Abstract
Integration of Metabolism and Survival
PP-1
The metabolic switch in liver methio nine
metabolism
T. K. Korendyaseva, V. A. Volkov, D. N. Kuvatov,
M. V. Martinov, V. M. Vitvitsky and F. I. Ataullakhanov
Laboratory of Physical Biochemistry, National Research Center
for Hematology, Moscow, Russia. E-mail: [email protected]
Methionine (Met) is an essential amino acid and the only sub-
strate for synthesis of S-adenosylmethionine (AdoMet) that is the
main substrate for multiple intracellular methylases. There are
two modes of Met metabolism in liver. In case of its dietary
restriction Met can be metabolized via conservative remethylation
cycle. In case of Met excess (high [Met]) it is mostly converted to
cysteine via transsulfuration pathway. Mathematical modeling of
methionine metabolism in liver (Martinov et al. 2000) predicts
that transition from Met conservation to Met consumption hap-
pens in narrow [Met] range and is accompanied by sharp 10-fold
increase in [AdoMet] and by significant increase in the rate of
Met consumption. To test model predictions we analyzed the
dependence of [AdoMet] and the rate of Met consumption on
[Met] in suspension of freshly isolated mouse hepatocytes. [Met]
varied from 40 to 400 lM. In the narrow [Met] range from 80 to
120 lM [AdoMet] sharply increased by eight times, while Met
consumption rate increased by six times in [Met] range from 40
to 150 lM. This data confirms the existence of the metabolic
switch in liver metabolism triggered by Met concentration.
PP-2
Effects of hyperthermia on mitochondrial
respiration and NAD(P)H fluorescence

response of liver and heart mitochondria was very different in
the range of temperature from 42 to 47 °C. Complete uncoupling
of oxidative phosphorylation and the inhibition of the respiration
was observed at 47 °C in isolated heart mitochondria. Respir-
ation was completely ceased in liver mitochondria, indicating that
their respiratory chain is more susceptible to higher temperature.
Increase of temperature to 47 °C was followed by NAD(P)H
fluorescence decrease both in heart and liver mitochondria.
Change of free Ca
2+
concentration in incubation medium from
5nM and 1 lM did not have significant effect on the observed
changes in mitochondrial respiration and NAD(P)H fluorescence;
however, Ca
2+
overload (10 lM Ca
2+
) drastically increased the
deleterious temperature effects in both types of mitochondria.
PP-3
The yeast Ccr4–Not complex controls
ubiquitination of the nascent-associated
polypeptide complex
O. Panasenko, E. Landrieux, M. Feuermann, A. Finka,
N. Paquet and M. Collart
Department of Microbiology and Molecular Medicine, University
of Geneva, CMU, Geneva, Switzerland.
E-mail: [email protected]
In this study, we determine that the Saccharomyces cerevisiae
Ccr4–Not complex controls ubiquitination of the conserved het-

,
A. Guevorkian
1
and A. Galoyan
2
1
Laboratory of Pathological Biochemistry, Institute of Biochem-
istry, Yerevan, Armenia,
2
Department of Neurohormones, Institute
of Biochemistry, Yerevan, Armenia.
E-mail: [email protected]
Trauma of skeletal muscle by long-lasting compression, is fol-
lowed by acute hemodynamic shock, myoglobinuria, acute renal
insufficiency, and lethal endotoxicity. There are numerous data
indicating that the main intoxication of the organism occurs dur-
ing decompression period, in which toxic metabolic products are
released into the blood and myocardium. Clinical data show that
death are most frequently depends on hyperkaliemia, starting
from the decompression. Natural cytokine – PRP was obtained
from both neurohypophysis and hypothalamic neurosecretory
Abstracts
77
granules by A. Galoyan. In the experiments 108 Wistar male rats
of 160–200 g mass were used. CS was induced by a compression
of femoral soft tissues using a special press. Common amount of
calcium ions was defined using crezolphtalein spectrophotometer
method. The results show the level of Ca
2+
in blood after 2 h

1
Genom Metabolism and Repair, Institute of Enzymology,
Biological Research Center-Hungarian Academy of Science,
Budapest, Hungary,
2
STAOK, Institute of Medical Biology,
Szeged, Hungary. E-mail: [email protected]
Uracil-free DNA is considered to be essential for most organ-
isms.
Fruit fly larvae present a very exceptional case, as the uracil-pre-
ventative dUTPase is restricted only to the imaginal discs, while
larval tissues associated with intensive DNA synthesis do not
contain it. Moreover, the gene of the major uracil-eliminating
UNG is missing, possibly leading to sustained presence of uracil
in DNA. Tissues containing uracil-DNA are pre-destinated to
death during metamorphosis, whereas imaginal discs survive.
Within this context, dUTPase gains importance beyond DNA
repair as a metamorphosis regulator factor.
In this study the subcellular localization of the two dUTPase iso-
forms were investigated.
They were expressed separately as fluorescent protein fused con-
structs in S2 cells and microinjected into early Drosophila
embryos.
The 23 kDa isoform, which contains a nuclear localization signal
(NLS), is present mainly in the nucleus. On the contrary, the
21 kDa isoform, lacking the NLS segment, remains in the cyto-
plasm. The 21 kDa shows an unexpected localization shift during
nuclear mitosis. In prophase, with nuclear envelope disintegrated,
this isoform accumulates in the karyoplasm. As nuclei enter telo-
phase, the 21 kDa isoform gets again excluded from the nuclei.

Short-term muscle fatigue and its restoration was analyzed in
rabbit muscle. Fast-twitch tibialis anterior was electrostimulated
at 10 Hz for 20 s, 1, 5, 15 and 30 min and then allowed to rest
for 30 min except for 30 min. Muscles stimulated for 30 min were
rested for 3 h.
Muscles were analyzed for ATP, creatine-P, glycogen, phosphor-
ylated glucose and fructose, and lactate. The fatigue index was
measured after rest periods.
The fatigue index decreased significantly after 15 and 30 min of
electrostimulation and did not recover after 30 min of rest. After
3 h of rest, muscle strength was nearly restored. Although all
metabolites were modified during fatigue, only ATP remained
significantly low after 3 h of rest, which prevented restoration of
muscle strength. The other metabolites were restored quickly.
ATP regulated the sarcolemma ionic channels. The chloride
channels (ClC-1) regulate the excitability of skeletal muscle. They
are inhibited by high ATP levels which decreases their sensitivity
to positive voltage. When ATP decreases, the activity of ClC-1
channels increases, reducing muscle excitability and inducing
muscle fatigue. Decrease of ATP protects muscle against sus-
tained contraction suggesting that changes in ATP concentration
could be decisive in the control of fatigue.
PP-7
Suppression of expression of
muscle-associated proteins by PPARa in
brown adipose tissue
T. Nakajima, N. Tanaka and T. Aoyama
Department of Metabolic Regulation, Shinshu University, Graduate
School of Medicine, Matsumoto, Japan.
E-mail: [email protected]

2
1
Faculty of Medicine, University of Latvia, Riga, Latvia,
2
Department of Medicinal Chemistry, Latvian Institute of Organic
Synthesis, Riga, Latvia. E-mail: [email protected]
Mildronate [3-(2,2,2-trimethylhydrazinium)propionate dihydrate]
is inhibitor of gamma butyrobetaine hydroxylase, an enzyme
which catalyses the synthesis of carnitine from gamma-butyrobe-
taine (GBB) in liver. It was found that mildronate ameliorates
cardiac function during ischaemia by modulating myocardial
energy metabolism. In this study we measured the changes in the
Abstracts
78
contents of carnitine and GBB in rat plasma, heart and brain tis-
sues during the long-term (28 days) treatment by mildronate (i.p.
120 mg/kg/daily). We used a HPLC set-up with pre-column deri-
vatization which allowed us to determine mildronate, carnitine
and GBB in a single run. Obtained data show that mildronate
caused the time-dependent significant decrease in carnitine con-
centration and increase of GBB concentration in rat tissues. We
detected about fivefold increase of GBB contents in plasma and
brain and sevenfold increase in rat heart. We also tested the car-
dioprotective action of mildronate in the experimental model of
heart infarction in isolated rat heart. Obtained results indicate
that the cardioprotective effect of mildronate develops in concert
with the induced changes in GBB and carnitine concentrations in
rat tissues. In conclusion, our study provides the experimental
evidence that the administration of mildronate not only decreases
the free carnitine concentration, but also brings about a signifi-

ences in glucose accumulation and CO
2
production were
observed in diabetic retinas. However, glycogen levels were 2.4-
fold higher and high lactate levels have been reported in diabetic
retina (Salceda et al. 1998).
Our results indicate an active oxidative metabolism in retina. The
low NAD/NADH ratios found at any glucose concentration tes-
ted suggested that the aerobic pathway should be rapidly satur-
ated. We proposed that gluconeogenesis could be a mechanism
for lactate removal during periods of high metabolic activity and
under pathological conditions.
PP-10
Phosphoinositides are involved in phagosome
formation and maturation in the ciliate
tetrahymena
D. Deli
1
, G. Leondaritis
2
, A. Tiedtke
3
and D. Galanopoulou
1
1
Laboratory of Biochemistry, Department of Chemistry, University
of Athens, Zografou, Athens, Greece,
2
Laboratory of Developmen-
tal Neurobiology and Neurochemistry, Institute for Biomedical

Tetrahymena. Ongoing studies with purified phagosomes of dif-
ferent maturation stages and in vivo visualization of PI redistribu-
tion during phagocytosis will clarify their exact targets.
PP-11
Contribution of cGMP signaling pathway(s) in
regulation of Leydig cell steroidogenesis
T. S. Tatjana and S. A. Silvana
Faculty of Science, University of Novi Sad, Novi Sad, Serbia and
Montenegro. E-mail: [email protected]
cGMP is formatted in Leydig cells but the role of this second
messenger in androgen (T + DHT) production have been incom-
pletely characterized. Here, we show presence of transcripts for
the all elements of cGMP signaling pathways, i.e. membrane-
bound guanylyl cyclase, NO synthethase (NOS), soluble guanylyl
cyclase, GMP-specific phosphodiesterase 5 (PDE 5), protein kin-
ase G (PKG I), multidrug resistance protein 5 (MRP5) as well as
cyclic nucleotide-gated channels (CNG; rode, olfactory and
cone). We also characterized effect of activation and inhibition of
different elements of cGMP signaling pathway(s) on androgen
production in static culture of purified adult rat Leydig cells
under basal conditions and in response to stimulation with hCG
and different steroidogenic substrates. In all treatments which rise
cGMP production stimulation of androgen production was
occurred and this phenomenon was more prominent in basal
than in receptor-controlled androgen production. Moreover, and-
rogen production was decreased in the presence of specific PKG
inhibitor, indicate that PKG-dependent phosphorylation take
place in regulation of Leydig cell steroidogenesis. Immunoprecipi-
tation study showed PKG-dependent phosphorylation of steroid-
ogenic acute regulatory protein (StAR), suggesting that both

5 (MRP 5), is gradually decreased during stress, while there were
no changes in the level of mRNA for other elements of NO-
cGMP signaling pathway(s). Results of this study, together with
those published, suggest that NO-cGMP signaling pathway(s) are
involved in stress-impaired testicular steroidogenesis.
PP-13
Estrogenic effects of natural and synthetic
compounds assessed in Sacch aromyces
cerevisiae
G. Hasenbrink
1
, L. Wildt
2
, J. Ludwig
1
and H. Lichtenberg-Frate
1
1
IZMB, Molecular Bioenergetics, University of Bonn, Kirschallee
1, Bonn, Germany,
2
Klinik fu
¨
r gyna
¨
kologische Endokrinologie und
Sterilita
¨
t, Universita
¨

YAP4P phosphorylation during yeast stress
response
J. Pereira, T. Nevitt and C. Rodrigues-Pousada
ITQB, UNL, Oeiras, Portugal. E-mail: [email protected]
YAP4 belongs to the YAP family of eight bZIP transcription fac-
tors. YAP4 has been described as a gene that confers resistance
to cisplatin and several antimalarial drugs. Recently, we were
able to associate YAP4 with the yeast stress response, showing
that its mRNA levels increase under osmotic and oxidative stress
and that Yap4 is induced and phosphorylated under these condi-
tions. By direct mutagenesis we show that Yap4 phosphorylation
is not involved in protein subcellular localization as the non-
phosphorylated mutants T192A- and S196A-Yap4 still give rise
to a nuclear resident protein. By blocking Yap4 transit to the
nucleus through mutation of its nuclear localization signal, we
observed that Yap4 phosphorylation is abolished. These results
suggest that Yap4 phosphorylation occurs in the nucleus and is
most probably related to its activation and/or stability. To
address this, Yap4 protein kinetics was analysed in the double
mutant T192A-S196A-Yap4. We observe that the mutant protein
is expressed but not phosphorylated during the time course
applied, suggesting that phosphorylation of T192 and S196 resi-
dues of Yap4 is not related to its stability under hyperosmotic
stress conditions. Band-shift analyses is being used to study the
role of Yap4 phosphorylation in its cis-element binding as well as
determine whether Yap4 can heterodimerize with Yap6, its clo-
sest family member, in vivo.
PP-15
Investigation of apoptotic gene expression
levels in multidrug-resistant MCF-7 cell lines

tinuous drug application in dose increments. According to cytotox-
icity analysis, developed cell lines were found to be resistant to
anticancer drugs used. Bcl-2 and Bax gene expression analysis was
performed by RT-PCR and, related protein levels were determined
by Western blot and immunostaining analysis. The results suggest
that differential expression levels of Bcl-2 and Bax genes may be
one of the mechanisms of acquired resistance in MCF-7 cells.
PP-16
Differential expression of isoforms of spleen
tyrosine kinase in tissues: effects of the
microbial flora
F. Duta
1
, M. Ulanova
1
, D. Seidel
2
, L. Puttagunta
3
,
S. Musat-Marcu
4
, K. S. Harrod
5
, A. D. Schreiber
6
, U. Steinhoff
2
and A. D. Befus
1

Western blot and real-time RT-PCR. Interestingly, Syk is present
in both germ-free and conventional mice and the microbial flora
has no major influence on overall expression of Syk.
We also investigated the distribution of Syk isoforms, long Syk
(L) and short spliced variant Syk (S), in tissues of germ-free and
conventional mice. They were widely expressed in mouse tissues,
although previously it was thought that Syk (S) was restricted to
bone marrow. Interestingly, Syk (S) protein was significantly ele-
vated in lung and spleen in germ-free mice.
Thus, Syk is widely distributed in various cells and tissues and is
likely involved in several pathways of development, and normal
and abnormal physiology.
Acknowledgment: Funded by CSACI/CAAIF/Merck Frosst,
Alberta Heritage Foundation for Medical Research and NIH.
PP-17
Expression of the human HSPA 2 gene in
cancer cell lines
W. Piglowski, A. Kwiecien, A. Mazurek, M. Jarzab,
Z. Krawczyk and D. Scieglinska
Department of Tumor Biology, Maria Skodowska-Curie Memorial
Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice,
Poland. E-mail: [email protected]
Heat-shock proteins are a group of highly conserved chaperone
proteins. The human Hsp70 family consists of at least eight mem-
bers that differ from each other by expression pattern. Many
types of cancer cells constitutively express elevated level of Hsp70i
protein which in normal cells is induced only by stress conditions.
The Hsp70i protein influences the phenotype of tumor cells ren-
dering them more resistant to agents inducing cell death. Another
member of Hsp70 family is the HSPA2 protein, which is a crucial

,
I. Vass
1
, P. Goloubinoff
3
, E. Vierling
2
and L. Vigh
1
1
Biol. Res. Center, Szeged, Hungary,
2
Univ. Arizona, Tucson,
AZ, USA,
3
Univ. Lausanne, Lausanne, Switzerland.
E-mail: [email protected]
The cellular pool of small heat shock proteins (sHsps) is divided
into a cytoplasmic subfraction responsible for regular chaperone
activity and a membraneous subfraction, involved in membrane
stabilization. We have isolated a series of Synechocystis Hsp17
mutants characterized with regard to in vivo thermotolerance,
in vitro chaperone activity and propensity to form oligomers. We
defined particular features of these mutants responsible for inter-
acting with membrane lipids, a potential determinant of their
membrane association. While causing destabilization of the oligo-
meric state, three mutations of Hsp17 caused no significant alter-
ations in the lipid and/or thylakoid-binding characteristics
compared to wild-type Hsp17. However, with a mutation at the
N-terminus (Q16R), a dramatic change in the association of

transcriptional activation of specific genes involved in the
response to several stress conditions such as oxidative, osmotic,
arsenic and heat stress, among others. The existing data concern-
ing the function of Yap proteins support both a degree of func-
tional overlap as well as distinct physiological roles.
Furthermore, data are beginning to emerge on the crosstalk
between the members of this family. Recent data obtained by us
strongly indicate that Yap8 and Yap1 are able to interact in
response to arsenic stress. This is the first evidence of the forma-
tion of heterodimers between bZIP transcription factors in yeast.
The generation of a strain deleted in all YAP genes is an invalu-
able tool in order to study the function of each member of the
Yap family individually. Thus, the main challenge of the present
study was the construction of the ‘YAP0 SUPER-MUTANT’
deleted in all YAP genes. The strategy used was a combination
of PCR-based gene disruption using the Cre/loxP system, tetrad
and phenotypic analysis. Experiments are being carried out in
order to understand the complex role of these transcription fac-
tors.
Rhythmic Signals: the Setting of Biological Time
PP-20
The effect of plant hormones (GA3, IBA and
ABA) on ARF1 and SAR1 expression in
Pisum sativum var. araka
O. Ertekin and A. R. Memon
TU
¨
B
_
ITAK – Research Institute for Genetic Engineering and

Laboratory of Neurochemistry, Institute of Physiology, Tbilisi,
GA, USA. E-mail: [email protected]
The Ras family of small GTP-binding proteins has been implica-
ted as a molecular switch that directs diverse cellular responses,
such as cell cycle progression, transformation, and cell death.
Ras is regulated by a series of post-translational modifications,
including farnesylation, palmitation, and nitrosylation, but the
role of these modifications on the regulation of downstream
effectors is not known. We investigated the effects of manumycin,
an inhibitor of farnesyltransferase and L-NAME, an inhibitor of
nitric oxide synthase on the activity of various transcription fac-
tors in mixed primary neuronal/glial cells. We have found that
both manumycin and L-NAME inhibit the DNA-binding activity
of NF-jB (50 kDa subunit). L-NAME also decreases the activity
of STAT and manumycin restore this inhibitory effect of
L-NAME. Both inhibitors raise the activity of c-Fos and only
manumycin elevate the DNA-binding activity of Sp1. Further-
more, manumycin, as well as L-NAME decrease the activity of
c-Jun, while in the presence of both inhibitors the DNA-binding
potency of this transcription factors does not change. It is
concluded that simultaneously (nitrosylated and farnesylated)
modified Ras alter the systems regulating the upstream pathway
of c-Jun and does not change the activity of the systems, control-
ling STAT, Sp1, NF-jB, CREB-1, ATF-2, and c-Fos.
Acknowledgment: This study was supported by INTAS 2001-
0666 grants.
PP-22
Treatment with substance P and caerulein
induces chemokine synthesis in pancreatic
acinar cells

PP-23
ERK and JNK activation differenti ally regulates
phosphatidic acid-induced matrix
metalloproteinase- 9 expr ession
S. H. Baek, J. G. Lee and C. H. Lee
Department of Biochemistry & Molecular Biology, College of
Medicine, Yeungnam University, Daegu, South Korea.
E-mail: [email protected]
Phosphatidic acid (PA) is implicated in pathophysiological pro-
cesses associated with cellular signaling events and inflammation,
which include regulating the expression of numerous genes. The
present study examined whether the temporal control of ERK
and JNK could differentially regulate the expression of NF-jB-
dependent gene, matrix metalloproteinase-9 (MMP-9). PA
induced the expression of MMP-9 in a dose-dependent manner,
but mRNA showed a biphasic increase by PA treatment. PA
induced phosphorylation of ERK1/2 and JNKs. Inhibition of
ERK1/2 with U0126 suppressed PA-induced MMP-9 expression,
whereas inhibition of JNKs with SP600125 enhanced cell migra-
tion, with strong increase of MMP-9 expression. PA activated
NF-jB pathway as measured by increased IjBa degradation,
promoter activity, and NF-jB-DNA binding. The expression of
MMP-9 and the cell migration was inhibited when NF-jB
activation was downregulated by SN-50, NF-jB inhibitor. In
addition, tumor necrosis factor-a antibody strongly suppressed
PA-induced MMP-9 expression, suggesting the involvement of
tumor necrosis factor-a pathway. Overall, these observations
demonstrate that activation of ERK1/2 and JNKs play a differ-
ent role in the activation of NF-jB and the subsequent regula-
tion of MMP-9.

highest level after trauma at the 48th hour.
Conclusion: Statistically the 24 and 48th hour CRP serum level
showed a meaningful increase compared to the 6th hour
(P < 0.01). These results make the measured IL-6 level after
trauma at the 24th hour helpful to estimating the tissue defeats
occurring based on trauma.
PP-25
Cytokine levels in the seminal plasma of fertile
and infertile men
N. Sayilir
1
, M. Tarakcioglu
2
and C. Karakaya
1
1
Department of Biochemistry, School of Medicine, Yuzuncu Yil
University,
2
Department of Biochemistry and Clinical
Biochemistry, School of Medicine, Gaziantep University.
E-mail: [email protected]
Aim: Cytokines are peptides used for the controlling of intracel-
lular activities and the in the cellular communication. They are
released from various specialized cells of urogenital systems of
men. These molecules are considered to have some effects on
sperm functions and fertility. In this study, examining the levels
of IL-6 and TNF-a in the seminal plasma of men who were
infertile due to various reasons, and correlations between various
sperm parameters and urogenital infections have become the

2
, R. Vilskersts
2
, R. Muceniece
2
and
M. Dambrova
1
1
Medicinal Chemistry, Latvian Institute of Organic Synthesis,
Riga, Latvia,
2
Faculty of Medicine, Latvian University, Riga,
Latvia. E-mail: [email protected]
In the recent years the nuclear factor kappa B (NF-jB) has
attracted considerable interest due to its key role in responses to
injury and inflammation, and regulation of a multitude of genes
of which several are shown to become activated during the
inflammation. We have shown earlier that the guanidine com-
pound ME10092 [1-(3,4-dimethoxy-2-chlorobenzylideneamino)-
guanidine] possesses a strong cardioprotective effect in an experi-
mental heart infarction model in the rat. We have also found
Abstracts
83
that the compound possesses a certain antioxidative profile, as
well as inhibition of activation of NF-jB in the rat cardiomyo-
cytes in simulated ischemia and reperfusion in vitro. In the pre-
sent study, we tested the activity of ME10092 in the
lipopolysacharide (LPS)-induced brain inflammation model in
mice in vivo. By electron paramagnetic resonance (EPR) we

types independently to the I-jB kinase signaling. TGase 2 induces
the polymerization of I-jB rather than stimulating I-jB kinase.
This polymerization of I-jB results in direct activation of NF-jB
in breast cancer cell lines. Consequently chemotherapeutic resist-
ance appears to be acquired in cancer cells due to TGase 2-medi-
ated NF-jB activation. We also found that TGase inhibition
reverses NF-jB activation concomitantly with drug resistance in
breast cancer cells. Taken together, developing TGase 2 inhibitors
will benefit on cancer therapy as chemotherapeutic sensitizers.
PP-28
Tracking NF-jB activation upon genotoxic
stress: a non-classical mechanism
S. C¸ o
¨
l Arslan and C. Scheidereit
Max Delbru
¨
ck Center for Molecular Medicine.
E-mail: [email protected]
The NF-jB family of transcription factors play multiple roles in
immune system, development and regulation of apoptosis. In the
basal state, NF-jB dimers are bound to the inhibitor IjB mole-
cules and kept in the cytoplasm. Upon receptor stimulation, the
kinase complex consisting of IKKa, IKKb and IKKc/NEMO
gets activated. The activated complex phosphorylates Ij B and
leads to its proteosomal degradation. The released NF-jB dimers
then translocate to the nucleus and regulate transcription.
In addition to well-described molecules like LPS, TNFa or IL-1,
genotoxic stress also activates NF-jB. The mechanism of this
activation has been proposed as sequential sumoylation, ATM

Division of Clinical Pathophysiology,
University Hospital, Lausanne, Switzerland.
E-mail: [email protected]
Introduction: Flagellin (FLAG), a 55 kDa monomer obtained
from the flagella of gram-negative bacteria, induces inflammatory
responses in vitro, mediated by Toll-like receptor 5 (TLR5).
Gram-negative sepsis is associated with myocardial failure, which
is related to myocardial cytotoxicity and inflammation triggered
by putative circulating mediators. Whether FLAG may exert
such a cytotoxic role during gram-negative sepsis has not been
evaluated. Thus, the aim of the present study was to explore a
potential role of FLAG as an inducer of cardiomyocyte inflam-
mation in vitro and in vivo.
Methods: In vitro, H9C2 rat cardiomyocytes were stimulated
with recombinant Salmonella FLAG (1–100 ng/ml, 10–24 h).
In vivo, BALB/c mice were injected (tail vein) with 1–5 l g FLAG.
Proinflammatory effects of FLAG were evaluated by its ability to
activate NFjB (monitored by degradation and phosphorylation
of IjB, nuclear p65 translocation, NFjB DNA binding and
NFjB-luciferase gene reporter), and to induce transcription and/
or expression of the inflammatory cytokines TNFa and MIP-2.
Results: FLAG-activated NFjB in a concentration-dependent
manner in cardiomyocytes both in vitro and in vivo, and also
upregulated the transcription and expression of TNFa and MIP-2.
Conclusion: Flagellin is a potent mediator of proinflammatory
signaling in cardiomyocytes and may represent a previously unrec-
ognized mediator of myocardial failure during gram-negative
sepsis.
PP-30
Regulation of antiviral response at the level of

A. Mialon
1,2
, M. Sankinen
1
,H.So
¨
derstro
¨
m
1
, T. T. Junttila
2,3,4
,
T. Holmstro
¨
m
1
, R. Koivusalo
4
, A. C. Papageorgiou
1
,
R. S. Johnson
5
, S. Hietanen
4,6
, K. Elenius
2,4
and
J. Westermarck

and c-Jun interact in transformed human cells in a manner that is
dependent on JNK activity. c-Jun target gene epidermal growth
factor receptor (EGFR) was identified as a novel gene whose
expression was specifically inhibited by topotecan. Moreover,
Topo I overexpression supported c-Jun-mediated reporter gene
activation and both genetic and chemical inhibition of c-Jun con-
verted cells resistant to topotecan-elicited EGFR downregulation.
Topotecan-elicited suppression of proliferation was rescued by
exogenously expressed EGFR. Furthermore, we demonstrate the
cooperation of the JNK-c-Jun pathway, Topo I, and EGFR in
the positive regulation of HT-1080 cell proliferation. Together,
these results have identified transcriptional coactivator Topo I as
a first endogenous cofactor for c-Jun in the regulation of cell pro-
liferation. In addition, the results of the present study strongly
suggest that inhibition of EGFR expression is a novel mechanism
by which topotecan inhibits cell proliferation in cancer therapy.
PP-32
Structural investigations of insect ecdysteroid
nuclear receptor with natural DNA response
element
M. Jako
´
b
1
, R. Koodziejczyk
1
, M. Orowski
1
, S. Krzywda
2

receptor. Until now most of the structures of nuclear receptors
were determined with artificial highly symmetric DNA response
elements, therefore we have purified and co-crystallised EcR and
Usp DNA binding domains from D. melanogaster with the 20 bp
natural response element hsp27. Crystals obtained by vapour dif-
fusion method diffracted synchrotron radiation to 1.95 A. Our
research show that both proteins use similar dimerisation surfa-
ces, and rely on the deformed DNA geometry to establish pro-
tein-protein contacts. We observe that in comparison to structure
with artificial DNA response element the main fold is preserved,
however the pattern of interactions differs which emphasizes the
previously suggested plasticity of ecdysteroid receptor.
Acknowledgment: Work is supported by a State Committee
for Scientific Research grant 3T09A04038.
PP-33
Molecular beacon for determining the hsp27
response element – ecdysteroid receptor
interaction
T. Krusin
´
ski, P. Dobryszycki, A. Kowalska and A. O
_
zyhar
Biochemistry Department, Wroclaw University of Technology,
Wroclaw, Poland. E-mail: [email protected]
The ecdysteroids are crucial during moulting and metamorpho-
sis processes among the insects. They act via a receptor, which
belongs to the nuclear receptors’ superfamily. Functional ecdy-
sone receptor consists of two proteins: the ecdysone receptor
(EcR) and the ultraspiracle (Usp). The EcR-Usp complex regu-

Abstracts
85
PP-34
Oestrogen receptor-alpha activates
transcription of the mammary gland
Na
+
/I
-
symporter (mgnis) gene
E. Yaman C¸ ankaya
1
, H. Alotaibi
1
, E. Demirpenc¸ e
2
and
U. H. Tazebay
1
1
Department of Molecular Biology and Genetics, Bilkent
University, Ankara, Turkey,
2
Department of Biochemistry, Faculty
of Medicine, Hacettepe University, Ankara, Turkey.
E-mail: [email protected]
Sodium Iodide Symporter (NIS) function in mammary gland
(mg) epithelial cells is essential for the accumulation of I- in
mother’s milk which is the newborn’s first source of I- for thy-
roid hormone synthesis. Furthermore, increased mgNIS expres-

Peptide (HARP) is an 18 kDa secreted polypeptide growth factor
with high affinity to heparin. HARP is mitogenic for endothelial
cells, stimulates angiogenesis in vitro and in vivo and plays a key
role in the progression of several types of tumours of diverse ori-
gin. In the present study we found that exogenous ATRA signifi-
cantly decreased human prostate cancer LNCaP cell
proliferation. Heparin affin regulatory peptide (HARP) seems to
be involved in the inhibitory effect of ATRA, because the latter
had no effect on stably transfected LNCaP cells that did not
express HARP. Moreover, ATRA significantly decreased HARP
mRNA and protein amounts in a concentration- and time-
dependent manner. These data suggest that ATRA affects pros-
tate cancer LNCaP cell growth through an effect on the expres-
sion of HARP and further studies are in progress to elucidate
mechanisms involved.
PP-36
Gene expression analysis of hedgehog
signalling pathway genes in breast cancer
O. Akilli-Ozturk
1
, B. Gur
1
, B. Bozkurt
2
, S. Seckin
3
and
I. G. Yulug
1
1

to show the expression profiling of the HH pathway genes in
breast cancer, which will help us to understand the initiation and
development mechanisms of this cancer.
PP-37
Regulatory role of FAK/PI-3k/actin signalling
in cancer cells
G. Kalergi
1
, D. Mavroudis
2
, V. Georgoulias
2
and C. Stournaras
1
1
Department Biochemistry, University of Crete Medical School,
Heraklion, Greece,
2
Department Clinical Oncology, University of
Crete Medical School, Heraklion, Greece.
E-mail: [email protected]
Recent findings in malignant MCF7 human breast epithelial- and
LNCaP human prostate cancer-cells suggested that actin cytoske-
leton reorganization regulated by activation of FAK and PI-3
kinase may regulate their phenotypic and metastatic profile. Here
we report that incubation of human A375 melanoma cells with
the opioid casomorphin induces activation of the same signalling
cascade FAK/PI-3K/Rac1, leading to potent actin reorganization
and inhibition of cell motility. To further assess the clinical
impact of these findings, cytospins of peripheral blood mononu-

as stress response. SAFB2, a protein highly homologous to
SAFB1 and also an ER-alpha corepressor, shares with it numer-
ous highly conserved domains. Their genes are localized head to
head on the same chromosome and their expression is regulated
by a common promoter. Although indirect evidence suggests that
SAFB1 and SAFB2 might have unique properties, any functional
differences especially regarding their corepressor activity are still
obscure. In this study, we have examined the interaction of
SAFB2 with SAFB1 molecular partners fished out by the yeast
two hybrid system. Among the clones tested only one clearly dis-
tinguishes between the two proteins in the yeast system and it
was chosen for further examination of its structural and func-
tional relation to SAFB1 and SAFB2.
PP-39
Clinicopathological study of survivin
expression in colorectal cancer
F. Shimamoto
1
, H. Tuncel
2
, S. Sai
1
, E. Aoki
1
, S. Tanaka
3
,
S. Oka
3
, R. Takahashi

were to investigate survivin expression in colorectal carcinoma
and to elucidate the relationships among the survivin, clinico-
pathological features and tumour progression. Immunohisto-
chemical analyses of 144 cases of advanced colorectal cancer
revealed 17 N+C+ cases with survivin (+) staining in both the
cytoplasm (C) and the nucleus (N), 92 N+C– cases with survivin
expression on only the nucleus, 12 N–C+ cases with survivin
expression on only one side of the cytoplasm, and 21 N–C– cases
that were (–) for survivin in both the cytoplasm and the nucleus.
The occurrence of metastasis was higher in the N–C+ group
than in the N+C– group, and the frequency of metastasis and
number of cases with stage D were lower in the N+ group than
in the N- group. Furthermore, the number of cases with stage D
was higher in the C+ group than in the C- group. The N+ cases
were associated with a better prognosis, while the C+ cases were
not. These findings suggest that the biological behaviour of colo-
rectal cancer may differ according to the localization of survivin
within the cancer cells.
PP-40
The JAK/STAT pathway constitutively
activated in cervical cancer cell lines is
inhibited by piceatannol
A. Valle-Mendiola, J. Mendoza-Rincon, B. Weiss-Steider and
I. Soto-Cruz
Lab. de Oncologı
´
a, Unidad de Investigacio
´
n en Diferenciacio
´

suggest that this pathway may play a role in the pathogenesis of
cervical cancer.
PP-41
The effect of simvastatin on signalling
pathways involved in pathoge nesis of
pancreatic cancer
H. Gbelcova
1
, T. Ruml
1
, Z. Knejzlik
1
, M. Lenicek
2
, J. Zelenka
2
and L. Vitek
2
1
Institute of Chemical Technology, Prague, Czech Republic,
2
Institute of Clinical Biochemistry and Laboratory Diagnostics,
Prague, Czech Republic. E-mail: [email protected]
Introduction: Inhibitors of HMG-CoA reductase are widely
used for treatment of hypercholesterolemia. However, inhibition
of this enzyme results also in depletion of intermediate biosyn-
thetic products contributing importantly to the cell proliferation.
In the present study, we investigated the effects of simvastatin on
the signalling pathways involved in the pathogenesis of pancre-
atic cancer.

tosis in T cells and numerous cancer cell lines. It can act by two
alternative mechanisms: regulation of target genes expression or
translocation from the nucleus to the mitochondria with subse-
quent release of cytochrome C. Thymic lymphoma VIII/d cell
line, derived from TCR transgenic mice, is resistant to Nur77-
mediated apoptosis, despite of unaffected expression and DNA-
binding activity of Nur77. We also observed mitochondrial trans-
location of this nuclear receptor. However, we found abrogation
of cytochrome C release in these cells. HA1004 (an inhibitor of
serine-threonine kinases) and FK506 (an inhibitor of calcineurin)
were shown to restore the sensitivity of examined lymphoma to
apoptosis induction. Here we show that apoptosis enhancement
by these agents correlated with increased cytochrome C appear-
ance in the cytosol. In conclusion, we show that despite of DNA-
binding and successful translocation to the mitochondria in VIII/
d thymic lymphoma cells, Nur77 is not able to trigger apoptosis.
The failure seems to be located at the level of cytochrome C
release and can be modulated by HA1004 or FK506 treatment.
This work was supported by grant 2/P05A/10929 from the Polish
State Committee for Scientific Research.
PP-43
Investigation of the effects of anastrozole and
quercetin on breast cancer in vitro
A. Demiroglu Zergeroglu
1
, E. Dulger
1
, E. Kilic
2
, O. G. Yildiz

levels on survival of lung cancer patients
M. Colakogullari
1
, E. Ulukaya
1
, A. Yilmaztepe
1
, G. Ocakoglu
2
,
M. Yilmaz
1
, M. Karadag
3
and A. Tokullugil
1
1
Department of Biochemistry, Uludag University Faculty of
Medicine, Bursa, Turkey,
2
Department of Biostatistics, Uludag
University Faculty of Medicine, Bursa, Turkey,
3
Department of
Chest Diseases and Tuberculosis, Uludag University Faculty of
Medicine, Bursa, Turkey. E-mail: [email protected]
Objective: As nitric oxide (NO) was proposed to be both an
upstream and downstream regulator of vascular endothelial
growth factor (VEGF), relationship between NO and VEGF
remains unclear.

, F. Sahin
1
, I. Pirim
2
, M. E. Yalvac
1
and
C. D.
_
Izbırak
1
1
Genetics and Bioengineering, Yeditepe University, Istanbul,
Turkey,
2
Medical Biology, Ataturk University, Erzurum, Turkey.
E-mail: [email protected]
Matrix metalloproteinase-1 (MMP-1) is an enzyme, which is
degrading extracellular matrix. Thus, MMP-1 is known to have a
contribution to tumour initiation and development due to alter-
ation of the cellular microenvironment that facilitates tumour
formation and angiogenesis. MMP-1 is thought to play a critical
role in tumour invasion and metastasis. Human MMP-1 has two
differently glycosylated proenzymes. Human MMP-1 gene is
Abstracts
88
expressed in a various physiological processes such as embryonic
development, and wound healing and number of pathological
processes, such as malignant tumours. The expression of MMP-1
is partly regulated by the upstream promoter sequences of the

4
and N. Atabey
3
1
Department of Pharmacology, Dokuz Eylul University School of
Medicine, Izmir, Turkey,
2
Department of Pathology, Dokuz Eylul
University School of Medicine, Izmir, Turkey,
3
Department of
Medical Biology and Genetics, Dokuz Eylul University School of
Medicine, Izmir , Turkey,
4
Institute of Oncology, Dokuz Eylul
University, Izmir, Turkey. E-mail: [email protected]
Aim: It is thought that HGF/c-Met signalling pathway has a
role in the invasion and metastasis processes of lung cancer. The
aim of this study is to determine the role of HGF/c-Met pathway
in NSCLC.
Methods: The expression of HGF and c-Met were determined
immunohistochemically in tissue samples of 63 NSCLC patients.
DNAs obtained from sections of the same tissues were amplified
by PCR using exon-14-specific primers that encode tyrosine kin-
ase domain of the c-Met receptor.
Results: C-Met and HGF expressions were determined in 81%
and 48% of NSCLC tissues, respectively, consequent to the im-
munohistochemical analyses. A correlation between the overex-
pression of HGF/c-Met and tumour size, tumour stage, lymph
node metastasis and relapse rate was not observed. C-Met was

G-protein beta-gamma-YFP subunit on the membrane translo-
cates to the golgi apparatus in less than 1 min. On deactivation
of the receptor with antagonist, it translocates back to the mem-
brane. This can be observed under the fluorescent microscope.
The translocation process takes place in seconds and can be
repeated several times. This sensor was used to elucidate the
receptor stimulated G protein activation mechanism. Rapid and
efficient screening of commercial drugs for receptors will also be
possible with this biological sensor.
PP-48
Towards understanding the structure and
function of g protein-coupled receptors: a
multidisciplinary approach
A. Shukla, C. Reinhart and H. Michel
Max Planck Institute of Biophysics.
E-mail: [email protected]
GPCRs are integral membrane proteins with seven transmem-
brane helices that regulate many cell signalling pathways via acti-
vation of G proteins. GPCR malfunction leads to several human
diseases and majority of commonly prescribed medicines act on
these receptors. However, structure based drug designing on
GPCRs has not been possible due to lack of high resolution
structure. Therefore, my efforts focus on expression, purification
and structural studies of selected GPCRs. Three different GPCRs
namely, bradykinin receptor, neuromedin receptor and angioten-
sin receptor, have been purified in milligram amounts and being
subjected to crystallization trials to obtain structural information.
In vitro and in vivo reconstitution of GPCR signalling complexes
(GPCR heterodimer, GPCR-arrestin and GPCR-G protein) is
also being pursued, which may provide insights into signalling

2
and W. Lee
1
1
Department of Biochemistry and Protein Network Research
Center, College of Science, Yonsei University, Seoul, Korea,
2
Department of Life Sciences, Division of Molecular Life Sciences
and Center for Cell Signalling Research, Ewha Womans Univer-
sity, Seoul, Korea,
3
Laboratory for Glycobiology, University of
Leuven & Flanders Interuniversity Institute for Biotechnology,
Leuven, Belgium,
4
Division of Biomedical Sciences, Faculty of
Medicine, Imperial College of Science, Technology and Medicine,
London, UK. E-mail: [email protected]
The syndecan transmembrane proteoglycans are involved in the
organization of the actin cytoskeleton and they have important
roles as cell surface receptors during cell-matrix interactions. Syn-
decan-4 can regulate cell-matrix interactions and it is enriched in
focal adhesions. We have shown that the syndecan-4 cytoplasmic
domain (4L) forms oligomeric complexes that bind to and stimu-
late PKCa activity in the presence of PtdIns(4,5)P2, emphasizing
the importance of multimerization in the regulation of PKCa
activation. Oligomerization of the cytoplasmic domain of synde-
can-4 is regulated either positively by PtdIns(4,5)P2, or negatively
through phosphorylation of serine 183 (Ser183). Phosphorylation
results in reduced PKCa activity by preventing PtdIns(4,5)P2-

(Turin), Italy,
2
Department of Molecular Immunology, Research
Institute for Microbial Diseases, Osaka University, Osaka, Japan.
E-mail: [email protected]
The secreted and membrane bound protein Semaphorin4D
(Sema4D) is endowed with angiogenic properties. Sema 4D binds
to its receptor, PlexinB1, and this interaction lead to the activa-
tion of Met, the tyrosine kinase receptor for the Hepatocyte
Growth Factor. Met activation induces cell proliferation, migra-
tion, prevention of apoptosis, and differentiation. To investigate
the in vivo angiogenic function of this Sema4D and its role in
tumour progression, we use Sema4D KO mice (kindly provided
by Dr Kikutami) that were injected with syngeneic tumourigenic
cells. KO animals showed an impairment of tumour growth and
a significant decrease of the number of lung metastases, com-
pared with Wt mice. Analysing the status of vessels inside the
tumours, KO animals displayed a five-time fold decrease in the
total vessel area, maintaining a similar vessel number. Tumour
vessels in KO animals seemed to be less well organized. Further
analyses would identify the host cell population producing
Sema4D and reveal how it activates endothelial cells and stimu-
lates the invasive/metastatic properties of tumour cells. Our pre-
liminary data suggest that Sema4D plays an important role in
the tumourigenic/metastatic process and that it is a likely candi-
date for an anti-neoplastic target therapy.
PP-51
CXCR4 expression in Ishikawa endometrial cell
lines
L. Kubarek and P. P. Jagodzinski

´
berova
´
, G. M. Shaik and P. Dra
´
ber
Institute of Molecular Genetics AS CR, Prague, Czech Republic &
3rd Medical Faculty, Charles University, Prague, Czech Republic.
E-mail: [email protected]
Changes in activity of the protein tyrosine phosphatase PTP-
PEST during mast cell activation through the high affinity IgE
receptor type I (FceRI) was studied. After antigen-mediated
aggregation of the IgE-FceRI complexes, the enzymatic activity
of PTP-PEST was rapidly enhanced with the peak at about
2 min after triggering. In cells with down-regulated expression of
a linker for activation of T-cells family member 2 (LAT2, for-
merly NTAL) by RNA interference, PTP-PEST was not activa-
ted by the FceRI-aggregation, probably due to the observed
absence of an increase of actin polymerisation after receptor trig-
gering. On the other hand, in cells with upregulated expression of
LAT2 after transfection of LAT2 cDNA under cytomegalovirus
promoter, the activity of PTP-PEST was increased in both resting
and activated cells. Enhanced activity of PTP-PEST in LAT2
overexpressors led to markedly decreased tyrosine phosphoryla-
tion of transmembrane adaptor protein PAG and decreased
association of Csk with PAG. This led to enhanced activity of
Lyn kinase and extremely hyperactive SHP-2 in both resting and
activated cells. Consequently FceRI was less phosphorylated,
causing the inhibition of phosphorylation of Syk kinase and
Abstracts

adenosine receptors, bound to a homogenous population of bind-
ing sites in rat striatal membranes with affinity K
d
= 0.14 nM
and density B
max
= 1620 fmol/mg protein. Similar binding prop-
erties have been also obtained for transfected CHO cell mem-
branes (K
d
= 0.23 nM and B
max
= 360 fmol/mg protein), but in
this case the pretreatment with adenosine deaminase (ADA) was
required to remove internal adenosine. The binding of [
3
H]
ZM241385 was fast and reversible, achieving equilibrium within
20 min at all radioligand concentrations. The analysis of the
obtained kinetic and saturation data indicated that the [
3
H]
ZM241385 binding might have at least two subsequent steps,
where a fast equilibrium is followed by a slow conformational
isomerization. The potency of ZM241385 to inhibit CGS21680-
induced cAMP accumulation in CHO cells (K
i
= 6.6 nM) was
considerably lower than its apparent affinity in binding experi-
ments, but in good agreement with the estimated equilibrium

Experimental Medicine, Hungarian Academy of Sciences,
Budapest, Hungary. E-mail: [email protected]
GABAB receptors are unique among G-protein coupled recep-
tors in their requirement for heterodimerization between two sub-
units, R1 and R2 for functional expression. Recent studies have
revealed that hetero-oligomerization between very different recep-
tors can also occur and this may profoundly change the binding
and signalling properties of the receptors. First we performed a
thorough characterization of the GABAB and CB1 cannabinoid
receptors by using ligand-stimulated [35S] GTPcS binding assays
in rat hippocampal membranes. Win55,212-2 (a CB1 agonist) dis-
played high potency (ED50 = 23.26 ± 1.2 nM) and efficacy
(148 ± 2.2%) in stimulating [35S] GTPcS binding. This effect
was completely blocked with SR141716 (a CB1 antagonist), prov-
ing that the CB1 receptors are fully functional. The GABAB
agonists baclofen and SKF 97541 also elevated [35S] GTPcS
binding by 149 and 186%, respectively. Interestingly, nanomolar
concentrations of the GABAB antagonist phaclofen slightly but
significantly lowered the maximal stimulation of [35S] GTPcS
binding compared to that obtained with Win55,212-2 alone.
These results can be interpreted to show an interaction, possibly
hetero-oligomer formation between CB1 and GABAB receptors
with altered functionality.
PP-55
Extracellular RNA in culture of transformed
and primary human cells
E. Morozkin, P. Laktionov, E. Rykova and V. Vlassov
Cellular Biology Group, Institute of chemical biology and
fundamental medicine, Novosibirsk, Russia.
E-mail: [email protected]

of Signals Transduction. E-mail: [email protected]
Skeletal muscle satellite cells are precursors of mammalian skel-
etal muscles. Differentiation of those precursors in vivo is regula-
ted by extracellular growth factors that transmit signals into the
cells. Extracellular ATP acting trough P2X and P2Y purinergic
receptors is also involved in this process. The effects of actin
cytoskeleton disruption by cytochalasin D on calcium signals
evoked by ATP, thapsigargin and caffeine were investigated in
C2C12 myoblasts and myotubes. In myoblasts the high and rapid
Ca
2+
response is generated mainly by P2X ion-gated receptors.
In myotubes, ATP generates weak Ca
2+
response acting through
P2Y metabotropic receptors only. Cytoskeleton disruption
strictly decreases general calcium response in myoblasts. Other-
wise, the calcium response evoked only by P2Y receptors seems
not to depend on cytochalasin D treatment. Thapsigargin, an
irreversible inhibitor of the SERCA ATPase, promotes the leak
of Ca
2+
from the ER. Caffeine acts through ryanodine receptors
releases Ca
2+
from internal stores. Both do not provoke any sec-
ond messenger formation. Ca
2+
mobilization is slightly decreased
after cytoskeletal disruption both in myoblasts and myo-

2
Department of Orthopaedic
Surgery, Clinique Saint Martin, Caen, France,
3
Department of
Anatomy, Faculty of medicine, Caen cedex, France.
E-mail: [email protected]
Interleukin-1b (IL1b) and Transforming Growth Factor-b
(TGFb) play a key-role in osteoarthritis (OA). In this present
study, we attempt to determine the influence of IL1b on TGFb
responsiveness in human articular chondrocytes (HAC). HAC
were treated with IL1b and TGFb-induced gene expression was
analysed through PAI1 and p3TPLux induction. R-Smads phos-
phorylation and TGFb receptors (TbRI, TbRII) and Smads
expression were defined by Western-Blot and real-time RT-PCR.
Transduction pathways (NO, MAPK, NFjB) were investigated
using specific inhibitors. Transfections of TbRII promoter or
expression vectors were performed to delineate DNA sequences
and to define transcriptional factors involved in IL1b effect. IL1b
pre-treatment inhibits TGFb-induced gene expression and
Smad2/3 phosphorylation, causes a dramatic decrease of TbRII
expression, and up-regulates Smad7. Interestingly, TbRII overex-
pression counteracts the loss of TGFb-responsiveness induced by
IL1b. TbRII downregulation is prevented by cycloheximide and
is attenuated by inhibition of NFjB pathway. This regulation
implicates TbRII core promoter that contains a putative binding
site for p65 and p65 overexpression decreases TbRII expression.
In conclusion, IL1b impairs TGFb signalling through TbRII
downregulation, which is probably, mediated by NFjB, and sec-
ondarily though Smad7 upregulation. These findings may

ing also suggests that level of DNA methylation can be respon-
sible for improper function of CD4+ T cells in patients with
autoimmune disease.
Acknowledgment: This work was supported by grant KBN
No. 2PO5B-019-27.
PP-59
Adenosine receptors in growth arrested
glioma cells
P. Krzeminski and J. Baranska
Nencki Institute of Experimental Biology, Department of
Molecular and Cellular Neurobiology, Laboratory of Signal
Transduction. E-mail: pkrzemin@nencki
Adenosine is final product of ATP and ADP metabolism that can
act on specific P1 receptors located mainly on plasma membrane.
Signal transduction through the group of four already known P1
receptors is tightly regulated not only by interaction of down-
stream proteins but also by the mechanisms of receptors desensiti-
zation and elimination of adenosine by specific nucleotide
transporter. Adenosine levels as well as ADP and ATP differs
among tissues and is elevated in many tumours. Stimulation of
nucleotide receptors can have various effects on cell faith what
depends on concentration, tissue model and growth conditions.
Recent result from our laboratory showed that level of ADP sensi-
tive receptor P2Y
1
is strongly decreased in growth arrest induced
by serum deprivation. As an ADP can be metabolised to the
adenosine by ectonucleases we decided to examine expression pro-
file and role in proliferation of P1 receptors of two glioma cell lines
c6 and T98g in serum deprivation induced growth arrest. Data

E-mail: [email protected]; [email protected];
[email protected]; [email protected]
Interferons accomplish their multiple biological activities by acti-
vating the STAT transcription factors, which are translocated to
the nucleus through specific nuclear localization sequence (NLS)
located in their ligands. Two putative NLS have been pointed
out in the human interferon gamma (hIFNc) spread over resi-
dues 83–89 and 124–132. To investigate the significance of the
putative upstream NLS for the biological activity of hIFNc we
have prepared a new construct of the hIFNc gene in which a
Gln codon was substituted for the Lys88 codon. The mutated
gene was cloned and expressed in E. coli LE392. This mutation
Abstracts
92
led to a 1000-fold decrease in both hIFNc antiviral and antipro-
liferative activities. When co-incubated with the wild type hIFNc
(standard), the mutant hIFNc competed for the cell receptors
that led to a 30% inhibition of the standard activity. This indi-
cates that the mutation does not interfere with the interaction of
the protein to its cell receptor but affects the intracellular trans-
duction in which Lys88 seems to play an important role. To
study the role of the C-terminal NLS, 21 C-terminal codons have
been deleted from the mutant hIFNc gene and this led to a
10 000-fold decrease in biological activity and 55% inhibition of
the standard activity in the competition assay. These data con-
firm our hypothesis that the lack of the C-terminus stabilizes the
hIFNc/receptor complex.
Acknowledgment: Supported by NSF, grant K-1405.
PP-61
Production of recombinant Go alpha protein

PP-62
Juvenile hormone binding protein from
G. mellonella binds to membrane protein
in the specific manner
M. Zalewska,A.O
_
zyhar and M. Kochman
Department of Biochemistry, Wrocaw University of Technology,
Wrocaw, Poland. E-mail: [email protected]
Juvenile hormone (JH) is essential for multiple physiological pro-
cesses: it controls larval development, metamorphosis and adult
reproduction. In the insect haemolymph, JH is in 99.9% in the
bound state with juvenile hormone binding protein (JHBP). This
protein protects JH molecule from non-specific hydrolases and
serves as a carrier to supply the hormone to the target tissues,
preventing its non-specific binding to hydrophobic surfaces.
However, mechanism describing the way in which JH enters the
target cells has not yet been elucidated. In this report we present
the studies on JHBP binding to membrane proteins. Membranes
isolated from VIIth instar larvae fat body of the G. mellonella
were incubated with increasing concentrations of radioiodinated
JHBP. We found that the interaction between JHBP and mem-
branes occurs with saturation kinetics and is specific and reversi-
ble. Specificity of binding was determinated by competitive
binding experiments in the presence of 100-fold excess of unlabe-
led JHBP or in the presence of non-specific protein (serum albu-
min). The above results indicate the existence of a membrane
protein, which recognizes JHBP molecule and perhaps may take
part in the transfer of JH to the target cells. Currently, investiga-
tions are being performed to identify the JHBP binding protein

neutrophils do that under some pathological conditions. Thus,
we have shown that neutrophils play role in response to allogeneic
tumour cells. Expression of costimulatory molecules suggests that
neutrophils can acquire properties of professional antigen-pre-
senting cells (APC) and their potential to polarize the immune
responses to tumour antigens.
PP-64
Screening for new proteins interacting with
endoglin, by the phage display technique
E. M. Garrido, F. J. Blanco, A. Fernandez-L, L. M. Botella and
C. Bernabeu
Centro de Investigaciones Biolo
´
gicas, Consejo Superior de
Investigaciones Cientı
´
ficas, Madrid, Spain.
E-mail: [email protected]
Endoglin, mainly expressed at the surface of the endothelial cells,
is one of the components of the transforming growth factor-beta
receptor complex and is involved in angiogenesis and cardiovascu-
lar development. Its mutation is responsible for Hereditary Haem-
orrhagic Telangiectasia, an autosomal dominant vascular disorder
characterized by arteriovenous malformations and telangiectases.
Due to the relatively high expression of endoglin at the surface of
Abstracts
93
endothelial cells, respect to other TGF-beta receptor components,
we postulate that endoglin might have other ligands unrelated to
the TGF-beta system. In order to identify new binding molecules,

Centro de Investigaciones Biolo
´
gicas, Consejo Superior de Investi-
gaciones Cientı
´
ficas. Madrid, Spain,
2
Departamento de Biologı
´
ay
Gene
´
tica, Facultad de Biologı
´
a, Universidad Autonoma de Madrid,
Madrid, Spain. E-mail: [email protected]
Hereditary Haemorrhagic Telangiectasia (HHT) is an autosomic
dominant vascular disease clinically characterized by spontaneous
and recurrent epistaxis, telangiectases, and arteriovenous malfor-
mations in internal organs. Mutations in Endoglin and ALK1
genes are responsible for HHT1 and HHT2, respectively. Both
genes are mainly expressed by endothelial cells and code for com-
ponents of the transforming growth factor b (TGF-b) receptor
complex. Blood outgrowth endothelial cells were obtained from
patient’s peripheral blood, characterized by specific endothelial
markers and functionally studied compared to endothelial cells
from healthy donors. HHT1 and HHT2 showed low levels of
endoglin expression. In the case of HHT1 it is due to endoglin
haploinsufficency, and in HHT2 it is probably due to endoglin
regulation by ALK1. Moreover, HHT cells showed impaired

cant up- or down-regulation with respect to their adjusted
P-values were selected for further functional studies. Out of 119
target genes containing receptors and ligands of Netrin, Ephrin,
Roundabout and Plexin families, nine were up-regulated, while
12 were down-regulated significantly. RNA interference was used
as a second filter for the selection of target molecules. As a first
attempt, we investigated the expression of slit-robo genes in HCC
cell lines and tumours. Our first results allowed us to hypothesize
that the members of this receptor family are differentially
expressed in HCC, according to differentiation status of HCCs.
PP-67
T-cadherin mediates low-density
lipoprotein–initiated mitogenic signalling
D. Kipmen-Korgun
1
, K. Osibow
2
, C. Zoratti
2
, J. Greilberger
3
,
G. M. Kostner
2
, G. Juergens
3
and W. F. Graier
2
1
Department of Biochemistry, Akdeniz University, Antalya,

overexpression of T-cadherin resulted in accelerated cell prolifer-
ation in a LDL dependent manner. Our data suggest that
T-cadherin serves as a signalling receptor for LDL that facilitates
a LDL-dependent mitogenic signal in the vasculature.
PP-68
CREB mediates arterial smooth muscle cell
migration via the regulation of osteopontin
gene transcription
S. Jalvy, M. A. Renault, L. Lam Shang Leen, I. Belloc,
A. P. Gadeau and C. Desgranges
INSERM U441, Atherosclerose, Pessac, France.
E-mail: [email protected]
The cAMP responsive element-binding factor (CREB) is activated
in arterial smooth muscle cells (SMC) by several growth factors
Abstracts
94
involved in arterial wall remodelling, such as PDGF-BB. The
extracellular nucleotide UTP, which induces SMC migration via
osteopontin (OPN) production, also increases CREB activation.
The aim of this study is to identify the mechanisms of CREB acti-
vation and its consequences on SMC migration and OPN regula-
tion. Stimulation of cultured SMC by UTP and PDGF-BB highly
induces CREB activation via ERK1/2 and p38, and via p38 and
PKA respectively. The role of CREB in SMC migration was
determined using the Transwell approach and a dominant negat-
ive form of CREB (A-CREB). A-CREB expression inhibits both
UTP- and PDGF-induced migration suggesting that the migratory
process is dependent on CREB activation. Moreover, using
A-CREB, we demonstrate that OPN expression, which is neces-
sary for UTP and PDGF migration, is also CREB-dependent. Gel

stimulates the release of TNF alfa. Its effect, being immediate
and long lasting, is mediated by specific receptors. Release of
IL-6 is not influenced. The data indicate selectivity of IL-1 beta
action on different groups of lymphocytes. Data suggest that the
mode of action of different humoural substances on cytokine
release from PBMC is mediated not only by activation of specific
receptors and by mechanisms stimulating protein synthesis, but
also by mechanisms facilitating exocytosis, probably due to
modulation of cell membrane properties.
PP-70
Cell surface hsp90 interacts with the
extracellular domain of her2 and contributes
to receptor activation
K. Sidera, M. Gaitanou, R. Matsas and E. Patsavoudi
Department of Biochemistry, Hellenic Pasteur Institute, Athens,
Greece. E-mail: [email protected]
HSP90 is a molecular chaperone that controls the folding assem-
bly intracellular disposition and proteolytic turnover of many
proteins most of which are involved in signal transduction pro-
cesses. HER2 is a 185-kDa receptor-like glycoprotein and a mem-
ber of the ErbB family of receptor tyrosine kinases that play a
central role in cellular proliferation differentiation and migration.
The role of HSP90 in the regulation of HER2 has been attrib-
uted to stabilization of the receptor at the cell surface via interac-
tion with its cytoplasmic kinase domain such that disruption of
the HER2/HSP90 association leads to receptor degradation. We
have previously demonstrated the cell surface localization of
HSP90 and its involvement in cell migration processes during
development of the nervous system. In the present work we show
using GST-pull down assays that surface HSP90 interacts with

_
Izmir Educational
Hospital, Izmir, Turkey,
2
Department of Biochemistry, Ataturk
Training and Research Hospital, Izmir, Turkey,
3
Department of
Biochemistry, Social Security Tepecik Educational Hospital, Izmir,
Turkey. E-mail: [email protected]
Diabetes mellitus is a metabolic disease caused by absence of
insulin or by insulin resistance that results in inappropriate insu-
lin action. We investigated the relationship between polimorfizm
of exon 31 of SUR1 gene with obese and non-obese Type II Dia-
betes Mellitus (DM). SUR1 gene codes the SUR1 protein that
takes part in secretion of insulin. Study is planned with 17
healthy persons, 20 non-obese Type II DM, 25 obese Type II
DM. We determined the serum glucose, cholesterol, trigiseride
levels, and blood (%) HbA1c. DNA is extracted from peripheral
blood. Single Nucleotide Polymorphisms (SNP) is determined by
Restriction Fragment Length Polymorphism (RFLP). We
observed a significant association between A allele and Type II
DM (P < 0.05) and this was stronger in obese Type II DM
patients while there were no association with the non-obese Type
II DM patients. The patients with hypertrigliseridemia showed
the same significant association with A allele frequency. This
study reports that SNP’s of exon 31 of SUR1 gene can be used
as a risk factor in Tip II DM, and in determining the other risk
factors, the genes that participate in obesity must be considered
more carefully.

Center for Molecular
Recognition, Columbia University College of Physicians and
Surgeons New York, NY, USA. E-mail: [email protected]
Dopamine transporter (DAT) is a member of monoamine trans-
porters family. DAT is the major target for psychostimulants like
cocaine and amphetamine (AMPH).The main effect of AMPH is
to induce DA efflux. It has recently been shown that phosphory-
lation of serines in N-terminal (N-T) tail of DAT regulates the
AMPH induced DA-efflux through an unknown mechanism. To
address this question we are establishing techniques to character-
ize conformational changes of the N-T tail of DAT. By applying
fluorescence resonance energy transfer (FRET) between YFP
fused in the N-terminal tail and a rhodamine labelled cocaine
analogue (JHC1-64) bound in the transmembrane domain of
DAT the movement of the tail relative to the fixed rhodamine
position could be monitored. To mimic the phosphorylated and
dephosphorylated state of the N-T serines we have mutated Ser7
and Ser12 to aspartate and alanines, respectively. These muta-
tions have been introduced in DAT constructs, one with YFP
fused to the N-T end and the one introduced in position 55 of
the N-T tail. FRET measurements between YFP in the YFPp1-
DAT construct and the bound JHC1-64 did not result in measur-
able energy transfer suggesting a long distance for FRET to
occur. StoD and StoA mutations did not result in measurable
energy transfer either. However, in the YFPp55-DAT construct
we found significant FRET. We are currently testing the effect of
StoD or StoA mutations in YFPp55-DAT construct by FRET as
an indication of movement of the N-T tail.
PP-73
Regulation of transcobalamin receptor

protein levels were elevated (2- to 3-fold) in the total membranes
of kidney, liver and intestine of rats made Cbl-deficient by either
TG (maintained for 2, 4, and 8 months) or feeding Cbl-deficient
diet for 12 months. However, elevation of TC-R levels in the spi-
nal cord was delayed and occurred after 8 months of TG or
12 months of feeding Cbl-deficient diet. Postoperative Cbl-
replacement treatment normalized the TC-R levels. Treatment of
human intestinal epithelial Caco-2 cells with TNF-a or addition
of HCY during culture resulted in 8- to 10-fold upregulation of
TC-R levels. These data indicate that in Cbl deficiency (however
induced): (a) TC-R is upregulated in most tissues, (b) increases in
TNF-a and HCY levels may be responsible for TC-R upregula-
tion and (c) TC-R upregulation may facilitate increased import
of Cbl in cells under stress of Cbl-deprivation.
PP-74
The role of protein kinase C in migration of
neuroblastoma cells
H. Stensman and C. Larsson
Laboratory Medicine, Molecular Medicine, Lund University,
Malmo
¨
, Sweden. E-mail: [email protected]
The capacity of cancer cells to migrate is crucial for its malig-
nancy. Here we demonstrate that stimulation with platelet-
derived growth factor (PDGF) induces an increased migration of
SK-N-BE(2)C neuroblastoma cells. Treatment with the general
PKC inhibitor GF109203X or the inhibitor of the classical iso-
forms Go
¨
6976 completely inhibits migration while an inhibitor of

2
and
J-C. do-Rego
2
1
Laboratory of Biomolecular Chemistry, Medical University of
Lodz, Lodz, Poland,
2
Laboratoire de Neuropsychopharmacologie
Expe
´
rimentale, CNRS-FRE 2735, IFRMP 23, Universite
´
de
Rouen, Rouen, France. E-mail: [email protected]
Endomorphin-2 (Tyr-Pro-Phe-Phe-NH
2
) and morphiceptin (Tyr-
Pro-Phe-Pro-NH
2
) are two structurally related endogenous opi-
oid peptides with high affinity and selectivity for the l-opioid
receptor. The aim of the present study was to examine the prop-
erties of endomorphin-2, morphiceptin, and their analogues,
modified in position 3 or 4 by introducing 3-(1-naphthyl)-D-alan-
ine (D-1-Nal) or 3-(2-naphthyl)-D-alanine (D-2-Nal), using a
functional [
35
S] GTPcS binding assay. Endomorphin-2, morphi-
ceptin, and their analogues were synthesized by a standard solid-

morphin-2.
Conclusion: The size and topographical location of the aroma-
tic ring of the position 3 and 4 amino acid residues seem to be
critical for the stimulation of the [
35
S] GTPcS binding and the
activation of the downstream effector systems.
PP-76
The effect of phospholipase C in angiotensin
II-induced p42/p44 MAPK phosphorylation in
cultured vascular smooth muscle cells
A. Cetin and A. Yesilkaya
Department of Biochemistry, Faculty of Medicine, Akdeniz
University, Antalya, Turkey. E-mail: [email protected]
Angiotensin II (Ang II) is the active component of the renin-an-
giotensin system, which has an important role in atherosclerosis,
hypertension, pathogenesis of cardiovascular diseases and regula-
ting blood pressure. It was shown that, in vascular smooth mus-
cle cells (VSMC), by stimulating Gq protein through AT1
receptors, Ang II activates highly complex intracellular signalling
pathways, which were known as ERK1-2 or p42/p44 Mitogen
Activated Protein Kinase (MAPK). These immediate signal trans-
duction processes are, G protein mediated activation of phosp-
holipase C (PLC), leading to phosphatidylinositol hydrolysis,
formation of inositol trisphosphate (IP3) and diacylglycerol accu-
mulation (DAG), increase in cytosolic free calcium concentration
(Ca
2+
), activation of protein kinase C (PKC), and vascular con-
structions/MAPK activations. This study was aimed to investi-

normal and transformed rat muscle cells and human sarcoma
cells by electron microscope technique and immunohistochemical
analysis. We compared interaction of cell surface enolase-like
receptor with plasminogen on sarcoma and normal rat muscle
cells in different conditions.
References
1. Ge J, Calt MD, Gregory RL. Infect Immun 2004; 72: 6748–
6752.
2. Merkulova T, Lucas M, Jabet N, Rouzeau JD, Gros F,
Lazar M, Keller. A. Biochem J 1997; 323: 791–800.
3. Pancholi V. Cell Mol Life Sci 2001; 58: 902–920.
PP-78
Establishment of genetically engineered neural
cells that express doxycycline-inducible TrkC
D. Klopotowska, L. Strzadala, E. Ziolo and J. Matuszyk
Institute of Immunology and Experimental Therapy, Polish
Academy of Sciences, Wroclaw, Poland.
E-mail: [email protected]
TrkC is a high affinity receptor for neurotrophin-3 (NT-3). The
goal of this study was to construct genetically engineered neural
cells that express TrkC under the control of a doxycycline
(DOX)-inducible promoter. MBG18 is a neural cell line derived
from brain of mouse embryos (BBRC 309:91), stably transfected
to express the reverse tetracycline-responsive transactivator
(rtTA) under the control of the EF1alpha promoter. The aim
was to engineer the cells (MBG18 and PC12 Tet-On) to express
the target gene (firefly luciferase, TrkC) at high level in response
to DOX and at low level in the absence of DOX. We studied the
expression of luciferase gene driven by either tetracycline-
response element (TRE) or modified TRE (TRE-tight). We found

Biology, University Paris, Paris, France.
E-mail: [email protected]
HARP (Heparin Affin Regulator Peptide) is an 18 kDa growth
factor (1) detected in various tissues and cell lines (2). HARP dis-
plays several biological actions, such as induction of cellular pro-
liferation, migration and angiogenesis, indicating its possible
involvement in carcinogenesis. Recently, we have identified and
characterized several HARP’s proteolysis fragments with either
similar or opposite to HARP’s biological activities (3). In the
present work, we investigated the biological activity of P10, a
synthetic peptide corresponding to HARP residues 122–131. Our
results suggest that P10 inhibits the in vitro proliferation, adhe-
sion, migration and anchorage independent cell growth of human
prostatic tumour cell lines PC3 and DU145. Using Western blot
analysis, we showed that SRC, AKT, ERK1/2 kinases and PTEN
Abstracts
97
phosphatase are activated following a treatment with P10. In
addition, studies of the mechanism of action indicated that P10
interfered with the binding of HARP receptors ALK and
RPTPb/z. Furthermore, we have shown that P10 inhibited in vivo
angiogenesis on the chicken embryo CAM assay. Taken together
these results indicated that P10 could be constitutes an interesting
tool for tumour therapy strategy.
References
1. Rauvala H. Embo J 1989; 8, 2933–2941.
2. Wellstein A et al. J Biol Chem 1992; 267: 2582–2587.
3. Polykratis A et al. Int J Biochem Cell Biol 2004; 36: 1954–
1966.
PP-80

ifications of proteins. Lysophosphatidic acid (LPA) is an import-
ant lipid regulator of fundamental cellular processes like
proliferation, apoptosis, differentiation and motility. Fatty alco-
hol phosphates, in which molecules the phosphate moiety is
directly attached to a hydrocarbon chain, represent the minimal
pharmacophores of LPA receptors as we have shown recently.
Here we have investigated whether farnesyl phosphates, which
are polyunsaturated fatty alcohol phosphates, can interact with
the cell surface and nuclear receptors for LPA. Both farnesyl
phosphate and farnesyl diphosphate potently and specifically ant-
agonized the LPA-elicited intracellular Ca
2+
-mobilization medi-
ated through the LPA3 receptor, while causing only modest
inhibition at LPA2 and had no measurable effect at LPA1. The
transcription factor peroxisome proliferator-activated receptor
gamma (PPARc) is activated by LPA and its mimetics including
fatty alcohol phosphates. We found that both farnesyl phosphate
and diphosphate compete with the binding of the synthetic
PPARc agonist (
3
H) rosiglitazone and weakly activate the
PPARc-mediated gene transcription. These results indicate new
roles for the oligoprenyl phosphates as endogenous modulators
of LPA receptors.
PP-81
Diverse effects of vascular endothelial growth
factor on pulmonary endothelial barrier
T. Mirzapoiazova, I. Kolosova, P. Usatyuk, V. Natarajan and
D. Verin

J. A. Ellerhorst, M. K. Johnson, C. P. Cooke and A. H. Diwan
Department of Experimental Therapeutics, M.D. Anderson Cancer
Center, Houston, TX, USA. E-mail: [email protected]
We have reported a high prevalence of hypothyroidism in the
cutaneous melanoma population, suggesting that the pathologic
hormonal environment of hypothyroidism promotes melanoma
growth. The objective of this study was to test the hypothesis
that thyroid-stimulating hormone (TSH), which is elevated in the
circulation of hypothyroid individuals, stimulates the growth of
melanoma cells. TSH receptors were detected by immunostaining
in all benign nevi, dysplastic nevi, and melanomas examined.
There was a trend toward increased staining intensity in melan-
omas when compared to benign nevi, suggesting that melanomas
may express high levels of the receptor. Melanoma cells and cul-
tured melanocytes both responded to physiologically relevant
concentrations of TSH by alterations in cAMP levels. In the
presence of TSH, melanoma cells activated the MAPK and PI3K
pathways as evidenced by phosphorylation of ERK and Akt.
These pathways were not activated in melanocytes. Furthermore,
melanoma cells, but not melanocytes, demonstrated a proliferate
response to TSH. We conclude that melanoma cells have pheno-
typic features similar to thyrocytes: they carry TSH receptors,
respond to TSH through cAMP, activate growth related signal
pathways, and proliferate. These findings support the hypothesis
that hypothyroidism promotes melanoma growth through TSH.
Clinical studies are warranted to examine the association of
hypothyroidism and elevated TSH levels with outcomes of mel-
anoma patients.
PP-83
Activation of mitogen activated protein

Investigation of the PKC and Raf-1 inter action
L. Sunesson and C. Larsson
Molecular Medicine, Laboratory Medicine, Malmoe, Sweden.
E-mail: [email protected]
Neuroblastoma is one of the most common solid tumours in
childhood and the malignancy has a rather poor prognosis.
Most neuroblastomas are undifferentiated tumours, consisting of
neuroblasts lacking neuronal processes. The protein kinase C
(PKC) family of protein kinases has been implicated to play
roles in many different cellular processes. For example, it has
been shown to be involved in the process of neurite outgrowth.
We have previously shown that introduction of the regulatory
domain of the novel PKC isoform e (PKCeRD) in neuroblasto-
ma cells induce neurite outgrowth. However, by which mechan-
ism PKCe accomplish this is still unknown. One way of
studying this effect has been to investigate different possible
interaction partners to PKCe. Raf-1 is another important kinase
involved in many different signal transduction pathways and it
has been shown to interact with PKC, especially with PKCe.In
our study, we have investigated the structures in PKC that
enable this interaction. By using co-immunoprecipitation tech-
niques, we have shown that both full-length classical and novel
PKC isoforms bind Raf-1, as well as the regulatory domains of
the different isoforms. Furthermore, our data suggests that
PKCe binds Raf-1 both via its regulatory as well as its catalytic
domain. Additional studies on the substructures of the regula-
tory domain of PKCe indicate that C1a and C2 bind Raf-1 bet-
ter than the C1b domain.
PP-85
Isolated native, oxidized LDL and HDL

_
Istanbul, Turkey.
E-mail: [email protected]
Background: LDL and oxidized LDL (ox-LDL) enhance plate-
let function via binding to its receptors on platelets, which are
different from the ‘classical’ receptors of the nucleated cells. The
present study was designed to observe the effects of isolated
LDL, ox-LDL and HDL to platelet binding properties of fibrin-
ogen and Glycoprotein (Gp) IIb/IIIa.
Methods: Washed platelets (WP) were prepared from of nine
healthy volunteers. Human LDL and HDL were separated by
density gradient ultracentrifugation and LDL was oxidized with
CuSO4 for 24h at 37°C. ADP (10 lM) induced WP were treated
with increasing concentrations of LDL/ox-LDL (7.5–400 lg/ml)
but HDL was added to final three concentrations. Expressions of
GPIIb/IIIa (CD41a) and antifibrinogen were measured by flow
cytometry. Results were converted to specific antibody binding
capacities per platelet (plt).
Results: Following ADP activation, levels of antifibrinogen/plt
and CD41a/plt increased significantly (P < 0.001). After treat-
ment with LDL/ox-LDL (7.5–400 lg/ml), levels of CD41a/plt
decreased significantly (P < 0.001) whereas levels of antifibrino-
gen/plt increased significantly in dose dependent manner
(P < 0.001), however, addition of HDL inhibited the increase in
antifibrinogen (P < 0.001).
Conclusion: These data showed that while LDL and ox-LDL
enhanced fibrinogen binding to platelets, they also abolished the
binding of antiGpIIb/IIIa to platelets dose dependently. HDL
reversed this effect of LDL/ox-LDL on only fibrinogen binding.
We concluded that GpIIb/IIIa might be a receptor for LDL and

AtoC contains two putative phosphorylation sites, i.e. a con-
served aspartic acid among the response regulators and a histi-
dine residue in an H box consensus sequence, normally common
to histidine kinases. We report here, that only phosphorylation-
competent AtoC can lead to enhanced production of cPHB in
E. coli, when overexpressed with AtoS. Specifically, upon aceto-
acetate induction, the mutation of Asp reduces cPHB accumula-
tion, compared with cells expressing wild-type AtoC. The
mutation of His residue has an even more pronounced effect.
The relative effects of these mutations on cPHB accumulation
are consistent with their effects on atoDAEB operon expression,
i.e. the mutation of Asp has a more potent phenotype than the
substitution of His, in the presence of spermidine. Introduction
of both AtoC mutations render the system unresponsive to
acetoacetate as well as polyamine, resulting in total abrogation
of the AtoS-AtoC/Az overexpression effect phenotype to cPHB
levels in E. coli.
Abstracts
99
PP-87
Reactivity of antibodies against
K. pneumoniae enolase and some cell wall
omp of K. pneumoniae and P. aeruginosa
strains
I. S. Bednarz, I. Ceremuga, J. Pietkiewicz and T. Banas
Department of Medical Biochemistry, Medical University,
Wroclaw, Poland. E-mail: [email protected]
The glycolytic enzyme alpha-enolase, despite its common cata-
lytic function in cytosol compartment of the cell, constitutes a
receptor for plasminogen at human, mammals and fungi bacterial

Bacteria as well as lower eukaryotes use phosphorylation cas-
cades in order to respond to changing environmental conditions.
A key mechanism of signalling pathways is the communication
of membrane integrated sensor kinases with cytoplasmatic effec-
tor proteins by complex reversible phosphorylation reactions
involving multistep phosphorelay systems. The Rcs regulatory
network is a global signalling system that controls a variety of
operons involved in capsule synthesis, virulence, motility or cell
division. The membrane bound sensor unit is formed by a het-
erodimer of the hybrid kinases RcsC and RcsD while the cyto-
plasmatic effectors RcsA and RcsB form a heterodimer upon
DNA-binding. The arrangement of enzymatic domains with histi-
dine kinase, phospho-receiver, phospho-transfer and DNA-bind-
ing activities is characteristic for the Rcs system and essential for
the modulation of signal transfer. We present the structural eval-
uation of three central functional domains of this signalling sys-
tem by heteronuclear high resolution NMR spectroscopy. We
further describe the so far unique structural fold of a newly iden-
tified domain integrated in the Rcs sensor kinases as well as in
kinases of other bacterial signalling systems. Phospho-transfer
mechanisms, the complex formation between sensor and effector
proteins and the phosphorylation dependent DNA binding activ-
ity of Rcs effector proteins has been analysed by multiple
approaches and will be presented.
PP-89
The RGD-independent signalling pathway in
response to fibronectin-tissue
transglutaminase complex
D. Telci
1

mediator by its association with fibronectin (FN). Formation of
a FN-TG2 complex provides pro-survival and distinct adhesive
characteristics. Here we show the RGD (Arg-Gly-Asp) independ-
ent signalling pathways in cell adhesion. To investigate the novel
RGD-independent mechanism, we inhibited the typical integrin-
mediated adhesion by using RGD peptides. The rescue of cell
adhesion does not depend on the binding of FN-TG2 complex to
a
4
b
1
integrins. However, RGD-independent cell adhesion is
inhibited by heparitinase digestion suggesting that FN-TG2 com-
plex interacts with a heparan sulfate proteoglycan receptor. The
cooperative effect of syndecan-2 with -4 during FN-TG2 medi-
ated RGD-independent cell adhesion was further investigated
using syndecan-4 null fibroblasts and siRNA technology. We pre-
viously showed that RGD-independent cell adhesion to FN-TG2
was linked to the activation of focal adhesion kinase. Here we
show that RGD-independent cell adhesion pathway by FN-TG2
is not functional in c-Raf-1 null fibroblasts. Moreover, the results
showing the activation of ERK and JNK suggest that the MAPK
pathway is involved during this process. This study indicates that
binding of TG2 to FN represents a novel cell adhesion signalling
mechanism through the MAPK survival pathways, which can
either act in synergy or as an alterative to integrin RGD-depend-
ent cell adhesion at sites of tissue injury.
PP-90
Regulation of M2 muscarinic rece ptor
expression in k562 cells by carbachol

EAL Turkey (UNESCO).
Keywords: Muscarinic receptors, carbachol, K562 cell.
PP-91
Structural and functional analysis of cell-free
expressed integral membrane proteins
D. Schwarz, C. Klammt, F. Loehr, A. Shypitsyna, S. Sobhanifar,
B. Schneider, A. Koglin, V. Doetsch and F. Bernhard
Institute of Biophysical Chemistry, University of Frankfurt,
Frankfurt, Germany. E-mail: [email protected]
Membrane proteins are involved in many human diseases but
extreme difficulties upon production of sufficient amounts have
excluded them so far almost completely from structural analysis.
Recent advances in the high-level cell-free production of integral
membrane proteins enable now production rates of several milli-
gram of protein per 1 ml of reaction and labelled samples suit-
able for analysis by NMR spectroscopy can be generated in as
fast as 24 h. The synthesized membrane proteins can furthermore
be inserted in the desired detergent micelles directly upon transla-
tion. We have produced a variety of structurally and functionally
diverse membrane proteins from prokaryotic and eukaryotic
origins including G-protein coupled receptors and multi-drug
transporters. Highly effective detergents for the solubilization of
cell-free produced membrane proteins have been selected and we
could establish modified protocols for the production of func-
tionally folded membrane proteins. The ligand binding activity,
oligomeric complex formation and functional reconstitution of
the endothelin B receptor and of the vasopressin receptor were
analysed by multiple approaches. We further demonstrate that
rationally designed combinatorial labelling schemes in combina-
tion with cell-free expression result in the rapid assignment of

metastasizes. Our results show that, in PC3 cells, SHP-1 associ-
ates, in a molecular complex, with focal adhesion kinase (FAK)
and Src, both of which are implicated in cell adhesion and migra-
tion processes, this association being regulated by collagen type
I. PC3 cell adhesion on this ECM component induces the release
of Src from the complex, coinciding with Src dephosphorylation
at the Tyr-416 but not Tyr-527 residue. Moreover, RNAi-medi-
ated gene silencing of SHP-1 in PC3 cells induces Src phosphory-
lation at both Tyr-416 and Tyr-527. In addition, SHP-1 knock
down decreases cell migration on collagen type I. These results
suggest that the tyrosine phosphatase SHP-1 plays a crucial role
in prostate cancer progression, regulating PC3 cell migration via
modulation of Src activity and its association to focal complexes.
Acknowledgment: Source of Funding: FIS (PI020964), C.S.
Castilla la Mancha (04077-00) & Fundacio
´
n MMA.
PP-93
The extracellular linker of transmembrane
neuregulins regulates their sorting and
juxtacrine function
J. C. Montero, R. Rodrı
´
guez-Barrueco, L. Yuste, P. P. Juanes,
J. da Silva Borges, L. Ferreira, A. Esparis-Ogando and
A. Pandiella
Centro de Investigacio
´
n del Ca
´

, H. E. Hamm
2
and Z. Georgoussi
1
1
Laboratory of Cellular Signalling and Molecular Pharmacology,
Institute of Biology, N.C.S.R. ‘Demokritos’, Athens, Greece,
2
Department of Pharmacology, Vanderbilt University School of
Medicine, Nashville, TN, USA.
E-mail: [email protected]
Opioid receptors modulate a variety of physiological responses in
the nervous system and belong to the superfamily of G protein
coupled receptors (GPCRs). Opioid receptor signalling mecha-
nisms have demonstrated that the third intracellular loop and the
carboxyl tail (CT) are critical in mediating the signal through
the G proteins and are also known to mediate protein-protein
Abstracts
101