme

´
nyi
1
, I. R. Korponay-Szabo
´
2
and
L. Fe
´
su
¨
s
1
1
Department of Biochemistry and Molecular Biology, Medical and
Health Science Center, University of Debrecen, Debrecen,
Hungary,
2
Heim Pa
´
l Children’s Hospital, Budapest, Hungary.
E-mail:
Enzyme functions of tissue transglutaminase (TG2) and their
modification by anti-TG2 autoantibodies may play a role in mani-
festations of coeliac disease, but the effects observed in previous
studies have been controversial. We aimed to evaluate the effect
of coeliac autoantibodies on transamidation, deamidation and
GTPase reactions catalyzed by TG2 by a systematic biochemical
approach 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 using
amine-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, immuno-
globulins from patients with severe malabsorption enhanced the
reaction velocity to 105.4–242.2% (mean: 189.5%) compared with
control antibodies. This activating effect was dose-dependent, and
most pronounced with immobilized glutamine-acceptor substrates,
and correlated inversely with the basal specific activity of the
enzyme. Immunoglobulins purified from the same patients after a
gluten-free diet did not affect transamidating activity significantly.
GTPase activity of TG2 decreased to 67.0–73.4% in the presence
of antibodies from all coeliac groups. In conclusion, an early and
severe malabsorptive clinical manifestation of celiac disease is
associated with the presence of autoantibody population that
enhances transamidation activity of TG2.
H1–013P
Calreticulin-bound transglutaminase 2 is
poorly accessible to celiac autoantibodies
I. Korponay-Szabo
´
1,2
, T. Halttunen
2
, K. Laurila
2
, I. Dahlbom
3
,

J. B. Kova
´
cs
4
and M. Ma
¨
ki
2
1
Dept. of Paediatrics, Univ. of Debrecen, Debrecen, Hungary,
2
Paediatric Research Centre, Univ. of Tampere, Tampere, Finland,
3
Research Unit, Pharmacia Diagnostics, Uppsala, Sweden,
4
Dept.
of Gastroenterology-Nephrology, Heim Pa
´
l Children
´
s Hospital,
Budapest, Hungary. E-mail: ilma.korponay-szabo@uta.fi
Background and aims: Tissue transglutaminase (TG2) is a
major autoantigen in celiac disease, an autoimmune disorder
induced by the ingestion of gliadin-containing cereal foods. Cal-
reticulin (CRT), another putative celiac autoantigen, acts as a
regulatory subunit for TG2 during receptor signaling. CRT has
some homology with gliadin and belongs to molecular chaper-
ones. We investigated whether CRT is able to interact directly
with TG2 also in vitro and whether this interaction influences

autoantigenic 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) to
establish 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 coated
TG2 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 the
presence of Ca
++
. Both free and CRT-bound TG2 were well
Abstracts
411
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:
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:
We have found that tissue-type transglutaminase (tTG), also
called as TGc, TGase2, and Ga
h
, has the activity of protein
disulfide isomerase (PDI). We have shown that tTG converts
completely reduced/denatured inactive RNase A molecule to the
native active enzyme. It was substantially inhibited by the simul-
taneous presence of other potential substrate proteins such as
completely reduced BSA, but not by native BSA. This activity
was especially high in the presence of GSSG, but not GSH. The
addition of GSH to the reaction mixture in the presence of
GSSG at a fixed concentration up to at least two hundred times
did not very substantially inhibit the PDI activity. It is possible
that tTG can exert PDI activity in fairly reducing environment

like cytosol, where most of tTG is found. It is quite obvious from
the following observations that PDI activity of tTG is catalyzed
by domain different from that used for the transglutaminase
reaction. Although the alkylation of Cys residues in tTG com-
pletely abolished the transglutaminase activity as was expected, it
did not affect the PDI activity at all. This PDI activity did not
require the presence of Ca
2+
. It was not inhibited by nucleotides
including GTP at all unlike the other activity of tTG. Our results
indicated that a disulfide bond(s) might serve as the active site.
We have been able to show the presence of the complex made up
with the enzyme and the substrate via mixed-disulfide bond(s).
We have started the investigation to elucidate the active site
disulfide bond. For this matter we synthesized a novel convenient
modification reagent for -SH groups. We have not completed the
elucidation 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. Sarang
1
, P. Molna
´
r
2
,T.Ne
´

meth
2
, S. Gomba
2
, T. Kardon
3
,
G. Melino
4
, S. Cotecchia
5
,L.Fe
´
su
¨
s
1
and Z. Szondy
1
1
Apoptosis Signaling Laboratory, Department of Biochemistry and
Molecular Biology, University of Debrecen, Debrecen, Hungary,
2
Department of Pathology, University of Debrecen, Debrecen,
Hungary,
3
Department of Medical Chemistry, Semmelweis Univer-
sity of Medicine, Budapest, Hungary,
4
IDI-IRCCS, University of

Tor Vergata, Rome, Italy,
5
Institute of Pharmacology and
Toxicology, University of Lausanne, Lausanne, Switzerland.
E-mail:
TG2 is a protein cross-linking enzyme known to be expressed by
hepatocytes, and to be induced during the in vivo hepatic apopto-
sis program. TG2 is also a G protein that mediates intracellular
signaling 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 mice
causes both disseminated endothelial cell apoptosis and fulminant
hepatic failure. Here we report that an intraperitoneal dose of
anti-Fas antibodies, which is sublethal for wild-type mice, kills
all the TG2 knock-outs within 20 h. While TG2
-/-
thymocytes die
in the presence of anti-Fas antibodies at the same rate as wild
types, TG2
-/-
hepatocytes have increased sensitivity towards anti-
Fas treatment both in vivo and in vitro, with no change in their
cell surface expression of Fas or the levels of FLIP
L
, but a
decrease in the Bcl-x
L
expression, and appearance of apoptotic
cells 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 of
chloroethylclonidine, an AR antagonist, to wild-type mice resul-
ted in down-regulation of Bcl-x
L
in the liver, and sensitized
hepatocytes for Fas-mediated apoptosis. Accordingly, mice defici-
ent in AR expression were also more sensitive to Fas-induced
killing, their liver was more sensitive to Fas-mediated apoptosis,
and expressed decreased levels of Bcl-x
L
. In conclusion, our data
Abstracts
412
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-x
L
.
Acknowledgment: This study was supported by OTKA
T034191, T034 918, ETT 48/2000, ETT 04/605-00 and QLK3-
CT-2002-02017.
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:
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 proteins 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:
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-expression
increases the GSH content in HeLa cells, implying the existence
of inter-compartmental regulatory pathways that control redox
signaling and homeostasis. Our results shed some light on the
molecular networks involved.
H2-003
Endoplasmic reticulum lumen: a Janus-faced
compartment
G. Ba
´
nhegyi

Endoplasmic Reticulum Research Group, Department of Medical
Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis
University, Budapest, Hungary. E-mail:
The 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 in
more oxidized state than the cytosolic ones. While the ratio
between reduced and oxidized glutathione is 100:1 in the cytosol,
this value is around 1–2:1 in the ER lumen. The redox potentials
calculated from these values are -0.24 and -0.18 V, respectively.
Generalizing the observations regarding the redox state of thiols
found in the ER of cells engaged in protein secretion, now it is
supposed that the redox conditions in the ER lumen are oxid-
izing. However, several intraluminal reactions have been des-
cribed, which require reducing equivalents. Such reactions
involve the isomerization of disulfide bonds, the steps of the vita-
min K cycle, the biotransformation of several xenogenous
ketones and aldehydes and the local activation of cortisone to
cortisol. This circumstance suggests that more redox systems exist
in the ER lumen. Since their redox state must be different, it can
also be suggested that these systems are functionally uncoupled.
While molecular oxygen as the ultimate electron acceptor can be
used 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. Ratcliffe
Henry Wellcome Building for Molecular Physiology, Oxford, Oxon
United Kingdom. E-mail:
Hypoxia inducible factor (HIF) is an alpha/beta heterodimeric
transcriptional complex that plays a key role in directing cellular
responses to hypoxia. Recent studies have defined novel oxygen
sensitive signal pathways that regulate the activity of HIF by
post-translational hydroxylation at specific residues within the
alpha-subunits. HIF prolyl hydroxylation governs proteolytic
regulation of HIF, whereas HIF asparaginyl hydroxylation
modulates interaction with transcriptional co-activators. These
hydroxylations are catalyzed by a set of non-haem Fe(II) 2-oxo-
glutarate (2OG) dependent dioxygenases. During catalysis, the
Abstracts
413
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 K
m
values of the HIF-P4Hs for O
2
were slightly above the concentra-
tion of dissolved O
2
in air, thus indicating that they are very effect-
ive oxygen sensors. The K
m
of FIH was distinctly lower, indicating
that a larger decrease in O
2
concentration 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:
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 probe
labels intracellular proteins, which become oxidized at tyrosine
residues 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 Western
blotting using the anti-fluorescein antibody. Identification of
labelled proteins were based on mass spectrometry. Exposure of
human fibroblasts to hydrogen peroxide (H
2
O
2
) resulted in the
specific labelling of endoplasmic reticulum resident proteins.
Photodynamic treatment (PDT) of these cells, loaded with the
photosensitizer 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 activates
apoptotic pathways, which are insensitive to hydrogen peroxide
treatment. 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 PDT
loaded 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 PDT
lysosomes disappeared, indicating that lysosomes were damaged.
We propose that the massive apoptosis observed is associated
with 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. Abramic
1
, U. Imaga
1
, M. Osmak
2
,L.E
`
Iicin-Ain
2
,
B. Vukelic
1
, K. Vlahovicec
3

and L. Dolovcak
1,4
1
Laboratory of Cellular Biochemistry, Department of Organic
Chemistry and Biochemistry, Rudger Boskovic Institute, Zagreb,
CROATIA,
2
Department of Molecular Medicine, Rudger Boskovic
Institute, Zagreb, CROATIA,
3
Protein Structure and Bioinformat-
ics Group, International Centre for Genetic Engineering and Bio-
technology, Trieste, ITALY,
4
Department of Molecular Biology,
University of Zagreb, Zagreb, CROATIA.
E-mail:
Dipeptidyl 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 of
DPP III originating from various species, the function of activity
important sulfhydryl groups is still inadequately understood. The
present study of the reactivity of these groups was undertaken in
order 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 the
enzyme’s residual activity with fluorimetric detection. Inactivation
of this human enzyme exhibited pseudo-first-order kinetics, sug-
gesting that all reactive SH-groups have equivalent reactivity,
and the second-order rate constant was calculated to be 3520

M-1min-1. Rat DPP III was hyperreactive to pHMB and showed
biphasic 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 sulfhydryl
were obtained from slopes of linear plots of pseudo-first-order
Abstracts
414
constants versus reagent concentration. Peptide substrates protec-
ted both mammalian DPPs III from inactivation by pHMB. Phy-
siological concentrations of biological thiols and H
2
O
2
inactivated the rat DPP III. Human enzyme was resistant to
H
2
O
2
attack 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:
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. Gil
1
, C. Ferreira-Batista
2
, R. Vazquez-Duhalt
1
and
B. Valderrama
1
1
Department of Cellular Engineering and Biocatalysis, Institute of
Biotechnology, National University of Mexico, Cuernavaca, More-
los Mexico,
2
Department of Molecular Medicine and Bioprocesses,
Institute of Biotechnology, National University of Mexico, Cuerna-
vaca, Morelos Mexico. E-mail:
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 per-
oxidase towards hydrogen peroxide is an intrinsic property of the
enzyme, not due to the presence of catalase activity.
H2-010P

Thionylation of PKC by reactive sulfur species:
disulfide S-monoxide and disulfide S-dioxide
F. L. Huang and K. P. Huang
Endocrinology and Reproduction Research Branch, NICHD, NIH,
Bethesda, Maryland United States of America.
E-mail:
Disulfide 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 H
2
O
2
in the presence of iron or
methyltrioxorhenium and the formation of DSOs was more
effective with free thiols than with their corresponding disulfides,
suggesting that sulfenic and sulfinic acids are the intermediates
for the formation of these compounds. Both DSMO and DSDO
were purified by HPLC and identified by mass spectrometry and
they were highly reactive toward thiol to form mixed disulfide.
The reaction rate of DSDO was at least an order of magnitude
faster 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 Western
blot with a specific antibody. The IC50 of GS-DSDO-mediated
inactivation of PKC (50 ll) was ~ 10-fold less than that of
GS-DSMO and mechanistically, the former mainly caused
thionylation and the latter both thionylation and polymerization
of PKC. DSOs derived from CPSH, CP-DSMO and CP-DSDO
were less potent as PKC inhibitors than their corresponding

derivatives from GSH, suggesting that the individual DSOs have
their distinct reactivities in spite of possessing similar functional
groups. Inactivation of PKC by DSOs could be reversed by DTT
at low levels of thionylation but became irreversibly inactivated
after extensive thionylation. These results suggest that DSOs
derived 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. Hayashi
1
, Y. Kojima
2
and S. Fujiwara
1
1
Department of Biochemistry, University of Kyushu Dental
College, Kitakyushu, Fukuoka Japan ,
2
Department of Pediatric
Dentistry, University of Kyushu Dental College, Kitakyushu,
Fukuoka Japan,
3
Department of Biochemistry, University of
Kyushu Dental College, Kitakyushu, Fukuoka Japan.
E-mail:
D-aspartate oxidase (D-AspO) is a flavoprotein that catalyzes

oxidative deamination of D-aspartate to oxaloacetate, H
2
O
2
and
NH
3
. D-AspO has been found in many mammal tissues, showing
that D-AspO differs from D-amino acid oxidase (DAO) and is
located in the peroxisomes of mammal liver and kidneys. In our
previous studies, we used rats to study the effects of low intakes
of zinc and calcium on the growth and development in tibia
metaphysis. However, we have no report on the enzyme for
cofactor zinc in rats salivary glands and brain stem. We aimed to
produce 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,
Abstracts
415
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:
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 x2of
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, restriction 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.
H2-013P
An in vitro study of effect of copper ion on
subspecies of lipoprotein(a) in human serum
M. Kadkhodaei-Elyaderani
1
and Z. Faezi Zadeh
2

1
Department of Biochemistry, Jundishapoor University of Medical
Sciences, Ahwaz, Iran,
2
Thalassaemia Research Centre,
Jundishapoor University of Medical Sciences, Ahwaz, Iran.
E-mail:
Lipoprotein(a) is one of the plasma lipoprotein that is composed
of an LDL core and glycoprotein apo(a). Lp(a) is present in
human plasma in four heterogenous subspecies (Lp(a)Lys-,
Lp(a)Lys+1, Lp(a)Lys+2, Lp(a)Lys+3). This subclassification is
made according to their ability to bind to lysine sepharose. Patho-
genicity of Lp(a) as a risk factor for cardiovascular disease, may
depend on its lysine binding site [LBS] activity, which imparts a
unique function to Lp(a), including a potential to inhibit fibrinoly-
sis. Previous studies have shown that Lp(a)Lys+, Lp(a)Lys+2
and 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 lysine
binding site activity of Lp(a) was investigated. For this purpose
four subspecies of Lp(a) were isolated using affinity chromatogra-
phy on lysine sepharose from pooled human serum. Serum lipids
were oxidized in the presence of 15, 30, 50, 75 and 100 m of
CuCl2. Lipid oxidation was measured as conjugated diene forma-
tion determined by spectrophotometric method at 245 nm. Four
species of Lp(a) in the oxidized serum was isolated. The results
showed 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 Haddad
1
, A. R. Rezaizadeh
2
, M. Mardani
3
,
G. R. Asadi Karam
4
and M. Mahmoodi
4
1
Department of Physiology, Kerman Medical University, Kerman,
Iran,
2
Department of Anatomy, Rafsanjan University of Medical
Sciences, Rafsanjan, Iran,
3
Department of Anatomy, Isfahan Univer-
sity of Medical Sciences, Isfahan, Iran,
4
Department of Biochemis-
try, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
E-mail:
It was reported that increased oxidative stress in diabetes mellitus
associated with decrease in collagen synthesis. In this present

study, the role of ascorbic acid on hydroxyproline and collagen
in diabetic rats is reported. This study was performed on 160
male 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 one
month before diabetic induction, until wound healing. Full thick-
ness incision induced on back skin of rat, 8 weeks after diabetic
induction. Content of collagen and biochemical measurement of
hydroxyproline at days 1, 3, 7, 11.15, and 20 after operation were
measured. The results showed, the mean of collagen content and
hydroxyproline in group I was more than all examined groups,
at days of 11, 15 and 20, in addition, in group III this indication
was less than group IV during all days. This data suggest that,
vitamin c supplementation in chronic diabetic rats impaired
Abstracts
416
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:

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:
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 additives
analyzed such as BHA and BHT. Results demonstrated that the

treatment 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. Nikolic
1
, D. T. Sokolovic
1
and B. J. Djindjic
2
1
Department of Biochemistry, University of Nis, Nis, Serbia and
Montenegro,
2
Department of Pathological Physiology, University
of Nis, Nis, Serbia and Montenegro.
E-mail:
Trauma, systemic inflammation, sepsis or toxins can modulate
tissue functions. Glutamine has been shown to have beneficial
effects in sepsis, trauma, infections, chemotherapy etc. The aim
of this study was to investigate the possible beneficial effect of
L-glutamine on acute hepatotoxicity induced by mercury chloride
(HgCl2). To study possible role of nitric oxide in mechanisms
toxicity of mercury chloride we have study interaction between
L-Glutamine and nitric oxide synthesis in tissue metabolism.
Male Spraque-Dawley rats weighing about 200 g. were used in
the experiment. Acute toxicity was induced by i.p. administration
of mercury chloride in a dose of 3 mg/kg. One group of animals
was pre-treated with L-glutamine (200 mg/kg) 1 h before mercury
chloride administration. Control group of animals was treated

with equal volume of saline. The animals were killed 24 h after
HgCl
2
administration. The level of citrulline, by product in nitric
oxide synthesis, was measured by diacetylmonoxime reaction.
Results of the study show that acute toxicity of mercury chloride
leads to elevation of tissue level of citrulline compared to control
group (P < 0.01). Administration of glutamine to control rats
decreases slowly citrulline level compared to control, while pre-
treatment of animals with glutamine before mercury chloride
administration leads to the most greater decreases in citrulline
level (P < 0.01). Heaving in mind that overproduction of nitric
oxide has cytotoxic effects administration of L-glutamine may be
beneficial in mercury toxicity through decreasing nitric oxide
level.
H2-018P
Peptides are formed when RNAase is treated
with Fenton reagent in the presence of copper
(II)
V. R. Oso
´
rio e Castro
1
and G. B. Domont
2
1
Laboratory of Biochemistry, Department of Biosciences,
Politechnic Institute of Castelo Branco, Castelo Branco, Beira
Interior Portugal,
2

Laboratory of Biochemistry, Department of
Biochemistry, Federal University of Rio Janeiro, Rio de Janeiro,
Rio de Janeiro Brazil. E-mail:
The inactivation of RNAase by Fenton reaction, within certain
conditions, 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 the
presence of copper (II). For example, with 4.4 x 10
–4
Cu
++
it
reaches 92% after 1 min and 98% after 30 min of reaction,
showing three fractions (F
1
Cu, F
2
Cu, F
3
Cu) by Sephadex G-75
gel 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 also
indicate peptides in fractions F
1
Cu and F
2

Cu. Particularly F
2
Cu
have, at least, seven peptides, well separated by high voltage
Abstracts
417
paper electrophoresis. In a control experiment with H
2
O
2
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:
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. Stark
1

,H.To
¨
rma
¨
2
and E. Oliw
1
1
Oliw Laboratory, Department of Pharmaceutical Biosciences,
Uppsala University, Uppsala, Sweden,
2
Dermatology, Department
of Medical Sciences, Uppsala University, Uppsala, Sweden.
E-mail:
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-hydroxy
metabolites were initially discovered in human seminal fluid and
are mainly formed by biosynthesis in seminal vesicles, presuma-
bly in series by cyclooxygenase type 2 (COX-2), cytochrome
P-450 4F8 (CYP4F8) and microsomal prostaglandin E synthase-1
(mPGES-1). Quantitation by real-time PCR suggested that PGH

synthase-2, CYP4F8, and microsomal PGE synthase-1 were
co-localized and abundant and correlated in seminal vesicles of
seven patients. (Stark et al. 2004). COX-2 is known to be up
regulated by inflammatory stimuli in many tissues, often together
with mPGES-1. COX-2 occurs along with CYP4F8 in non-lesion-
al epidermis, and the latter is strongly up regulated in psoriatic
lesions (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 in
human epidermis and CYP4F8 has been found to be strongly
upregulated 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 progress
investigating if CYP4F8 may metabolize other fatty acids then
Arachidonic 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 epidermis
may have a similar function.
Acknowledgments: This work was supported by Vetenskapsra
˚
-
det Medicin (03X-06523), Edvard Welanders Stiftelse and Finsen
stiftensen
H2-021P
Implication of oxidative stress in protein
degradation, acetylcholinesterase, (Na
+
,K
+

)-
ATPase and Mg
2+
-ATPase activities in rat brain
after forced swimming
T. Tsakiris
1
, P. Angelogianni
1
, C. Tesseromatis
2
, S. Tsakiris
1
and
C. Tsopanakis
1
1
Laboratory of Experimental Physiology, Medical School,
University of Athens, Athens, Greece,
2
Laboratory of Experimental
Pharmacology, Medical School, University of Athens, Athens,
Greece. E-mail:
The aim of this study was to investigate, whether exercise stress
[short (2 h) or prolonged (5 h) forced swimming in rats] could
modulate brain total antioxidant status (TAS), tissue protein con-
centration and the activities of acetylcholinesterase (AChE),
(Na
+
,K

+
)-ATPase and Mg
2+
-ATPase. Protein concentration,
TAS and enzyme activities in homogenized rat brain were deter-
mined spectrophotometrically. Protein concentration was
decreased by 15% (P < 0.01) and by 30% (P < 0.001) after 2 h
and 5 h of forced swimming, respectively. TAS was decreased by
20–25% 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 forced
swimming, respectively. In contrast, (Na
+
,K
+
)-ATPase and
Mg
2+
-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 of
exercise. Control values in non-treated rats were unaltered
(P < 0.05). In conclusion, short or prolonged forced swimming
induces oxidative stress in rats, probably resulting in a reduction
in brain protein concentration and AChE activity. In addition, a
(Na
+
,K
+
)-ATPase and Mg
2+

-ATPase activation was observed
under the above mentioned experimental conditions. This stress
condition may modulate brain intracellular Mg
2+
concentration,
neural excitability, metabolic energy production and neurotrans-
mission.
Abstracts
418
H2-022P
Low total antioxidant status is implicated with
degradation of erythrocyte membrane protein
content in patients with classical
galactosaemia
S. Tsakiris
1
, H. Michelakakis
2
, T. Tsakiris
1
and K. H. Schulpis
2
1
Laboratory of Experimental Physiology, Department of Medical
School, University of Athens, Athens, Greece,
2
Aghia Sophia’ Chil-
dren
´
s Hospital, Institute of Child Health, Athens, Greece.

E-mail:
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
compared with those of group A (1.41 ± 0.11 mmol/l,
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
H
2
O
2
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 H
2
O
2
application. Survival was determined
for 2 h after the completion of H
2
O
2
exposure, 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 H
2
O
2
stress. This 6AN-mediated protective
effect was associated with a modest increase (by up to 55%) of

the cytosolic free Ca
2+
, 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 Ca
2+
-independ-
ent isoforms of PKC. The longest of the 6AN treatments tested
(18 or 23 h) induced the Unfolded Protein Response (UPR) in
H9C2 cells. UPR is an adaptative, transcriptional upregulation
of S/ER-resident chaperones caused by a disturbance of the
S/ER lumenal environment, and is highly conserved in eukary-
otic cells. Although in some systems UPR has been shown to
exert an antioxidative protection, this appeared not to be the
case in the present work, as the protective effect of 6AN was
only slightly affected by a complete inhibition of protein synthe-
sis, which did abolish UPR. We propose that 6AN induced an
oxidative shift in the cytosol redox status owing to an impaired
glutathione regeneration via the oxidative pentose pathway,
leading to an increased Ca
2+
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 ear-
lier described ‘‘Ca
2+
pre-conditioning’’ of cardiomyocytes.
H2-024P
Heroin abuse caused oxidative stress and
oxidative damages of protein

Q. Zheng
1
,B.Xu
1
,G.Li
1
, X. Sun
1
and Z. Wang
1
1
Ocean School, Yantai University, Yantai, Shandong PR China,
2
Pharmacy School, Yantai University, Yantai, Shandong PR
China. E-mail:
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 stress
involved in hepatotoxicity induced by heroin.
Methods: A dose-increasing method was used to develop the
heroin-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) by
DTNB method and percentage of cell with damaged DNA by
comet method, and the activity of alanine aminotransferase
(ALT) by commercial kit in hepatocytes of heroin administered
mice. Coefficient of linear pair wise regression was used to evalu-
ate the correlation between oxidative damage and hepatotoxicity
induced by heroin.
Results: The oxidative damaged products, MDA, protein car-

bonyl and the percentage of cells with damaged DNA increased
in hepatocytes of heroin administered mice, while the antioxid-
ant enzyme and substances, activities of superoxide dismutase
(SOD), catalase (CAT), and glutathione peroxidase (GPx) as
well as the content of GSH decreased, on the other hand, the
hepatotoxic index, activity of ALT in serum of heroin adminis-
tered mice elevated remarkably. Positive correlations between
ALT 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 GSH
content in hepatocytes (r = –0.6648, n = 15) were found. How-
ever, there is no close correlation between ALT activity and
DNA damage.
Conclusions: Increase of oxidative damaged DNA, protein and
lipid with decrease of antioxidant activities implied that there
was a seriously oxidative stress in mice liver induced by heroin.
Oxidative stress may cause hepatotoxicity.
Abstracts
419
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:
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
in the hippocampus.
Acknowledgment: This work was supported by NIH grant
NS34007.
H3-002
Modulation of mitochondrial oxidant signals

I. J. Reynolds
Department of Pharmacology, University of Pittsburgh, Pittsburgh,
PA, USA. E-mail:
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 thereby
stimulate oxidant generation. In this way, zinc provides an exam-
ple of an important neurotoxin that is mobilized as the result of
the modulation of mitochondrial oxidant signals, and which can
also modulate mitochondrial itself, possibly in a feed-forward
loop. This kind of oxidant signaling could prove to be of great
importance in neurodegenerative disease.
H3-003
Functional consequences of protein tyrosine
nitration and oxidation

H. Ischiropoulos
Stokes Research Institute and Department of Biochemistry and
Biophysics, Children’s Hospital of Philadelphia, and The University
of Pennsylvania, Philadelphia, PA, USA.
E-mail:
A better understanding for the role of oxidants in human dis-
ease may be derived by the identification of specific protein tar-
gets that are post-translationally modified by oxidants and by
examining the effect of these selective modifications upon pro-
tein function. Tyrosine nitration is now recognized as a covalent
protein modification derived from the reaction of proteins with
nitrating agents. Biological protein nitration appears to be a
rather selective process; not all tyrosine residues in proteins and
not all proteins are nitrated in human and model systems of
disease. Published studies and work in progress has revealed
that the presences of specific tyrosine residues in local environ-
ments of proteins are the primary determinants of the site of
nitration. Proteomic analysis, specific monoclonal antibodies
and immunoprecipitation methodologies have identified a num-
ber of proteins that are nitrated under physiological conditions
and in human pathologies. Protein tyrosine nitration does not
always modify protein function, despite alterations in secondary
structure, and quite often the coexistence of other amino acid
oxidation precludes from attributing changes in function to
tyrosine nitration. To gain a better understanding of the biolo-
gical consequences of protein tyrosine nitration we investigated
the role of this modification on the induction of inappropriate
protein aggregation. The data indicated that protein tyrosine
nitration 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 human
neurodegenerative diseases such as Parkinson’s disease and for
the association between nitrative stress and risk for coronary
artery disease will be discussed.
H3-004
H
2
O
2
signaling through cysteine modifcations
L. Monceau
1
, S. Fourquet
1
, A. Sekowska-Danchin
1
, F. Tacnet
1
,
G. Rousselet
1
, E. Hidalgo
2
and M. B. Toledano
1
1
Laboratoire Stress Oxydants et Cancer, Service De Biologie
Mole
´
culaire Syste

´
mique, Commissariar a l’Energie Atomique,
Gif-sur-Yvette, France,
2
Cell Signalling Unit, Departament de
Cie
`
ncies Experimentals i de la Salut, Universitat Pompeu Fabra,
Barcelona, Spain. E-mail:
Microbial H
2
O
2
sensors are master regulators of cellular H
2
O
2
homeostasis. They are activated by oxidation when intracellular
levels of H
2
O
2
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 essential
for aerobic life and has the hallmarks of a homeostatic control.
Abstracts
420
In mammals, regulators activated by reversible oxidation with
mechanisms similar to those of microbial H

2
O
2
sensors are
being described. However their physiological function is not to
regulate H
2
O
2
homeostasis. Rather they respond to the second
messenger H
2
O
2
and regulate specific cellular pathways. The
yeast S. cerevisiae Orp1-Yap1 and S. pombe Tpx1-Pap1 two-
component H
2
O
2
sensors share an overall common mechanism
of activation involving reversible disulfide bond formation.
H
2
O
2
-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 H
2
O
2
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
H
2
O
2
redox signaling and suggest the existence of specific path-
ways of cysteine oxidation by H
2
O
2
.
H3-005
Calcium release mediated by redox activation
of ryanodine receptors induces CREB and ERK
phosphorylation in N2a cells and hippocampal
neurons
M. A. Carrsaco
1
, U. Kemmerling
1,2

, P. Mun
˜
oz
3
and C. Hidalgo
1
1
FONDAP CEMC, Facultad de Medicina, ICBM, Universidad de
Chile, Santiago, RM Chile,
2
FONDAP 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:
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. Marengo
1
, C. De Ciucis
1
, L. Raffaghello
2
, V. Pistoia
2
,

D. Cottalasso
1
, M. A. Pronzato
1
, U. M. Marinari
1
and
C. Domenicotti
1
1
Department of Experimental Medicine, University of Genoa,
Genoa, Italy,
2
Laboratory of Oncology, Gaslini Hospital, Genoa,
Italy. E-mail:
The by-product of biological processes is represented by reactive
oxygen species (ROS) which have high chemical reactivity and
can damage a variety of biomolecules, such as lipids, proteins,
carbohydrates and nucleic acids. Recent studies imply that ROS
are not only destructive elements for cells, but also they are
essential 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 signa-
ling molecules. Among these signal proteins, a crucial role in cell
responses is played by protein kinase C (PKC), a family of isoen-
zymes also implicated in tumorigenesis. Oxidative regulation of
delta and epsilon PKCs has been demonstrated to be important
in the development of cancer preventive or therapeutic agents.
Neuroblastoma is characterised by the production of oxygen
intermediates 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. In
our study, we have found that the production of ROS induced
by BSO is able to trigger mitochondrial pathway of apoptosis in
neuroblastoma cells with MYCN amplification. This process is
mediated by the oxidative activation of the pro-apoptotic PKC
delta which might be involved also in the production of ROS
amplifying the apoptotic cascade. Future studies will be
addressed to alter the expression of PKC isoenzymes in order to
induce cancer cell death; this offers new strategies for increasing
the efficacy of chemiotherapy in neuroblastoma clinical treat-
ment.
Acknowledgments: This work was supported by grants from
Genoa 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. Anatolievich
1
, P. L. Anatolievna
2
, T. T. Nikolaevna
3
and
L. E. Anatolievna
4
1
Pathophysiology, Academy of Medical Sciences of Ukraine, Insti-
tute of Gastroenterology, Dnepropetrovsk, Ukraine,
2

Biochemistry,
Academy of Medical Sciences of Ukraine, Institute of Gastroenter-
ology, Dnepropetrovsk, Ukraine,
3
Pathophysiology, Academy of
Medical Sciences of Ukraine, Institute of Gastroenterology,
Dnepropetrovsk, Ukraine,
4
Biochemistry, Ukrainian State
Scientific Research Institute of Medical and Social Problems of
Disability, Dnepropetrovsk, Ukraine. E-mail:
Investigation of oxidant-prooxidant status of rats’ blood by
introduction of medical bile in the stomach resulted in intensifica-
tion of free radical process in the blood of the animals. So the
level of the thiobarbituric acid (TBA) active products in plasma
of blood increased on 94.6%. Observable phenomena are intensi-
fied after the rats’ immobilization and subjection to cold
(4 ± 1 °C) stress for 1 h. Similar tendencies were valid for the
erythrocytes considering as a model of general cells reactions.
Decrease of antioxidant status was simultaneously observed,
activity of catalase, SOD, glutathione peroxidase, glutathione
reductase and a level of restored glutathione were reduced.
Administration of l-arginin-l-glutamat (20 mg/100 g) resulted in
Abstracts
421
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-indu ced erythrocyte damage in
rats
O. A. Adaramoye
1
and O. Akinloye
2
1
Laboratory of Drug Metabolism, Department of Biochemistry,
University of Ibadan, Ibadan, Oyo Nigeria,
2
Laboratory of
Metabolic Research, Department of Chemical Pathology, Ladoke
Akintola University of Technology, Osbongo, Osun Nigeria.
E-mail:
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 (CCl
4
) (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 CCl
4

and KV when compared
to CCl
4
- only treated rats. Similarly, the significant increase
(P < 0.05) in membrane cholesterol levels observed in CCl
4
-
treated rats was significantly decreased (P < 0.05) in rats trea-
ted simultaneously with CCl
4
and KV. Therefore, there was no
significant difference (P < 0.05) in cholesterol–phospholipids
molar ratio (C/P) of rats treated with CCl
4
+ KV, and con-
trols. Thus, KV normalizes the CCl
4
-induced change in erythro-
cyte membrane lipid composition. In addition, KV antagonizes
the effect of CCl
4
on the activity of the membrane bound
enzyme, Ca
2+
-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-Katic
1
, K. Stankov
1
, Z. Kovacevic
2
, I. Milenkovic
1
and
V. Mijatovic
1
1
Department of Biochemistry, University of Novi Sad, Novi Sad,
Serbia and Montenegro,
2
Serbian Academy for Sciences and Arts,
Branch in Novi Sad, Novi Sad, Serbia and Montenegro.
E-mail:
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 carbohy drate
moieties in glycoproteins?
V. Martinek
1
, J. Sklenar
1,2
, M. Sulc
1,2
, K. Bezouska
1,2
, E. Frei
3
and M. Stiborova
1
1
Faculty of Science, Department of Biochemistry, Charles Univer-
sity Prague, Prague, Czech Republic,
2
Institute of Microbiology,
Academy of Sciences, Prague, Czech Republic,
3
Division of
Molecular Toxicology, German Cancer Research Center,
Heidelberg, Germany. E-mail:
Glycosylation is one of the most important post-translational
modification of protein [1]. It has been suggested previously that

carbohydrate moieties may be involved in glycoprotein folding,
protection against proteolysis, and participate in specific recogni-
tion functions [2]. We observed that radicals generated from two
xenobiotics, 1-phenylazo-2-hydroxynaphthalene (Sudan 1) and
ellipticine, by horseradish peroxidase readily attach to non-gly-
cosylated proteins such as HSA, while glycosylated proteins such
as the peroxidase itself were not modified. Sudan I is a liver and
urinary bladder carcinogen in mammals known to be metaboli-
cally activated by both cytochromes P450 and peroxidases to
reactive species that bind covalently to nucleic acids and proteins
[3]. Ellipticine is an alkaloid with antineoplastic and anti-HIV
activities that is oxidized by peroxidases generating radicals parti-
cipating in its pharmacological efficiency [4]. We established a
remarkable correlation between the resistance of glycoproteins
(asialofetuin, fetuin, ovomucoid, Tamm-Horsfall glycoprotein
and alpha1-acid glycoprotein) towards modification by the above
radicals, and the degree of glycosylation. Comparison of these
activities 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. Our
findings indicate a novel role for protein glycosylation, namely
the protection of protein against the attack by radicals generated
from a one-electron oxidation of the xenobiotics.
Acknowledgments: This work was supported by Ministry of
Education of Czech Republic (MSM 0021620808), by the Institu-
tional Research Concept (AVOZ5020963), and by the Grant
Agency 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 Res 2001; 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. Behne
Department of Molecular Trace Element Research in the Life
Sciences, Berlin, Germany. E-mail:
The lung and trachea are constantly exposed to many gases and
particular matter present in the atmosphere. They therefore
require a specific defence system against oxidants and free radi-
cals. The trace element selenium seems to play an important
role in this defence system. In the form of selenocysteine it is
an essential component of glutathione peroxidase, an enzyme
involved in the detoxification of peroxides. In addition, several
Abstracts
422
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. Dillioglugil
1
, H. M. Kir
1
, M. D. Gulkac
2
, A. O. Kanli
2
,
S. Kuskay
1
, H. K. Ozdogan
1

and C. Eraldemir
1
1
Department of Medical Biochemistry, Kocaeli University, Faculty
of Medicine, Kocaeli, Turkey,
2
Department of Medical Biology,
Kocaeli University, Faculty of Medicine, Kocaeli, Turkey.
E-mail:
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. Strocchi
Department of Pharmacology, University of Bologna, Bologna,
Italy. E-mail:
Alpha-synuclein is known to be a brain pre-synaptic protein and
to be a structural component of the filaments in Lewy bodies in
Parkinson’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 pro-
motes fibril formation in vitro, suggesting the importance of
phosphorylation of the filamentous protein in the pathogenesis of
Parkinson’s disease.
Recent findings of abnormal protein folding, coupled with oxida-
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 the
phosphorylation state of alpha-synuclein as detected by Western
blot analysis using specific phospho-antibodies and modified the
intracellular distribution of this protein in differentiated SH-SY5Y
cells. These results demonstrated that oxidative stress induced
post-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
of Bologna, Funds for Selected Research Topics.
H3-014P
Increased cardiac activity enhances RyR2
S-glutathionylation
P. Donoso
1,2
,G.Sa
´
nchez
1,2
, Z. Pedrozo
1
, R. Domenech
1
and
C. Hidalgo
1,2
1
Instituto de Ciencias Biome
´
dicas, Universidad de Chile, Santiago,

Chile,
2
Calcium Signaling Group, FONDAP CEMC, Santiago,
Chile. E-mail:
Reactive oxygen species (ROS) have been involved in the develop-
ment of ischemic preconditioning in the heart but the source of
ROS and their mechanisms of action are uncertain. Short episodes
of tachycardia or exercise can also induce preconditioning, and
both maneuvers increase the activity of ryanodine receptors (RyR)/
calcium release channels in sarcoplasmic reticulum (SR) fractions
isolated from dog heart. Since RyRs are redox sensitive and their
activity increases by oxidation, we investigated whether the
NAD(P)H oxidase, a ROS producing enzyme, played a role in the
increase in cardiac RyR2 activity produced by tachycardia or exer-
cise. We found that tachycardia, as well as exercise, increased the
association of rac1 and of the NADPH oxidase cytosolic regulatory
subunit p47phox to the isolated cardiac SR fraction. Both tachy-
cardia and exercise increased twofold the production of superoxide
anion 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 S-glutathiony-
lation, with a concomitant increase in RyR2 activity. These results
suggest that the increased NAD(P)H oxidase activity induced by
tachycardia and exercise is responsible for the increase in RyR2
redox modifications (S-glutathionylation) over the endogenous lev-
els. This mechanism may be one of the primary factors that cause
the higher RyR2 activity found in these conditions.
Acknowledgments: This work was supported by Fondecyt
grants 1030446, 1030449 and FONDAP 15010006.

Abstracts
423
H3-015P
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. Demasi
1
, G. M. Silva
2
, F. P. Cavalher
2
,J.A.Ba
´
rcena
3
,
C. R. A. Bertoncini
4
and L. E. S. Netto
2
1
Instituto Butantan, Sao Paulo, SP Brazil,
2
Universidade de Sa
˜
o
Paulo, Sao Paulo, SP Brazil,

3
Universidad de Co
´
rdoba, Co
´
rdoba,
Spain,
4
Universidade Federal de Sa
˜
o Paulo, Sao Paulo, SP Brazil.
E-mail:
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 modifications,
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. Erdelyi
1
, E. Bakondi
1
, C. Szabo
2
, P. Gerely
1
and L. Virag
1
1
Department of Medical Chemistry, University of Debrecen, Med-
ical and Health Science Center, Debrecen, Hungary,
2
Inotek Phar-
maceuticals, Beverly, MA, USA. E-mail:
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 of
phosphorylation of the inhibitor of NF-kappaB (I-kappaB).
Regarding the AP-1 signaling pathway, GT induced a maximal
phosphorylation 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 the
NF-kappaB pathway (proximal to IkappaB phosphorylation)
and inhibits the p38-ATF-2 pathway and/or the DNA binding of
AP-1 despite of activation of the JNK-c-jun pathway.
H3-017P
Protective effect of green tea on erythrocyte
membrane of different age rats intoxi cated

with ethanol
Z. A. Figaszewski
1
, I. Dobrzyn
˜
ska
1
, B. Szachowicz-Petelska
1
,
J. Ostrowska
2
and E. Skrzydlewska
2
1
Laboratory of Electrochemistry, Department of Biology and
Chemistry, University of Białystok, Bialstok, Poland,
2
Department
of Analytical Chemistry, Medical Academy of Białystok,
Białystok, Poland. E-mail:
It is known that ageing is characterized by changes in cell metabo-
lism resulting e.g. in modification of the structure and function of
cell membrane components being mainly the consequences of
reactive oxygen species action. These disturbances are also
enhanced by different xenobiotics e.g. ethanol. Therefore the aim
of this paper has been to examine green tea influence on total anti-
oxidant status and on composition and electric charge of erythro-
cyte membrane phospholipids in ethanol intixicated rats of
various ages. Antioxidant abilities of erythrocytes were estimated

by measuring total antioxidant status (TAS). Qualitative and
quantitative composition of phospholipids in the membrane was
determined by HPLC, while the extent of lipid peroxidation was
determined by spectrophotometric measurement of the level of
lipid peroxidation products as thiobarbituric reactive substances
(TBARS). Electrophoresis was used to determine the surface
charge density of the rat erythrocyte membrane. It was shown that
the process of ageing was accompanied by a decrease in TAS and
in the total amount of phospholipids as well as by increase in lipid
peroxidation and surface charge density of erythrocyte membrane.
However ethanol administration caused decrease in TAS and
increase in the amount of all phospholipids and in the level of
lipid peroxidation products. Ethanol as well significantly enhanced
changes in surface charge density of erythrocyte membrane. The
ingestion of green tea partially prevented decrease in erythrocytes
antioxidant 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 process
and/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. Sumegi
Departments of Biochemistry and Medical Chemistry, University
of Pecs, Pe
´
cs, Hungary. E-mail:
Activation of poly(ADP-ribose) polymerase (PARP), a high

copy-number nuclear enzyme, was reported to have a significant
Abstracts
424
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. Giricz
1
, M. M. Lalu
2
, C. Csonka
1
, P. Bencsik
1

, R. Schulz
2,3
and P. Ferdinandy
1,4
1
Cardiovascular Research Group, Department of Biochemistry,
University of Szeged, Szeged, Hungary,
2
Cardiovascular Research
Group, Department of Pharmacology, University of Alberta,
Edmonton, Alberta Canada,
3
Cardiovascular Research Group,
Department of Pediatrics, University of Alberta, Edmonton,
Alberta Canada,
4
PharmaHungary 2000 Co., Szeged, Hungary.
E-mail:
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 preconditioned 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. Huang
Microbial Proteomics Laboratory, Department of Life Science,
National Central University, Jhongli City, Taiwan PR China.
E-mail:
The octylphenol polyethoxylates (OPEOn) are recalcitrant non-

ionic surfactants. The release of OPEOn has resulted in concern
due to their metabolites in the environment showing estrogenic
activity. However, little is known about the degradation path-
ways, microorganisms and enzymes involved in the degradation of
OPEOn. A Gram-negative rod, Pseudomonas nitroreducens TX1,
was shown to be able to grow on 0.05–20% (v/v) of OPEOn as
sole carbon source. An OPEOn-dependent oxygen consumption
activity was observed to be induced in strain TX1 when grown
solely on OPEOn. An oxygen consumption enzyme was isolated
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 and
metals. In addition, the functional proteomic study also found
that this enzyme was up-regulated for 3–5-fold in OPEOn-grown
proteome compared to succinate-grown one by 2D-PAGE. The
gene of dihydrolipoamide dehydrogenase and its promoter in
P. nitroreducens TX1 were also cloned and sequenced. The amino
acid sequence of dihydrolipoamide dehydrogenase showed a high
identity (94%) to those from P. aeruginosa PAO1 and P. fluores-
cens. The sequence motifs in the promoter of dihydrolipoamide
dehydrogenase gene showed the binding sites for several regulator
proteins involved in many stress responses such as heat shock,
oxidative, alcohol and osmotic stresses. This is the first report on
such a novel function of dihydrolipoamide dehydrogenase, which
may 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. Harandi
1
, A. Allameh
1
and P. J. O’Brien
1
1
Department of Biochemistry, Ahvaz Medical Science University,
Ahwaz, Ahvaz Iran,
2
Dr Allameh Laboratory, Department of
Biochemistry, University of Trabiat Modares, Tehran, Iran,
3
Dr O’Brien Laboratory, Department of Pharmacy, University of
Toronto, Toronto, Ontario Canada.
E-mail: a.aminiharandi.utoronto.ca
Iron is toxic to the liver as well as to other organs and tissues of
the body. This toxicity is most likely related to enhancement of
oxidative stress and free radical reaction stimulated by Iron. Major
pathophysiological consequences of iron on the liver are the devel-
opment of hepatic fibrosis and cirrhosis, porphyries cutanea tarda
and development of hepatocellular carcinoma. Suspension of
hepatocytes 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 reac-
tion, lipid peroxidation, reactive oxygen species in (ROS) and
glutathione as an effective factor to follow up oxidative stress were
determined. Decreased glutathione (>90%) as index for oxidative
stress and increase of ROS in incubated isolated hepatocytes by
iron show that toxicity of iron is related to oxidative stress that

generated by iron overload. Lipid peroxidation of hepatocytes by
free radicals that produce in Fenton reaction was responsible for
hepatocytes damage and finally caused cell death.
Abstracts
425
H3-022P
Diversity of glutathione S-transferases in
bovine liver
B. S. Isgor
1
, N. Coruh
2
and M. Iscan
3
1
Chemistry Laboratory, Department of Material Engineering,
Atilim University, Ankara, Turkey,
2
Biochemistry Laboratory,
Department of Chemistry, Middle East Technical University,
Ankara, Turkey,
3
Biochemistry Laboratory, Department of Biolo-
gical Sciences, Middle East Technical University, Ankara, Turkey.
E-mail:
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. Leus
1
, E. D. Zabitskaya

1
, N. I. Shtemenko
1
and
A. V. Shtemenko
2
1
Laboratory of Biochemistry, Department of Biophysics and
Biochemistry, Dnepropetrovsk National University,
Dnepropetrovsk, Ukraine Ukraine,
2
Laboratory of Synthesis of
Metal-organic Substances, Department of Inorganic Chemistry,
Ukranian State University of Chemical Technology,
Dnepropetrovsk, Ukraine Ukraine. E-mail:
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 of
both enzymes in healthy animals that confirmed low toxicity of

these substances. Being introduced to tumor-bearing animals
CROL 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 RBC
SOD is strongly dependent from the structure of radical in the
molecule of CROL. Possible mechanism of these cell-specific
actions of CROL is speculated.
H3-024P
Cambialistic-superoxide dismutase from
diatom: cloning, expression, and enzyme
stability
C T. Lin
1
, Z X. Huang
1
and R H. Juang
2
1
Institute of Bioscience and Biotechnology, National Taiwan Ocean
University, Keelung, Taiwan 202, Keelung, Taiwan ROC,
2
Department of Biochemical Science and Technology, National
Taiwan University, Taipei, ROC. E-mail:
A cDNA clone of 1.1 kb encoding a putative Mn-superoxide
dismutase (Mn-SOD) from diatom was cloned by the PCR tech-
nique. Nucleotide sequence analysis of this cDNA clone revealed
that it was translated into 286 amino acid residues. When the
truncated sequence containing 217 amino acid residues was com-
pared with Mn-SODs from V. mimicus and E. coli, as well as
two Fe-SODs from E. coli and P. leiognathi, this SOD showed

higher homology to Mn-SOD. The amino acid residues required
to coordinate the single manganese ion were conserved in all
reported Mn-SOD sequences. This cDNA was introduced in an
expression vector, pET-20b(+) and transformed into E. coli. The
expressed 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 K
d
was 3.21 · 10
–2
/min at 65 °C heating.
The enzyme was inhibited either in acidic pH (below 5.0) or in
the presence of imidazole (above 1.6 m), and had only moderate
effect under SDS (above 4%), whereas it was not affected under
an alkaline pH (above 9.0). The atomic absorption spectrometric
assay showed that 0.3 atom of iron/manganese (2:1) was present
in each subunit of SOD, reconstituted activity suggesting that
diatom SOD was cambialistic -SOD. The finding of this SOD
cDNA could be used for a reference in comparing the differences
among marine phytoplankton species and as a probe to detect
the transcription level of this enzyme, which can be applied in
cosmetics for skin protection caused by oxygen-containing free
radicals.
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-Klita
1

, M. Chelchowska
1
and P. Kubik
2
1
Department of Biochemistry, Institute of Mother and Child,
Warsaw, Poland,
2
Department of Obstetrics and Gynecology,
Institute of Mother and Child, Warsaw, Poland.
E-mail:
The 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 pregnant
women. Zinc, copper and selenium as cofactors for these
enzymes, were also determined (by ICP MS method). Activities
of SOD during pregnancy were similar in supplemented and
Abstracts
426
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 (Vibovit
Ò
mama,
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:
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 H
2
O
2
and prior treatment of cyclohexamide.
Cyclohexamide treated cells were found significantly more sensi-
tive to the cytotoxic effect of H
2
O

2
, 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 H
2
O
2
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 obes e 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:
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
patients).
Results: Obese diabetic patients had lower glutathione
(6.11 ± 0.82 vs. 7.32 ± 0.62 nmol/g Hb in the controls,
P < 0.04), and higher MDA (18.64 ± 2.7 vs. 15.71 ± 1.5 nmol/g
protein in the controls, P < 0.01). The activity of superoxide dis-
mutase (SOD) was reduced in the obese diabetic group (312.52 ±
49 vs. 390.01 ± 71 U SOD/g Hb in the controls, P < 0.05). There
were no significant differences in the concentration of plasma thiols
(7.37 ± 0.4 vs. 7.63 ± 0.4 nmol/g protein in the controls) and
plasma total antioxidant capacity (1.58 ± 0.1 vs. 1.55 ± 0.09 mM
trolox in the controls) measuring the combined free radicals scav-
enging ability of non-enzymatic antioxidants. This reduction in
superoxide dismutase activity and the imbalance in the redox status
of the plasma demonstrated is consistent with an increase in free
radical 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. Milnerowicz
1
, M. Jablonowska
1
and S. Milnerowicz
2
1

Departament of Biomedical and Environmental Analyses, Wroc-
law University of Medicine, Wroclaw, Poland,
2
Department and
Clinic of Gastointestinal and General Surgery, Wroclaw University
of Medicine, Wroclaw, Poland. E-mail:
Introduction: The imbalance of reactive oxigen species (ROS)
generating and ROS scavengic processes is thought to lead to the
damage of pancreatic cells that initiate autodigestion of the entire
organ. Antioxidative enzymes play an important role in the regu-
lation of ROS action. The aim of this study was to determined
serum activity of Cu/Zn SOD and plasma activity of GPx, and
also the blood level of GSH.
Methods: The activity of Cu/Zn SOD (Superoxide Dismutase
Assay-IBL, Germany) and GPx (Glutathione Peroxidase Colo-
rim/Kinetic Assay-IBL, Germany), and the blood GSH level were
measured in: 35 patients with chronic pancreatitis (CP), 20
patients with chronically exacerbated pancreatitis (CEP), 15
patients with acute pancreatitis (AP), 22 normal healthy subject.
Results: In all examined groups of patients a decrease in GSH
level was observed, in comparison to control group
(448.0 ± 82.7 nmol/ml), furthermore, the highest decrease was
noticed among patients with AP (135.8 ± 46.5 nmol/ml). Addi-
tionally, together with decrease in GSH level, the highest increase
in activity of GPx was observed in patients with AP
(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 the
GSH/GPx system plays the role of the first line of defence
against 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. Martin
Laboratory of Biochemistry, Department of Research, Hospital
Ramon y Cajal, Madrid, Spain. E-mail:
During the oxidative stress generated by hydrogen peroxide
(H
2
O
2
) in nerve growth factor (NGF)-differentiated PC12 cells,
Abstracts
427
eIF4E-binding protein (4E-BP1) and initiation factor 4E
(eIF4E) are significantly dephosphorylated, and an enhance-
ment of the association of 4E-BP1 to eIF4E which in turn
decreases eIF4F formation, is observed. The treatment with
N-acetyl-cysteine (NAC) completely abolishes eIF4E dephosph-
orylation, whereas 4E-BP1 dephosphorylation and eIF4F activ-
ity inhibition are significantly but not fully reversed.
Rapamycin, the mammalian target of rapamycin (FRAP/
mTOR) inhibitor, induces similar effects on 4E-BP1 phosphor-

ylated levels to that produced by H
2
O
2
. In addition, rapamy-
cin abolishes the effect of NAC on H
2
O
2
-induced eIF4F
complex inhibition. Nevertheless, the NAC-induced recovery of
phosphorylated eIF4E levels is independent to rapamycin.
Neither the MAP kinase inhibitors, PD98056 and SB203580,
or the protein phosphatase 2A inhibitor, okadaic acid, mimic
NAC effect on H
2
O
2
-induced eIF4E dephosphorylation.
Altogether our findings suggest that NAC reversion of H
2
O
2
-
induced effects on eIF4s factors depend on two MAP kinase-
independent signal transduction pathways, being at least one
of them rapamycin-dependent.
Acknowledgments: This work was supported by grants FIS02/
0304 and 03/0469.
H3-030P

Reduced and oxidized forms of human serum
albumin in sports, aging and disease
K. Oettl
1
, S. Hallstro
¨
m
1
,G.Ju
¨
rgens
1
, D. Fuchs
2
, H. Blomster
3
,
O. Schmut
4
, K. Sudi
5
and J. Greilberger
1
1
Institute of Physiological Chemistry, Medical University Graz,
Graz, Austria,
2
Division of Biological Chemistry, Medical Univer-
sity Innsbruck, Innsbruck, Austria,
3

Department of Ophthalmology,
Kuopio University, Lahti, Finland,
4
Department of Ophthalmology,
Medical University Graz, Graz, Austria,
5
Institute for Sport
Science, Karl-Franzens University, Graz, Austria.
E-mail:
Human serum albumin, the major protein in serum, has a
cystein residue in position 34 which is not involved in a disul-
fide bond and may exist in different oxidation states: as a fully
reduced sulfhydryl group, as a mixed disulfide with, e.g.,
cystein, glutathione or homocystein, or in higher oxidation
states like sulfenic, sulfinic or sulfonic acid. Serum albumin is
known to act as an antioxidant, the oxidation state of human
serum albumin is discussed as a marker for systemic oxidative
stress and it was shown that the disulfide content of serum
albumin is increased by strenuous exercise, in older people and
during the course of different diseases. We could confirm the
antioxidative action of serum albumin with different methods.
However, for quantification the method used has to be consid-
ered in detail. Despite the increase of the disulfide proportion
during strenuous exercise, the value was lower in trained ath-
letes compared to sedentary controls. Patients with Diabetes
mellitus type I and II have elevated disulfide levels in serum
albumin compared to healthy controls and non-diabetic
patients, respectively. In contrast to oxidative stress parameters
like the carbonyl content of serum proteins the disulfide level of
albumin highly correlates with age. The proportion of disulfide

ranged from 17.9 ± 2.5 % in healthy children
(12.4 ± 0.5 years) up to 61.4 ± 10.9% in otherwise healthy
patients with senile cataract (75.7 ± 13.1 years). The redox state
of serum albumin cannot be used as a marker for oxidative
stress without consideration of age.
H3-031P
Influence of
L-arginin-L-gluthamate on
the system of glutathione of stressed
rats’ brain.
A. L. Ponomarenko
1
, A. A. Ponomarenko
2
, G. L. Ruslanovna
3
,
L. E. Anatolievna
4
and R. A. Ivanovich
5
1
Laboratory of Biochemistry, Academy of Medical Sciences of
Ukraine, Institute of Gastroenterology, Dnepropetrovsk, Ukraine,
2
Laboratory of Pathophysiology, Academy of Medical Sciences of
Ukraine, Institute of Gastroenterology, Dnepropetrovsk, Ukraine,
3
Laboratory of Pathophysiology, Academy of Medical Sciences of
Ukraine, Institute of Gastroenterology, Dnepropetrovsk, Ukraine,

4
Laboratory of Biochemistry, Ukrainian State Scientific Research
Institute of Medical and Social Problems of Disability, Dneprope-
trovsk, Ukraine,
5
Laboratory of Pathophysiology, Academy of
Medical Sciences of Ukraine, Institute of Gastroenterology,
Dnepropetrovsk, Ukraine. E-mail:
Stressing organism is known to result in intensifying of free rad-
ical processes in the brain. Modeling rats’ stress reactions was
carried out by immobilizational cold influence and introduction
of medical bile in the stomach. Increase of thiobarbituric acid
(TBA) active products by three times in this rats’ brain tissues
was found out. Stress influence resulted in significant decrease of
regenerated glutathione level on background of weak inactivation
of glutathione peroxidase (GP), glutathione reductase (GR) activ-
ity was not changed. Registered catalase activation by 1.7 times
did not compensate registered free radical process strengthening.
Injection of l-arginin-l-gluthamat (20 mg/100 g) resulted in
reduction of active processes in the brain tissues, more significant
activation of catalase (by 2.1 times), strengthening GR and GP
activity in comparison with control parameters by 1.3 and 1.4
times correspondingly. Thus influence of l-arginin-l-gluthamat
results in brain antioxidant system stabilization after stress of the
rats. This phenomena allows for organism to respond more ade-
quately to unfavorable endogenous and exogenous influences.
H3-032P
Mitochondrial oxidative stress induced by
hyperglycemia mediates increased renal
expression of transforming growth factor-beta

1 in anesthetized rats
S. S. Prabhakar
Department of Internal Medicine, Texas Tech University Health
Sciences Center, Lubbock, TX, USA.
E-mail:
Oxidative stress plays a major role in the development and progres-
sion of diabetic nephropathy by inducing cytokines that promote
matrix expansion and glomerulo-sclerosis. To examine the mecha-
nisms of renal effects of hyperglycemia, we studied the effects of
acute high glucose on systemic oxidative stress and renal TGF-beta
1 expression. Three groups (n = 6 each) of Sprague–Dawley rats
(8–12 weeks old) were anesthetized and given a 12 h intravenous
infusion of either 5 mm glucose (control), or 20 mm glucose (HG)
or HG given after pretreatment with alpha-tocoferol given intra-
peritoneally at 20 mg/kg body wt. (HG + AT). After the infu-
sions, the rats were euthanized, kidneys harvested and sections of
renal tissue analyzed for TGF-beta 1 immunostaining using anti-
bodies specific for TGF-beta 1 and quantified by imaging software
using the staining in control as a reference (1.00). Urinary excretion
of 8-hydoxy-deoxyguanosine (8-OHdG), a product of reactive oxy-
gen species (ROS)-induced DNA damage and a sensitive marker of
systemic oxidative stress was measured by ELISA (expressed as ng/
day/kg body wt). Rats infused with high glucose had increased
Abstracts
428
urinary excretion of 8-OHdG (2165 ± 278* vs. 810 ± 59 in con-
trol, P < 0.01) and TGF-beta 1 immunostaining (5.65* vs. 1.00
control, *P < 0.01). However these changes induced by high glu-
cose were attenuated with pre-administration of alpha-tocoferol,
an anti-oxidant. The 8-OHdG excretion in HG+AT group

decreased to 1165 ± 176 (P = NS vs. control) and TGF-beta 1
immunostaining in the kidney was reduced to 2.40 (P < 0.05 vs.
HG). These observations indicate that stimulation of TGF-beta 1
in the kidney by hyperglycemia may be mediated by increased
mitochondrial oxidative stress.
H3-033P
Effect of cigarette smoke on plasma and
salivary proteins
A. Ripsky Totan
1
, M. Greabu
1
, C. Totan
2
and T. Spinu
3
1
Department of Biochemistry, UMF Carol Davila, Bucharest,
Romania,
2
OMF Surgery, UMF CarolDavila, Bucharest, Romania,
3
Department of Prosthodonics, UMF Carol Davila, Bucharest,
Romania. E-mail:
Cigarette smoke contains stable oxidants that cause oxidative
damage and increased proteolysis of proteins Whole phase cigar-
ette smoke contains about 4000 components, which from around
3000 are present in its gas phase and 1000 components in the
particulate, tar phase. Cigarette smoke has been implicated with
degenerative pulmonary and cardiovascular disease: bronchitis,

emphysema, myocardial infarction, lung cancer and other malig-
nancies. We have incubated human whole saliva and plasma with
whole phase and tar phase cigarette smoke, in presence and
absence of vitamin C, respectively B6. After incubation we have
analyzed the following salivary enzymes: amylase, lactic dehy-
drogenase, alkaline phosphatase, aspartate aminotranspherase.
We have also analyzed the content of protein thiols. To illustrate
the oxidative stress we have measured the salivary uric acid, the
most important salivary antioxidant. Plasma proteins were ana-
lyzed after incubation by electrophoresis in agarose gel. We have
also analyzed the content of protein thiols after the different
types of incubations. Our results suggest that adequate intake of
antioxidants may help smokers to avoid cigarette smoke-induced
oxidative damage and to prevent degenerative diseases.
H3-034P
Thermotolerant monomeric chloroplastic Cu/
Zn superoxide dismutase from Chenopodium
murale
S. Sabarinath
1
, S. Khanna
1
and R. K. Chopra
1
1
Stress Physiology Lab, Water Technology Centre, Indian Agricul-
tural Research Institute, New Delhi, India,
2
Biotechnology Lab,
School of Biotechnology, Thaper Institute of Engineering and

technology, Patiala, Punjab, India,
3
Stress Physiology Lab, Water
Technology Centre, Indian Agricultural Research Institute,
New Delhi, India. E-mail:
Chenopodium murale is a weed species having wide adaptation to
different climatic regimes and experiences a temperature range of
5–45 °C during its life span. Chenopodium species are often used as
a model plant to study the mechanism of thermotolerance. Super-
oxide dismutase enzyme (SOD, EC.1.15.1.1) is ubiquitous, being
widely distributed among O
2–
consuming organisms and is the first
line of defense against oxidative stress. The thermostability of the
SOD isozymes from C. murale was characterized in vitro. The leaf
protein extracts, thylakoidal and stromal fractions were subjected
to elevated temperatures ranging from 50 °C to boiling and ana-
lyzed for activity and isoform pattern of SOD. Out of six SOD iso-
forms present in leaf protein extract, SOD V showed stability even
after boiling the extract for 10 min. Under high temperature treat-
ment (>60 °C) there was an appearance of a new SOD band with
higher electrophoretic mobility. The inhibitor studies and sub cel-
lular analysis revealed that the SOD V isoform was a chloroplastic
Cu/Zn SOD. The stromal Cu/Zn SOD was more stable than the
co-migrating thylakoidal isozyme at 80 °C and at boiling for
10 min. The thermostable chloroplastic Cu/Zn SOD was purified
to homogeneity and further characterized. The thermostable chlo-
roplastic Cu/Zn SOD was a monomer with a molecular weight of
24 kDa, pI of 5.6 and a half-life of 128 min. The presence of higher
metal content and the intra subunit interactions may contribute to

the high stability of the enzyme.
H3-035P
Paraquat-induced genes in horseweed Conyza
canadenesis (L.) Cronq
V. Soo
´
s
1
,B.Jo
´
ri
1
, D. Szeg
}
o
1
, Z. Szigeti
1
,E.Pa
´
ldi
2
,I.Ra
´
cz
1
and
D. La
´
sztity

1
1
Department of Plant Physiology, Eo
¨
tvo
¨
s Lora
´
nd University,
Budapest, Hungary,
2
Agricultural Research Institute of the
Hungarian Academy of Sciences, Martonva
´
sa
´
r, Hungary.
E-mail:
The non-selective bipyridyl herbicide paraquat (Pq) exerts a phy-
totoxic effect by diverting electrons from PSI to molecular oxy-
gen, thus generating toxic oxygen forms. Extensive use of Pq has
led to the worldwide incidence of resistant weeds. Pq-resistant
biotypes of horseweed have also been found in Hungary. It has
been suggested that Pq-inducible protein(s) may play a role in
resistance and these presumably function by transporting Pq to
metabolically inactive compartment. Studies on the expression of
the genes upregulated by Pq treatment in susceptible and resist-
ant biotypes have revealed an increase in the level of ferritin2
gene and of a PotE-like putative cationic amino acid transporter
gene. Ferritins, the multimeric iron storage proteins, are the main

regulators of the cellular level of uncomplexed iron. Iron home-
ostasis control is essential since toxic effect of paraquat-generated
oxygen radicals is aggravated by the production of hydroxyl radi-
cals by uncomplexed iron. The primary structure of the ferritin2
gene from resistant and susceptible biotypes of horseweed was
determined. Ferritin2 genes had identical primary structure in the
two biotypes and were found to exhibit great similarity and pos-
sess all the structural characteristics of known plant ferritin2
genes. The enhanced expression level in both the susceptible and
the resistant biotype after Pq treatment is probably connected
with defence reactions and can be regarded as part of a general
stress response reaction. The PotE-like putative amino acid trans-
porter may directly take part in the cellular transport and/or
sequestration of Pq into a metabolically inactive compartment.
H3-036P
Inhibition of free radical processes by the total
peroxidases of some plant extracts
A. Sukiasyan and A. Zakaryan
Department of Biophysics, Yerevan State University, Yerevan,
Armenia. E-mail:
Lipid peroxidation in lipid structures is accompanied with storage
of different oxidative products, which help us to worth the direc-
tion of this process. Development of different pathologies first of
all leads to violation of balance between level of lipid peroxida-
tion of lipid structures and bio-antioxidant system, including
proteins-enzymes. In our experiments was investigated the influ-
ence of distilling water extracts of following drug plants Quercus
cortex, Artemisia absinthium, and Salvia officinalis on lipid com-
ponents of biological target (homogenate of a cow brain in Tris-
HCl buffer with pH = 7.4) by methods of chemiluminescence

and determining the activity of total peroxidases of drug extracts.
Abstracts
429
These plants use in traditional Armenian medicine as anti-inflam-
matory preparation. The dark background of luminometer is
30 ± 5 impulse per 10 s, and a level of spontan chemilumines-
cence of biological target is 138 ± 17 impulse per 10 s by tem-
perature of measuring at +40 °C. An evaluation of antioxidant
activity of those extracts by chemiluminescence analyses was
showed that extracts inhibited the level of chemiluminescence of
biological target on 49% (Quercus cortex), 33% (Artemisia absin-
thium) and 8% (Salvia officinalis), and by decreasing of the total
activity of peroxidase per mg of total protein for each plant
extract (Quercus cortex 0.077 ± 0.004, Artemisia absinthium
0.040 ± 0.002, Salvia officinalis 0.020 ± 0.002). These received
results can be explained by function of antioxidant enzyme sys-
tem of plant extracts, in particular, a total peroxidase.
H3-037P
Effects of the antitumoral dequalinium on
K562 and NB4 human leukemia cell lines
mitochondria implication in cell death
P. Sancho
1
, E. Galeano
1
, M D. Delgado
2
and
A. I. Garcı
´

a-Pe
´
rez
1
1
Bioquı
´
mica y Biologı
´
a Molecular, Alcala
´
de Henares, Alcala
´
De
Henares, Madrid Spain,
2
Biologı
´
a Molecular, Cantabria,
Santander, Cantabria, Spain. E-mail:
The antitumour agents used in chemotherapy are aimed to eradi-
cate the tumor by inducing malignant cell death, thus limiting its
growth and spreading. However, the lack of specificity for tumor
cells exhibited by these agents cause undesirable side effects that
have led to the investigation of new therapeutic strategies designed
to specifically target malignant cells. It is well established that the
efficacy of conventional antitumor drugs is due to their ability to
induce apoptosis. Mitochondria are now known to play a critical
role in initiating apoptotic cell death. The agent dequalinium
(DQA), as other delocalized lipophylic cations, is accumulated and

retained in mitochondria of carcinoma cells due to a higher negat-
ive mitochondrial transmembrane potential in tumor cells than in
normal cells. This behavior provides an attractive basis for the use
of DQA as selective antitumor activity. To understand the antitu-
mor properties of DQA, we are studying its effects on two different
leukemia cell lines: NB4, derived from acute promyelocytic leuke-
mia, and K562, derived from chronic myeloid leukemia. Up to
now, we have shown that DQA displays differential cytotoxic
activity in these cell lines. NB4 cells died by apoptosis and/or nec-
rosis while K562 showed a resistance to apoptosis at all time-peri-
ods and DQA concentrations assayed and cell death was always by
necrosis. This apoptotic resistance may be reversed in the presence
of the bcr/abl inhibitor protein STI571. In both cell lines, the cell
death seems to be mediated by early alterations of mitochondrial
function as evidenced by loss of mitochondrial transmembrane
potential, O
2
accumulation, SOD activity and ATP depletion.
These studies open the possibility of DQA application for leukemia
cells treatment as well as to study the molecular mechanism
responsible of the observed mitochondrial alterations.
H3-038P
The effect of l-arginine on catalase and
arginase enzyme activity levels in
sreptozotocin induced diabetic rats
S. Temy
´
zer Ozan
1
and B. Tasdemir

1
1
Department of Biochemistry, Firat University, Elazig, Turkey,
2
Department of Biochemistry, Firat University, Elazig, Turkey.
E-mail:
Diabetes mellitus is a group of metabolic diseases characterized
by hyperglycemia resulting from defects in insulin secretion,
insulin action, or both. Oxidative stress has an important role on
diabet and pathogenesis of the further complications of diabetes.
The levels of induced free-radicals and decline of antioxdant
defence mechanisms can lead to damage of enzymes and cell
organelles, increased of lipid peroxidation and development of
insulin resistance. In the present study, it was aimed to investi-
gate that the effect of l-arginine given intrapertoneally on the
arginase, and erythrocyte catalase enzyme activities in streptozot-
ocin-induced diabetic rats. Twelve-week-old 48 Wistar albino
male rats were used for the study. The rats were seperated in to
four groups as following: control, diabetic, diabetic + l-arginin
and l-arginine. The control group was induced by the injection
of only phosphate-citrat buffer intraperitonelly (0.5 ml/rat) in
rats. The diabet group was caused by the injection of streptozot-
ocin intraperitoneally (55 mg/kg body weight, in phosphate-citrat
buffer). Diabet + l-arginin and l-arginin groups were given by
the injection of 10 mml-arginine (0.5 ml/each rat) intraperitone-
ally. In the present study was investigated the erythrocyte cat-
alase and arginase enzyme activities in liver and kidney tissues.
Erythrocyte catalase enzyme activities reduced significantly in the
diabetic group (40.72 ± 9.82) in comparison with the control
(59.41 ± 13.95) group. No significant difference was observed

between the control and diabetic + l-arginine group (49.9 ±
12.2) in the erythrocyte catalase activities levels (P < 0.05). The
activities of arginase enzyme in liver and kidney tissues of dia-
betic group (266.9 ± 18.45 and 8.86 ± 1.01) were increased sig-
nificantly in comparison with the control groups (159.61 ± 11.97
and 7.81 ± 0.82 P < 0.05). In conclusion, the effect of l-argin-
ine on the erythrocyte catalase and liver and kidney arginase
activities were observed significantly to improve in comparison
with the control groups. l-arginin is also metabolized to l-ornith-
ine, which can be processed to polyamines and proline. As poly-
amines are important mediators of cell growth and l-proline is a
substrate for collagen synthesis, it is thought that both pathways
have an important role on pancreatic repair processes.
H3-039P
Investigation of cancer cell growth inhibitory
effects of new selenium phenolic succinate
antioxidants
P. S. Vraka, A. D. Keramidas, C. Drouza, M. Rikkou and
A. D. Odysseos
Inorganic and Bioinorganic Chemistry, Chemistry, University of
Cyprus, Nicosia, Cyprus. E-mail:
Selenium and vitamin E are two antioxidants that have attracted
great attention due to a strong synergism resulting cancer chemo-
preventive activity. Previous studies on the inhibitory effects of
cell growth by vitamin E compounds and its derivatives have
shown that RRR-a-tocopheryl succinate (VES) has the most
potent apoptotic effect in vitro. Accumulated evidence supports
the chemical form of selenium as an important factor in eliciting
defined cellular responses in the in vitro system. Herein, we report
the synthesis and assessment of the antioxidant activity and of

the in vitro cancer cell growth inhibitory properties of novel vita-
min E-selenium esters. These compounds are designed to (a) pro-
vide a better understanding of vitamin E synergism with
selenium, (b) enable the synthesis of antioxidants combining both
selenium and vitamin E antioxidant activity, and (c) elucidate the
mechanisms underlying the potent apoptotic effects of succinate
esters. They have been evaluated and characterized by NMR
spectroscopy. Antioxidant activity was assessed by determining
glutathione peroxidase (GPx) catalytic activity using benzenethiol
as a glutathione alternative and H
2
O
2
as an oxidant. Free radical
scavenging capacity was established using 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH) assay. Reactions of organic peroxides with
Abstracts
430
the antioxidants were evaluated by NMR study to determine the
mechanisms. Redox potentials were determined using cyclic vol-
tammetry. The molecules exhibit both the antioxidant activity of
selenium and vitamin E. They have shown significant antiprolif-
erative, growth inhibitory and apoptotic effects on breast and
prostate cancer cell lines in vitro. These anti-cancer properties
were tested against p-dodecyl-phenol and its succinylated com-
pound. Interestingly, whereas succinate-p-dodecyl-phenol has an
IC
50
comparable to that of VES, p-dodecyl-phenol has a statisti-
cally significant lower effect, compared to RRR-a-tocopherol.

These data suggest that the cancer cell growth inhibitory proper-
ties of these compounds are mainly attributed to the succinate
moiety.
H3-040P
Luteococcus japonicus subsp. casei possesses
antioxidant properties
N. V. Vorobjeva and L. I. Vorobjeva
Department of Physiology of Microorganisms, Biology faculty,
Lomonosov Moscow State University, Moscow, Russian
Federation. E-mail:
Bacteria are particularly useful for conducting studies on stress
factors and responses to them. The data obtained by us demon-
strated that Luteococcus casei are SOD- and catalase-positive.
Apart from the individual protection and reactivation system
L. casei are characterized by the synthesis of stress-related exo-
metabolite that promotes the viability of the other bacterial and
yeast cells subjected to different stress factors. A protein exome-
tabolite (M.m. 8–9 kDa) isolated from the supernatant of a
L. casei culture resuscitates producer cultures under H
2
O
2
-or
paraquat-induced oxidative stress. The protein exometabolite
caused a systemic resuscitating effect under UV-irradiation, ther-
mal inactivation, and oxidative stress. We succeeded in isolating
the protein exometabolite from yeasts and establishing cross-
effects of a proteinaceous extracellular factors of adaptation on
eukaryotes and prokaryotes under stress.
H3-041P

Some plasma oxidative stress parameters in
obesity patients with or without diabetes
mellitus
B. Virgolici, M. Mohora, F. Paveliu and I. Stoian
Department of Biochemistry, University of Medicine, Bucharest,
Romania. E-mail:
Oxidative stress and inflammation are involved in the initiation
and progression of obesity and diabetes mellitus. The aim of our
study was to find out some markers of oxidative stress in 20
obese patients with type 2 diabetes mellitus (group D) and twenty
age-matched obese subjects (group O) and compare the results
with the control values from twenty matched healthy subjects
(group H). Spectrophotometric methods were used. For the fol-
lowing parameters: plasma ceruloplasmin, d-ROM (determinable
Reactive Oxygen Metabolites), a-dicarbonyls, and the activity of
erythrocyte superoxide dismutase (SOD) the values were modified
in the same way for the groups of patients versus healthy sub-
jects. The patients had lower a-dicarbonyls levels than the con-
trols (for D vs. H, P < 0.047 and for O vs. H, P < 0.043).
Also, the activity of SOD was significantly lower in patients than
in controls (for D vs. H, P < 0.05 and for O vs. H, P < 0.05).
There were not significant differences for plasma ceruloplasmin
and d-ROM levels. Comparing group O vs. D, all the above
parameters had very close values. The antioxidant capacity (AC)
was higher in group O vs. group H (P < 0.001) and higher in
group O vs. D (P < 0.02). In both groups of patients, D and O,
the ceruloplasmin values were positively correlated with the AC
levels. A negative correlation between AC and d-ROM was
observed for group D. Our data underline that in type 2 diabetes
mellitus and obesity, the markers of oxidative stress are modified

in the same way. The high AC for obese patients may be due to
hyperuricemia. Improving the plasma antioxidants defenses in
diabetes mellitus may have benefic effects.
H3-042P
Glutathione biosynthesis in Pasteurella
multocida is accounted for by a bifunctional
protein catalyzing both c-glutamylcysteine
synthetase and glutathione synthetase
activities
B. Vergauwen, D. De Vos, F. Pauwels and J. J. Van Beeumen
Protein Biochemistry and Protein Engineering, Biochemistry,
Ghent University, Ghent, Belgium.
E-mail:
It is generally accepted that glutathione (c-l-Glu-l-Cys-Gly) bio-
synthesis occurs in a two-step process catalyzed by two unrelated
ATP-dependent ligases, c-glutamylcysteinyl synthetase (GshA)
and glutathione synthetase (GshB), for which the genes are gen-
erally not in close proximity on either prokaryotic or eukaryotic
genomes. GshA is a member of the glutamine synthetase family,
while GshB groups together with highly diverse sequences
belonging to the ATP-grasp superfamily of amide-bond forming
ligases. The genome of Pasteurella multocida encloses a typical
GshA homologous sequence which, however, exhibits noncon-
formity in having a C-terminal extension with similarities to
ATP-grasp sequences such as those of cyanophycin synthetases
and d-Ala-d-Ala ligases, although no primary structure similarity
appears when comparing the C-terminal extension to typical
GshB sequences from either prokaryotic or eukaryotic origin. On
the other hand, a prokaryotic GshB homologous sequence is
untraceable from the P. multocida genome. We now show that

the putative GshA hybrid protein sequence of P. multocida can
catalyze both typical ATP-dependent ligase reactions leading to
the formation of glutathione out of its constituent amino acids.
This result strongly suggests that the N-terminal GshA-like
sequence catalyses c-glutamylcysteine synthetase activity and that
the C-terminal ATP-grasp homologous stretch catalyses typical
glutathione synthetase activity. It remains to be determined, how-
ever, whether there actually is a division into two functionally
separated domains and, if so, where in the sequence the two
domains are fused together. The kinetic parameters for the two
separate enzymatic activities were determined using purified
protein.
H3-043P
Neuronal nitric oxide template: useful for
virtual screening
O. Vajragupta
1
, C. Boonyarat
2
, G. M. Morris
3
, R. Huey
3
,C.Li
3
and A. J. Olson
3
1
Department of Pharmaceutical Chemistry, Mahidol Universit,
Bangkok, Thailand,

2
Department of Pharmaceutical Chemistry,
Konkean University, Konkean, Thailand,
3
Department of
Molecular Biology, The Scripps Research Institute, La Jolla,
CA 92037-1000, USA. E-mail:
Neuronal nitric oxide synthase (nNOS) is a homodimer enzyme
that is responsible for catalyzing the production of nitric oxide
(NO). As such, nNOS has been proposed as a potential target
for drug design. The present study reports the development of a
Abstracts
431
template of active binding site of nNOS. This template will be
useful for structure-based drug design. A crystallographic struc-
ture of nNOS bound with the selective nNOS inhibitor, l-N
x
-nit-
roarginine-(4R)-amino-l-proline amide, ligand I, was selected to
construct the nNOS template. The developed template of nNOS
was validated by redocking with native ligand, ligand I, and by
docking the other three nNOS inhibitors, namely l-N
x
-nitroargi-
nine-2,4-l-diaminobutyramide (ligand II), d-phenylalanine-
d-nitroarginine amide (ligand III), and l-N
x
-nitroarginine (ligand
IV). The 3D-orientations of the four inhibitors obtained from
docking with the developed nNOS template were compared with

the crystallographic poses. The results showed the same orienta-
tion with RMSD less than 2.0 A
˚
. The new nNOS template repor-
ted here should prove to be a useful tool for virtual screening in
searching for new nNOS inhibitors.
H3-044P
Proteomic analysis of leukemic cell line U937
treated with hypoxia and mimetic hypoxia
L S. Wang
1
, Y H. Han
1
, L. Xia
1
, Y. Zheng
1
, W L. Chen
1
,
L. Zhang
1
, L P. Song
1
and G Q. Chen
1,2
1
Department of Pathophysiology, Shanghai Institute of
Hematology, Rui-Jin Hospital, Shanghai Second Medical
University, Shanghai, PR China,

2
Health Science Center, Shanghai
Institutes for Biological Sciences and graduate school, Chinese
Academy of Sciences, Shanghai, PR China.
E-mail:
The role of hypoxia in the pathophysiology of solid tumors has
been widely reported, but few clues were reported for the poten-
tial effects of hypoxia on leukemic cells. We reported recently
that moderate hypoxia and hypoxia-mimetic agents can induce
differentiation in leukemic cells, but the mechanism remains
largely unknown. In this study, human acute monomyelocytic
leukemic cell line U937 were treated with 50 mm CoCl
2
or incu-
bated in 2% O
2
for 24 h, that induced the accumulation of
HIF-1a protein. Then, cell lysates were subjected to two dimen-
sional gel electrophoresis (2 DE ) coupled with MALDI-TOF-
TOF (PMF and MS/MS) to identify the protein spots. As a
result, 40 of dysregulated proteins were identified, most of them
were HIF-1a-dependent, because these proteins were also modu-
lated in inducible HIF-1a-expressing U937 cell line. Hypoxia
related genes could be arbitrarily classified into following groups:
glucose and energy metabolism (including the well established
HIF-1a target genes endolase I and LDHA), oxidative stress
response, apoptosis, cell cycle and transcriptional regulation. Our
study provided an insight into the effect of hypoxia on leukemic
cell line in a global means, and several candidate target gene of
HIF-1a were found out as well.

H3-045P
Determination of superoxide dismutase (SOD),
Ggutathione peroxidase (GSH-Px) activities
and MDA levels in the erythrocytes treated
with H
2
O
2
in the presence and absence of
2,6- diisopropyl phenol
G. Yucebilgic, G. Gursoy, S. Tukel and R. Bilgin
Department of Chemistry, University of Cukurova, Adana, Turkey.
E-mail:
Superoxide dismutase (SOD) and glutathione peroxidase (GSH-
Px) activities, malondialdehyde (MDA), reduced glutathione
(GSH) and methemoglobin (MetHb) levels were determined in
normal erythrocytes and H
2
O
2
treated erythrocytes in the pres-
ence and absence of 2,6- diisopropyl phenol (Propofol). In H
2
O
2
treated erythrocytes; MDA levels, Met Hb %, and GSH-Px
activities were increased whereas SOD activity was decreased as
compared with the corresponding values of normal erythrocytes.
However, no significant change was determined in GSH values.
In H

2
O
2
+ propofol treated erythrocytes MDA levels, MetHb
% and GSH-Px activity were decreased almost to the levels of
normal erythrocytes. On the other hand, in the presence of prop-
ofol SOD levels were even higher than that of normal erythro-
cytes. Besides that GSH level was not changed significantly.
These results showed that propofol exhibit significant antioxidant
activity in vitro.
H4–Protein Networks in Cellular Functions
H4-001
Quantitative proteomics and systems biology
R. Aebersold
Molecular Systems Biology, ETH Zurich, Zu
¨
rich, Switzerland.
E-mail:
Systems biology is the science of dynamic networks of interacting
biomolecules. It is based on the insight that such networks have
intrinsic properties determining their structure and function that
are not apparent from the analysis of the isolated components
that constitute the system and that are critical for an understand-
ing of the function and control of the system as a whole. Systems
biology was made possible by the availability of the complete
genome sequence of the human and other species and by advan-
ces in biology, engineering and computer science that have collec-
tively catalyzed the emergence of technologies for the systematic
and quantitative measurement of genomic and proteomic profiles
and the integrative analysis of the obtained results. Most biologi-

cal networks involve proteins. Proteomics, the systematic analysis
of proteins is therefore an important component of systems
biology. In this presentation, we will discuss advances in quanti-
tative proteomics as a genomic science and its application to the
analysis of biological networks.
References
1. Ranish JA, Yi EC, Leslie DM, Purvine SO, Goodlett DR,
Eng J, Aebersold R. The study of macromolecular complexes
by quantitative proteomics. Nat Genet 2003; 33(3): 349–355.
2. Aebersold R, Mann M. Mass spectrometry-based proteomics.
Nature 2003; 422(6928): 198–207.
3. Ranish JA, Hahn S, Lu Y, Yi EC, Li XJ, Eng J, Aebersold R.
Identification of TFB5, a new component of general transcrip-
tion and DNA repair factor IIH. Nat Genet 2004; 36(7): 707–
713.
4. Giglia-Mari G, Coin F, Ranish JA, Hoogstraten D, Theil A,
Wijgers N, Jaspers NG, Raams A, Argentini M, van der Spek
PJ, Botta E, Stefanini M, Egly JM, Aebersold R, Hoeijmakers
JH, Vermeulen W. A new, tenth subunit of TFIIH is respon-
sible for the DNA repair syndrome trichothiodystrophy group
A. Nat Genet 2004; 36(7): 714–719.
Abstracts
432