Báo cáo khoa học: All Other Topics pdf

All Other Topics
F1-1
Effects of atorvastatin on oxidative stress in
L-NAME- treated rats
R. Gelisgen
1
, D. Konukoglu
1
, H. Uzun
1
, R. Kalayci
2
, N. Arican
3
and M. Kaya
4
1
Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul
University, Istanbul, TURKEY,
2
Istanbul Medical Faculty Research
Institute for Experimental Medicine, Istanbul University, Istanbul,
TURKEY,
3
Istanbul Medical Faculty, Forensic Medicine, Istanbul
University, Istanbul, TURKEY,
4
Department of Physiology, Istanbul
Medical Faculty, Istanbul University, Istanbul, TURKEY
Current evidence suggests that the beneficial effects of statins seem
to be mediated not only by their lipid-lowering properties but also

by improving vascular endothelial cell function. Chronic inhibition
of endothelial nitric oxide synthesis by oral administration of
N w-nitro-L-arginine methyl ester (L-NAME) to rats induces early
vascular inflammation and subsequent arteriosclerosis. To test the
relationship between statin (atorvastatin) therapy and oxidative
stress, we determine the levels of plasma oxidative stress markers
(protein carbonyl; PCO, lipid hydroperoxides; LPH, and oxidized
LDL; ox-LDL) and plasma antioxidants (Paraoxonase -1; PON-1
and total thiol; T-SH) in L-NAME induced endothelial dysfunc-
tion rat models. Adult male Wistar rats were divided into three
groups, as saline (n: 8, controls), L-NAME (1 mg/ml of drinking
water for three weeks, n: 8), and Atorvastatin plus L-NAME
(4 mg/kg/day for 1 week during the third week of L-NAME treat-
ment, n: 11). Plasma oxidative marker levels were higher and anti-
oxidative marker levels were lower in L-NAME group than
controls (for each, P < 0.01). Atorvastatin plus L-NAME group
have lower plasma ox-LDL and LPH levels and higher PON-I
activities than L-NAME group (for each, P<0.01). Therefore
statins are likely to restore the L-NAME-induced inhibition of
endothelial NO synthase activity by lowering oxidative stress.
F1-2
Circulating oxidized LDL and antibodies against
oxidized LDL in type 2 diabetes patients with
and without coronary artery disease
R. Gelisgen
1
, B. Cimen
1
, V. Yumuk
2

, M. Bolayirli
3
,
M. Hacibekiroglu
3
and G. Burcak
1
1
Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul
University, Istanbul, TURKEY,
2
Department of Internal Medicine,
Cerrahpasa Medical Faculty, Istanbul University, Istanbul, TUR-
KEY,
3
Central Research Laboratory, Cerrahpasa Medical Faculty,
Istanbul University, Istanbul, TURKEY
We aimed in this study to investigate firstly whether ox-LDL and
oLAB are increased in patients with type 2 diabetes mellitus com-
pared with control subjects and secondly whether they differ
between diabetic patients with and without coronary artery disease
(CAD) .We studied type 2 diabetic patients (n=60) and nondia-
betic control subjects (n=27). We divided the patients according
to the presence of CAD. CAD was present in 29 diabetic patients.
We assessed ox-LDL in plasma and oLAB, glucose, HbA1c, creati-
nine, high density lipoprotein cholesterol (HDL-C), low density
lipoprotein cholesterol (LDL-C), total cholesterol, triglycerides,
total protein and albumin in serum. ox-LDL and oLAB were
measured with ELISA. In diabetic group ox-LDL,ox-LDL/LDL-C
and oLAB were significantly higher than the control

group.(P<0.001, P<0.001, P<0.05).Significantly higher val-
ues for ox-LDL(P<0.001, P<0.001)and ox-LDL/LDL-C
(P<0.001, P<0.001)were observed in both of the diabetic
groups with and without CAD in comparison to the control group.
oLAB level was found significantly higher only in the diabetic
group with CAD than the control(P<0.01).ox-LDL concentra-
tions did not display any significant difference between diabetic
groups with and without CAD, but oLAB and ox-LDL/LDL-C
were higher(P<0.05, P<0.05) in the diabetic group with CAD.
F1-3
Decrease in lipid peroxidation as a result of
black tea antioxidant action.
W. Luczaj, E. Zapora and E. Skrzydlewska
Medical University Of Bialystok, Bialystok, POLAND
It has been widely postulated that reactive oxygen species (ROS)
play an essential role in the aging process. The increased genera-
tion of ROS during aging results in oxidative stress which causes
changes in structure and function of important cellular compo-
nents, especially lipids. Black tea has been recently proved to be a
source of water-soluble antioxidants that may enhance cellular
antioxidant abilities. The present study has been designed to
investigate the efficiency of preventive effect of black tea on liver
lipids oxidative modifications in 2-, 12-, 24-month-old rats. Lipid
peroxidation was estimated by measurement of lipid hydroperox-
ides, malondialdehyde, 4-hydroxynonenal and 8-isoprostaglandin
F
2a
by high-performance liquid chromatography (HPLC) and by
spectrophotometric determination of conjugated dienes as well as
phospholipase A

2
. Aging process causes the significant increase in
the level of lipid oxidative modification products (6–53%). Admin-
istration of black tea remarkably prevented the age-related increase
in concentrations of all measured lipid peroxidation markers. In
comparison to 2-month-old rats, the preventive effect of black tea
causes decrease (6–20%) in the examined markers in the liver of
12- and 24-month-old subjects. Our findings indicate that black tea
protects lipids against age-related oxidative modification.
F1-4
Oxidation of serum low density lipoprotein in
experimental hyperthyroidism; role of
paraoxonase
G. Simsek
1
, R. Gelisgen
2
, R. Yucel
1
, N. Dariyerli
1
, H. Genc
2
,
Y. Karter
3
, C. Simsek
4
and H. Uzun
2

1
Department of Physiology, Cerrahpasa Medicine Faculty, Istanbul,
TURKEY,
2
Department of Biochemistry, Cerrahpasa Medicine Fac-
ulty, Istanbul, TURKEY,
3
Department of Internal Medicine, Cer-
rahpasa Medicine Faculty, Istanbul, TURKEY,
4
Department of
Public Health, Cerrahpasa Medicine Faculty, Istanbul, TURKEY
Increased oxidative stress has been reported in hyperthyroid
patients. It is known that oxidative stress causes atherosclerosis.
Atherogenesis may be induced hyperthyroidism due to free radical
mediated damage, e.g., lipid peroxidation, because lipid peroxida-
tion plays a prominent role in lipoprotein oxidation. While oxid-
ized low density lipoprotein (LDL) has atherogenic effect, high
density lipoprotein (HDL) – associated paraoxonase1(PON1) pre-
vents oxidation of LDL. This study was undertaken to compare
the LDL oxidation and PON1 activity in hyperthyroid and control
rats. Hyperthyroidism was induced by administration of L-thyrox-
ine, 0.4 mg/100 g food, for 5 weeks. Plasma T3, T4, TSH, mal-
ondialdehyde (MDA), total cholesterol, LDL cholestrol, HDL
cholestrol, apolipoprotein A-1, triglycerides, oxidized LDL levels
and PON1 activity were measured in hyperthyroid (n=12) and
control (n=12) rats. Significantly increased MDA (P<0.001),
oxidized LDL levels (P<0.01), decreased HDL-cholestrol
(P<0.01), apolipoprotein A-1 levels (P<0.05) and PON1 activ-
ity ( P<0.01) were found in hyperthyroid rats in comparison to

control rats. There was no significant change in total cholestrol,
triglycerides and LDL cholesterol levels. Our data indicate
increased lipoprotein oxidation and reduced PON1 activity,
thereby atherogenesis may be induced in hyperthyroidism.
ª 2007 The Authors Journal compilation ª 2007 FEBS 265
Abstracts
F1-5
Effects of hyperbaric oxygen treatment on
glutathione and superoxide dismutase levels
indistracted rat muscle
S. Civelek
1
, S. Toklu
2
, Z. Kasymova
3
, H. Uzun
1
and A. Kaynar
3
1
Department of Biochemistry, Cerrahpasa Medical Faculty,
University of Istanbul, Istanbul, TURKEY,
2
Department of
Underwater and Hyperbaric Medicine, Medical Faculty Istanbul,
University of Istanbul, Istanbul, TURKEY,
3
Istanbul University,
School of Dentistry, Istanbul, TURKEY

The aim of the study was to evaluate the effects of HBOT therapy
on the muscle tissues surrounding the distracted rat tibia via bio-
chemical parameters. The distraction protocol was carried out in
optimum (0.5 mm/day) and hyperphysiologic (1 mm/day) rates. A
total of 46 age-matched male Wistar rats were randomly divided into
5 groups; Group 1 (n=10) was distracted at 0.5 mm/day, Group 2
(n=10) was distracted at 0.5 mm/day and received HBOT treat-
ment, Group 3 (n=10) was distracted at 1 mm/day, Group 4
(n=10) was distracted at 1 mm/day and received HBO treatment,
and Group 5 was the sham operated control. At the end of experi-
mental period of 5th and 9th days for 0.5 mm/day and 1 mm/day
respectively, GSH levels and SOD activity were spectrophotometri-
caly determined. Both GSH levels and SOD activity decreased with
osteodistraction in muscle tissues. GSH levels and SOD activity in
the in distracted rat muscle were higher in the HBO treated groups
(Groups 2 and 4) than those in the untreated groups (Groups 1 and
3). Our preliminary data suggests that HBO may augment antioxid-
ant levels in distracted rat muscle at both optimum and hyperphysio-
logic rates. The implication of these findings is not yet completely
known and therefore we are currently studying the effects of HBO
therapy on bone healing and inflammatory cell recruitment in ani-
mals, which underwent similar distraction conditions. Our findings
may also elucidate the role of HBO therapy on soft and hard tissue
healing in clinical applications of distraction osteogenesis.
F1-6
Advanced glycation endproducts (AGEs) and
antioxidant status in STZ induced diabetic rats:
effects of copper supplementation
S. Civelek, R. Gelis¸ gen, G. Andican, A. Seven, S. Ku
¨

cu
¨
k,
M. Ozdogan and G. Burcak
Department of Biochemistry, Cerrahpasa Medicine Faculty, Istanbul
University, Istanbul, TURKEY
We aimed to investigate the effects of Cu(II) supplementation on
glycemic parameters, advanced glycation end products (AGEs),
antioxidant status (GSH and total antioxidant capacity, TAOC)
and lipid peroxidative damage (TBARS) in streptozotocin (STZ)
induced diabetic rats and controls. Wistar albino rats were grouped
as ‘control’, ‘STZ administered’, ‘Cu(II)supplemented’ ‘STZ admin-
istered and Cu(II)supplemented’. STZ was administered (ip) at a
single dose of 65 mg/kg and Cu II, 4 mg/kg (sc) every two days for
60 days. At the end of 60 days, glucose, Cu II, TBARS, TAOC
were measured in plasma, GSH in erythrocytes and GHb in blood.
Utilizing a flow system with two dedectors connected on line AGEs
were measured spectrofluorometrically (k
ex
= 247 nm,
k
m
= 440 nm) and low molecular peptides spectrophotometrically
(k = 280 nm). In ‘STZ administered’ group plasma glucose,
(P<0.01) GHb (P<0.05) and AGE(P<0.01) levels were
higher than the control group. ‘Cu II supplemented’ group had
lower plasma glucose (P<0.05) higher GHb (P<0.001), TAOC
(P<0.05) and TBARS levels (P<0.001). In ‘STZ administered
and Cu (II) supplemented’ group TAOC level was higher than the
‘STZ administered ‘group (P<0.01). Plasma AGE levels did not

display any significant differences between Cu II supplemented and
unsupplemented groups. The contribution of Cu II supplementa-
tion to oxidative stress and AGE formation in diabetes remains to
be elucidated.
F1-7
Characterization of antioxidant capacities of
13 medicinal plant extracts
F. X. Malcata
1
, A. Pereira
1
, I. Leita
˜
o
1
, M. Gia
˜
o
1
, J. Fernandes
1
,
M. Pintado
1
, L. Belo
2
and A. Santos-Silva
2
1
College of Biotechnology, Porto, PORTUGAL,

2
Department
Bioquı
´
mica, Faculdade de Farma
´
cia-Inst. de Biologia Molecular e
Celular, UP, Porto, PORTUGAL
Cardiovascular diseases (CVD) – mainly caused by atherogenesis,
are the major cause of mortality and morbidity in the Western
world. Infusions prepared with plant leaves are particularly rich in
antioxidants, which seem to play a crucial protection role therein.
Our aim was to evaluate the antioxidant features of 13 medicinal
plant extracts: Agrimony, Avocato, Eucalyptus, Heath, Myrtle,
Raspberry, Sage, Savory, Spanish wood Marjoram, Sweet amber,
Thyme, Walnut-tree and Yarrow. Said features were determined
by the ability to protect normal human red blood cells (RBC)
against hydrogen peroxide – mediated oxidative damage in vitro.
Our results showed that oxidative hemolysis of RBCs was sup-
pressed by pre-treatment with all tested extracts (0.005%, w/v). In
general, the protective effect increased in a dose-dependent manner
(0.005–0.1%, w/v) – except for Heath, which exhibited a pro-
hemolytic effect at 0.1% (w/v). The plant extracts with the highest
protective effect, at the concentrations tested, were: Savory, Sage,
Agrimony, Myrtle, Yarrow and Walnut-tree. These six extracts are
thus potential nutraceutical ingredients towards CVD prevention.
F1-8
On the dual nature and code of free radicals in
biological systems
S. Volovyk

1
and J. Siedow
2
1
Duke University Medical Center, Durham, NC, USA,
2
Duke
University, Durham, NC, USA
Free radicals, primordial ‘sea’ for the life origin and existence,
induced by terrestrial and extraterrestrial radiation, are evolutional-
ly archetypal, ubiquitous, and omnipotent in physiological-patho-
physiological dichotomy in living systems. Delicate borderline
norm-pathology with continuity-discontinuity dichotomy is a func-
tion of immanent dual electronic nature and physiological func-
tional ambivalence and complementarity of free radicals based on
their dynamic charge transfer (CT)/redox ambivalence, correspond-
ing code of chemical reactivity and selectivity and dynamic free-
radical homeostasis. Subtle perturbations in radicals CT spatiotem-
poral homeodynamics, in responsive signaling/controlling net-
works, concomitant alterations in genes expression, transcription,
and apoptosis, CT telomere/telomerase balance, DNA CT, hemi-
spheric biochemical dominance/accentuation, including alteration
of nitric oxide-superoxide complementarity, neurotransmission pat-
tern, synaptic circuitry, etc have more fundamental impact on all
organism systems functioning and deterioration than simple radi-
cals inflicted oxidative cellular damage. This conceptualization of
free-radical paradigm constitutes new dimension in deciphering
molecular mechanisms of organism systems disorder – CT(redox)o-
mics, that penetrates and links all ‘omics’.
Acknowledgment: This material is based upon work supported

by DOE under Award Number DE-FC01–06EH06028.
266 ª 2007 The Authors Journal compilation ª 2007 FEBS
Abstracts
F1-9
Ethanol-induced replication of hepatitis B virus
requires IL-6 and JAK2 signaling pathway
B. Kim, J. Kim and Y. Park
Department of Biochemistry and Division of Brain Korea 21
Program for Biomedical Science, Korea University College of
Medicine, Seoul, REPUBLIC OF KOREA
In patients infected with hepatitis B virus (HBV), alcohol intake
may exacerbate clinical course of acute or chronic HBV infection
and make effects on the susceptibility to HBV-related disease, such
as cirrhosis and hepatocellular carcinoma. In this study, we studied
the effect of ethanol on HBV replication and the signaling pathway
involved in the event. In HepG2.2.15 cells, which are known to
produce hepatitis B virus particles, the level of HBV transcripts
and HBV DNA by ethanol treatment was increased in a dose- and
time-dependent manner. In contrast, acetaldehyde as a product of
the ethanol metabolism occurred in hepatocyte had no effect on
the synthesis of HBV mRNA and ethanol-induced synthesis of
HBV mRNA was not affected by pretreatment with 4-Methylpy-
razole hydrochloride, alcohol dehydrogenase inhibitor. The synthe-
sis of IL-6 mRNA was induced by ethanol treatment. Also,
ethanol exposure induced the phosphorylation of JAK2, STAT3
and STAT5. Ethanol-induced phosphorylation of JAK2 was inhib-
ited by pretreatment with IL-6 neutralizing antibody. Most import-
antly, pretreatment with IL-6 neutralizing antibody reduced the
ethanol-induced synthesis of HBV mRNA. Increase of HBV pro-
moter activity by ethanol was abolished by pretreatment with the

inhibitor of JAK2 (AG490) in HepG2 cells. Furthermore, ethanol-
stimulated synthesis of HBV mRNA was completely inhibited by
pretreatment with AG490 and by transfection of dominant negat-
ive JAK2 plasmid. The level of HBV DNA enhanced by ethanol
treatment was also blocked by pretreatment with AG490 in
HepG2.2.15 cells. These results suggest that ethanol-enhanced rep-
lication of HBV requires production of IL-6 and activation of
JAK2.
F1-10
Biochemical analysis of HCV E1E2 proteins
containing entire and truncated ectodomains
J. D. Tello,J.Go
´
mez-Gutie
´
rrez, B. Ye
´
lamos, M. J. Feito and
F. Gavilanes
Facultad de Ciencias Quı
´
micas. Universidad Complutense de Madrid,
Madrid, SPAIN
The hepatitis C virus (HCV) encodes two glycoproteins, E1 and
E2, which are components of the virus envelope. Their N-terminal
ectodomains (residues 192–340 for E1 and 383–661 for E2) are
thought to be responsible for the interaction between the virus and
its receptor as well as for the fusion of the viral and cellular mem-
branes. Using a baculovirus expression system we showed that,
whereas E2 ectodomain could be successfully obtained with native-

like properties, the efficient production of E1 was only achieved as
a fusion protein with E2, either at the N-terminal (E1E2) or at the
C-terminal (E2E1) end. Moreover, we analyzed several E1E2 chi-
meras having the E2 ectodomain truncated at different positions in
order to define the requirements for the expression of properly
folded E1. Thus, we observed that the deletion of the C-terminal
portion of E2 (residues 494–661) was dispensable, since protein
E1E2–493 was correctly processed; however, the additional
removal of the E2 473–493 fragment resulted in a missfolded poly-
peptide, indicating that this region of E2 is necessary for the acqui-
sition of the correct conformation of E1. The availability of
different chimeras containing either the entire or truncated ectodo-
mains has allowed us to develop comparative studies of their fuso-
genic properties, by determining their ability to interact and
destabilize phospholipid vesicles, and their interaction with hepatic
cells.
F1-11
Regulation of proteolytic processing of
non-structural polyprotein in alphaviruses
A. Lulla
1
, A. Golubtsov
2
, T. Ahola
2
and A. Merits
1
1
University of Tartu, Tartu, ESTONIA,
2

Institute of Biotechnology,
Helsinki, FINLAND
Semliki Forest virus (SFV) replication strategy relies on the pro-
duction of replicase proteins in the form of non-structural polypro-
tein precursor. Processing of the ns-polyprotein is performed by
viral non-structural protease. During the course of infection poly-
protein processing leads to rearrangements of the replication com-
plex so that its RNA template preference is changed in favor of
minus-strands over plus-strands. Previous results indicate that the
cleavage sites represent a compromise between protease recognition
and other requirements of the virus life cycle. The determinants of
cleavage efficiency are located in the region preceding the cleavage
site and the protease recognizes at least residues P4 to P1’. Ran-
dom mutagenesis analysis revealed that amino acid residues P4,
P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much vari-
ation in contrast to residue P5, also mutation of residues P4 and
P1’ had a significant effect on cleavage efficiency. However, it was
found that only two cleavage sites are recognized by their short
amino acid sequence, whereas processing of 2/3 site requires
domain of nsP3 which is used for precise positioning of recognition
sequence at the catalytic center of protease. Interestingly, the direct
comparison of the requirements for the processing of cleavage sites
in two prototype alphaviruses, SFV and Sindbis virus, indicates
that in alphaviruses at least two different mechanisms of regula-
tion, which lead to the similar outcome in the regulation of viral
RNA synthesis. The results of current research significantly
improve our understanding of the regulation of alphaviral replica-
tion and reveal the principally new role of alphaviral nsP3 protein
in the virus life cycle.
F1-12

Effects of the Semliki Forest virus temperature
sensitive mutations on the functions of
virus-encoded proteins
V. Lulla
1
, T. Ahola
2
and A. Merits
1
1
Institute of Molecular and Cell Biology, Tartu, ESTONIA,
2
Institute of Biotechnology, University of Helsinki, Helsinki,
FINLAND
We have sequenced the nonstructural protein coding region of Sem-
liki Forest virus temperature-sensitive (ts) mutant strains ts1, ts6,
ts9, ts10, ts11, ts13, and ts14. In each case, the individual amino
acid changes uncovered were transferred to the prototype strain
background and thereby identified as the underlying cause of the
altered RNA synthesis phenotype. Several assays were used at dif-
ferent temperatures to verify the phenotypic effects of the revealed
mutations: titration of virus stocks, analysis of viral RNA synthesis,
labeled polyprotein processing and other enzymatic assays. All
mutations mapping to the protease domain of non-structural pro-
tein nsP2 caused defects in nonstructural polyprotein processing
and subgenomic RNA synthesis, and all mutations in the helicase
domain of nsP2 affected subgenomic RNA production. These types
of defects were not associated with mutations in other nonstructural
proteins. Two mutations mapping to nsP1 caused defects in RNA
synthesis and did not affect the methyltransferase and guanylyl-

transferase activities of nsP1 in vitro.
ª 2007 The Authors Journal compilation ª 2007 FEBS 267
Abstracts
F1-13
Changes of Ca
2+
affinity to membrane proteins
of the sarcoplasmatic reticulum at experimental
crash syndrome
G. A. Kevorkian, L. H. Melkonyan, H. L. Hayrapetyan,
A. G. Guevorkian, H. F. Khachatryan and L. N. Arakelyan
H.Buniatian Inst. of Biochemistry, Yerevan, ARMENIA
The experimental crash syndrome induced by crash of the hip soft
muscle depends on two periods of the pathogenesis development:
compression period (2 and 5 h) and decompression period (2, 4, 24,
48 h). Compression period was taken as control, and respectively,
the decompression period was studied as experimental one. Our
observations indicate that at decompression there take place a gen-
eral intoxication of the organism and an infarction of myocardium.
The experimental data coincide with clinical results obtained in the
period of the 1988 earthquake in Armenia. By our results the devel-
opment of the infarction of myocardium depended on toxic peptides
released to blood from the randomized muscles and injured kidneys.
In 2 h after decompression the membrane proteins of the sarcoplas-
matic reticulum began to change Ca
2+
affinity, data of which were
plotted in a Scatchard coordinate system. In 24 h after decompres-
sion 5 acidic proteins and calsequestrin affinity almost wholly loose
Ca

2+
affinity. Simultaneously the membrane protein with 32 kDa
molecular weight, which does not possess Ca
2+
affinity, begins to
bind Ca
2+
. It is shown that Ca
2+
is bound in 2 points: with high
and low affinity. Hence, in 48 h after decompression the concentra-
tion of Ca
2+
in high affinity part (Bmax and Kd) does not change,
while Bmax and Kd of the low affinity part increase. Absolutely
identical changes took place with the affinity to membrane proteins
of the sarcoplasmatic reticulum at adrenal necroses, at injuries of
myocardium at experimental pancreatitis, as well as at pancreatitis
and crash syndrome. The problem of general mechanism of myocar-
dium injury at pathologies with different etiology is represented.
F1-14
A model for the influence of the polarizability
on the ions solvation in the lipid membrane
D. Ionescu
1
and R. A. Ionescu
2
1
University of Medicine and Pharmacy, Bucharest, ROMANIA,
2

National Institute for Physics and Nuclear Engineering ‘Horia
Hulubei’, Bucharest, ROMANIA
We discuss about conductance mechanisms at the level of lipid
artificial membranes, taking into consideration the influence of the
properties of the ions, emphasizing on their polarizability. One of
the properties of the lipid membrane which is changed due to the
presence of the ions in the bordering aqueous phase is the mem-
brane dipole potential and this can be macroscopically seen by the
amplitude of the diffusion currents through the lipid membrane
given by differently charged ions. Our model takes a step deeper in
the structure of the membrane and we intend to explain the man-
ner in which the polarizability of the ions influences their solvation
in the lipid membrane, supposing that they diffuse through the
lipid phase surrounded by water molecules. Our calculations con-
firm that the total energy of an ion into the lipid phase, which is
given by the electrostatic and surface contributions, has a mini-
mum at a radius of a few Angstrom. This means that it is energet-
ically favorable for an ion to diffuse through the membrane
together with water molecules. Because the polarizability of the ion
plays an important role at the interface, we investigate the role of
the hydrated ion total polarizability on the ion-lipid-water parti-
tion.
F1-15
Elongation and desaturation of fatty acids are
critical in lipid metabolism, growth, and
ontogeny of Caenorhabditis elegans
K. Sakamoto, M. Horikawa, T. Hashimoto and T. Nomura
University of Tsukuba, Tsukuba, JAPAN
Obesity is a main causative factor in human lifestyle-related
disease. Recently, it was reported that a deficit in the mouse stea-

royl-CoA desaturase 1 (Scd1) gene decreases biosynthesis and
accumulation of fatty acid and revitalizes the b-oxidation of fatty
acid. To examine the physiological role of fatty acid desaturase
(fat) and elongase (elo) in ontogeny, fatty acid accumulation, and
individual lifespan, we performed bacteria-mediated RNA interfer-
ence (RNAi) in the nematode Caenorhabditis elegans. Suppression
of the expression of elo-2 gene mRNA caused a drastic decrease in
the amount of body fat, miniaturization of the body, defects in
egg-laying, and increased lifespan. The amount of body fat was
markedly decreased, and body size reduced, by downregulation of
fat-6 and fat-7—a functional homolog of the mouse Scd1 gene—-
whereas lifespan was drastically reduced. RNAi of fat-6 decreased
the mRNA levels of the genes involved in fatty acid synthesis, and
RNAi of fat-7 increased the mRNA levels of b-oxidation-related
genes. Additionally, RNAi of the elo-2 gene caused a remarkable
decrease in fatty acid biosynthesis-related gene expression. These
results indicated that the elongation and desaturation of fatty acids
are integral to various phenomena such as ontogeny and lifespan
and play important roles in fatty acid accumulation and consump-
tion.
F1-16
Anions modulate the interaction of hypericin/
BSA or amitriptyline complex with lipid
membrane
D. Ionescu, M. Dragusin, M. Dima, A. Popescu and C. Ganea
Universtity of Medicine and Pharmacy ‘Carol Davila’, Bucharest,
ROMANIA
By the means of an electrophysiological method, Black Lipid
Membrane (BLM), we studied the manner in which the electrical
capacitance and conductance of the lipid membrane vary in the

presence of some lyotropic anions, which are lately frequently
encountered in various foods as additives, when the bovine serum
albumin (BSA) – Hypericin complex was added. We previously
reported (Ionescu and Ganea 2005) the increase in the conductance
and the capacitance of the membrane in the presence of lyotropic
anions when hypericin only is added and our recent results show
that in the presence of perchlorate, acetate and chloride the electri-
cal properties of the lipid membrane vary also when BSA –
Hypericin complex is added, but with a different profile: the aug-
mentation of the electrical conductance and capacitance of the
lipid membrane when hypericin is added is steeper than the one
obtained when adding BSA – Hypericin complex, suggesting a
slower incorporation of the complex in the membrane. On the
other hand, an increasing concentration of tricyclic antidepressant
amitriptyline also modifies the insertion kinetics of hypericin in the
lipid membrane in the presence of lyotropic anions showing a sat-
uration of the binding process.
Reference
1. Ionescu D, Ganea C. The Hofmeister effect of anions on the
insertion of hypericin in lipid bilayers. Eur. Biophys. Journal
2005; 34: 699.
268 ª 2007 The Authors Journal compilation ª 2007 FEBS
Abstracts
F1-17
Effect of alpha-lipoic acid on myeloperoxidase
activity and lipid peroxidation level in
carrageenan-injected rats
F. Odabasoglu
1
, H. Aygun

2
, Z. Halici
3
, Y. Bayir
1
, A. Cakir
4
,
E. Cadirci
5
and F. Atalay
1
1
Department of Biochemistry, Faculty of Pharmacy, Ataturk
University, Erzurum, TURKEY,
2
Department of Orthopedics and
Traumatology, Medical Faculty, Ataturk University, Erzurum, TUR-
KEY,
3
Department of Pharmacology, Medical Faculty, Atatu
¨
rk Uni-
versity, Erzurum, TURKEY,
4
Department of Chemistry, Education
Faculty, Atatu
¨
rk University, Erzurum, TURKEY,
5

Department of
Pharmacology, Faculty of Pharmacy, Ataturk University, Erzurum,
TURKEY
Alpha-lipoic acid (ALA) is a dithiol that is found naturally in mito-
chondria as the coenzyme for pyruvate dehydrogenase and alpha-ke-
toglutarate dehydrogenase. We have investigated alterations in the
activity of myeloperoxidase (MPx) and level of lipid peroxidation
(LPO) following oral administration of ALA, indomethacine (IND)
and diclofenac (DIC) in rats with carrageenan-induced paw edema.
Fortytwo Dawley rats divided into seven groups, of which paws of
rats in six groups were injected with carrageenan (CAR). Then, they
received ALA (50, 100 and 200 mg/kg), IND (25 mg/kg) and DIC
(25 mg/kg) orally. Rats from the other group served as control. Fol-
lowing euthanasia, paw tissues were scrapped and then ground
within liquid nitrogen for activities of enzymes and GSH level. In the
present study, we found that (i) ALA reduced the development of
CAR-induced paw edema, at a smaller magnitude for ALA than for
IND and DIC; (ii) All doses of ALA and IND have significantly
decreasing effect on amplification in MPx activity resulting from
induced paw edema; and (iii) All doses of ALA, IND and DIC ameli-
orated amplifications in the LPO level caused by CAR injection.
These results suggest that the anti-inflammatory effect of ALA on
CAR-induced acute inflammation can be attributed to its amelior-
ating effect on lipid peroxidation level and myeloperoxidase activity.
F1-18
A Chinese hamster ovarian cell line imports
cholesterol by high density lipoprotein
degradation
H. Stangl
1

, T. Pagler
1
, S. Golsabahi
1
, H. Laggner
1
,
A. Lohininger
1
, S. Rhode
2
, G. J. Schu
¨
tz
2
, M. Pavelka
1
,
C. Wadsack
3
and W. Strobl
1
1
Medical University of Vienna, Vienna, AUSTRIA,
2
Institute of Bio-
physics, Johannes Kepler University Linz, Linz, AUSTRIA,
3
Clinic
of Obstetrics and Gynecology, Medical University of Graz, Graz,

AUSTRIA
Plasma high density lipoprotein (HDL) is inversely associated with
the development of atherosclerosis. HDL exerts its atheroprotec-
tive role through involvement in reverse cholesterol transport in
which HDL is loaded with cholesterol and transports its lipid load
back to the liver. Thereby HDL is not dismantled but transfers its
lipids to the cell. Here we present evidence that a Chinese hamster
ovarian cell line (CHO7) adapted to grow in lipoprotein deficient
media degrades HDL and internalizes HDL-derived cholesterol.
Delivery of HDL cholesterol to the cell was demonstrated by a
down-regulation of cholesterol biosynthesis, an increase in total
cellular cholesterol content and by stimulation of cholesterol esteri-
fication after HDL treatment. This HDL degradation pathway is
distinct from the LDL receptor pathway, but also degrades LDL.
25-OH cholesterol, a potent inhibitor of the LDL receptor path-
way, down-regulated LDL degradation in CHO7 cells only in part,
and did not down-regulate HDL degradation. Dextran sulfate
released HDL bound to the cell surface of CHO7 cells and heparin
treatment released protein(s) contributing to HDL degradation.
The involvement of heparan sulfate proteoglycanes and lipases was
tested by two inhibitors genistein and tetrahydrolipstatin. Both
blocked HDL degradation significantly. Thus, these CHO7 cells
degrade HDL and LDL to supply themselves with cholesterol via
a novel degradation pathway. HDL degradation with similar prop-
erties was also observed in a human placental cell line.
F1-19
Structural modeling and site-directed
mutagenesis of Zea mays
phosphomethylpyrimidine kinase/thiamine
phosphate synthase

M. Rapala-Kozik
1
, M. Olczak
2
, A. Starosta
1
, J. Soroka
1
,
K. Ostrowska
3
and A. Kozik
1
1
Jagiellonian University, Faculty of Biochemistry, Biophysics and
Biotechnology, Krakow, POLAND,
2
University of Wroclaw, Faculty
of Biotechnology, Wroclaw, POLAND,
3
Jagiellonian University,
Faculty of Chemistry, Krakow, POLAND
Vitamin B1 (thiamine) can be synthesized by bacteria, fungi and
plants from two precursors: 4-amino-5-hydroxymethyl-2-methylpy-
rimidine phosphate (HMP-P) and 4-methyl-5-(2-hydroxyethyl) thi-
azole phosphate (HET-P). HMP-P is then phosphorylated to
HMP-PP which condenses with HET-P into thiamine phosphate
(TMP). To perform two last steps, the higher plants, unlike other
thiamine synthesizing organisms, use bifunctional enzymes with
both HMP-P kinase and TMP synthase activities. These unique

plant enzymes had not been structurally or kinetically character-
ized until our recent isolation of recombinant Zea mays protein. In
this work we present structural models of that protein. The pres-
ence of separate HMP-kinase- and TMP-synthase domains was
deduced from sequence comparisons with other enzymes with these
activities. Based on the sequence similarity of N- and C-terminal
domains to two bacterial enzymes with known three-dimensional
structures, Salmonella typhimurium HMP-P kinase and Bacillus
subtilis TMP synthase, respectively, we modeled their overall struc-
ture and arrangement of active site residues, using the SWISS-
MODEL server. Like the bacterial prototypes, the plant HMP-P
kinase domain had a ribokinase fold and TMP-synthase form a
typical TIM barrel. Site-directed mutagenesis was used to experi-
mentally verify the model-predicted active site residues including
G65, A72, D77, T96, Q98, M134, V161 and C268 at the HMP-P
binding region and R375, K377, N405, D406, S444, T470, K473,
G500, V523, S524 at TMP synthase center.
F1-20
Structural and functional characterization of
CheY from Helicobacter pylori
K. H. Lam
1
, T. K. W. Ling
2
and S. W. N. Au
1
1
Department of Biochemistry, Centre for Protein Science and Crys-
tallography, Faculty of Science, The Chinese University of Hong
Kong, Hong Kong SAR, HONG KONG,

2
Department of Microbio-
logy, The Chinese University of Hong Kong, Prince of Wales Hospi-
tal, Hong Kong SAR, HONG KONG
Helicobacter pylori is the human pathogen that causes gastritis,
duodenal ulcer and gastric cancer. About 50% of the world popu-
lation has been infected with H. pylori. Motility is an important
virulence trait for H. pylori infection. From animal model in previ-
ous studies, cheA, cheW and cheY mutants are non-chemotactic
and show attenuated phenotype, suggesting that chemotaxis is
essential in colonization. CheY belongs to response regulator
superfamily that controls the clockwise or counter-clockwise move-
ment of flagella. The process is determined by phosphorylation
and dephosphorylation of CheY. Phosphorylation increases the
affinity of CheY to FliM, a component of switch protein complex,
altering the flagella rotation to opposite direction. To study the
underlying mechanism of chemotactic regulation in H. pylori,we
have expressed and purified recombinant CheY. Here, we report a
1.8
o
A crystal structure of CheY. Structural comparison between
CheY in H. pylori and other species will be discussed. The in vitro
interaction between CheY and FliM will be investigated.
ª 2007 The Authors Journal compilation ª 2007 FEBS 269
Abstracts
F1-21
Crystal structure of D-psicose 3-epimerase from
Agrobacterium tumefaciens and its complex
with true substrate D-fructose
K. Kim, W. Jung and S. Rhee

Seoul National University, Seoul, REPUBLIC OF KOREA
D-Psicose, a rare sugar produced by the enzymatic reaction of D-
tagatose 3-epimerase (DTEase), has been used extensively for the
bioproduction of various rare carbohydrates. Recently character-
ized D-psicose 3-epimerase (DPEase) from Agrobacterium tumefac-
iens was found to belong to the DTEase family and to catalyze the
interconversion of D-fructose and D-psicose by epimerizing the C3
position, with marked efficiency for D-psicose. The crystal struc-
tures of DPEase and its complex with the true substrate D-fructose
were determined; DPEase is a tetramer and each monomer belongs
to a TIM-barrel fold. The active site in each subunit is distinct
from that of other TIM-barrel enzymes, which use phosphorylated
ligands as the substrate. It contains a metal ion with octahedral
coordination to two water molecules and four residues that are
absolutely conserved across the DTEase family. Upon binding of
D-fructose, the substrate displaces water molecules in the active
site, with a conformation mimicking the intermediate cis-enedio-
late. Subsequently, Trp112 and Pro113 in the b4-a4 loop undergo
significant structural changes, sealing off the active site. Structural
evidence and site-directed mutagenesis of the putative catalytic res-
idues suggest that the metal ion plays a pivotal role in catalysis by
anchoring the bound D-fructose, and Glu150 and Glu244 carry
out an epimerization reaction at the C3 position.
F1-22
Crystallization and structure solution of major
grass pollen allergen Phl p 3
V. Devanaboyina
1
, W. Keller
1

, A. Nandy
2
and H. Fiebig
2
1
University of Graz, Graz, AUSTRIA,
2
Allergopharma J.Ganzer
KG, Reinbek, GERMANY
Ig-E mediated allergies represent a major health problem in the
industrialized world as they affect almost 25% of the population.
For the precise analysis of the surface exposed IgE epitopes, the
three dimensional structure of allergens are the most important
source of information. The detailed knowledge derived from the
three dimensional structure together with immunological data
allows for the rational development of strategies to convert aller-
gen molecule in to hypoallergenic derivatives through destruction
or reorientation of IgE epitopes, such allergen derivatives may act
as potent hypoallergenic vaccines for immunotherapy.
The allergens from grass pollen are amongst the most potent and
frequent elicitors of allergic disease. Phl p 3 is a timothy grass pol-
len allergen with a molecular weight of 10 959 kDa. Phl p 3 crys-
tallizes in two different forms at pH of 5.5 with PEG as
precipitant. The Phl p3 sequence has a 57% sequence identity with
Phl p 2, whose X-ray structure has been solved (PDB: 1WHO). A
data set was collected to 2.3A
˚
at DESY, Hamburg. Structure is
solved by Molecular replacement method. Phl p 3 has a FN-type
III fold and it is beta sheet protein.

Acknolledgement: This work is generously supported by Aus-
trian science Foundation. FWF-SFB F018
F1-23
Reconstitution of azurocidin proteolytic activity
by site-directed mutagenesis
M. Olczak and T. Olczak
University of Wroclaw, Wroclaw, POLAND
Azurocidin (HBP or CAP37) is a 37-kDa polypeptide produced
mostly in human neutrophils. The protein is proteolytically proc-
essed during maturation by removal of 19-aa signal peptide and
then two short 5-aa and 2-aa peptides from the N-terminus and
3-aa peptide from the C-terminus. Azurocidin belongs to serine
proteinase family, closely related to neutrophil elastase, cathepsin
G, proteinase 3, chymase and granzymes. However, the proteolytic
activity of azurocidin was lost due to a substitution of His41 and
Ser175 residues from catalytic triad by Ser and Gly residues,
respectively. Using site-directed mutagenesis and non-lytic insect
cell expression system we designed and expressed a double azuroci-
din mutant with reconstituted catalytic triad and single mutants
with Ser41 replaced by His or Gly175 substituted by Ser. All pro-
teins contained respective protease cleavage site, inserted down-
stream of a signal peptide sequence, allowing production of mature
azurocidin after activation with specific protease (enterokinase, fac-
tor Xa, or protease TEV). We showed that azurocidin with recon-
stituted catalytic triad exhibited proteolytic activity when
incubated with fluorescently labeled casein. Preliminary results also
suggest autocatalytic removal of a tripeptide at the C-teminus of
the protein. In contrast, single mutations of azurocidin has no
effect on recovery of proteolytic activity. Our results confirms that
the proteolytic mechanism of pro-peptide removal and azurocidin

activation is preserved in the double mutant.
F1-24
Study of operation of the molecular hinges in
human PGK using site-directed mutagen esis
B. Flachner, J. Szabo
´
, A. Varga, P. Za
´
vodszky and M. Vas
Institute of Enzymology HAS, Budapest, HUNGARY
The glycolytic enzyme 3-phosphoglycerate kinase (PGK) contains
two structural domains. The domains each bind one of the two
substrates: 1.3-BPG and MgADP or 3-PG and MgATP in the for-
ward or reverse reaction. The transfer of the phospho group
between the two substrates requires closure of the two domains.
This motion brings the substrates into optimal distances for the
reaction. Molecular graphical comparison of the open and closed
crystal structures suggested the location of the main hinge region
in b strand L. It may also regulate the other hinges at the ends of
helix 7. In order to identify the side chains responsible domain clo-
sure we constructed the following mutants of human PGK: S392A
and T393A (bL), S398A (a14), N336A (bJ) as well as E192A (a7).
The mutants were characterised by biophysical tests (CD, DSC)
and by enzyme kinetic and substrate binding experiments. T393 is
important in receiving the transmitted effect of 3-PG from the N-
domain. The side-chain of N336 is located in the nucleotide-bind-
ing site. It has an essential role in the enzyme activity and mediates
the effect of the nucleotide substrate towards the main hinge. The
side-chain of E192 connects a7tobL and has a role in the con-
formational stability on the whole molecule. It also mediates the

effect of 3-PG towards the bL. Based on these results a compre-
hensive picture about operation of the main molecular hinge of
PGK is presented.
270 ª 2007 The Authors Journal compilation ª 2007 FEBS
Abstracts
F1-25
The role of metal ion on active site in
Pseudomonas stutzeri L-rhamnose isomerase
H. Yoshida, M. Yamaji, M. Yamada, G. Takada, K. Izumori,
T. Ishii and S. Kamitori
Kagawa University, Kagawa, JAPAN
L-rhamnose isomerase from Pseudomonas stutzeri (P. stutzeri
L-RhI) is homo tetrameric enzyme and can catalyze the reversible
isomerization between various aldoses and ketoses in the presence
of appropriate metal ions. Each monomer contains two metal ions,
‘structural’ and ‘catalytic’ metals. L-RhI shows metal dependency
and has the highest catalytic activity in the presence of Mn
2+
. The
relative activity for Cu
2+
,Co
2+
,Zn
2+
compared to that of Mn
2+
is ca. 50, 40, 10%, respectively.
To investigate the role of metal ion on active site in P. stutzeri
L-RhI, we determined the crystal structures of P. stutzeri L-RhI in

the presence of Zn
2+
(L-RhI_Zn), Mn
2+
(L-RhI_Mn), and in
complex with a substrate, D-psicose (L-RhI_Mn/D-psi). In the
electron density map of L-RhI_Mn, the ‘catalytic’ metal could be
located at two positions, implying that Mn
2+
possibly moves
between the two positions in each monomer, whereas the electron
density maps of L-RhI_Zn and L-RhI_Mn/D-psi showed obvi-
ously one position for ‘catalytic’ metal in each monomer. The
movement of Mn
2+
as a catalytic metal might be related to cata-
lytic activity of P. stutzeri L-RhI.
F1-26
Gene expression profiling in the bone tissue of
osteoporotic mice
I. Orlic, F. Borovecki, P. Simic and S. Vukicevic
Medical School, Zagreb, CROATIA
Osteoporosis is a major public health problem that is characterized
by microarchitectural deterioration, low bone mass and increased
risk of fractures. Although widely studied, complex etiology of
osteoporosis has still not been fully clarified. Ovaricetomized
(OVX) mice represent an optimal animal model to investigate bone
loss in osteoporosis. To further elucidate the underlying mecha-
nisms of decreased bone formation and increased bone resorption
following ovariectomy, we conducted gene expression profiling

experiments using bone samples of OVX C57BL/6J mice.
At 21 days following OVX we observed deregulation of genes
involved in bone resorption, suggesting that this time point is very
close to the peak of osteoclastic activity. Following OVX, genes
involved in immune response, cell cycle regulation, growth, apop-
tosis and bone resorption were upregulated, while genes that are
important for mitosis, metabolism of carbohydrates, extracellular
matrix structure, angiogenesis, skeletal development and morpho-
genesis were downregulated. Among bone specific genes we
observed upregulation of interleukin 7 (IL-7), IL-7 receptor and
matrix metallopeptidase 8, while TGFb-3, procollagen type I and
VI exhibited marked decrease in expression. We also discovered
downregulation of two genes, parathyroid hormone receptor 1 and
WD repeat domain 5, that are involved in skeletal development
but were not previously reported to be altered in osteoporosis.
In conclusion, OVX greatly influences expression of various genes
involved in diverse biological processes confirming the notion that
numerous pathways play an important role in pathophysiology of
osteoporosis.
F1-27
Effect of alloxan on urea, creatine and bilirubin
levels in serum of rats
A. Cebi, S. Yasar, G. Oto and H. Demir
Yuzuncu Yil University, Van, TURKEY
This study was carried out to investigate whether alloxan could
affect biochemical parameters (urea, creatine, total bilirubin and
direct bilirubin) in serum of rats or not. Twelve Sprague-Dawley
albino rats were divided into two experimental groups; control and
study groups. A single dose (100 mg/kg) of alloxan was injected in-
traperitonealy to the study group rats. Same amount of physiologi-

cal saline was injected to the control group rats. Various
biochemical constituents were measured first, third and sixth hour
after the alloxan injection. All biochemical parameters were meas-
ured using an autoanalyzer (BNN/Hitachi-911) and the corres-
ponding kit (DPC, Diagnostic Products Corporation, USA). One
way ANOVA test was performed for statistical analysis. After all-
oxan injection, urea levels increased significantly in all the meas-
urements. Total bilirubin and direct bilirubin levels decreased as
regard to control group in all the measurements after the alloxan
injection whereas creatine levels decreased only in the measurement
of first hour. It is concluded from this study that alloxan differ-
ently effect urea, creatine and bilirubin levels in rats.
F1-28
Differential regulation of homocysteine
transport in vascular endothelial and smooth
muscle cells
X. Jiang
1
, F. Yang F
1
, E. Brailoiu
1
, H. Jakubowski
2
, N. Dun N
1
,
A. I. Schafer
3
, X. Yang

1
, W. Durante
4
and H. Wang
1
1
Temple University School of Medicine, Philadelphia, PA, USA,
2
UMDNJ-New Jersey Medical School, Newark, NJ, USA,
3
University of Pennsylvania School of Medicine, Philadelphia, PA,
USA,
4
University of Missouri School of Medicine, Columbia, MO,
USA
We previously reported that homocysteine (Hcy) inhibits endothel-
ial cell (EC) growth and promotes vascular smooth muscle cell
(VSMC) proliferation. To begin to elucidate the underlying bio-
chemical basis for these disparate effects, this study compared Hcy
transport in cultured human aortic EC (HAEC) and smooth mus-
cle cells (HASMC). L-Hcy (10 lM) was transported into both cell
types in a time dependent fashion but was approximately 4-fold
greater in HASMC. Hcy transport in HAEC had a Michaelis con-
stant (Km) of 39 lM and a maximal transport velocity (Vmax) of
873 pmol/mg protein/min. In contrast, Hcy transport in HASMC
had a lower affinity (Km=106 lM) but a higher transport capacity
(Vmax = 4192 pmol/mg protein/min). Competition studies
revealed that the small neutral amino acids tyrosine, cysteine, gly-
cine, serine, alanine, methionine and leucine inhibited Hcy uptake
in both cell types but the inhibition was greater for tyrosine, serine,

glycine and alanine in HAEC. Sodium-depletion reduced Hcy
transport to 34% in HAEC and 63% in HASMC. Increases in pH
from 6.5 to 8.2 or lysosomal inhibition blocked Hcy uptake only
in HAEC. In addition, Hcy shares carrier systems with cysteine, in
a preferable order of ASC > XAG = L in HAEC and
ASC > L > XAG in HASMC. The sodium-dependent system
ASC is responsible for approximately 90% of Hcy uptake in
HAEC and 65% in HASMC. The predominant role of system
ASC and the lysosomal regulated feature of Hcy transport in EC
may contribute to its cell specific proatherogenic effect and cardio-
vascular disease.
ª 2007 The Authors Journal compilation ª 2007 FEBS 271
Abstracts
F1-29
Ciliated epithelial- and regional-spe cific
expression and regulation of the estrogen
receptor
b
2 in rat fallopian tubes
R. Shao, Sr. and H. Billig
Neuroscience And Physiology, Go
¨
teborg University, Go
¨
teborg,
SWEDEN
In the present study, we have investigated the intracellular localiza-
tion and regulatory patterns for ERb isoforms in rat fallopian
tubes. Western blot analysis reveals that two ERb isoforms corres-
ponding to ERb1 and ERb2 are expressed in rat fallopian tubes.

However, ERb2 is the predominant form of ERb in this tissue.
High-resolution confocal imaging and immunohistochemical analy-
sis provide ample evidences that ERb expression is limited almost
exclusively to the ciliated epithelial cells in contrast to ERa, which
is widely distributed. Furthermore, within the ciliated epithelial
cells, ERb is colocalized with b-tubulin IV at stem portion of the
cilia. We show that ERb2 protein expression is tightly regulated by
17b-estradiol (E2) or diarylpropionitrile (DPN) in a time-depend-
ent manner without changes in ERb1 expression. These estrogenic
effects are inhibited by an ER antagonist ICI 182.780. In addition,
significant alteration of ERb immunoreactivity is only detected his-
tologically in the ampullary region. As the cilia are considered an
essential determinant of tubal transport, we further demonstrate
that E2- or DPN-induced ERb2 activation is associated with alter-
ations in tubal protein expression crucial for the regulation of cal-
cium-dependent ciliary beating. Given the coordinated regulation
and interaction of ER and progesterone receptor in the cilia, we
hypothesize that tubal ERb2 may facilitate the estrogen-mediated
transport process by processing protein-protein interaction under
physiological and/or pathological conditions. This study shows for
the first time that a previously unrecognized localization of ERb
isoform in rat fallopian tubes.
F1-30
Recombinant starmaker
T. M. Kapon
1
, G. Rymarczyk
1
, M. Nocula-Ługowska
1

,
M. Jako
´
b
1
, Z. Szewczuk
2
and A. O
_
zyhar
1
1
Department of Biochemistry, Wroclaw University of Technology,
Wrocaw, POLAND,
2
Group of Chemistry and Stereochemistry
of Peptides and Proteins, Faculty of Chemistry, University of
Wroclaw, Wrocaw, POLAND
Otoliths are essential elements in sensory system of the zebrafish
(Danio rerio). These structures enable fishes to sense gravitational
forces and linear accelerations. The biomineralization of the oto-
liths is controlled by Starmaker (Stm) protein, which determines
their morphology and even their crystal lattice structure. To facili-
tate understanding of the molecular role Stm protein plays in bio-
mineralization, it is necessary to obtain large amounts of pure Stm
for in vitro studies. In this report, we describe bacterial expression
system that allows to obtain about 1 mg of protein from 1 l of cul-
ture. We have also elaborated purification procedure based on salt-
ing out, as well as size exclusion and hydroxyapatite
chromatography. Consequently, we have purified Stm devoid of

any peptide tag, and the identity of the protein was confirmed by
ESI MS. Subsequent gel filtration revealed extended conformation
of Stm in solution, showing Stokes radius of 78.6 A
˚
, which is
much higher than expected for a globular protein of the molecular
mass of Stm (64.5 kDa). Such extended conformation, which is
characteristic for intrinsically unstructured proteins, might result
from high mean net charge of Stm combined with its low mean hy-
drophobicity.
Acknowledgement: Supported by a grant from the State Com-
mittee for Scientific Research.
F1-31
Prenatal cocaine exposure alters coronary
artery reactivity in adult offspring
L. Zhang
1
, D. Xiao
1
and S. Yang
2
1
Loma Linda University, Loma Linda, CA,
2
California State
University, San Bernardino, CA, USA
The present study tested the hypothesis that prenatal cocaine expo-
sure alters myogenic reactivity of the coronary artery in adult off-
spring. Pregnant rats received cocaine (30 mg/kg/day) or saline
from days 15–21 of gestational age. Coronary arteries were isolated

from 3 month-old offspring, and pressure-dependent myogenic
tone was measured simultaneously with vessel wall intracellular
Ca
2+
concentrations ([Ca
2+
]
i
). Prenatal cocaine exposure signifi-
cantly decreased coronary artery myogenic responses without
affecting [Ca
2+
]
i
in adult male rats, resulting in a decrease in the
ratio of changes in diameter to changes in [Ca
2+
]
i
. In contrast,
cocaine treatment caused a significant increase in pressure-induced
myogenic tone and the ratio of changes in diameter to changes in
[Ca
2+
]
i
in coronary arteries of female offspring. Inhibiton of eNOS
with L-NNA did not alter coronary artery myogenic responses in
either saline or cocaine-treated offspring. The results suggest that
prenatal cocaine exposure reprograms coronary artery function in

a gender specific manner, resulting in a downregulation of pres-
sure-dependent myogenic tone in male adult offspring, but an up-
regulation in female offspring, which is caused by changes in the
Ca
2+
sensitivity of myogenic mechanism.
Acknowledgement: Supported in part by USA NIH grants
HL82779 and S06GM073842.
F1-32
Depolarization-evoked increase of cytosolic
Ca
2+
: a new fluorescent assay in rat cortical
neurons
S. Francisconi, P. Salvati and C. Caccia
Newron Pharmaceuticals SpA, Bresso (MI), ITALY
Most of the studies in the literature on depolarization-induced
Ca
2+
increase have been performed using synaptosomes and Fura2
as Ca
2+
indicator while only a few studies have been carried out
in cultured neurons. In contrast to synaptosomes, which allow
pharmacological studies only of presynaptic effects, neuronal cul-
tures show intact neural circuits and synaptic integrity (pre-and
postsynaptic cross-talk). Aim of the present study was to develop a
fluorescence assay using a new and stable Ca
2+
dye (Fluo4) to

measure the depolarization-induced Ca
2+
influx in cultured rat
cortical neurons. In our experimental conditions, high K
+
depolar-
ization induced a very fast increase in [Ca
2+
]
i
, mainly due to Ca
2+
entry through voltage-dependent calcium channels (no increase in
absence of Ca
2+
ions in the medium) and partly to the depletion
of intracellular Ca
2+
storages (ryanodine receptor activation).
Using specific toxins able to block different calcium channel sub-
types, it was confirmed that R-, L- and P-type calcium channels
played the major role in KCl-mediated Ca
2+
influx. The rise in
Ca
2+
evoked by veratridine has to be ascribed only to Ca
2+
influx
through voltage-dependent calcium channels, as it was completely

abolished in absence of extracellular Ca
2+
. Our rat cortical neuron
preparations respond to pharmacological characterization with ref-
erence calcium and sodium channel blockers and therefore it could
be a suitable model for screening novel compounds with therapeu-
tic potential for channelopathies.
272 ª 2007 The Authors Journal compilation ª 2007 FEBS
Abstracts
F1-33
AGEs in experimental diabetic nephropathy: are
pyridoxal phosphate and thiamine
pyrophosphate beneficial ?
G. Burcak, M. Ozdogan and S. Ku
¨
c¸ u
¨
k
Department of Biochemistry, Cerrahpas¸ a Medical Faculty,
_
Istanbul
University,
_
Istanbul,
_
Istanbul, TURKEY
Increased advanced glycation end product (AGE) formation is the
major mechanism implicated in diabetic nephropathy (DN). In this
study we questioned the benefit of pyridoxal phosphate (PLP) and
thiamine pyrophosphate (TPP) in DN. In Wistar albino rats

grouped as ‘Diabetic’,’Diabetic+PLP’,’Diabetic+TPP’, ‘Dia-
betic+Insulin’ and ‘Control’, glucose HbA1c, AGE-peptides
(plasma and kidney), aldose reductase (AR) activity (kidney), 8-epi
PGF
2a
(urine), creatinine clearance, microalbuminuria and
b
2-
microglobulinuria were measured. Our data revealed the estab-
lishment of DN. AGE-peptides increased both in the kidney and
in the plasma of diabetic rats (P<0.05, P<0.01) and were
found to be correlated (P<0.01). AR acitivity did not display
any significant difference between the groups. 8-epi PGF
2a
was
higher in diabetic rats than in control (P<0.001). Insulin treat-
ment caused significant decreases in all parameters except renal
hypertrophy and plasma AGE-peptide levels. PLP supplementation
caused reduction in microalbuminuria and 8-epi PGF
2a
lev-
els.(P<0.01,P<0.05). TPP treatment appeared to have no
effect on the measured parameters. To conclude, our results sug-
gest that AGE-peptides in plasma reflect the AGE content in kid-
ney. PLP treatment slowed the progression of DN and decreased
glomerular injury. Considering the inhibitory effects of TPP and
PLP on advanced glycation and oxidative stress, further studies
addressing their role in DN are needed.
F1-34
The relationship of bone metabolism with nitric

oxide and cytokines in chronically ethanol
treated rats
S. Kaya
1
, I. Guner
2
, I. Birincioglu
3
, V. Sozer
4
, H. Uzun
1
,
S. Aydin
1
, Y. Karter
5
, C. Simsek
6
, G. Yigit
2
and G. Simsek
2
1
Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul
University, Istanbul, TURKEY,
2
Department of Physiology, Cer-
rahpasa Medical Faculty, Istanbul University, Istanbul, TURKEY,
3

The Republic of Turkey Ministry of Justice Council of Forensic
Medicine, Istanbul, TURKEY,
4
Yıldız Technical University Arts and
Science Faculty Department of Chemistry, Istanbul, TURKEY,
5
Department of Internal Medicine, Cerrahpasa Medical Faculty,
Istanbul University, Istanbul, TURKEY,
6
Department of Public
Healty, Cerrahpasa Medical Faculty, Istanbul University, Istanbul,
TURKEY
We investigated the effects of NO, IL-lb, IL-6 and TNF-a on bone
metabolism in chronically ethanol treated rats. Chronic ethanol
intake was produced in 6 months old rats by gradual substitution
(within 3 weeks) of tap water in diet for 5, 10, 15 and finally 20%
of ethanol after which the animals were maintained under these
conditions for 4 months. After 4 months of ethanol administration
N
w
-nitro-L-arginine methyl ester (L-NAME) which is a NOS inhib-
itor was given for three weeks along with ethanol to the same rats.
Blood samples takes from the tail vein of rats were analysed for
serum IL-lb, IL-6, TNFa, NO, Ca, P, PTH, 25(OH) D
3
, B-ALP.
Ethanol treatment increased both cytokine and NO levels. Ca
decreased. P, PTH and 25(OH) D
3
did not change. While the b-

ALP decreased there was no change in T-ALP. ALT, AST and
GGT increased. Simultaneous administration of ethanol and L-
NAME in the same rats increased IL-6 and TNF-a levels whereas
IL-lb were unchanged. Ca and P increased while PTH and
25(OH)D
3
decreased. T-ALP, b-ALP, ALT, AST and GGT
increased. In this study we observed that ethanol suppressed bone
turnover. Antiremodeling effect of ethanol may be mediated by NO
F1-35
Transgenic rabbit as a source of recombinant
human TNAP for pharmaceutical application
L. Bodrogi
1
, R. Brands
2
, W. Raaben
3
, W. Seinen
2
, M. Baranyi
1
,
D. Fichter
3
and Z. Bo} sze
1
1
Agricultural Biotechnology Center, Godollo, HUNGARY,
2

IRAS,
University of Utrecht, Utrecht, NETHERLANDS ANTILLES,
3
AM-Pharma B.V., Bunnik, NETHERLANDS ANTILLES
Transgenic rabbits are excellent bioreactors for the production of
recombinant proteins in their milk both on an experimental and
commercial scale. Alkaline phosphatases (AP) are promising thera-
peutic agents in the Gram negative bacterial lipopolysaccharide
(LPS) mediated diseases. LPS is dephosphorylated and thereby
detoxified by placental AP at physiological pH. In our earlier
experiments calf intestinal AP prevented 80% of mice from lethal
Escherichia coli infection and attenuated LPS toxicity in piglets.
Purified tissue non-specific AP (TNAP) is not available in large
quantities from tissue sources which would enable to analyse its
efficacy in animal sepsis models. We created two transgenic rabbit
lines by pronuclear microinjection with the whey acidic protein
promoter- humanTNAP minigene. Lactating females of both lines
produced enzymatically active human TNAP which was two orders
of magnitude higher compared to normal human serum levels and
the molecular weight of recombinant protein was compliant with
the authentic human form. After purification of the recombinant
human TNAP from rabbit milk its effectiveness will be tested in
animal sepsis models and it can be a valuable option with import-
ant implication in attenuating LPS mediated inflammatory
responses.
Acknowledgement: This work was supported by the grants
NWO-OTKA N37293 and GVOP-AKF 71/2004.
F1-36
Expression of osteonectin correlates with levels
of fin regeneration in zebrafish (Danio rerio)

A. Brito, L. Cancela and P. Gavaia
CCmar, University of Algarve, Faro, PORTUGAL
Mammals have the ability to regenerate some tissues such as blood
and liver, but the majority of organs fail to regenerate. In contrast,
zebrafish is capable of regenerating complex organs/tissues such as
optic nerve, scales, heart and fins, and is presently one of the most
used metazoan in regeneration research. Zebrafish fin is composed
of multiple fin rays with bony parts (lepidotrichia) originated by
intramembraneous ossification. Fin regeneration is an epimorphic
process dependent on formation of a specialized structure (blas-
tema), consisting of mesenchymal-like cells located between stump
tissues and the wounded epidermis, This mass of proliferative, plu-
ripotent progenitor cells is the key intermediate for strict growth
control and cell reprogramming leading to faithful restoration of
lost parts. Osteonectin, a glycoprotein that complexes with colla-
gen fibres and hydroxyapatite, was suggested to be involved in ini-
tiation of active mineralization, found in regenerated tissues and
its levels appeared to increase in tissues undergoing remodelling.
Our main goal was to determine the pattern of osteonectin expres-
sion during the first 96 h post amputation in zebrafish fin through
real-time PCR and whole-mount in situ hybridization. The results
showed a clear correlation between osteonectin expression and
bone formation.
Acknowledgements: This work was funded by FCT/POCI/
MAR/60883/2004 (XenoFish). AB Brito and PJ Gavaia are recipi-
ents of FCT fellowships CCMAR/BTI/0041/2006 and SFRH/BPD/
14528/2003.
ª 2007 The Authors Journal compilation ª 2007 FEBS 273
Abstracts
F1-37

Antihypertensive effect of a novel ACE-I
inhibitory peptide from bigeye tuna dark
muscle in spontaneously hypertensive rats
S. Kim, Z. Qian, M. Kim and S. Lee
Pukyong National University, Busan, REPUBLIC OF KOREA
To produce bioactive peptides of fish processing by-products, big-
eye tuna dark muscle was hydrolyzed using various enzymes (Alca-
lase, a-chymotrypsin, Neutrase, papain, pepsin, and trypsin) for
extraction of ACE I inhibitory peptide. Pepsin-proteolytic hydroly-
sates exhibited the highest ACE I inhibitory activity among the
others tested. An ACE I inhibitory peptide was purified by con-
secutive chromatographic methods using a Hiprep 16/10 DEAE
FF anion exchange column and an octadecylsilane (ODS) C18
reversed phase column. In the result of N-terminal amino acid
sequencing analysis, the peptide purified from pepsin-digests of
bigeye tuna muscle protein (BTMP-Pepsin) is a dodeca-oligopep-
tide with rich trypsin in c-terminus (WPEAAELMMEVDP;
1.5 kDa of molecular weight). The ACE I inhibitory peptide,
BTMP-Pepsin, exhibited potent inhibitory activity with 21.6 lMof
ACE IC
50
value, and it was evaluated as a non-competitive inhib-
itor against ACE I in the assay for inhibitory pattern by Lineweav-
er-Burk plotting. Antihypertensive effect in spontaneously
hypertensive rats (SHR) also revealed that oral administration of
BTMP-Pepsin can decrease systolic blood pressure significantly
(P<0.05). In addition, MTT assay showed no cytotoxicity on
human embryonic lung fibroblasts cell line (MRC-5). The result of
this study suggests that ACE inhibitory peptides derived from
BTMP could be potential candidates to develop nutraceuticals and

pharmaceuticals.
F1-38
Genetic variation in rocky mouse, Apodemus
mystacinus (Danford and Alston, 1877)
(Mammalia: Rodentia) in Turkey
R. C¸ olak
1
, G. Olgun
1
, I. Kandemir
2
,E.C¸ olak
1
and N. Yig
˘
it
1
1
Ankara University, Faculty of Science, Department of Biology,
Ankara, TURKEY,
2
Karaelmas University Faculty of Science &
Art, Department of Biology, Ankara, TURKEY
Apodemus mystacinus is widely distributed from Balkans to Middle
East and Caucasus. Three subspecies of A. mystacinus are distribu-
ted in Turkey. Despite of several previous studies, the taxonomic
status of A. mystacinus in Turkey is problematic. The aim of the
this study was to survey genetic structure based on DNA markers
and to contribute to the taxonomy and population genetics of
A. mystacinus in Turkey. A total of 22 specimens were collected

from 11 locations in Turkey. To explore the extent of genetic var-
iation in A. mystacinus populations, a Randomly Amplified Poly-
morphic DNA marker system was used. The estimates of NEI’s
standart genetic identity and genetic distance were calculated to
show the genetic relationships between studied populations. All
estimations were calculated with the POPGENE software. With
the 60 RAPD markers tested, 14 of them yielded 147 polymorphic
DNA bands. The estimated genic diversity for A. mystacinus popu-
lations was ranged from H = 0.0227 (P=4.55) in Trabzon to
0.2045 (P = 40.91%) in Mugla population. The total gene diver-
sity was calculated as H
T
= 0.3087 in A. mystacinus populations.
G
ST
value calculated was high (0.7438) indicating that genetic dif-
ferentiation among the studied populations was substantial. Dend-
ogram constructed with genetic distance data contained 3 clusters.
The groupings in the A. mystacinus cluster were consistent with the
previously assigned subspecific categories. The implication of the
results with respect to the genetic variation of subspecies was also
discussed.
F1-39
Immunohistochemical distribution of the novel
peptide alarin in the adult rat brain
N. Eberhard
1
, S. M. Schmidhuber
1
, I. Rauch

1
, G. Sperk
2
and
B. Kofler
1
1
Department of Pediatrics, Paracelsus Private Medical School, Salz-
burg, AUSTRIA,
2
Department of Pharmacology, University of Inns-
bruck, Innsbruck, AUSTRIA
Recently, the expression of a splice variant of the Galanin-like pep-
tide (GALP) gene, named Alarin, was observed in gangliocytes of
human neuroblastic tumors and different neuronal tissues. Given
that GALP has potent species-specific and time-dependent effects
in the central nervous system and has been suggested to constitute
a link between metabolism and reproduction, we aimed to deter-
mine the distribution of Alarin in the rat brain. For immunohisto-
chemical studies, an affinity purified polyclonal antibody directed
against synthetic murine Alarin peptide was used. Alarin-like-im-
munoreactivity (Alarin-LI) was observed mainly in the basal gan-
glia of the rodent brain, which was further confirmed by RT-PCR.
Additional immunostained areas were observed in the amygdala,
in the pirifom cortex, and in the CA1, stratum lacunosum molecul-
are of the hippocampus. Relatively dense staining was also noted
in the tuberomammillary nucleus (TM), a cluster of magnocellular
cells in the posterior hypothalamus, which is the main source of
neuronal histamine in the brain. The expression of Alarin in cells
of neuronal origin, mainly in the basal ganglia and the TM indi-

cates possible association in motor control, emotion, cognition and
learning.
Acknowledgement: Supported by the Paracelsus Private Medical
University
F1-40
Response of gingival mast cells in
streptozotocin-induced type 2 diabetic rats
N. Ozsoy
Department of Biology, Faculty of Science, Ankara University,
Ankara, TURKEY
Diabetes is a complex disease, which triggers various complications
including tissue destruction mechanisms. One of these complica-
tions is gingivitis. Occurrence of gingival tissue damage in diabetes
is well known, but its mechanism has not yet been adequately
explained at cellular and molecular levels. Although type 2 diabetes
comprises 80% of all diabetics, type 1 diabetic cases are more fre-
quently investigated. Cytological studies in especially type 2 diabet-
ics and their conclusions are far less than desired. Experimental
diabetes should be a logical method for investigating oral changes
due to diabetes. In this study, diabetes has been induced in neona-
tal male rats by intraperitoneal injection of 90 mg/kg streptozoto-
cin (STZ). The effects of experimental diabetes on gingival mast
cell responses were investigated ultrastructurally. The ultrastructur-
al study of the mast cell demonstrated mast cells had evidence of
secretion. Gingival biopsies obtain from rats with type 2 diabetes
mellitus showed more mast cell secretion than control biopsies.
Mast cell exhibited an atypical degranulation, which included cyto-
plasmic granules filled by partially or fully dissolved granular mat-
rices. It was found that the integrity of the membrane of some
granules deteriorated and a common membrane surrounded some

of them. There was pycnosis in the nuclei of some mast cells, and
granules having less dense dissolved granule matrix material filling
were noted around them. Some mast cells presented a number of
cytoplasmic secretory granules containing less dense, irregular
threads. These ultrastructural features suggest that diabetes causes
abnormal mast cell degranulation which in turn triggers gingival
tissue breakdown associated with diabetes mellitus.
274 ª 2007 The Authors Journal compilation ª 2007 FEBS
Abstracts
F1-41
Effects of vitamin C on muscle ultrastructure in
experimental diabetes
S. Cebesoy,N.O
¨
zsoy and N. Gu
¨
l
Ankara U
¨
niversity, Ankara, TURKEY
The diabetic effects of streptozotocin (STZ) were investigated in
gastronecmius muscle. In the study, 38 male adult Wistar Albino
rats with weights 200 ± 20 g were used by separating them into
four groups: control, vitamin C, diabetes, diabetes + vitamin C.
Vitamin C is an important antioxidant. Streptozotocin in sterile
physiological saline was injected into animals intravenously. The
ultrastructure of type IIa, type IIb and type I fibers in gastronecm-
ius muscle of control and STZ diabetic rats and STZ diabetic rats
with vitamin C was studied. In diabetic animals the mitochondria
of type IIa and type I fibers showed a loss of cristae. There was

also an increased number of lipid droplets. The shape of the nuclei
was highly irregular in these fibers. The type IIb fibers showed
some disorientation of the T-tubuler system. STZ-diabetes has dif-
ferential effect on the fine structure of the three fiber types
&#305;n rat skeletal muscle. &#304;n diabetic rats with vitamin C
mitochondria of type IIa and type I showed alittle a loss of cristae
.The muscle fibers showed normal orientation of T-tubuler system.
In this fibers there was also decreased number of lipid droplets.
F1-42
Involvement of gastrin peptides in iron
metabolism
S. Kovac
1
, K. Smith
1
, J. R. Burgess
2
, A. Shulkes
1
and
G. S. Baldwin
1
1
University of Melbourne, Melbourne, AUSTRALIA,
2
Royal Hobart
Hospital, Hobart, AUSTRALIA
We have reported that circulating gastrin concentration is
increased in plasma of mice and humans with HFE-related hemo-
chromatosis. Due to the intriguing relationship between circulating

gastrins and iron status, the aim was to investigate whether gastrin
peptides are involved in iron homeostasis. Using CCK2 receptor
knockout mice (CCK2R
-/-
) which are hypergastrinemic, we ana-
lyzed liver iron and the status of several proteins involved in iron
homeostasis in 4 and 10 week old mice. While no significant differ-
ence was observed in 4 week old mice, hepatic iron concentration
was significantly higher in 10-week-old mice compared to the con-
trol mice. Using real-time PCR we analysed the mRNA expression
of hepcidin, a putative iron homeostasis regulator and duodenal
DMT-1, the divalent metal transporter. Although no difference
was found in hepcidin mRNA expression, the DMT-1 mRNA
expression was significantly increased in the 10-week-old mice com-
pared to control mice. Next we investigated the serum transferrin
saturation (sTS) and observed a 50% increase in CCK2R
-/-
mice.
This increase in sTS was also correlated with circulating gastrin
concentration in patients presenting with hypergastrinemia due to
multiple endocrine neoplasia type 1. Our data reveal a previous
unrecognised reciprocal relationship between circulating gastrins,
sTS and iron status. The role of gastrins as novel iron homeostasis
regulators requires further investigation.
F1-43
Inhibition of amyloid fibrillation of lysozyme by
indole derivatives
D. Morshedi
1
, N. Rezaei-Ghaleh

1
, A. Ebrahim-Habibi
1
,
A. Ghasemi
1
, S. Ahmadian
1
and M. Nemat-Gorgani1
1,2
1
Institute of Biochemistry and Biophysics, University of Tehran,
Tehran, ISLAMIC REPUBLIC OF IRAN,
2
Stanford Genome
Technology Center, Stanford University, Palo Alto, CA, USA
Amyloid aggregation of polypeptides is related to some pathologic
states known as amyloidoses. There is a great deal of interest in
developing small molecule inhibitors of the amyloidogenic proces-
ses. In the present communication, the inhibitory effect of a num-
ber of indole derivatives on hen egg white lysozyme (HEWL)
fibrillization is reported. Acidic pH and high temperatures were
used to drive HEWL toward amyloid formation A variety of
techniques, ranging from ThT fluorescence and Congo red absorb-
ance assays to far-UV CD and transmission electron microscopy
were employed to characterize the HEWL fibrilization process.
While Indole-3-acetate (IAA) did not protect the HEWL native
state from conformational changes, it was found effective in dimin-
ishing the HEWL amyloid fibril formation, delaying both the
nucleation and elongation phases of fibrilization. Disaggregation of

previously formed HEWL amyloid fibrils was also enhanced by
IAA. Amongst the other indole derivatives tested, Indole-3-Carbi-
nol (I3C) and tryptophol were found superior to IAA regarding
their inhibitory effect,. These observations, taken together, suggest
that the indole ring is likely to play the main role in inhibition and
the side chain hydroxyl group may also contribute positively, in
contrast to the side chain carbonyl and intervening methylene
groups.
F1-44
Brain aromatase of gilthead seabream (Sparcus
aurata) using dibenzylfluoresce in
D. Hancer and A. Sen
Department of Biology, Faculty of Arts and Sciences, Pamukkale
University, Denizli, TURKEY
Family 19 of the P450 super family is responsible for the conver-
sion of androgens into estrogens. Data on native aromatase
enzyme kinetics and thus actual catalytic activity are scarce in fish.
In the present study, fluorescence aromatase assay was optimized
to measure aromatase activity (AA) in the gilthead seabream (Spa-
rus aurata) using dibenzylfluorescein (DBF) as a fluorometric sub-
strate in brain and liver microsomes. Brain and liver AA have
showed lineerity until 20 and 40 lg protein concentration through-
out 30 min, respectively. Optimum pH of brain and liver AA have
found to be 6.50 and 8.25, respectively and optimum temperature
have found to be 30°C for two of them. Brain and liver enzyme
activities have saturated at and above 2 lM DBF concentrations.
Apparent V
max
and K
m

values of brain and liver aromatase using
Lineweaver-Burk graph are calculated 10.5 and 10.0 pmol/min/mg
protein and 1.49 and 1.38 lM, respectively. Testosterone appeared
to competitively inhibit the fish brain and liver aromatase. Our
laboratory has been interested in the identification of Turkish tra-
ditional dietary plants that can effect AA. These findings could
lead us to identify the plants that could be a potential dietary
source for novel aromatase inhibitors. Immunochemical characteri-
zations are underway using anti-rat aromatase IgGs. The kinetic
parameters of this assay can be useful to measure AA in other
species.
ª 2007 The Authors Journal compilation ª 2007 FEBS 275
Abstracts
F1-45
Inhibition of bovine liver glutathione reductase
with nickel ions
B. Tandog
˘
an and N. N. Ulusu
University of Hacettepe Faculty of Medicine, Ankara, TURKEY
The homodimeric FAD-containing glutathione reductase (GR)
belongs to the family of NADPH-dependent oxidoreductases and
is present in many pro- and eukaryotic organisms. GR is import-
ant enzyme in maintaining the reduced state of the cell and in spe-
cialized pathways. Inhibition of the enzyme has been attractive
approach for various purposes such as the development antimalar-
ial and anticancer agents. In this study we have tested effects of
the some metal ions on the purified bovine liver GR. Among these
metals, calcium (Ca
2+

), lithium (Li
+
) have no effect on bovine
liver GR but nickel (Ni
2+
) showed inhibitory effect on the enzyme.
The obtained IC
50
value of Ni
2+
is 0.8. Kinetic characterization of
the inhibition is also investigated. Ni
2+
inhibition is non-competit-
ive with respect to GSSG (Ki
GSSG
0.313 ± 0.01 mM) and uncom-
petitive with respect to NADPH (Ki
NADPH
0.932 ± 0.03 mM).
Nickel, is the most powerful metal carcinogen, is capable of in vivo
binding with the cell nucleus and causing promutagenic damage.
Keywords: bovine liver, glutathione reductase, inhibition, kinet-
ics, metal ions, nickel
Acknowledgement: This work is a part of the project (02 G085)
supported by Hacettepe University Scientific Research Unit.
F1-46
Inhibition of bovine liver glutathione reductase
with cadmium ions
B. Tandog

˘
an and N. N. Ulusu
University of Hacettepe Faculty of Medicine, Ankara, TURKEY
Glutathione reductase (GR, NADPH: oxidized glutathione oxido-
reductase, E.C 1.6.4.2) is a flavoprotein that catalyzes the
NADPH-dependent reduction of oxidized glutathione (GSSG) to
reduced glutathione (GSH). In this study we have tested effects of
the some metal ions on the purified bovine liver GR. Among these
metals, aluminium (Al
3+
), barium (Ba
2+
), have no effect on
bovine liver GR but cadmium (Cd
2+
) showed inhibitory effect on
the enzyme. The obtained IC
50
value of Cd
2+
is 0.08. Kinetic char-
acterization of the inhibition is also investigated. Cd
2+
inhibition
is non-competitive with respect to both GSSG (Ki
GSSG
0.221 ± 0.02 mM) and NADPH (Ki
NADPH
0.113 ± 0.008 mM).
Because of GR is a crucial enzyme in the antioxidant system; this

study may be a useful for understanding the mechanisms for oxi-
dative damage associated with heavy metal toxicity.
Keywords: bovine liver, glutathione reductase, inhibition, kinet-
ics, metal ions, nickel
Acknowledgement: This work is a part of the project (02 G085)
supported by Hacettepe University Scientific Research Unit.
F1-47
Kinetic properties of glutathione reductase from
bovine liver
N. N. Ulusu and B. Tandogan
Hacettepe University, Ankara, TURKEY
Glutathione reductase is a crucial enzyme, catalyzing the NADPH-
dependent reduction of GSSG to GSH. GR is mainly localized in
cytosol, mitochondria, and chloroplast. Glutathione reductase is
essential for the antioxidative system that maintains adequate lev-
els of reduced cellular GSH. Because of glutathione reductase that
protect cells from ROS-induced damage, knowing molecular and
catalytic properties of this enzyme have a vital role for the organ-
ism. Kinetic characterization of bovine liver GR was also investi-
gated, Km(NADPH) 0.063 ± 0.008 mM and Km(GSSG)
0.154 ± 0.015 mM were determined. It is accepted that parallel
lines observed in these double reciprocal plots obeys Ping Pong
mechanism and we have showed this in our steady state study.
According to our results of statistical analysis, the Ping Pong
mechanism is a suitable model since the loss function is less than
the other mechanisms. However, competitive inhibition by a prod-
uct could be accepted in sequential mechanisms but not in a Ping
Pong mechanism. In this study, kinetic data are consistent with a
branching reaction mechanism previously proposed for GR from
other sources by other studies.

Keywords: branched mechanism, glutathione reductase, kinetic
mechanism, Km, Vm
Acknowledgement: This work is a part of the project (02 G085)
supported by Hacettepe University Scientific Research Unit.
F1-48
Purification and some molecular properties of
bovine liver glutathione reductase
N. N. Ulusu and B. Tandogan
Hacettepe University, Ankara, TURKEY
Glutathione reductase (GR, NAD(P)H: oxidized glutathione ox-
idoreductase, EC 1.6.4.2) catalyses the reduction of oxidized gluta-
thione (GSSG) to reduced glutathione (GSH) using NADPH as
reducing cofactor. GRs had been purified from a variety of
sources, from bacteria to mammalian cells, show great similarity
both in physical and kinetic parameters, with different purification
folds and yields. Glutathione reductase from the liver of bovine
was purified to homogeneity by means of two subsequent chroma-
tography steps using 2’ 5’ ADP-Sepharose 4B and DEAE-Seph-
arose fast flow columns. The enzyme has been purified 5456-fold
and 38.4 yields from the bovine liver. The molecular properties of
bovine liver glutathione reductase have been studied under a vari-
ety of experimental conditions. Optimum pH and temperature was
found to be 50°C and 70, respectively. The activation energy of the
reaction catalyzed by the enzyme was 9.065 kcal/mol. The molecu-
lar weight of the enzyme was found to be 55 kDa by SDS-PAGE.
Keywords: bovine liver; glutathione reductase; molecular proper-
ties, purification; SDS-PAGE
Acknowledgement: This work is a part of the project (02 G085)
supported by Hacettepe University Scientific Research Unit.
276 ª 2007 The Authors Journal compilation ª 2007 FEBS

Abstracts
F1-49
Chemical modification of Lys residues prevents
TFE-induced aggregation of
a-chymotrypsin
A. Ghasemi
1
, N. Rezaei-Ghaleh
1
, A. Ebrahim-Habibi
1
and
M. Nemat-Gorgani
1,2
1
Institute of Biochemistry and Biophysics, Tehran, ISLAMIC
REPUBLIC OF IRAN,
2
Stanford Genome Technology Center,
Stanford University, Palo Alto, CA, USA
Protein aggregation has been shown to be a prominent feature of
many neurodegenerative diseases. On the other hand, this process
is also a plague in almost all stages of protein drug development
and industrial processes involving proteins. In this regard, studies
on model proteins which could be driven toward aggregation states
become more interesting. One of the issues addressed in these stud-
ies is the investigation of methods to prevent aggregation. In the
present study, modification of lysine residues in a-chymotrypsin
was carried out using acetic anhydride and the number of modified
residues was determined by employing fluorescamine. There are 14

lysine residues in this enzyme, from which a maximum number of
8 have been modified. By using different amounts of acetic anhy-
dride, modified enzyme preparations were obtained containing 6, 7
and 8 acetylated lysine residues with no changes of concomitant
catalytic activity. While extensive aggregation was observed in the
native structure upon incubation in% 32 TFE (30 min), no aggre-
gates could be detected for any of the modified forms. Changes in
the structural properties of the protein were investigated by intrin-
sic fluorescence and some preliminary circular dichroism. These
studies show that acetylation of lysine residues prevents TFE-
induced aggregation of a-chymotrypsin.
F1-50
Effect of amino acids on thermal aggregation of
glutamate dehydrogenas e
M. Sabbaghian
1
, A. Ebrahim-Habibi
1
and M. Nemat-Gorgani
1,2
1
Institute of Biochemistry and Biophysics, Tehran, ISLAMIC
REPUBLIC OF IRAN,
2
Stanford Genome Technology Center,
Stanford University, Palo Alto, CA, USA
Protein aggregation is a major problem both in laboratories and
industries since it results in the formation of insoluble and inactive
aggregates in vitro. Moreover some neurodegenerative diseases are
related to deposition of these structures. Thus finding general strat-

egies to prevent the inactivation and aggregation of proteins is of
great interest. One of the major approaches in this regard is the
use of small molecular additives. In this study thermal aggregation
of GDH was investigated in the presence of a number of amino
acids. Among these, Gly and Ala were moderately effective while
Lys, Leu, Arg and Pro were found to have noticeable inhibitory
effects. These amino acids were also effective in protecting the
enzyme against loss of activity. While total loss of activity was
observed for the control in less than 10 minutes by heating at
50°C, 80% of activity remained in the presence of arginine and
lysine and 40% in the presence of proline. It thus appears, as also
supported by the intrinsic fluorescence data, that these amino acids
may protect the protein structure against thermal denaturation
leading to aggregation. Lysine and arginine were also found useful
in stopping protein aggregation when added after the process was
initiated. Based on these observations it is clear that Arg, Pro and
Lys protect the enzyme against thermal inactivation by stabilizing
protein structure.
F1-51
Characterization of DHHC6, a novel human
putative palmitoyl-transferase
M. Corral-Escariz and I. Rodrı
´
guez-Crespo
Universidad Complutense de Madrid, Madrid, SPAIN
Recent studies have determined that the human DHHC9/hErf4
complex has palmitoyl-transferase activity for human H-Ras. Fol-
lowing this initial discovery, palmitoyl-transferase activity has also
been observed for several members of the DHHC-CRD (Asp-His-
His-Cys-Cysteine Rich Domain) palmitoyl transferases using PSD-

95 and eNOS as acceptor proteins. This ability to transfer palmi-
tate has been assigned to proteins that display a catalytic DHHC
motif that using palmitoyl-CoA as substrate, transfer the palmitate
moiety to various substrates. Inspection of the human genome
reveal that 23 different genes with DHHC-CRD motifs are present.
We have cloned DHHC6 a human member of the DHHC-CRD
family of proteins that displays a consensus N-terminal sequence
for N-myristoylation. Site directed mutagenesis followed by label-
ling with radioactive fatty acids allowed us to conclude that
DHHC is also palmitoylated at position Cys5. Immunofluores-
cence studies reveal that the dually acylated protein (myristoylated
plus palmitoylated) is not enriched in caveolae/rafts. In addition,
wild-type DHHC6 becomes concentrated in perinuclear areas, dis-
playing significant colocalization with the Golgi marker ß-cop.
Since the protein is predicted to possess four transmembrane heli-
ces, we cloned the first 11 amino acids of DHHC6 an determined
the targeting properties of the acylation motifs individually. RT-
PCR experiments indicate that DHHC6 is expressed in numerous
tissues and cell lines. In order to identify substrates of DHHC6,
proteins known to become palmitoylated in vivo were cotransfected
with this palmitoyl-transferase and the incorporation of palmitate
was determined.
F1-52
Activity determination of enzymedextran
sulphate conjugates
C. Arıo
¨
z,O
¨
.O

¨
ztolan, M. C¸ elebi and H. Kuzu
Yildiz Technical University, Istanbul, TURKEY
a-Amylases (a-1.4 glucan-4-glucanohydrolase, EC 3.2.1.1) are
monomeric enzymes that catalyse the hydrolysis of internal a-D-
(1.4) glucosidic linkages in starch and related oligo- and polysac-
charides with release of malto-oligosaccharides and glucose in the
a-anomeric form (1), they do not attack to a-D- (1.6) glucosidic
bonds. This enzyme is used in starch, detergent, paper and fabric
industries; also in production of beer and fruit juice. Proteases are
one of the most important enzymes which have been used in the
daily industry as milk-clotting agents (rennet) for the manufacture
of cheese (2). Mucor miehei rennet (proteinase) which is specific
cleavege of the casein micelle-stabiling protein j-casein, at the phe-
nylalanin (105)-methionine (106) peptide bond. In food industry,
to produce good Quality product and prevent some difficulties,
water soluble conjugates of mucor miehei rennet with dextran sul-
phate would benefit. Protein-polysaccharide conjugates, that is nat-
ural, non-toxic, and have improved functional properties, have
significant potential for use in the food and health industries. In
this study, commercial enzymes are purified with gel filtration
chromatography at +4°C. Covalent conjugates of these purified
enzymes with dextran sulphate in different ratios were synthesized.
Activities of purified enzyme and conjugates measured in different
pHs and temperatures.
Keywords: a-amylase, dextran sulphate, mucor miehei rennet
ª 2007 The Authors Journal compilation ª 2007 FEBS 277
Abstracts
F1-53
Activity determination of HRP-Dextran

conjugates in different pH values
Y. Bas¸ aran, M. Altıkatog
˘
lu, C. Arıo
¨
z, Y. Sarıc¸ ay and H. Kuzu
Yildiz Technical University, Istanbul, TURKEY
Horseradish peroxidase (HRP) is an important heme enzyme with
enormous medical diagnostic, biosensing and biotechnological
applications. The enzyme has been characterized as a single poly-
peptide chain consists of 308 residues, with an N-terminal residue
blocked by pyroglutamate. It is heavily glycosylated and contains
a single protoporphyrin IX as a prosthetic group, two calcium
ions, four disulfide bonds, and eight N-linked carbohydrate chains.
The structure can be modified by using polysaccharide derivatives
containing aldehyde groups prepared via periodate oxidation of
the parent polymers. These polyfunctional polymers can react with
primary amino groups placed in enzymes. In general, polysaccha-
rides are so much effective in stabilizing enzymes because of the
increased rigidity as well as hydration of the enzyme molecule
caused by multi attachment of polyhydroxyls. In this study, HRP
was purified by Affinity chromatography with con-A (concanavalin
A). HRP-Dextran conjugates in different ratios were synthesized
and the synthesized conjugates were characterized by Viscotek.
One of these synthesized conjugates was chosen for determining
activities in different pH values and the activities were also deter-
mined for pure HRP (Horseradish peroxidase). The results for
both were compared with each other and the thermal stability for
the chosen ratio was also measured.
Keywords: conjugation, dextran, different pH activities, HRP

F1-54
Thermal stability of HRP-dextran conjugates
M. Altikatoglu, C. Arioz, Y. Basaran and H. Kuzu
Yildiz Technical University, Istanbul, TURKEY
Peroxidases are heme enzymes that participate as catalysts in oxi-
dative reactions where hydrogen peroxide is commonly the electron
acceptor. HRP (Horseradish Peroxidase) is a well-known and
highly investigated member of the peroxidase superfamily I. Horse-
radish peroxidase (HRP, E.C.1.11.1.7) catalyzes the oxidation of
aromatic hydrogen donors by hydrogen peroxide through a two-
electron transfer pathway. In biotechnology, stabilities of the
enzymes in vitro are still being one of the most important issues.
Storage and operational stabilities are both important for the
usage of enzymes. Different approaches have been utilized to
improve enzyme stability, including genetic engineering, chemical
modification, inclusion of additives and immobilization to various
matrices. Covalent conjugation of the enzymes with water soluble
polymers can lead to improvement of the stability. In this study,
HRP (Horseradish Peroxidase) were purified with affinity chroma-
tography. HRP (Horseradish Peroxidase)-dextran covalent conju-
gates were prepared in different ratios. Thermal stability of these
conjugates and pure HRP were studied. The activity results for
thermal stability of these conjugates were compared with pure
enzyme activities.
Keywords: dextran, HRP, peroxidase, thermal stability
F1-55
Trehalases from the midgut of the Lepidoptera
Spodoptera frugiperda (Sf)
M. C. P. Silva, W. R. Terra and C. Ferreira
Universidade de Sao Paulo, Sao Paulo, BRAZIL

Trehalase mobilizes the circulating sugar trehalose that insects use
as energy. It is present in every insect tissue and might be a good
target for insect control. There is no 3D structure or definition of
catalytical groups for trehalases of any source. As part of a wider
study, our aim here is to compare the soluble(ST) and membrane-
bound trehalase (MBT) from Sf larvae. We had already purified
and characterized the ST(Insect Biochem. Molec. Biol. 2004; 34:
1089).MBT-activitity recovery is improved when PMSF is added
to the homogenizing media, suggesting that MBT is solubilized by
an endogenous serine proteinase during preparation. MBT was
purified by a series of chromatographic steps and has 67 kDa, a
specific activity of 10 U/mg and is less thermaly stable them ST.
The corresponding cDNAs of both enzymes were cloned and se-
quenced. The deduced sequences of ST and MBT have, respect-
ively, 578 and 652 amino acid residues, a signal peptide of 23 and
18 residues, and both present six potential N-glycosylation sites.
MBT shows a transmembrane region spanning from S 586 to M
607. Recombinant ST was expressed and purified, showing the
same properties of the enzyme purified from insect midgut. Anti-
body raised against ST do not recognize MTB. The most parcimo-
nious tree using insect trehalase sequences shows that membrane
bound and soluble enzymes are separated in two groups and that
Sf ST and MBT are more similar with Bombyx mori soluble and
membrane-bound enzymes, respectively, than with each other.
Acknowledgement: Supported by FAPESP and CNPq.
F1-56
Two digestive cysteine proteinases from
Dysdercus peruvianus
T. D. Bifano and W. R. Terra
Universidade de Sa

˜
o Paulo, Sa
˜
o Paulo, BRAZIL
Hemipterans and most coleopterans are the only insects that have
cysteine proteinase (Cys) as a digestive enzyme. The midgut of D.
peruvianus have three sections (V
1
,V
2
and V
3
). V
2
contains most
Cys activity. It was homogenized, centrifuged and the supernatant
was applied onto an anion-exchange column. The eluted active
fractions were pooled and, after a concentration step, the pool was
submitted to gel filtration. Two activity peaks were resolved and
separately loaded onto an affinity column. Cys activity was monit-
ored with carbobenzoxy-Phe-Arg-7 amido 4 methyl coumarin (Z-
FR-MCA) as substrate. Two Cys were purified. Cys 1 and cys 2
have 32 and 45 kDa, respectively, the same pH optimum of 6.3
and show identical thermal inactivation profiles, following appar-
ent first-order kinetics with a half-life of 90 min at 40ordm;C. Both
Cys are inhibited by E-64 with a K
D
of 17.3 nM (Cys 1) and
7.11 nM (Cys2). The kinetical parameters of Cys 1 and Cys 2 on
two substrates are (Km, lM): Cys 1 on Z-FR-MCA (6.9) and on

Z-RR-MCA (9.1) and Cys 2 on Z-FR-MCA (5.3) and on Z-RR-
MCA (6.8). Both Cys are more active on Z-FR-MCA than on Z-
RR-MCA, suggesting they are cathepsins-L (CALs). Four
sequences were identified by random sequencing of a normalized
expression midgut cDNA library as CALs, being 3 contigs with
869, 818, and 812 bp and one singlet with 711 pb. The search for
correlation between the purified enzymes with those sequences is in
progress.
Acknowledgement: Supported by FAPESP and CNPq.
278 ª 2007 The Authors Journal compilation ª 2007 FEBS
Abstracts
F1-57
Role of the glucose-6-phosphate translocase
in the short-term regulation of
glucose-6-phosphatase by cAM P
M. Soty, A. Gautier-Stein and G. Mithieux
INSERM U855, Lyon, FRANCE
Glucose-6-phosphatase (G6Pase) catalyses the hydrolysis of glu-
cose-6-phosphate (G6P) in glucose. This enzymatic system consists
in two major proteins: a G6Pase catalytic subunit (G6PC) and a
G6P translocase (G6PT). The genetic studies showed the import-
ance of these two proteins for the optimal functioning of the sys-
tem. However, the relative contribution of each and the role of
their association are incompletely understood. We investigated the
role of cAMP, the second messenger of glucagon, in a short-term
regulation mechanism of G6Pase activity, and the potential role of
the two subunits in this regulatory process. Caco-2 cells were cho-
sen because they are known to exhibit the capacity of glucose pro-
duction, including expression of endogenous G6Pase gene. We
studied the effects of G6PC overexpression by adenoviral treat-

ment (AdCMV-G6PC) in the presence of cAMP and/or of a G6PT
specific inhibitor (S4048). These experiments were performed at
37°C and 21°C. After viral treatment, there was a 17-fold increase
in enzymatic activity compared to control cells. 1 h treatment of
10
-4
M forskolin (FK) (adenylate cyclase activator) promoted a
70% stimulation of G6Pase activity in transduced cells (untreated,
2.80 ± 0.51; FK-treated, 4.73 ± 0.92 lMol/h/mg de prot), with
no increase in the amount of G6PC protein. This effect of FK was
abolished at 21°C, a temperature known to inhibit membrane flu-
idity. Moreover, S4048 inhibited the G6Pase activation stimulated
by FK in filipin-treated cells (a treatment preserving intracellular
membranes) and after membrane solubilization by CHAPS. We
report the first evidence of the existence of a post-traductional
mechanism of regulation of G6Pase by cAMP and that this pro-
cess was dependant on both the membrane fluidity and the pres-
ence of functional G6PT.
F1-58
Identification of a novel CoA synthase isoform
I. Nemazanyy, O. Breus and V. Filonenko
Institute of Molecular Biology and Genetics, Kyiv, UKRAINE
CoA synthase is a bifunctional enzyme which mediates the final
stages of CoA biosynthesis. CoA and its derivatives are important
players in cellular metabolism and signal transduction. In previous
studies, we have reported molecular cloning, biochemical charac-
terization, and subcellular localization of CoA synthase (CoASy).
We described the existence of a novel CoA synthase isoform,
which is the product of alternative splicing and possesses a 29 aa
extension at the N-terminus. We termed it CoASy beta and origin-

ally identified CoA synthase, CoASy alpha. The transcript specific
for CoASy beta was identified by electronic screening and by RT-
PCR analysis of various rat tissues. The existence of this novel iso-
form was further confirmed by immunoblot analysis with antibod-
ies directed to the N-terminal peptide of CoASy beta. In contrast
to CoASy alpha, which shows ubiquitous expression, CoASy beta
is primarily expressed in the brain. Using confocal microscopy, we
demonstrated that both isoforms are localized on mitochondria.
The N-terminal extension does not affect the activity of CoA syn-
thase, but possesses a proline-rich sequence which can bring the
enzyme into complexes with signalling proteins containing SH3 or
WW domains.
F1-59
A study of thermally induced changes in the
structure and activity of yeast hexokinaseII
H. Ramshini
1
, N. Rezaei-Ghaleh
1
, A. Saboury
1
and
M. Nemat-Gorgani
1,2
1
Institute of Biochemistry and Biophysics, Tehran,
ISLAMIC REPUBLIC OF IRAN,
2
Stanford Genome Technology
Center, Stanford University, Palo Alto, CA, USA

Hexokinase is the first enzyme in glycolytic pathway but despite
it’s importance, relatively few thermal studies have been carried
out on this protein. In the present study, various spectroscopic
techniques have been employed to investigate thermal stability of
the yeast hexokinase II. Our results indicate that the enzyme
undergoes an irreversible conformational transition between 60
and 65ordm;C, when it is heated up to 65ordm;C. However, the
structural transition proved to be significantly reversible when the
heating process is stopped at temperatures lower than 60ordm;
C. The reversible character of thermal unfolding, and the fact that
this process was not kinetically controlled allowed us to calculate
some thermodynamic parameters related to this process. Our find-
ings demonstrate that the purely conformational transition of yeast
hexokinase II is reversible, and irreversibility of the process is
caused by side reactions. The possibility of chain fragmentation/
cleavage was excluded, but protein aggregation may be involved
and contribute to the mechanism. Thermoinactivation studies also
indicated that the enzymatic activity significantly diminishes after
10 min, with much greater loss of activity observed at temperatures
above 55ordm;C, possibly due to rapid deamidation of a critical
unprotected Asn.
F1-60
The functional roles of allosteric site glutamate 59
involving the human malic enzyme regulation
H. Hung
1
, K. Chang
1
and G. Liu
2

1
National Chung-Hsing University, Taichung, TAIWAN,
2
Chung-Shan Medical University, Taichung, TAIWAN
Human mitochondrial NAD(P)
+
-dependent malic enzyme is a reg-
ulatory enzyme allosterically activated by fumarate. Structural
studies have revealed that Arg67 and Arg91 are the direct ligands
for fumarate in the allosteric site. Site-directed mutagenesis further
demonstrates that Glu59, a structural neighborhood of Arg67, also
play an important role on the enzyme activation. In the present
study, Glu59 is substituted to Asp, Gln, Arg and Leu. The kinetic
experiments demonstrate that the K
m
and k
cat
values did not show
significant changes among the WT and E59 mutant enzymes in the
absence of fumarate. Substitution of Glu to Arg and Leu causes
the enzyme insensitive to fumarate. The wild-type enzyme is fully
activated by fumarate about 1.4-fold. The E59Q enzyme can be
activated similar to WT but occurs in higher fumarate concentra-
tion. The E59D mutant enzyme, however, can be significantly acti-
vated by fumarate more than WT several times. We further
examine the activation constants of these enzymes. The K
act
and
aK
act

values in E59D and E59Q were notable higher than WT,
indicating that the binding affinities of fumarate in the E59D and
E59Q enzymes were lower than WT. The b and bV
max
values of
E59D enzyme are significantly higher than those of WT, suggesting
that the super activation of E59D enzyme may be due to the con-
formational flexibility in the fumarate binding site.
ª 2007 The Authors Journal compilation ª 2007 FEBS 279
Abstracts
F1-61
Different types of KIT and PDGFRA mutations in
gastrointestinal stromal tumors
N. N. Mazurenko
1
, I. Belyakov
1
, I. Tsiganova
1
, O. Anurova
1
,
P. Snigur
1,2
and V. Selchuk
1,2
1
NNBlokhin Cancer Research Center, Moscow, RUSSIAN
FEDERATION,
2

Moscow Medical Stomatological University, Mos-
cow, RUSSIAN FEDERATION
A variety of mutations leading to constitutive, ligand independent
activation of the tyrosine kinases KIT and PDGFR-a have been
identified in gastrointestinal stromal tumors (GISTs) We have ana-
lyzed KIT and PDGFRA mutations and their diagnostic and prog-
nostic value in 63 GISTs. All tumors were CD117 positive. Genomic
DNA was extracted from tumor cells from paraffin sections and
used for PCR followed with direct sequencing. Ninety percents of
GISTs revealed mutation. Most of GISTs (71.4%) had mutation of
KIT exon 11, predominantly in-frame deletions within codons 557–
560. One GIST had a deletion of the splice acceptor site for exon 11.
These mutations abrogate juxtamembrane autoinhibition of the KIT
kinase. There were also 6 GISTs with deletions involving the autop-
hosphorylation sites Y568 and Y570 and 7 GISTs with duplications
of 2–12 amino acids in the 3¢-end of KIT exon 11. The majority of
GISTs with exon 11 deletions had metastases to liver or omentum
while GISTs with duplications developed without metastases pre-
dominantly in women over age 65. Duplications of 502–503 codons
of KIT exon 9 were found in 5 GISTs in small intestine (8%). One
GIST had mutation in KIT exon 17, 6 cases lack any mutation.
PDGFRA mutation was detected in 5 (8%) GISTs and only one
tumor had typical substitution D842V. Among 19 GIST patients
with metastases that received Gleevek three tumors were primarily
resistant (two had no mutation and one had KIT D820V). Several
patients developed secondary resistence and showed mutations in 17
exon after imatinib treatment. Mutation analysis is a tool in GIST
diagnosis and in assessment of sensitivity to kinase inhibitors.
F1-62
Breast and ovarian cancer in Greek population.

Functional study of various BRCA1 protein
mutants
I. Drikos and C. Vorgias
Faculty of Biology, Department of Biochemistry, Athens, GREECE
Breast cancer (BC) is, together with lung cancer, the most common
malignancy among women in the Western world. One third of the
familial cancer cases can be attributed to mutations of certain genes
(hereditary breast cancer syndromes). Mutations of BRCA1 gene
predispose to hereditary breast and ovarian cancer. The breast can-
cer tumor suppressor gene BRCA1 encodes an 1863-amino acid nuc-
lear protein with C-Terminal tandem BRCA1 C-terminal (BRCT)
motifs. The BRCT repeats consist of 95–100 residues and are essen-
tial for the tumor suppressor function of BRCA1. In human cells,
BRCA1-BRCT interacts with phosphorylated BACH1 helicase to
activate cell cycle checkpoint in response to DNA damage. For this
interaction, the motif pSer-X-X-Phe (where X could be any amino
acid) of BACH1 binds with high affinity to BRCT-tan. Over 100
missense polymorphisms located in BRCT domain. Among thirteen
mutations valuable in our laboratory, five mutations of BRCA1-
BRCT domain have been selected for extended biochemical, bio-
physical and functional analysis, rely on cancer susceptibility. In
order to be able to characterize these mutations on a protein level,
the carboxy-terminal of BRCA1 gene was cloned in a suitable vector
and the corresponding protein was obtained in a highly soluble and
stable form. Furthermore, the secondary and tertiary structure of
the domain were analysed using circular dichroism and the thermo-
dynamic stability of the domain was measured using differential
scanning calorimetry. Additionally, the affinity parameters of the
complex between BRCA1–BRCT domain as well as proteins
involved in DNA repair, such as BACH1 and CTIP, is under investi-

gation by employing isothermal titration calorimetry (ITC).
F1-63
Novel expression patterns of organic anion
transporting polypeptide 2 in hepatocellular
carcinoma cells
C. Huang
Fooyin University, Taipei, TAIWAN
Organic anion transporting polypeptide 2 (OATP2) is involved in
the hepatocellular uptake of a variety of endogenous and xenobiot-
ic substances. We hypothesized that the expression of OATP2 may
differ among normal liver cells, liver cancer cell lines, and hepato-
cellular carcinoma (HCC) tissues. Reverse transcription polymerase
chain reaction was used to clone full-length OATP2 in one normal
and seven liver cancer cell lines, and 40 HCC tissues. OATP2
RNA expression was not detected in the Hep-G2 and Mahlavu
liver cancer cell lines. Unexpectedly, several original splicing forms
of OATP2 were identified. The J5 and HA22T/VGH HCC cell
lines contained more splicing forms of OATP2 than SK-Hep-1 and
HA59T/VGH variants. The results of immunoblot immunofluores-
cence and immunocytochemistry analysis were consistent with the
above findings. OATP2 RNA expression was present in peritumor-
ous non-neoplastic liver tissues in all 40 of our HCC patients,
while it was strongly reduced or even absent in 13 of the corres-
ponding HCC samples. Sixteen OATP2 splicing forms were
observed in both peritumorous non-neoplastic and/or HCC tissues
in our sample population. In conclusion, analysis of the data sug-
gests that OATP2 expression may be reduced in liver cancer cells,
and that the splicing forms may play an important role in HCC.
F1-64
Combined detection of GSTP1

hypermethylation and hepsin activity for
prostate non-invasive cancer diagnostics
E. Vassileva
1
, M. Savvateeva
2
, E. Kuznetsova
2
, D. Fiev
1
,
O. Abakumova
3
, A. Lyashenko
4
, A. Z. Vinarov
1
and
E. S. Severin
5
1
Moscow Medical Academy, Moscow, RUSSIAN FEDERATION,
2
Moscow Institute of Medical Ecology, Moscow, RUSSIAN
FEDERATION,
3
Institute of Biomedical Chemistry RMAS,
Moscow, RUSSIAN FEDERATION,
4
Center of Medical

Biotechnologies, Moscow, RUSSIAN FEDERATION,
5
Institute of
Molecular Diagnostics, Moscow, RUSSIAN FEDERATION.
We assess the feasibility of a urinary test for prostate cancer (PCa)
detection in a high-risk patient group based on methylation-specific
PCR (met-PCR) analysis of P-class glutathion S-transferase
(GSTP1) gene promoter and on the analysis of amydolytic activity
of hepsin, a type II transmembrane serine protease. Other cDNA
microarray studies have shown that hepsin is one of the highly
overexpressed genes in tissue compared with nonmalignant and
benign prostatic hyperplasia PCa tissue. Also it is known that pro-
moter hypermethylation is a common epigenetic alteration affect-
ing cancer-related genes. We collected the urine specimens from
the patients with suspicious on PCa immediately after DRE (digi-
tal rectal examination). Genomic DNA was isolated and analysed
by met-PCR. We have analyzed hepsin activity with chromogenic
substrate Spectrozyme
Ò
. We correlated our data with clinical infor-
mation obtained from the patient record. Our results demonstrated
that a screening test based on GSTP1 methylation and hepsin
detection in the urine specimens of patients with suspected prostate
malignancy may be a useful adjunct or probable substitution to
serum screening tests. This molecular assay has potential applica-
tion for distribution of patients into low- and high-risk groups for
surveillance versus repeat biopsy.
280 ª 2007 The Authors Journal compilation ª 2007 FEBS
Abstracts
F1-65

The effects of glycosaminoglycans/
proteoglycans on FGF-2 induced growth on two
human colon cancer cell lines
G. Chatzinikolau, D. Nikitovic and G. Tzanakakis
Medical School, University of Crete, Heraklion, GREECE
Basic fibroblast growth factor (FGF-2) and its respective tyrosine
kinase receptors (FGFRs) are widely expressed by both normal
and cancer tissues. Heparan sulphate- glycosaminoglycan chains
(HS) are accessory molecules that modulate FGF-2-signaling. The
aim of the present study, was to examine the possible participation
of various glycosaminoglycan (GAGs) i.e. chondroitin sulphate
(CS), dermatan sulphate (DS) and heparin on basal and FGF-2-
induced growth of HT29 and HCT116 human colon cancer cell
lines. Treatment with exogenous GAGs, inhibition of endogenous
GAGs/proteoglycans (PGs) synthesis and specific enzymic diges-
tions to cleave cell-associated GAGs were utilized. Our results
demonstrated differences in FGF-2 expression profiles as well as in
the mitogenic response to FGF-2 among the HT-29 moderately
and the HCT116 poorly differentiated colon cancer cell lines. CS/
DS treatment caused a prominent inhibition of HCT116 cell
growth whereas the cleavage of membrane bound CS/DS chains
strongly stimulated FGF-2-induced growth. On the other hand
heparin increased both the basal and the FGF-2-dependent growth
of HT29 cells and no effects of CS/DS were found. In conclusion,
both CS/DS and HS affect basal and FGF-2-induced colon cancer
cell growth in a distinct manner.
F1-66
Identification of a novel peptide ligand of
human vascular endothelia growth factor
receptor 3 for targeted tumor diagnosis and

therapy
X. Qin, M. Li, X. Xue, Z. Yan, W. Han and Y. Zhang
Biotechnology Center of The Fourth Military Medical University,
Xi’an, CHINA
Human vascular endothelia growth factor receptor 3 (VEGFR-3)
is a recently identified number of III receptor tyrosine kinase fam-
ily. It is found upregulated in a variety of human cancers and is
believed to play important role for tumor growth and metastasis.
It is a potentially rational target for drug delivery. To identify
novel ligands with specific binding capabilities to VEGFR-3, we
screened a phage display peptide library and found a consensus
motif of the peptides which is displayed by the positive phages
CSDxxHxWC (x is any amino acid). The phage displaying peptide
CSDSWHYWC (designated as P1) exhibited the highest affinity to
VEGFR-3 in phage ELISA and the chemically synthesized P1
could bind to VEGFR-3 specifically in a dose dependent manner.
What’s more the flow cytometry assay and immunoflorenscence
showed that the FITC labeled P1 could bind to VEGFR-3 positive
carcinoma cells with specificity. In the competition assay, the phag-
es displayed P1 could specifically inhibit the binding of P1 and
VEGFR-3 and the P1 could specifically inhibit the FITC-P1 bind
to VEGFR-3 positive carcinoma cells. Our data indicated that P1
has potential to bind to human VEGFR-3 with specificity. Such a
novel peptide ligand of VEGFR-3 holds considerable promise for
tumor diagnosis and therapy. Further studies to verify whether P1
could home anti-tumor drugs to the tumor tissue in animal tumor
model is underway. This study provides a basis for further devel-
opment of peptide ligand-based, human VEGFR-3-targeted cancer
therapy.
F1-67

Expression of the hypoxia-related genes OPN,
CA9, VEGF and EPO in human asctrocytic brain
tumors in vivo and in vitro
H. M. Said, C. Hagemann, J. Stojic, M. Flentje, K. Roosen,
G. H. Vince and D. Vordermark
University of Wu
¨
rzburg, Wu
¨
rzburg, GERMANY
Human glioblastoma cells display of a hypoxia-specific pattern of
carbonic anhydrase 9 (CA9) gene expression in vitro was recently
demonstrated (Said et al., J Neurooncol 2007). Hypoxia-related
expression of osteopontin (OPN), CA9, erythropoietin (EPO) and
vascular endothelial growth factor (VEGF) in vitro in glioblastoma
cell lines and in vivo in human tumor samples – low-grade astrocy-
toma (LGA) and glioblastoma (GBM) has not been examined
extensively. Expression of 4 different hypoxia induced genes (OPN,
CA9, EPO and VEGF) and one hypoxia dependent transcription
factor (HIF-1 a) were analysed in vivo in 2 patient groups with
LGA and GBM and in vitro in 3 glioma cells lines, GaMG, U373
and U251, on protein level or mRNA. No elevated HIF-1a mRNA
expression was shown, & an increase of at least 2-fold in CA9
mRNA was shown in 3/15 patients (LGA) and 12/15 patients in
GBM, while in VEGF it was 2/15 LGA and 7/15 GBM, in OPN
7/15 patients LGA and 15/15 patients in GBM,& in EPO 10/15
LGA and 12/15 GBM. In vitro, EPO was overexpressed only at
moderate hypoxia 1%O
2
while CA9 was overexpressed in all 3 cell

lines with the highest in GaMG (3 fold). On protein level, OPN,
CA9, EPO and HIF-1a were expressed in GBM at a higher rate
than in LGA. OPN was 3-fold overexpressed in GBM compared
to LGA. In vitro, CAIX and HIF-1a showed a clear hypoxia regu-
lated protein overexpression pattern. EPO was less induced than
CAIX in all three cell lines. OPN and CA9 mRNA were clearly
upregulated at 0.1% O
2
in all cell lines. EPO was upregulated at
1%O
2
. Combined OPN and CA9 overexpression is a clear pheno-
type associated with GBM, but also appears at a lower frequency
in LGA, rendering OPN and CA9 as optimal diagnostic markers
or targets for tumor-specific treatment approaches.
F1-68
Association between angiotensin converting
enzyme polymorphism, obesity and
hypertension in postmenopausal Turkish
women
F. Ciloglu
GENLAB Medical Diagnostics and Research Laboratory, Istanbul,
TURKEY
The renin-angiotensin system plays an important role in blood
pressure regulation, vascular and cardiac modification well as adip-
ocytosis and possibly adipocyte metabolism. The aim of this study
was to investigate the association of angiotensin converting enzyme
(ACE) insertion (I)/deletion (D) polymorphism with obesity, body
mass index, waist circumference and hypertension in postmenopau-
sal women. For this purpose, in 186 women between the ages of

45–65, body weight, height, waist circumference and body fat mass
as well as blood pressure values were measured. Those taking
blood pressure medications were noted and blood samples were
obtained from all for PCR determination of ACE genotype. The
statistical analysis of the results showed that there was no signifi-
cant association between having II, ID or DD genotypes of the
ACE gene and weight, body mass index, body fat mass, waist cir-
cumference and the incidence of hypertension. Therefore we can
conclude that ACE polymorphism is not a significant factor for
obesity and hypertension in postmenopausal women.
ª 2007 The Authors Journal compilation ª 2007 FEBS 281
Abstracts
F1-69
The immunohistochemic al assa y of MnSOD in
heart muscle of patients operated on with used
ECC and cardioplegic arrest
M. Duman
´
ska
1
, J. Saczko
1
, A. Duman
´
ski
1
, M. Daczewska
2
,
M. Pelczar

1
, M. Szapka
1
and W. Kustrzycki
1
1
Wroclaw Medical University, Wroclaw, POLAND,
2
Wroclaw
University, Wroclaw, POLAND
Ischemic heart disease is a term for clinical syndromes caused by sig-
nificantly reduced blood flow to a region of the heart. During cardiac
surgery heart action is arrested which may lead to ischemia. Their
potentially harmful effects in tissue are limited through quenching
by endogenous antioxidant – superoxide dismutase. There is consid-
erable evidence that exposure to an oxidative stress can induce anti-
oxidant enzymes, such as catalase and Mn superoxide dismutase, in
a variety of systems. Current techniques of myocardial protection
during cardiac surgery are evolving with the use of less conventional
modalities of cardioplegia and have reduced the morbidity and mor-
tality during cardiac operations. Blood cardioplegic solutions appear
superior to cold cardioplegia in terms of myocardial protection. The
aim of the study was to investigate the expression of MnSOD in
heart muscle by immunohistochemical assay. Fifteen patients indica-
ted for heart surgery due to various heart diseases were included in
this study. During cardiac surgery heart action of eight patients was
arrested by hyperthermic blood cardioplegia. In seven patients heart
action was arrested by cold cardioplegia. The time of heart arrest
was from 28 to 105 min. Biopsy specimens were taken before and
after heart arrest. Expression of MnSOD in heart muscle samples

was evaluated by immunohistochemical assay. Expression of
MnSOD in heart muscle biopsy specimens taken before heart arrest
were on physiological level in contrast to those taken after restor-
ation of heart action, where MnSOD level was significantly higher.
F1-70
Altered expression of UDP-N-acetyl-D-
galactosamine:polypeptide N-acetyl-
galactosaminyltransferases in cervical cancer
J. Kopitz
University, Heidelberg, GERMANY
The most abundant form of O-glycosylation of proteins in man is
initiated by the polypeptide N-acetyl-a-galactosamininyltransferases
(ppGalNAcTs), a family of glycosyltransferases that attach a-N-
acetylgalactosamine to the hydroxyl groups of Ser/Thr side chains.
We now tested whether expression of ppGalNAcT isoforms is
altered during cervical cancer development, thereby paving the way
for altered O-glycosylation. For this purpose seven cell lines derived
from cervical cancer were analysed for the expression of nine
enzymes (ppGalNAc-T1–T9) using RT-PCR and immunohisto-
chemistry. Among the ppGalNAc-Ts expressed by cell lines (-T1,
-T2, -T3, -T4, -T6, -T7 and -T9) the best specificity was shown for
ppGalNAc-T6. Thus, we selected this enzyme as a target gene for
further evaluation. 199 samples derived from normal cervical epi-
thelia (n=36), low-grade (LSIL, n=39), high-grade (HSIL
n = 88) and cervical cancer (n = 36) lesions selected from an
ongoing epidemiological study were analysed for the expression of
ppGalNAc-T6. ppGalNAc-T6mRNA was found in 1/36 (2.7%)
normal, 9/39 (23%) LSIL, 33/88 (37.5%) HSIL and 34/35(97%)
cervical carcinoma samples. In pre-neoplastic lesions a significant
increase of ppGalNAc-T6 expression was observed in the HPV-pos-

itive group, compared with the HPV-negative group. RT-PCR of
matched 11 normal/tumor pairs confirmed ppGalNAc-T6 mRNA
expression only in tumor samples. Immunohistochemistry using
monoclonal antibodies directed against ppGalNac-T6 validated the
RT-PCR findings. Thus our data provide for the first time evidence
for tumor specific ppGalNAc-T6 expression in cervical cancer and
suggest that ppGal-Nac-T6 may give rise to specific changes in
O-glycosylation already detectable during precancerous neoplasia.
F1-71
The effects of renal replacement therapy on
plasma asymmetric dimethylarginine, nitric
oxide and sensitive C-reactive protein levels
H. Uzun
1
, D. Konukoglu
1
, M. Besler
2
, F. Erdenen
2
, C. Sezgin
1
and
C. Mu
¨
derrisoglu
2
1
Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul
University, Istanbul, TURKEY,

2
Department of Internal Medicine,
Istanbul Education and Research Hospital, Istanbul, TURKEY
Asymmetric dimethylarginine (ADMA),endogenous inhibitor of
endothelial (NO) synthase, and high sensitive C-reactive protein
(hsCRP) recently were recognized as risk factors for atheroscler-
osis. We evaluated the effects of renal replacement therapy on
plasma of NOx, ADMA and hsCRP levels in patients with end
stage renal diseases (ESRD, n: 30), dialysis patients (n: 58),and age-
matched healthy controls (n: 20). ESRD patients have significantly
lower NOx levels than controls (P<0.01). Plasma hsCRP and
ADMA levels higher in dialysis patients group than in ESRD
group (for each, P<0.001). Haemodialysis(HD)decreased ADMA
levels (P<0.01) and increased both NOx (P = 0.03) and hsCRP
levels (P<0.001). Plasma ADMA and NOx levels were not differ-
ent in peritoneal dialysis(PD) from ESRD patients. PD patients
have higher hsCRP levels than ESRD patients(P<0.001).PD
patients have lower plasma both NOx and hsCRP levels and higher
ADMA levels than HD patients (for each, P<0.001). Age of
dialysis was 11.60 ± 6.59 and 6.03 ± 2.54 years in HD and PD
patients, respectively. Dialysis dose were positively correlated with
ADMA levels (P<0.01) and negatively correlated with both
plasma NOx and hsCRP, as well as age of dialysis (P<0.01, for
each). In conclusion, cardiovascular risks are prevalent in ESRD
patients than in the general population. Dialysis may play increase
in cardiovascular risk. Different dialysis treatment strategies differ-
entially affect on endothelial dysfunction-induced atherosclerosis.
F1-72
Plasma protein carbonyl and thiol stress in
obese patients after gastric band applications

H. Uzun
1
, D. Konukoglu
1
, R. Gelisgen
1
, K. Zengin
2
and
M. Taskin
2
1
Department of Biochemistry, Istanbul University, Cerrahpasa Med-
ical Faculty, Istanbul, TURKEY,
2
Department of Surgery, Istanbul
University, Cerrahpasa Medical Faculty, Istanbul, TURKEY
The aim of this study is to examine relationship between oxidative
plasma protein and thiols stress and weight loss by laparoscopic
gastric band applications. Plasma protein carbonyl (PCO) concen-
tration, marker of protein oxidation, plasma thiol (P-SH) and
erythrocyte glutathione (GSH, major intracellular thiol) concentra-
tion, and antioxidants, and metabolic markers, Homeostatic Model
Assessment -Insulin resistant (HOMA-IR), body mass index (BMI)
and plasma lipids were determined in morbidly obese patients (n:
22, BMI: 48.4 ± 6.4 kg/m
2
) baseline and 1 and 6 months after
operations and age-matched controls (n: 20, BMI: 21.3 ± 1.8 kg/
m

2
). Plasma PCO levels were significantly higher and plasma and
erythrocyte thiols concentrations were significantly lower in mor-
bidly obese patients than in controls (for each, P<0.01). BMI,
plasma triglycerides and HOMA-IR was positive correlated with
plasma PCO and negative correlated with plasma P-SH and eryth-
rocyte GSH (for each, P<0.01). Plasma HDL-cholesterol levels
were positive correlated with plasma erythrocyte GSH (r: 0.405,
P<0.01) and negative correlated with plasma PCO (r:-0.273,
P<0.01). Plasma PCO concentrations were decreased and P-SH
and erythrocyte GSH concentration were elevated following weight
loss (for each, P<0.01). In conclusion, protein and thiol oxida-
tive stress was improved by weight loss after laparoscopic gastric
banding in short period.
282 ª 2007 The Authors Journal compilation ª 2007 FEBS
Abstracts
F1-73
Oxidized LDL (OxLDL) and anti-oxLDL antibody
(oxLDL Ab) levels in peripheral atherosclerotic
disease
G. Andican
1
, A. Seven
1
, M. Uncu
1
, M. Cantas¸ demir
2
, F. Numan
2

and G. Burc¸ ak
1
1
Department of Biochemistry, Cerrahpasa Tıp Fakultesi, Istanbul
University, Istanbul, TURKEY,
2
Department of Radiology,
Cerrahpasa Tıp Fakultesi, Istanbul University, Istanbul, TURKEY
Oxidative modification of LDL (oxLDL) plays an important role
in atherogenesis and is proposed as a useful marker for identifying
patients with coronary artery disease. Antibody against oxLDL
(oxLDL Ab) is detected in human sera, although its biological sig-
nificance is not well established. We aimed, in this study, to meas-
ure oxLDL and oxLDL Ab in the plasma of healthy controls
(n=21) and peripheral atherosclerotic disease (PAD) patients
(n=21). Total risk for atherosclerosis was estimated by Global
Risk Assesment Score (GRAS) calculated on the basis of age, total
cholesterol, HDL-cholesterol (HDL-C), diabetes, hypertension and
smoking. Total, LDL and HDL-cholesterol levels were determined
by enzymatic methods. Levels of circulating oxLDL were measured
by monoclonal antibody 4E6-based competition ELISA. IgG class
oxLDL Ab titer was measured by ELISA. Compared with healthy
controls, PAD patients had higher levels of oxLDL (P<0.05),
oxLDL Ab (P<0.05), LDL cholesterol (LDL-C) (P<0.05),
total cholesterol (P<0.05) and lower HDL-C (P<0.05). Ox-
LDL was found to be positively correlated with total cholesterol
and LDL-C and GRAS but not with oxLDL Ab in PAD patients.
These findings might indicate that high LDL-C levels influence
the oxidation of LDL and oxLDL is possible marker of PAD.
However, the role of oxLDL Ab in atherosclerosis remains contro-

versial.
F1-74
The importance and predictive value of
extracellular matrix proteins for preeclampsia
H. Uzun
1
, D. Konukoglu
1
, A. Benian
2
, R. Madazli
2
,
M. Albayrak
2
, H. Genc
1
, S. Aydin
1
, M. Oncul
2
and S. Uludag
2
1
Department of Biochemistry, Cerrahpas¸ a Medical Faculty, Istanbul
University, Istanbul, TURKEY,
2
Department of Obstetrics and Gyn-
ecology, Cerrahpas¸ a Medical Faculty, Istanbul University, Istanbul,
TURKEY

The purpose of this study was to examine the relationship between
fibronectin(FN), as a marker of endothelial activation, and hyalu-
ronic acid(HA) and hydroxyproline(HP), as markers of extracellu-
lar matrix degradation in normal (n : 17) and preeclamptic
pregnancies (n: 29). We obtained the samples from studied group
before delivery. FN and HA concentrations in maternal and cord
bloozd and placenta were determined by enzyme-linked immuno-
sorbent assay. HP content in placental tissue was determined by
using a colorimetric assay. FN levels of all samples species were
significantly higher in preeclamptic patients than in controls. HA
concentrations of cord blood and placenta were found to be eleva-
ted in patients with preeclamsia. Preeclamptic patients had signifi-
cantly higher placental HP levels than controls. Maternal serum
FN levels were also correlated with placental levels of HA and
HP. Maternal and cord blood FN levels show high sensitivity
(89.7% and 86.2%) and specificity (92.9% and 92.9%) for pree-
clampsia. Sensitivity (79.3%) and specificity (85.7%) of plasental
HP were significantly higher than values of plasental FN and HA.
Serum FN levels were elevated in patients with preeclampsia com-
pared to normotensive pregnant women. Maternal FN has high
predictive value and it may be accepted as a marker of ECM dis-
modelling, as well as endothelial dysfunction.
F1-75
The influence of clozapine treatment on plasma
levels of reactive oxygen species and
antioxidant enzymes in patients with
schizophrenia
S. Yabanoglu
1
, A. Ertugrul

2
, K. Basar
2
, B. Demir
2
, G. Ucar
1
and
B. Ulug
2
1
Department of Biochemistry, Faculty of Pharmacy, Hacettepe Uni-
versity, Ankara, TURKEY,
2
Department of Psychiatry, Faculty of
Medicine, Hacettepe University, Ankara, TURKEY
In the present study the influence of 8 weeks of clozapine treat-
ment on plasma levels of reactive oxygen species in schzophrenics
were investigated. Twenty subjects who met DSM-IV criteria for
schizophrenia and 20 healthy controls who were matched for age
and sex status were recruited for the study. Psychopathology,
plasma levels of thiobarbituric acid reactive substances (TBARS)
as an indicator of plasma lipid peroxidation, reduced (GSH) and
oxidized (GSSG) glutathione levels and antioxidant enzyme activit-
ies were determined in the study groups at baseline and 8 weeks
after initiation of clozapine treatment. Psychopathology was
assessed by Positive and Negative Syndrome Scale (PANSS) and
Clinical Global Improvement. Baseline plasma GSH level, catalase,
superoxide dismutase, glutathione reductase, and glutathione
S-transferase activities were found to be significantly lower, and

plasma TBARS and GSSG contents significantly higher than those
of controls whereas no difference was detected in plasma glutathi-
one peroxidase activity. At 8th weeks of clozapine treatment, the
results of patients approached to those of controls and the differ-
ence between patients and controls regarding to the above parame-
ters were not statistically significant anymore. The increase in
plasma antioxidant enzyme activities, and the decrease in plasma
TBARS level were found to be well correlated with the improve-
ment in PANSS general psycopathology in the patients after
8 weeks of clozapine treatment suggesting that clozapine treatment
may restore the oxidative damage occurred in schizophrenia.
F1-76
Variation of serum level of proinflammatory
cytokines after mud therapy in patients with
ankylosing spondylitis
D. Profir
1
, V. Marin
1
, O. Surdu
1
and N. Rosoiu
2
1
Balneary and Rehabilitation Sanatorium Techirghiol, Constanta,
ROMANIA,
2
Biochemistry Department, Faculty of Medicine,
Ovidius University Constanta, Constanta, ROMANIA
The study aims to evaluate anti-inflammatory activity of cold mud

ointment and mud bath in patients with ankylosing spondylitis
(AS). The applied treatment: progressive heliotherapy, cold mud
ointment and swimming into the salted water of the lake, in addi-
tion to electrotherapy, kinetotherapy and massage. The studied
group included 25 patients (6 female and 19 male), 13 of them with
AS (following ACR criteria for diagnosis) and the other 12 with
osteoarthritis. 20 patients underwent cold mud ointment and 5
heated mud packing. The patients were clinically and paraclinically
evaluated before and after mud treatment. Were determined serum
level of TNFa, IL-1b and IL-6. After cold mud ointment all three
cytokines decreased. After heated mud packing interleukines serum
level decreased, meanwhile TNFa serum level increased. After mud
bath all three cytokines increased. Complex variation of proinflam-
matory cytokines serum level after mud therapy suggests the bene-
fits of sapropelic mud of Techirghiol in progression of the disease
in patients with AS.
ª 2007 The Authors Journal compilation ª 2007 FEBS 283
Abstracts
F1-77
Biochemical exploration in ovary cancer
N. Rosoiu
1
and I. Verman
2
1
Biochemistry Department, Faculty of Medicine, ‘Ovidius’ University
Constanta, Constanta, ROMANIA,
2
Biochemistry Department, Fac-
ulty of Medicine, ‘Ovidius’ University Constanta, Constanta,

ROMANIA
The oxidative stress, defined as the imbalance between the produc-
tion and degradation of the reactive oxygen species (ROS) that
induce oxidations, damage of membranes, and modification of pro-
teins and DNA, is considered to play an important role in cancer
pathogenesis. The antioxidant defense system is sophisticated and
adaptive, the reduced glutathione (G-SH) being a central constitu-
ent of this system. Redox phenomena are intrinsic to life processes
and G-SH is a major pro-homeostatic modulator of intracellular
-SH groups of proteins. The paper presents blood values of hemo-
globin, ESR, fibrinogen, glucose, alkaline phosphatase, alanin ami-
notransferase and reduced glutathione in a study group consisted
of 20 patients diagnosed with ovary cancer. Reduced glutathione
concentration was dramatically decreased, signifying a low level of
the antioxidative protection in all investigated patients. Thus our
study intended to evaluate the useful information for the diagnosis,
prognostic and therapy monitoring of ovary cancer aiming to posi-
tively influence the survival rate and the patients’ quality of life.
Keywords: ovary cancer, reduced glutathione
F1-78
Reduced glutathione and certain antioxidant
enzymes in schizophrenia
R. D. Rosoiu
1
, N. Rosoiu
2
, A. Danulescu
3
and M. Basa
4

1
Faculty of Biology, ‘Ovidius’ Univeristy Constanta, Constanta,
ROMANIA,
2
Biochemistry Department, Faculties of Medicine and
Biology, ‘Ovidius’ University Constanta, Constanta, ROMANIA,
3
Constanta County Emergency Hospital, Psychiatric Clinic,
Constanta, ROMANIA,
4
Constanta Military Emergency Hospital,
Constanta, ROMANIA
Oxidative stress-mediated cell damage has been considered in the
pathophysiology of schizophrenia. Due to the fact that the bio-
chemical mechanisms in oxidative stress involved in schizophrenia
are not completely understood, in the present research we have
chosen to study reduced glutathione (GSH) and antioxidant
enzymes such as glutathione-reductase (GR), superoxide dismutase
(SOD) and total antioxidant status (TAS) in blood. For this pur-
pose we have chosen: two groups of schizophrenic subjects,
15 medicated and 15 non-medicated patients, and a third group of
15 healthy subjects as the control group. It was observed an
important decrease below the normal limit of the reduced glutathi-
one and glutathione-reductase. The superoxide dismutase which
metabolizes superoxide radicals was found increased, possibly as
an adaptive response to free radical overload. The low level of the
GSH and GR in patients with schizophrenia reveals the existence
of dysfunctions in the defence mechanisms against the free radicals
and ROS, which leads to the acceleration of the degenerative mor-
phofunctional processes, with negative effects at cerebral level.

Because GSH, the main non-protein antioxidant, is a systemic pro-
tectant against oxidative stress and free radical damage, GSH and
the antioxidant enzymes deficit in patients with schizophrenia, may
lead to membrane peroxidation and microlesions of dopaminergic
terminals, resulting in loss of connectivity.
F1-79
Enzyme activity of serum dipeptidyl peptidase
IV (DPP IV/CD26) in rheumatoid arthritis
L. Baticic
1
, D. Detel
1
, T. Kehler
2
, N. Varljen
2
and J. Varljen
1
1
School of Medicine, University of Rijeka, Rijeka, CROATIA,
2
Tha-
lassotherapia Opatija, University of Rijeka, Rijeka, CROATIA
Introduction: The dipeptidyl peptidase IV (DPP IV/CD26) is a
multifunctional glycoprotein present both in a soluble form in
many biological fluids and on the surface of different cells type. It
could play an important role in the modulation of immune and
inflammatory processes. A number of different proinflammatory
peptides involved in rheumatoid arthritis pathogenesis, like RAN-
TES and stromal cell-derived factor (SDF)-1a are substrates for

DPP IV.
Aim: The aim of this study was to assess the serum DPP IV activ-
ity in patients with rheumatoid arthritis, psoriatic arthritis, and
osteoporosis, compared to healthy controls.
Material and methods: According to their clinical and laborat-
ory data, patients were divided in three groups: patients with rheu-
matoid arthritis, psoriatic arthritis and patients with osteoporosis.
The control group included 65 healthy blood donors. The serum
DPP IV activity has been measured spectrofotometrically using
Gly-Pro p-nitroanilide as substrate.
Results: A statistically significant decrease in serum DPP IV was
found in patients with rheumatoid arthritis (30.45 ± 3.04 U/l)
compared to the control group (48.37 ± 1.10 U/l) (P<
0.001). In contrast, serum DPP IV activities in patients with
psoriatic arthritis (49.96 ± 3.80 U/l) and osteoporosis (43.11 ±
3.9 U/l) were not statistically significantly different from healthy
controls.
Conclusion: The decreased serum DPP IV activity in patients
with rheumatoid arthritis suggests its possible involvement in the
regulation of immune processes, which could lead to the disease
occurrence. It could likewise be possibly involved in the disease
progression.
F1-80
Polymorphism in the promoter regions of IGF I
gene in elite athletes of different sport
disciplines
K. Krych
1
, H. Mizgajska-Wiktor
1

and A. Goz´ dzicka-Jo
´
zefiak
2
1
Department of Biology and Environmental Protection, University
School of Physical Education, Poznan
´
, POLAND,
2
Department of
Molecular Virology, Adam Mickiewicz University, Poznan
´
,
POLAND
IGF I (insulin-like growth factor) is a protein involved in adapta-
tion of human body to physical activity through its influence on
development, growth and differentiation of skeletal muscles. This
protein has an influence on metabolism of muscular proteins, glu-
cose and regeneration of muscle cells. Such features made IGF I to
be commonly used as a doping substance. The aim of the study
was searching the genetics determinants to achieve success in elite
sport. In this purpose polymorphism in the promoter regions of
IGF I gene was examined. Athletes (400) of 14 different sport dis-
ciplines were tested. The control group represented 100 students.
Three out of four P1 promoter regions were analyzed using PCR,
single-strand conformation polymorphism (SSCP) analysis. In all
three studied promoter regions nucleotide sequence changes were
observed but with different frequency. The mutations in first pro-
moter region were noticed in sportsmen represented all 14 disci-

plines, in the second region changes were observed in sportsmen of
four disciplines and in the third region changes in sportsmen of
eight disciplines. So, the first region was the most polymorphic (for
control group it was the only region with mutations). The results
suggest, that nucleotide sequence changes may play some role in
adaptation of human body to physical activity.
284 ª 2007 The Authors Journal compilation ª 2007 FEBS
Abstracts
F1-81
Comparison with plasma xanthine oxida se and
purine metabolites in normal and preeclamptic
pregnancies
Y. Aliciguzel, Sr.
1
, O. Elmas
1
and T. Simsek
2
1
Akdeniz University Medical Faculty, Department of Biochemistry,
Antalya, TURKEY,
2
Akdeniz University Medical Faculty,
Department of Gynecology, Antalya, TURKEY
Preeclampsia is a syndrome that is found about 1–5% percent in
all pregnancies, which is characterized with hypertension and pro-
teinuria. Especially in last 15 years, lots of studies are experienced
related with free radical theory, for explaining the preeclampsia. In
our study, we detected the roles of hypoxanthine, xanthine, uric
acid, nitrite and xanthine oxidase activity which are related to

endothelial dysfunction, in normal and preeclamptic pregnants.
Nitrite was measured via an electrochemical method and other
parameters were detected via HPLC method. Hypoxanthine, xan-
thine, uric acid, allantoin and xanthine oxidase activity has been
found with higher levels in preeclampsia than normal pregnants
levels. Other parameters are not changed in both groups. Accord-
ing to our results, we concluded increased xanthine oxidase activity
may play a role for endothelial dysfunction in preeclampsia, and
higher levels of hypoxanthine, uric acid and allantoin are strong
evidences of free radical induced injury in preeclampsia
F1-82
Croatian report on mutations and
polymorphisms related to congenital disorder
of glycosylation Ia (CDG-Ia)
S. Supraha, S. Dabelic and J. Dumic
Faculty of Pharmacy and Biochemistry University of Zagreb,
Zagreb, CROATIA
Congenital disorder of glycosylation Ia (CDG-Ia) is the most com-
mon type of CDGs, a growing group of rare genetic disorders
characterized by deficient protein glycosylation. It is caused by spe-
cific mutations in phosphomannomutase 2 gene (PMM2); the most
frequent ones in European population are R141H and F119L.
However, the variations in the frequencies of these mutations were
observed among the populations of different geographic origin.
Although no CDG-Ia patient is detected in Croatia so far, the
necessity of revealing genetic characteristics of our population
encouraged us to determine the frequencies of mutations R141H
and F119L and intragenic SNPs, IVS5+19T/C and IVS5+22T/A
in PMM2 gene in Croatian population.
For screening target mutations, single-strand conformation poly-

morphism (SSCP) analysis was used. All samples showing aberrant
SSCP patterns and adequate number of randomly chosen ones
with normal pattern were additionally sequenced.
No mutations R141H and F119L neither allele IVS5+22A have
been detected. The estimated frequency of IVS5+19T allele was
0.958 and IVS5+19C allele was 0.042, while the estimated inci-
dence of heterozygotes for IVS5+19T/C was 0.06. Although the
absence of both mutations (R141H and F119L) and the estimated
allele frequencies of intragenic SNP IVS5+19T/C in analyzed
series suggest specificity of the Croatian population regarding
these mutations/SNP comparing to other European populations,
bigger population sample has to be analyzed to provide the final
conclusion.
F1-83
TPMT deficient alleles correlate with higher
incidence of 6-MP toxic effects in children with
ALL
N. Karas Kuzelicki
1
, J. Jazbec
2
and I. Mlinaric Rascan
1
1
Faculty of Pharmacy, University of Ljubljana, Ljubljana,
SLOVENIA,
2
University Children’s Hospital, Ljubljana,
SLOVENIA
It has been postulated that childhood acute lymphoblastic leuke-

mia (ALL) patients with thiopurine S-methyltransferase (TPMT)
deficiency tend to have better response to 6-mercaptopurine (6-
MP) therapy, but are at higher risk of developing toxic effects dur-
ing the consolidation and maintenance phase of the treatment.
TPMT methylates 6-MP, thereby preventing cytotoxic thioguanine
nucleotides formation. In a retrospective study, therapy data for
295 patients with childhood ALL were obtained from protocol
phases consisting exclusively of 6-MP and methotrexate. DNA was
extracted from bone marrow smears by means of 6100 Nucleic
Acid PrepStation. TPMT activity deficient TPMT*3 alleles were
determined using ABI Prism
Ò
7700 Sequence Detection system.
TPMT*3 heterozygotes (20 of 295 patients) tolerated lower doses
of 6-MP than wild-type patients (P=0.003). TPMT*3 patients
had higher incidence of toxic effects, which led to a discontinu-
ation of the therapy, than wild-type patients: bone marrow depres-
sion (70% vs. 12%, P<0.001), stomatits (25% vs. 8%,
P=0.030) and recurrent infection or sepsis (45% vs. 7%,
P<0.001). There was no difference between the TPMT*3 and
wild-type patients in the incidence of the ALL relapse (25% vs.
33%, P=0.622), which could be explained by karyotypic abnor-
malities in cancer cells. In conclusion, TPMT deficient alleles corre-
late with a higher incidence of 6-MP toxic effects.
F1-84
Phylogenetic analysis of the predominant
mtDNA variant, haplogroup H
L. Pliss
1
, V. Baumanis

1
, A. Krumina
2
, A. Puzuka
2
, K. Tambets
3
and R. Villems
3
1
Latvian Biomedical Research and Study Centre, Riga, LATVIA,
2
Riga Stradins University, Riga, LATVIA,
3
Estonian Biocentre, Uni-
versity of Tartu, Tartu, ESTONIA
Mitochondrial DNA haplogroup H (mtDNA hg H) is by far the
most frequent maternal lineage cluster in Europe, less so in South
Asians and in Native Siberians.
A phylogeographic analysis of mtDNA hg H, covering almost half
(44.5%) of the mtDNA genomes in the Latvian population, was
undertaken. 133 samples were screened for 10 polymorphisms in
the mtDNA coding region and two polymorphisms in HVS-II in
addition to HVS-I sequence variation. The statistical significance
of population differences was evaluated using the chi-square test.
The phylogeny of the samples was studied by the construction of a
reduced median network.
Ten different sub-haplogroups were detected among 133 analyzed
samples. Forty-five per cent of the classified hg H genomes
belonged to sub-hgs H1 and H5. Sub-hg H1b, which occurs more

frequently in Eastern and North-Central Europe, was the most
abundant type of sub-hg H1 among Latvians, also being frequent
among Estonians. In the Latvian gene pool, H1b was significantly
more frequent than among Eastern Slavs. The frequency of H1a in
the Latvian population was similar to that of other North-Eastern
and Eastern European populations. Significant differences of H1a
frequencies were observed between Latvians and Finns. Sub-hg
H5, the second largest in the Latvian mtDNA pool, was found to
be significantly more frequent among Latvians than in Eastern
Slavs. We can conclude that the vast majority of H genomes
belonged to H*, H5, and H1b, and mostly haplotypes were shared
with Finno-Ugric populations.
ª 2007 The Authors Journal compilation ª 2007 FEBS 285
Abstracts
F1-85
Plasma, whole blood, erythrocyte and urine free
choline concentrations in Turkish healthy
subjects
Y. O. Ilcol, B. Z. Hizli, E. Eroz and I. H. Ulus
Uludag University Medical School, Bursa, TURKEY
Background: Plasma choline constitutes an important source of
choline for neuronal acetylcholine synthesis and for producing phos-
pholipids and an essential component of all membranes. In blood,
choline is concentrated within erythrocytes through the action of an
uptake molecule that is unsaturated at normal plasma choline con-
centrations. Free choline is also found in other biological fluids.
Methods: This study was undertaken to determine the concentra-
tions of plasma, erythrocyte, whole blood and urine free choline in
50 healthy adults ages between 18–45 years old and to determine
the relations between these parameters. Whole blood, plasma,

erythrocyte and urine free choline levels were measured by high
performance liquid chromatography.
Results: The mean values for concentrations of plasma, whole
blood and erythrocyte free choline were 10.1 ± 0.45, 27.2 ± 3.6
and 20.1 ± 2.0 lmol/l, respectively. There was a highly significant
and positive correlation between whole blood and erythrocyte
(r=0.939, P<0.0001) free choline concentrations. There were a
significant and positive correlation between plasma and whole
blood (r=0.322, P<0.05) and plasma and erythrocyte
(r=0.297, P<0.05) free choline concentrations. The mean val-
ues for concentrations of 24 h collected and spot urine free choline
were 17.4 ± 1.9 and 25.3 ± 3.1 lmol/l respectively.
Conclusions: This study provides the normal values for plasma,
whole blood, erythrocyte and urine free choline in Turkish popula-
tion. The data can be used for comparison with the value obtained
from other populations.
F1-86
PON1 192RR genotype is a risk factor for
ischemic stroke
B. Can Demirdo
¨
gen
1
, A. Turkanoglu
1
, S. Bek
2
, S. Demirkaya
2
,

O. Vural
2
, E. Arinc¸
1
and O. Adali
1
1
Middle East Technical University, Ankara, TURKEY,
2
Gu
¨
lhane Mili-
tary Medical Academy, Department of Neurology, Ankara, TURKEY
Background and aim: Oxidation of low-density lipoprotein
(LDL) is a process presumed to be important in the initiation and
progression of atherosclerosis. Human paraoxonase 1 (PON1) is
known to protect high density lipoprotein and LDL from oxidative
modifications and thus is protective against the development of
atherosclerosis. Since carotid atherosclerosis is a risk factor for
stroke, we investigated the effects of activity and genetic polymor-
phisms of PON1 in ischemic stroke.
Methods and results: Serum paraoxonase (PON), arylesterase
(ARE) and diazoxonase (DIA) activities and PON1 192Q/R geno-
types for 106 ischemic stroke patients and 68 controls were deter-
mined. PON (238.7 ± 148.4) and ARE (115.8 ± 34.7) activities of
stroke patients were found to be slightly lower than PON
(250.2 ± 136.7, P=0.60) and ARE (118.9 ± 35.2, P=0.56)
activities of controls. There was no difference in DIA activities of
patients and controls (P=0.76). The prevalence of the PON1
192RR genotype was significantly increased among patients

(17.0%) as compared to controls (15.9%; P=0.03; OR = 3.27
95% CI = 1.06–10.13). PON, ARE and DIA activities of patients
and controls did not predict case status by use of logistic regression
with current age included as a covariate. When PON1 192Q/R
genotype was added into the model, 192RR genotype significantly
(P=0.01, OR = 5.31, 95% CI = 1.46–19.3) predicted stroke ver-
sus control status. The Hosmer-Lemeshow goodness-of-fit test
showed that our model has a good fit with the data (P=0.49).
Conclusion: Our results suggest that 192RR genotype is import-
ant in susceptibility to ischemic stroke.
Keywords: 192RR, PON1, Stroke
F1-87
Evaluation of tubular damage in children with
vesicoureteral reflux
J. Basic, E. Golubovic, P. Miljkovic, G. Bjelakovic, T. Cvetkovic
and V. Milosevic
Medical faculty, Nis, SERBIA AND MONTENEGRO
Vesicoureteral reflux (VUR) is a common congenital anomaly of
the urinary tract characterized by reflux of infected urine, with the
potential for compromise of renal function. The aim of the study
was to investigate the eventual influence of different grades of
VUR on proximal tubule cell damage using urinary enzymes alka-
line phosphatase (AP) and c-glutamyl transpherase (c-GT).Chil-
dren with VUR detected by voiding cystourethrography were
investigated. According to a grade of VUR, patients were separ-
ated into three groups. The first group included 12 children with
VUR grade I-II. The second group was consisted of 12 children
with grade III of VUR. Patients with VUR grade IV-V (n: 11)
were members of the third group. Control group was consisted of
17 healthy children. AF and c-GT activities were examined in sam-

ples of morning urine specimens using standard biochemical analy-
sis.The main value of urinary AF activity in third group showed
statistically significant increase compared to control values. Urin-
ary c-GT activity in the third group was decreased in comparison
to a group of healthy children, but not statistically significant. We
discussed increase of AF activity in children with high grade of
VUR as a consequence of retrograde urine flow (intrarenal reflux)
and consecutive proximal tubule cell damage. On the contrary,
decreased c-GT activity might be caused by some specific inhibitor
in urine.
F1-88
Analysis of seasonal changes of antioxidants
and other active substances in breast milk in a
sample of Czech population
I. Marova, S. Macuchova, I. Ceskova and A. Halienova
Faculty of Chemistry, Brno, CZECH REPUBLIC
The aim of presented work is to study seasonal differences in con-
tent of some active substances in breast milk in a sample of Czech
population. This study was realized during the period from April
2006 to March 2007. 118 subjects were enrolled in the volunteer
group. In these subjects early breast milk was taken (usually 5th day
after childbirth). In all human milk samples levels of selected anti-
oxidants (e.g. tocopherols, ascorbate, carotenoids – RP-HPLC-MS),
total antioxidant status (TRAP), fatty acid levels (GC-MS) and pro-
tein profiles (PAGE-SDS, microfluidic electrophoresis) were meas-
ured. Monthly changes of all parameters were evaluated and
average values were compared with completed nutritional question-
naires. Breast milk protein profiles were very similar among all sub-
jects and relatively independent on diet of mothers. The main
protein fractions were lactalbumine and caseins. On contrary, lipid

composition of breast milk was more variable and depended on
dietary intake. While levels of saturated and monounsaturated fatty
acids as well as arachidonic acid were quite similar among all sub-
jects and among average monthly values, the levels of some of the
PUFA were highly variable. Monthly average values of individual
antioxidants significantly differed according to composition of com-
mon local food sources; the highest differences exhibited carote-
noids and retinol. TRAP values varied in range from 0.55 mmol/l
(March, April) to 0.95 mmol/l (September, October). According to
our findings, in Czech population levels of antioxidants, antioxidant
status and fatty acid composition in breast milk most likely reflect
habitual seasonal dietary differences.
286 ª 2007 The Authors Journal compilation ª 2007 FEBS
Abstracts
F1-89
Molecular and phenotypic characteristics of
patients with phenylketonuria in Serbia and
Montenegro
M. Stojiljkovic
1
, J. Jovanovic
1
, M. Djordjevic
2
, S. Grkovic
2
,
M. Cvorkov-Drazic
2
, B. Petrucev

1
, N. Tosic
1
,
T. Karan-Djurasevic
1
, L. Stojanov
2
and S. Pavlovic
1
1
Institute of Molecular Genetics and Genetic Engineering, Belgrade,
SERBIA,
2
Mother and Child Healthcare Institute ‘Dr Vukan Cupic’,
Belgrade, SERBIA
Phenylketonuria (PKU) is an inborn metabolic disease caused by
deficiency of hepatic enzyme, phenylalanine hydroxylase (PAH).
We analyzed 34 unrelated patients with PKU from Serbia and
Montenegro and identified 19 mutations in PAH gene. By combi-
ning PCR-RFLP, DGGE and sequencing analysis, mutation detec-
tion rate of 97% was achieved. The most frequent mutations were:
L48S (21%), R408W (18%), P281L (9%), E390G (7%) and
R261Q (6%), accounting for 60% of all mutant alleles. The
remaining ones occurred at frequency less than 5%. The homozyg-
osity value was rather low (0.10), indicating the heterogeneity of
population. This finding reflects numerous migrations over the
Southeastern Europe. According to pretreatment serum phenylal-
anine level, patients were assigned to classic PKU (65%), mild
PKU (35%) and MHP (0%). The genotype-phenotype correlation

was studied in homozygous and functionally hemizygous patients.
Although previous studies reported phenotypic inconsistency of
L48S, the mutation was exclusively associated with the classical
PKU in this population. Recent studies show the connection
between L48S and a good response to therapy with PAH enzyme
cofactor tetrahydrobiopterin. We speculate that consistent severe
phenotype of L48S could be a consequence of independent modu-
lator genetic factors specific for the population. The characteriza-
tion of PAH mutations created the base for molecular diagnostics
of phenylketonuria in Serbia and Montenegro.
F1-90
Aluminium oxide hydroxyapatite composites
I. Peker
1
, O. Gunduz
1
, F. Oktar
1
, S. Salman
1
, S. Agathopoulos
2
and L. Ozyegin
1
1
Marmara University, Istanbul, TURKEY,
2
Ioannina University, Io-
annina, GREECE
Because of its superb biocompatibility to the human body,

hydroxyapatite [HA, Ca
10
(PO
4
)
6
(OH)
2
] ceramics have been used as
implants in bone and teeth restorations. Nevertheless, its poor
mechanical properties have restricted wider applications in load-
bearing devices. Hence, composite materials of HA, with ceramic
oxides or metallic dispersants as reinforcing agents, have been pro-
posed. In this study the effect of sintering on the mechanical prop-
erties HA-Al
2
O
3
composites, where the source of HA is natural
from bovine bones (BHA) was investigated. Microstructural SEM
analysis indicated typical densification behaviour, similar to that
observed in earlier studies on the sintering behaviour of HA. This
study showed that the addition of 5% Al
2
O
3
results in increase of
mechanical strength of BHA. It is very surprising to get lower
mechanical values for increasing content of Al
2

O
3
. Further studies
must be achieved with 15 and 20% Al
2
O
3
why the adding Al
2
O
3
cause decrease at the mechanical values. But it will be also interest-
ing to conduct some cell culture studies.
F1-91
Cell adhesion and cytoskeleton organization on
zirconia surfaces
V. Mitran
1
, A. Cimpean
1
, R. N. Ciubar
1
, C. M. Ciofrangeanu
1
,
B. Vladila
2
, P. Gautier
3
and D. Iordachescu

1
1
Biology Faculty, Bucharest University, Bucharest, ROMANIA,
2
Zirimplant Ltd., Bucharest, ROMANIA,
3
HTI Technologies, Lyon,
FRANCE
The purpose of this study was to explore the biocompatibility of a
material implant based on zirconia (ZrO
2
+ HfO
2
+Y
2
O
3
) with
human gingival fibroblasts (HGF-1 cell line). The cells were grown
on zirconia discs and plastic supports and comparatively studied
for cytotoxicity, cell adhesion and organization of the cytoskeleton.
One protein that is of particular interest in determining the rela-
tionship between cells and their substrate is fibronectin as it repre-
sents one of the major molecules mediating cell attachment. Its
expression was determined by immunofluorescence demonstrating
a similar pericellular distribution on the plastic and dental material
supports. An other objective of this study was to investigate the
association of the cell orientation with expression of the cytoskele-
tal proteins. We immunolocalized by fluorescence two proteins that
are numerous in focal adhesions namely vinculin and talin. Cyto-

skeleton orientation was studied by immunostaining of the vimen-
tin intermediary filaments and a-tubulin, one of the tubulin
subunits in microtubules. Immunocytochemical analyses of these
cytoskeletal proteins did not reveal any differences between the
cells grown on zirconia discs and plastic supports suggesting that
the tested material is biocompatible and an adequate substrate for
HGF-1 fibroblasts. This biocompatibility was also demonstrated
by LDH and MTT-based cytotoxicity assays.
F1-92
Fluvastatin restores endothelial dysfunction
caused by collar in rabbit carotid artery
M. Arun
1
, G. Sevin
1
, C. Gonen-Korkmaz
1
, G. Yetik-Anacak
1
,
E. Ozer
2
and L. Ustunes
1
1
Department of Pharmacology, Faculty of Pharmacy, Ege
University, Izmir, TURKEY,
2
Department of Pathology, Faculty of
Medicine, Dokuz Eylul University, Izmir, TURKEY

Statin therapy appears to exhibit pleiotropic effects on many com-
ponents of atherosclerosis including endothelial function. We
hypothesized that fluvastatin may have effects on early atheroscler-
osis. Rabbits were treated with fluvastatin (8 mg/kg/day, p.o.,
n=4) and placebo (n=6) for 3 weeks. On the 8th day, a soft
silicon collar was placed around the left carotid artery for 14 days
and then both carotid arteries (ca) were isolated and cut into rings.
The rings were used for isolated organ chamber experiments and
histological examinations.
Results: In placebo group, collar decreased contractile responses
(E
max
, g) to agonists (KCl: 1.9 ± 0.45 and 0.4 ± 0.06; phenyleph-
rine (Phen): 3.7 ± 0.65 and 1.31 ± 0.104, P<0.001; serotonin
(5-HT): 4.2 ± 0.39 and 1.75 ± 0.17, P<0.001, sham vs. collar).
Collar treatment also increased sensitivity to 5-HT (6.35 ± 0.37
and 6.9 ± 0.18 P<0.001) and decreased it to Phen (6.14 ± 0.09
and 5.78 ± 0.07 P<0.01). The collar dependent decrease in%
relaxation response to acethylcholine (ACh) (66.4 ± 5.4 and
45.3 ± 2.7, P<0.05) was significantly increased by fluvastatin
treatment (45.3 ± 2.7 and 82.5 ± 9.85 P<0.05, placebo vs. flu-
vastatin). Fluvastatin also increased sensitivity to muscarinic recep-
tors in both normal (6.40 ± 0.13 and 6.88 ± 0.16, P<0.05) and
collared arteries (6.34 ± 0.15 and 6.87 ± 0.21, P<0.05). The
beneficial effects of fluvastatin were dependent neither on the con-
tractile mechanisms nor collar induced histological changes.
Conclusion: The present study demonstrates that chronic fluvast-
atin treatment restores endothelial dysfunction in
collar-induced early atherosclerosis.
ª 2007 The Authors Journal compilation ª 2007 FEBS 287

Abstracts
F1-93
The effects of different doses of diclofenac on
viability and proliferation of lymphocyte cell
culture
T. Dujic, A. Jevric-Causevic and M. Malenica
Department of Biochemistry and Clinical Analysis, Faculty of
Pharmacy, University of Sarajevo, Sarajevo, BOSNIA AND
HERZEGOVINA
Nonsteroidal antiinflammatory drugs, including diclofenac, have
been reported to induce apoptosis in a variety of cell lines (colorec-
tal cancer, oral cavity cancer, promyelocytic leukemia, neuroblasto-
ma and other cell lines). In this study, the effects of diclofenac on
the growth and apoptosis of human peripheral blood lymphocytes
were examined. After separation from blood, lymphocytes were
suspended in RPMI 1640 medium, transferred to a 96-well micro-
plate and then cultured at 37ordm;C. Solution of diclofenac was
added to cultures after 24 h, in final concentrations of 10, 20, 50,
100 and 300 lmol/l. After 48 h, proliferative response was evalu-
ated by WST-1 assay.
There was no significant difference in the absorbance between con-
trols and cell cultures treated with five different concentrations of
diclofenac (P>0.05). The doses applied did not cause any signifi-
cant alterations in proliferation of cultured human lymphocytes.
F1-94
Antigenic properties of recombinant BBK32
protein fragment in Lyme borreliosis patients
V. Baumanis, R. Ranka and E. Verdins
LU Biomedical Research and Study Centre, Riga, LATVIA
Truncated borrelial surface-exposed fibronectin-binding lipoprotein

protein BBK32 was evaluated as an antigen in the serodiagnosis of
Lyme borreliosis (LB). The borrelial fibronectin-BBK32 interaction
is likely to be important in Borrelia burgdorferi-specific pathogenic
mechanisms, particularly in the context of dissemination and/or
persistence. B. burgdorferi express surface-exposed fibronectin bind-
ing lipoprotein BBK32 during infection in mammals. This protein
has immunological activity and was evaluated as antigen in the
serodiagnosis of LB. Moreover, fibronectin-BBK32 interactions
seem to be important in the pathogenesis of Lyme disease. The
recombinant fragment of BBK32 from B. afzelii was cloned and
expressed in E. coli with six histidine tag. The expression of protein
was proved by Western Blot analysis with anti-His antibodies. The
protein was purified with Ni-NTA Spin Kit and used in further
applications. Serum samples of patients with proved LB diagnosis
were used for immunoglobulin G (IgG) Western blotting (WB) or
enzyme-linked immunosorbent assay (ELISA). Immunologic assays
showed that almost all samples were positive for our recombinant
truncated BBK32 protein. Our findings indicate that the BBK32
proteins are promising serodiagnostic antigens for the detection of
LB and may serve as useful tool in Lyme disease pathogenesis
studies.
Acknowledgement: This work has been supported by European
Social Fund.
F1-95
Guanosine treatment modulat es glutamate
transport capacity in neonatal rats when
administered after hypoxia-ischemia
M. B. Moretto
1
, B. Boff

2
, D. Lavinsky
2
, C. A. Netto
2
,
J. B. Rocha
1
, S. Wofchuk
2
and D. Souza
2
1
Universidade Federal de Santa Maria, Santa Maria, BRAZIL,
2
Universidade Federal do Rio Grande do Sul, Porto Alegre,
BRAZIL
Hypoxia-ischemia (HI) is a leading cause of morbidity and mortal-
ity in the perinatal period. Biochemical events such as energy fail-
ure, membrane depolarization, brain edema, increase of
neurotransmitter release, increase of intracellular Ca(
2+
), produc-
tion of oxygen-free radicals, and decrease of blood flow are trig-
gered by hypoxia-ischemia and may lead to brain dysfunction and
neuronal death. The nucleoside guanosine (Guo) have been shown
to exert neuroprotective effects against glutamatergic excitotoxic
events. A model of HI was prepared by ligating the left common
carotid artery in 7-day-old rats, followed by 8% hypoxia exposure
and the effect of intraperitoneal injection guanosine was investi-

gated in several protocols. In this work, we demonstrated that
guanosine avoided the decrease in the glutamate uptake by hippo-
campal slices measured 3 days after HI insult. This effect was
achieved when administrated in three doses, i.e.: (i) immediately,
24 and 48 h after the insult; (ii) 3, 24 and 48 h after HI; (iii) 6, 24
and 48 h after the injury. We conclude that guanosine might
enhance glutamate transport capacity in early stage, and could
contribute to the maintenance of extra cellular glutamate in phy-
siological concentrations. Such effect may play an important role
in the regulation of central nervous system development, suggest-
ing the potential value of initiating clinical studies.
F1-96
Targeting of a diabetogenic T cell population by
DNA vaccination
E. Rivas
1
, F. Rodriguez
2
and T. Stratmann, Jr.
1
1
University of Barcelona, Barcelona, SPAIN,
2
CReSA, Barcelona,
SPAIN
Type I diabetes (T1D) is caused by the selective destruction of the
insulin-producing beta cells of the pancreatic islets by autoreactive
T cells. BDC2.5 is a diabetogenic, autoreactive T-cell clone that
recognizes an unidentified autoantigen expressed by beta cells. By
using tetramers of I-Ag7, the restricting MHC class II molecule of

BDC2.5, and a mimotope that is recognized by BDC2.5, we have
previously identified a T cell population in the NOD mouse model
that shares its antigen specificity with BDC2.5. This CD4 T cell
population, termed 2.5mi+, was already identified in 4 week-old
NOD mice, before either insulitis or T1D are established, and
therefore might represent an ideal target for immunotherapy. To
test this hypothesis, DNA vaccines were generated in order to tar-
get a mimotope peptide – 2.5mi – recognized by 2.5mi+ T cells to
lysosomes, the MHC class II loading compartment. Repeated
immunization lead to the expansion of 2.5mi+ T cells. No signs of
long-term antigen-specific T cell tolerance were detected. Neverthe-
less, diabetes was prevented in the majority of animals following
treatment. Therefore, mechanisms different from T cell tolerance
need to be responsible for diabetes prevention in this experimental
setup.
288 ª 2007 The Authors Journal compilation ª 2007 FEBS
Abstracts
F1-97
Zoledronic acid resistance mechanisms of
osteosarcoma cell lines
M. Padrines
1
, B. Ory
1
, G. Moriceau
1
, F. Redini
1
, M. J. Rogers
2

and D. Heymann
1
1
INSERM ERI 7, Nantes, FRANCE,
2
Institute of Medical
Sciences, Aberdeen, UK
We recently demonstrated selective and original anti-tumor effects
of zoledronic acid (Zol) on several osteosarcoma cell lines. The
present study investigated the potential Zol-resistance developed by
osteosarcoma cells after prolonged treatment. Long-term treatment
with 1 lM Zol reduced the sensitivity of osteosarcoma cells to high
concentrations of Zol. XTT assays demonstrated that the Zol-
resistant cells were always sensitive to conventional anti-cancer
agents such as methotrexate, mafosfamide and doxorubicin and
that the resistance process was not associated with the multidrug
resistance (MDR) phenotype. However, other experiments revealed
that this drug resistance was restricted to the nitrogen containing
bisphosphonates. This resistance was also correlated with a higher
transcript level and enzymatic activity of farnesyl diphosphate syn-
thase (FPPS). To demonstrate the involvement of FPPS in the
Zol-resistance mechanism, the Zol-resistant cells were transfected
with a siRNA for FPPS. This transfection strongly increased their
sensitivity to Zol. This study confirms the therapeutic potential of
Zol for the treatment of bone malignant pathologies but reveals
the possibility that the treatment regimen may be important in
terms of duration and dose to avoid the development of drug
metabolic resistance.
F1-98
Expression of 11b-hydroxysteroid

dehydrogenase type 1 (11HSD1) and
glucocorticoid synthesis in immune cells
P. Ergang
1
, P. Leden
1,2
, K. Vagnerova
´
1
, K. Vitackova
3
,
I. Miksik
3
, M. Kment
4
and J. Pacha
3
1
Academy of Sciences, Prague, CZECH REPUBLIC,
2
Third
Faculty of Medicine, Charles University, Prague, CZECH
REPUBLIC,
3
Academy of Sciences of Czech Republic, Prague,
CZECH REPUBLIC,
4
Third Faculty of Medicine, Charles
University, Prague, CZECH REPUBLIC

11HSD1plays an important role in the prereceptor modulation of
glucocorticoids by locally converting less active cortisone and
11-dehydrocorticosterone into active cortisol and corticosterone.
As endogenous glucocorticoids seem to play an important role in
regulation immune system, we hypothetized that the local metabo-
lism of glucocorticoids via 11HSD1 is involved in modulation of
local concentration of glucocorticoids in immune cells and
inflamed tissues. Expression of 11HSD1 mRNA and enzyme activ-
ity were assessed in colon, mesenteric lymphatic nodes (MLN) and
intraepithelial lymphocytes (IEL) obtained from rats and mice
without and with experimental colonic inflammation. We demon-
strated that inflammation stimulated 11HSD1 mRNA and enzyme
activity in colon and that this up-regulation of 11HSD1 was asso-
ciated with induction of 11HSD1 in LMN and IEL. Inflammation
was accompanied by elevated levels of pro-inflammatory cytokines
TNF-a and IL-1b and COX 2 in both colon and MLN. Using the
colonic organ culture system in vitro, we showed that incubation
with TNF-a increased the expression of 11HSD1 whereas treat-
ment with IL-1b was without any effect. These data indicate that
development of inflammation and activation of immune cells pro-
motes local production of biologically active glucocorticoids from
their inactive 11-oxo derivatives and thus 11HSD1 might uncouple
the local concentration of glucocorticoids from their plasma level.
Acknowledgement: Supported by Ministry of Health of the
Czech Republic (NR/8576–3).
F1-99
Functional activity of adipose- and bone
marrow derived mesenchymal stromal cells
under hypoxia and inflammation in vitro
A. Efimenko, N. Kalinina and V. Tkachuk

Lomonosov Moscow State University, Moscow, RUSSIAN
FEDERATION
Adult mesenchymal stromal cells derived from adipose tissue
(ADSC) and bone marrow (BMDSC) secrete a various number of
angiogenic cytokines and improve vascularization of ischemic hind-
limbs being injected in damaged muscle or intravenously. But the
exact changes of their functional activity after delivery to ischemic
area remain to be cleared. We directly compared in vitro the reac-
tions of ADSC and BMDSC to hypoxic and inflammatory condi-
tions which are the main factors affecting cells in ischemic area.
ADSC and BMDSC were isolated from C57BL/6 mice and expan-
ded with serial passages. Then cells were cultivated for 48 h under
1% (hypoxia) or 20% (normoxia) O
2
or in the presence of TNFal-
pha (T) or conditioned medium (CM) from THP-1 cells stimulated
by TNFalpha (T+). Cell apoptotic rates analyzed by quantitative
TUNEL in hypoxia groups were similar to normoxia groups, but
significantly increased (up to 70%) both for T- and T+-groups.
Gene expression and levels of angiogenesis-related cytokines
(VEGF, HGF, its receptor c-met, PlGF) in CM increased under
hypoxia and slightly changed or decreased in T- and T+-groups.
This effect was higher for ADSC than for BMDSC. Similar chan-
ges were observed for endothelial tube formation on Matrigel in
response to CM from different cells groups. The expression of
CXCR4 was significantly elevated in response to hypoxia on
ADSC surface, but slightly on BMDSC. In conclusion hypoxia,
but not inflammatory factors, stimulates angiogenic activity of
ADSC and BMDSC in vitro. ADSC seem to be more respondent
to hypoxia in secreting angiogenic growth factors and homing

toward SDF-1 than BMDSC. Considering their isolation, cultiva-
tion and expansion simplicity ADSC have a great potency in cell-
based therapy of ischemic diseases.
F1-100
The master crosstalk for protein kinase A
type II: cyclic AMP vs. H
2
O
2
M. Zentella
1
, H. Vazquez
1
, J. P. Pardo
1
, J. L. Rendon
1
,
R. Villalobos
1,2
, H. Riveros
1
and E. Pin
˜
a
1
1
UNAM, Me
´
xico, D.F., MEXICO,

2
Unidad de Biomedica, Facultad
de Estudios Superiores-Iztacala, Unam, MEXICO
In adipose tissue, fasting or stress increases lipolysis rate via c
AMP whereas feeding promotes lipogenesis using insulin signaling;
a convergent point of molecular control to choose which one of
either pathways will be activated is presented here. H
2
O
2
added
generated by insulin inhibited cAMP-dependent PKA activation
and its amplification cascade until lipolysis. Once activated with
cAMP, PKA was insensitive to H
2
O
2
. Impediment of PKA activa-
tion by H
2
O
2
remained after its elimination, but it is reverted by
incubation with thioredoxine-thioreductase. H
2
O
2
prevented cAMP
activation of PKA holoenzyme formed with purified wild type cat-
alytic-a and regulatory-RIIa subunits, the holoenzyme was insen-

sitive to H
2
O
2
when catalytic-a subunit was replaced by C199A
mutant subunit. It is demonstrated that in presence of H
2
O
2
, Cys
199 fromcatalytic-a and probably Cys 97 from regulatory-RIIa
subunits, form a disulfide bond preventing cAMP-dependent PKA
activation, yhusdisplaying a novel mechanism for its regulation. In
conclusion, specific PKA oxidationstops, whereas specific PKA
reduction allows, cAMP amplification chain cascade. Based in
modeling and phylogenetic analysis, this regulation mechanism
involving RIIa subunit might be extensive to RIIb subunits. This
redox control of PKA might be shared for other physiological
responses.
ª 2007 The Authors Journal compilation ª 2007 FEBS 289
Abstracts