Tài liệu Báo cáo khoa học: Oral Presentations Integration of Metabolism and Survival pdf

Oral Presentations
Integration of Metabolism and Survival
OP-1
The ankyrin repeat and SOCS box-containing
protein Asb-9 targets creatine kinase B for
degradation
M. A. Debrincat
1
, J. G. Zhang
2
, T. A. Willson
3
, B. T. Kile
3
,
S. L. Masters
2
, L. M. Connolly
4
, R. J. Simpson
4
, H. M. Martin
2
,
N. A. Nicola
2
and D. J. Hilton
3
1
Cancer and Haematology/Molecular Medicine, The Walter
and Eliza Hall Institute of Medical Research, Melbourne,

Australia,
2
Cancer and Haematology, The Walter and Eliza Hall
Institute of Medical Research, Melbourne, Australia,
3
Molecular
Medicine, The Walter and Eliza Hall Institute of Medical
Research, Melbourne, Australia,
4
The Joint Proteomics Laboratory
of the Walter and Eliza Hall Institute of Medical Research and
Ludwig Institute for Cancer Research, Melbourne, Australia.
E-mail:
The suppressors of cytokine signalling (SOCS) proteins inhibit
cytokine signalling by direct interaction with Janus kinases
(JAKs) or activated cytokine receptors. In addition to the N-ter-
minal and SH2 domains that mediate these interactions, SOCS
proteins contain a C-terminal SOCS box. Evidence suggests
SOCS box-containing proteins act as part of an elongin-cullin-
SOCS (ECS) E3 ubiquitin ligase complex, marking target pro-
teins for degradation. The specificity of the complex is deter-
mined by the protein interaction motif located upstream from the
SOCS box. A number of other protein families that possess a
SOCS box have been identified, the largest of which are the ank-
yrin repeat and SOCS box-containing Asbs. While it is known
that the SOCS proteins are involved in the negative regulation of
cytokine signalling, the biological and biochemical functions of
the Asbs are undefined. To understand the functional role of Asb
proteins, a proteomic approach was implemented and creatine
kinase B (CKB) was identified as a specific binding partner of

Asb-9. Transfection of increasing concentrations of a tagged
Asb-9 construct into 293T cells increased the polyubiquitination
of CKB and resulted in a concomitant decrease in total CKB
levels within the cell. The targeting of CKB for degradation by
Asb-9 was entirely SOCS box-dependent. The interaction has
been confirmed in vivo and suggests that Asb-9 may act as a
specific ubiquitin ligase-regulating CKB abundance.
OP-2
Signal transduction of cytokinine
M. Gilmanov
1
, S. Ibragimova
1
, Sv. Sadykova
1
, Zh. Basygaraev
1
and A. Sabitov
2
1
Laboratory of Enzyme Structure and Regulation, Aithozhin’s
Institute of Molecular Biology and Biochemistry,
2
Department of
Chemistry, Al-Faraby’s Kazakn National University.
E-mail:
The cytokinine the most important phytohormone, which is con-
trolling the division of the plant, cells. We carried out the investi-
gation of cytokinine signal transduction. We were discovered the
most important participant of cytokinine signal transduction.

This participant was purified from germinated wheat grains by
hydrophobic chromathography on octyl-sepharose 4B CL and by
reversed-phase chromatography on RP-18 type column. Thus we
were named the isolated substance as secondary cytokinine hor-
mone or shorter mediator of cytokinine (MC). The MC is very
powerful phytohormone as it shows the physiological activity at
concentration 1000 times less than cytokinine. In contrast of
cytokinine the MC appears own biochemical activities. For exam-
ple, MC is activated NADP-glutamate dehydrogenase and
H+ATP-ase, while the cytokinine not be able to activate this
enzymes. The purified MC is competitive repressed the binding
of tritium-labeled fusicoccine with fusicoccine receptors on plas-
matic membrane winch were isolated from roots of Zea mays
sprouts. It was shown that the binding of MC with fusicoccine
receptors led to increasing the level of cytoplasmic calcium ions.
Then calcium ions by participation of inositides is activated the
proteinkinase C. Proteinkinase C was isolated by gel chromatog-
raphy on sephacryle S-300 column and by ion-exchange chroma-
tography on DE-52 column. This enzyme is the last participant
of the signal transduction of cytokinine.
OP-3
Skeletal muscle elongation factor 2
phosphorylation during contraction:
mechanism of regulation
A. J. Rose, J. B. Kobberø, T. J. Alsted, T. E. Jensen and
E. A. Richter
Copenhagen Muscle Research Centre, Department of Human
Physiology, Institute of Exercise & Sport Sciences, Copenhagen,
Denmark. E-mail: arose@ifi.ku.dk
Very little is known about the effect of exercise on the molecular

regulation of polypeptide synthesis in skeletal muscle. Here, the
effect of contractions on skeletal muscle eukaryotic elongation
factor 2 (eEF2) phosphorylation and eEF2 kinase activity was
investigated. In response to contractions in situ, there was a rapid
(i.e. 15s) fivefold increase in eEF2 phosphorylation at Thr56 in
the contracting gastrocnemius muscle of rats that was maintained
at this level during 30 min of contractions, with no change in the
non-stimulated contralateral muscle. Furthermore, eEF2 phos-
phorylation was higher in both soleus and extensor digitorum
longus muscles of mice when contracted ex vivo, indicating that
the mechanism behind this increase is related to local factors. No
change in in vitro eEF2 kinase activity was observed in the con-
tracted rat muscles at any time-point or when measured at pH
6.8 versus 7.2. Furthermore, the increase in eEF2 phosphoryla-
tion occurred at a time before any change in AMPK activity was
observed and was normal in contracting muscles of mice expres-
sing non-functional AMPK. However, Ca
2+
-calmodulin potently
increased the activity of skeletal muscle eEF2 kinase when meas-
ured in vitro. Taken together, these data indicate that the inhibi-
tion of protein synthesis in contracting muscle may arise from
phosphorylation of eEF2 via a Ca
2+
-calmodulin-eEF2 kinase
cascade.
Abstracts
42
Integration of Defence and Survival
OP-4

Investigation of multidrug resistance in
docetaxel and doxorubicin-resistant MCF-7 cell
lines
O
¨
. Darcansoy
_
Is¸ eri
1
, M. Demirel Kars
1
, U. Gu
¨
ndu
¨
z
1
and
F. Arpacı
2
1
Department of Biological Sciences, Middle East Technical
University, Ankara, Turkey,
2
Department of Oncology, Gu
¨
lhane
Military Academy School of Medicine, Ankara, Turkey.
E-mail:
Ineffectiveness of anticancer drugs during chemotherapy or recur-

rence of malignancy after therapy is a frequently observed situation
in cancer chemotherapy. Multidrug resistance (MDR) phenomenon
is defined as the resistance of tumor cells to various cytotoxic drugs.
It is a major impediment to successful treatment of breast cancer
using chemotherapy. Cancer cells either strengthen the already pre-
sent systems necessary for the removal of toxins from cells or acquire
resistance to cytotoxic drugs. Members of the ATP-binding cassette
(ABC) transporter superfamily have an important role in MDR.
Among these, proteins coded by the ABCB1 (MDR1), ABCC1
(MRP1), and ABCG2 (BCRP) genes are the most important trans-
porters related to MDR phenotype. In this study, effects of expres-
sion levels of the MDR1, MRP1, BCRP genes on the development
of docetaxel and doxorubicin resistance using a model MCF-7
breast carcinoma cell line is evaluated. Docetaxel and doxorubicin
were applied to cell culture in dose increaments and resistant sub-
lines were developed. Cytotoxicity analysis of drugs was performed
in wild type and developed resistant sublines to test development
of resistance. Total RNA was isolated from cells, converted to
cDNA and amplified by using gene (MDR1, MRP1, and BCRP)-
specific primers by RT-PCR. Western blot analysis and immuno-
staining were performed to determine the related protein levels.
OP-5
GSK3: identification of a novel mechanism
controlling inflammation in the brain
E. Beurel
1
, S. Michalek
2
and R. Jope
1

1
Department of Psychiatry and Behavioral Neurobiology,
University of Alabama at Birmingham, Birmingham, AL,
USA,
2
Department of Microbiology, University of Alabama at
Birmingham, Birmingham, AL, USA. E-mail:
Controlling inflammation is a major challenge in the brain where
inflammation has devastating consequences because, since dam-
aged neurons cannot be replaced, neuroinflammation contributes
to neurodegenerative diseases. In response to lipopolysaccharide
(LPS), brain microglia, and astrocytes activate cytokines produc-
tion, such as IL-6. Glycogen synthase kinase-3 (GSK3) regulating
transcription factors, was studied as a regulator of neuroinflam-
mation. In mouse primary astrocytes, we examined if GSK3-
regulated IL6 production stimulated by LPS and its amplified
production caused by co-administered interferon-c. Both LPS-
induced IL-6 production and also its potentiation by interferon-c
are highly dependent on GSK3. With both IL-6 production was
abolished by GSK3 inhibitors, demonstrating the key role of
GSK3 in neuroinflammation. The mechanism of LPS-induced
IL-6 production potentiated by interferon-c was due to GSK3
activation and nuclear depletion of GSK3. This suggests that
interferon-c plus LPS potentiates IL-6 production by activating
cytosolic GSK3 which can control the activation of transcription
factors that activate IL-6 expression. Chromatin immunoprecipi-
tation is being used to identify the transcriptional targets of
GSK3, such as NF-jB that regulate IL-6 production. This study
identified GSK3 as a key regulator of neuroinflammation, estab-
lishing that GSK3 inhibitors provide a new strategy to counteract

the devastating effects of neuroinflammation.
Rhythmic Signals: the Setting of Biological Time
OP-6
Computational search of the interaction
between melanopsin and cryp tochrome-2
proteins
E. B. Unal
1
, B. Erman
2
and I. H. Kavakli
2
1
Computational Sciences and Engineering, Koc University, Istan-
bul, Turkey,
2
Chemical and Biological Engineering, Koc University,
Istanbul, Turkey. E-mail:
Circadian rhythms are the biological processes that oscillate in the
biochemical, physiological and behavioral functions of organisms
with a periodicity of approximately 24 h without any external cues.
In mammals, circadian rhythm is generated by molecular clock,
which is located at suprachiasmatic nuclei (SCN) part of brain.
Circadian rhythm is reset by external factors such as light. The
cryptochromes,which was first discovered in Arabidopsis,are the
blue-light photoreceptors. They absorb light and transmit the elec-
tromagnetic signal to blue-light dependent of signal transduction.
In mammals, the cryptochromes and melanopsin have been pro-
posed as circadian photoreceptor pigments that exist in the inner
retina to transmit signal to the SCN to tell the time of day. Both

humans and mice have two cryptochrome proteins; CRY1 and
CRY2. CRY2 is mostly expressed in retinal cells. Based on current
evidence we propose that CRY2 may interact with melanopsin to
mediate the light dependent signal-transduction in mammals.We
have taken both computational and experimental approaches in
order to show possible interaction between them during the circa-
dian photoreception. First, we have predicted 3-D structure of
cryptochrome using EsyPred, Robetta programs.Then, we have
shown that both cryptochrome and melanopsin may interact in
silico using various computational softwares, such as AUTO-
DOCK, HEX.We are currently verifying our computational data
taking experimental approach, specifically the FRET.
OP-7
Mitochondrial electron transport chain
reactivity in the brain and eye tissues under
circadian rhythm alterations
M. B. Yerer
1
, M. P. Alcolea Delgado
2
, S. Aydog
˘
an
1
,
F. J. Garcia-Palmer
2
and P. R. Salom
2
1

Faculty of Medicine, Department of Physiology, University of
Erciyes, 38039 Kayseri, Spain,
2
Department of Biochemistry and
Molecular Biology, University of Balearic Islands, Mallorca,
Spain. E-mail:
Mitochondria plays a central role in energy-generating processes
within the cell thorough the electron transport chain (ETC),
the primary function which is ATP synthesis via oxidative
Abstracts
43
phosphorylation (OXPHOS) which is shown to be related to age-
ing and apoptosis when this balance is destroyed under different
circumstances. This study is performed to investigate the effects
of alterations in the physiological melatonin levels via the circa-
dian rhythm changes, on the mitochondiral ETC in brain and
eye and how these changes are correlated to the pineal gland
melatonin receptor expressions. Fifty Sprague–Dawley male rats
weighing 200–250 g were used in five groups of different circa-
dian rhythms. The control group was 12/12 h of light/dark (L/D)
cycle. Different circadian rhythms of 24/0 h L/D, 0/24 h L/D,
16/8 h L/D and 8/16 h L/D cycles were applied to the groups for
1 week, respectively, in special cages where the duration of the
light and the climate can be adjusted. The melatonin receptors,
MEL1 and MEL2 expressions were determined by real-time PCR
in the pineal gland. The mitochondria of the brain and eye tis-
sues were isolated from the homogenates and the activation of
the mitochondrial OXPHOS complexes were determined by spec-
trophotometric micro-methods described before. Plasma melato-
nin levels were also determined by ELISA kit (IBL, Turkey).

Related to circadian rhythms, the plasma melatonin levels were
the highest in the 0/24 L/D group compared to the other groups
(p < 0.05) and the MEL1 and MEL2 receptor expressions were
also altered significantly by the circadian rhythms (p < 0.05).
The Complex I activity is found to be decreased significantly in
the 24/0 L/D group compared to the control and the 0/24 h L/D
group (p < 0.05) in the brain mitochondria whereas it was sig-
nificantly higher in the eye mitochondria compared to the control
(p < 0.05). Complex III activities were slightly lower in the 24/
0 h L/D group, whereas there was a significant increase in the
eye mitochondria in all the groups compared to the control
(p < 0.05). Furthermore, there was a significant increase in the
Complex IV and V activities in the brain mitochondria were sig-
nificantly higher 24/0 L/D group compared to its control
(p < 0.05) whereas they were found to be unaffected in the eye
mitochondria. As a consequence, this is the known first report to
show the MEL1 and MEL2 receptor expressions by real-time
PCR under different circadian rhythms. These alterations both in
the receptors and the plasma melatonin levels are found to be
correlated with the mitochondrial respiratory chain complexes
which are directly related to the energy metabolism in the cells
during the ageing process and the apoptosis. This study was
granted by NATO Science Fellowships A2 Programme of TUBI-
TAK.
Signaling and Cancer: Nuclear Receptor Connection
OP-8
Dysregulated Msx and Dlx gene expre ssion in
epithelial odontogenic tumors
S. Ghoul-Mazgar
1

, B. Ruhin
2
, D. Hotton
3
and A Berdal
3
1
Laboratoire de Biologie Oro-Faciale et Pathologie, INSERM
U714-IFR-58, Universite
´
s Paris 7 and Paris 6 Laboratoire
d’Histologie-Embryologie, Faculte
´
de Me
´
decine Dentaire de
Monastir, Tunisia,
2
Stomatology and Maxillofacial Surgery
Department, Pitie
´
Salpe
ˆ
trie
`
re University Hospital, Paris Cedex 13,
France,
3
Laboratoire de Biologie Oro-Faciale et Pathologie
INSERM U714-IFR-58, Universite

´
s Paris 7 and Paris 6, France.
E-mail:
Odontogenic tumors are rare pathologies, mostly benign, located
in maxillary area. The most frequently observed benign epithelial
odontogenic tumors is called ameloblastoma and may although
give rise to the extremely rare malignant epithelial odontogenic
tumors, usually named odontogenic carcinomas.The differential
diagnosis between these tumors is so difficult regarding the
diverse clinical prognosis and therefore management. Homeodo-
main proteins comprise transcription factors that are essential in
many developmental processes. Homeodomain is encoded by a
highly conserved 60 amino acid sequence called homeobox that is
responsible for specific interactions with DNA. Non-clustered ho-
meobox genes are called non-HOX and include the Msx and Dlx
gene family. In this study, we examined the Dlx and Msx gene
expression by RT-PCR and in situ hybridization in recurrent 13
benign ameloblastomas and one malignant clear cell odontogenic
carcinoma (CCOC). Our data show specific expression pattern of
Msx and Dlx gene in the CCOC compared with benign amelobl-
astomas. Furthermore, exploring the expression pattern of signal
molecules by RT-PCR, Bmp2 was shown to be inactivated in the
carcinoma, but not Bmp4. Malignancy of epithelial odontogenic
carcinoma seems to be a multistep and highly heterogeneous pro-
cess requiring activation and deactivation of multiple and specific
genes suggesting exploration of homeogene expression to discrim-
inate benign ameloblastomas and odontogenic carcinomas.
OP-9
Ligand-specific dynamics of the androgen
receptor on its target promoter in living cells

T. I. Klokk
1
, P. Kurys
1
, C. Elbi
2
, A. K. Nagaich
2
,
A. Hendarwanto
2
, T. Slagsvold
1
, C. Y. Chang
3
, G. L. Hager
2
and F. Saatcioglu
1
1
Department of Molecular Biosciences, University of Oslo, Oslo,
Norway,
2
Laboratory of Receptor Biology and Gene Expression,
National Cancer Institute, Bethesda, USA,
3
Department of
Pharmacology and Cancer Biology, Duke University Medical
Center, Durham, MD, USA. E-mail:
Androgen receptor (AR) mediates the action of androgens, which

are important in the development and maintenance of the male
reproductive system and in pathologic conditions such as pros-
tate cancer. This is the basis for the routine use of antiandrogens
to block AR function in disease states, but little is known on the
mechanisms involved. We studied ligand-dependent AR interac-
tion with a target promoter in vivo, using photobleaching micros-
copy, kinetic modeling, FRET analysis, and in vitro chromatin
remodeling. The interaction of agonist-bound AR with the
MMTV promoter was rapid and transient. In the presence of
antagonist, and with a transcriptionally impaired AR mutant, an
even faster interaction was seen due to decreased residence time
on the promoter. The short residence times seen for AR in
response to all ligands support the ‘hit-and-run’ model and three-
dimensional genome-scanning hypothesis of transcription factor
action. Furthermore, agonist and partial antagonists, but not
pure antagonists, induced the recruitment of a chromatin-re-
modeling complex to the HRE. Finally, FRET analysis in vivo
demonstrated both intermolecular and intramolecular interac-
tions between the N- and C- termini of AR at the HRE. Thus,
three-dimensional scanning of the genome space, ligand-depend-
ent modulation of AR kinetic properties, recruitment of chroma-
tin remodeling complexes and proper intermolecular and
intramolecular interactions are all critical for the in vivo function
of AR on its target promoter.
Abstracts
44
Cell Surface Receptors and Downstream Targets
OP-10
Mutations of the growth hormone receptor
gene in Turkish patients with Laron-type

dwarfism
A. Arman
1
, N. Yordam
2
, A. Ozon
2
, P. Isguven
3
, A. Coker
4
and
I. Peker
1
1
The Faculty of Engineering, Marmara University, Istanbul,
Turkey,
2
The Department of Pediatrics, Division of Endocrinology,
Hacettepe University, Ankara, Turkey,
3
The Department of
Endocrinology, Goztepe Government Hospital, Istanbul,
Turkey,
4
The Department of Biology, Art and Sciences, Marmara
University, Istanbul, Turkey. E-mail:
Growth hormone (GH) mediates its growth, fat and carbohy-
drate metabolism through insulin-like growth factor-I (IGF-I).
Interaction of GH with the GH receptor (GHR) is necessary for

systemic and local production of IGF-I that mediates GH
actions. Mutations in the GHR cause severe postnatal growth
failure and the disorder is an autosomal recessive genetic disease
called Laron-type dwarfism, characterized by elevated serum GH
associated with low levels of IGF-I. In this research, our purpose
was to analyse the GHR gene for mutations in eight patients
with Laron-type dwarfism. Eight patients were selected based on
their phenotypic characteristics and their genomic DNAs were
isolated from bloods of eight laron children. Exon 2–9 specific
polymerase chain reactions (PCRs) and their flanking splice sites
were amplified PCR. The PCR products were purified and se-
quenced. We defined three missense mutations (S40L, V125A,
I506L), one nonsense mutation (W157X), one sense mutation
(G168G), one exon 3 deletion (exon 3 deletion) and one frame-
shift mutation (G insertion in exon 2) located in the extracellular
domain of GHR in eight patients. Acknowledgment: This project
was supported by Turkish Republic State Planning Organization.
OP-11
Human XLas signals more efficiently than the
a-subunit of the stimulatory G protei n (GSa)
in vitro
A. Linglart
1
, M. J. Mahon
2
, T. Dean
2
, T. J. Gardella
2
,

H. Jueppner
2
and M. Bastepe
2
1
Pediatric Endocrinology and INSERM U561, Saint Vincent de
Paul Hospital, Paris, France,
2
Endocrine Unit, Department of
Medicine, Massachusetts General Hospital and Harvard Medical
School, Boston, MA, USA. E-mail:
harvard.edu
XLas is partly identical to the a-subunit of the stimulatory G pro-
tein (Gsa) with an extended N-terminus. Using adenoviral expres-
sion and cells that endogenously lack Gsa and XLas (GnasE2–/–
cells), we investigated human XLas (hXLas). Immunofluorescence
microscopy showed membrane and punctate perinuclear staining
for hXLas. On metal affinity chromatography, hXLas co-purified
with a histidine-tagged Ga1. Furthermore, a PTH(1–15) analog,
which normally prefers binding to a Gsa-coupled form of
PTHR1, bound to membranes from GnasE2–/– cells co-expres-
sing PTHR1 and hXLas. Also, hXLas was able to mediate basal
and agonist-induced cAMP accumulation. However, while hXLas
was expressed at lower levels than Gsa in transduced cells, basal
cAMP level in cells expressing hXLas was ~twofold higher than
in cells expressing Gsa. Similarly, basal ERK1/2 phosphorylation
in GnasE2–/– cells transiently expressing hXLas was markedly
enhanced. Isoproterenol treatment also resulted in significantly
higher levels of cAMP accumulation in hXLas-expressing cells
than Gsa-expressing cells, whereas PTH-induced cAMP accumu-

lation appeared similar in cells co-expressing PTHR1 and either
hXLasorGsa. These findings indicate that hXLas can act as a
more potent signaling protein than Gsa, which may have implica-
tions in responses mediated typically by Gsa.
OP-12
Growth inhibition of c6 glioma cells in
monolayer and spheroid cultures by the
combination of carvedilol and imatinib
M. Erguven
1
, A. Bilir
2
, S. Tuna
2
and N. Akev
1
1
Department of Biochemistry, Faculty of Pharmacy, Istanbul
University, Istanbul, Turkey,
2
Department of Histology and
Embryology, Istanbul Faculty of Medicine, Istanbul, Turkey.
E-mail:
Rat C6 glioma is a chemoresistant experimental brain tumour,
which is difficult to treat with a combination of drugs. A new
tyrosine kinase inhibitor, imatinib (Gleevec), has recently been
found efficacious in the pre-clinical trials for glioblastoma (GBM).
Carvedilol (Dilatrend), antihypertensive drug, has been demon-
strated to reverse multidrug resistance (MDR) to anticancer drugs
in several tumor cell lines in vitro. However, any possible modula-

tory effect of carvedilol on brain tumours and on the efficacy of
imatinib has not yet been evaluated experimentally. In the present
study, we have investigated whether carvedilol provides synergistic
or antagonistic effect on imatinib-induced cytotoxicity in mono-
layer and spheroid cultures of malignant C6 glioma cells. C6
glioma cells in monolayer and spheroid cultures were treated with
the combination of carvedilol and imatinib in concentration of
10 lM. The cell proliferation, morphology, spheroid volumes,
bromodeoxyuridine-labelling index (BrdU-LI) were evaluated.
The expression levels of caspase-3, caspase-9, hypoxia inducible
factor-1 (HIF-1), Bcl-2 and platelet-derived growth factor receptor
alpha (PDGFRa) were examined by Western blot analysis. The
statistical significance was analysed by using the Student’s t-test.
The results demonstrated that carvedilol and imatinib in combina-
tion display enhanced antitumour activity in vitro against experi-
mental rat C6 glioma in monolayer and spheroid cultures.
OP-13
Non-traditional ways of B lymphocyte
regulation: receptors for acetylcholine and
thrombin
S. Komisarenko
1
, R. Grailhe
2
, Y. Petrova
1
, J. P. Changeux
2
,
W. Bahou

3
and M. Skok
1
1
Palladin Institute of Biochemistry, Kiev, Ukraine,
2
Pasteur
Institute, Paris, France,
3
State University of New York, Stony
Brook, NY, USA. E-mail:
Immune system is highly specialized to recognize and destroy the
invading pathogens and transformed self-cells. For this purpose,
it is equipped with unique antigen-specific receptors and employs
specific ways of cell-to-cell communication. However, both the
development and functioning of immune cells are regulated by
various external factors including those of nerve and blood coagu-
lation systems. Correspondingly, lymphocytes express receptors
for non-immune mediators, such as acetylcholine and throm-
bin. We demonstrated the presence of nicotinic acetylcholine
Abstracts
45
receptors (nAChRs) and protease-activated receptors type 3
(PAR3) on mouse B lymphocytes using specific antibodies gener-
ated to functionally important parts of these receptors: compo-
nents of acetylcholine-binding site or thrombin-cleavage site. It
was shown that signaling through nicotinic receptors stimulated
proliferation of B lymphocyte-derived cell lines, supported B-lym-
phocyte survival in the bone marrow, but limited its activation in
mature state. In contrast, PAR3 activation inhibited B-cell line

proliferation, but enhanced CD40-mediated B-lymphocyte prolif-
eration. It is concluded that signaling through nAChR and PAR3
affects basic vital functions of B lymphocytes, such as prolifer-
ation and survival, and interferes with their activation pathways.
These data show the ways, by which acetylcholine, nicotine or
thrombin can regulate the humoral branch of immunity.
OP-14
Identification and characterization of a novel
transcriptionally active domain in the linker
region of the TGFb-regulated Smad3 protein
M. Siderakis, V. Prokova, S. Mavridou, P. Papakosta and
D. Kardassis
Department of Medicine, University of Crete and IMBB-FORTH,
Heraklion, Crete, Greece. E-mail:
Our structure–function analysis of human Smad3 protein, a key
mediator of TGFb signaling in mammalian cells revealed that the
middle, non-conserved, linker domain has an autonomous and
potent transactivation function. The region with the maximal
transactivation capacity was the 143–248 that consist of almost
the entire linker domain and the first 18 amino acids of the MH2
domain. The corresponding regions in Smad4 as well as in
Smad1, which is a key mediator in Bone Morphogenetic Protein
(BMP) signaling, are also transcriptionally active in mammalian
cells further supporting the important role of this domain for
Smad function. Smad3 mutants bearing an internal deletion of
the 200–230 region or single amino acid substitutions in two
highly conserved residues of this region (Q222A and P229A) had
severe defects in oligomerization and transcriptional activation of
target promoters. In contrast, mutagenesis of a non-conserved
amino acid residue in the same region (N218A) did not affect

any Smad function examined. Using a protein–protein interaction
assay based on biotinylation in vivo, we were able to show that
the Smad3 mutant with the internal deletion of amino acids 200–
230 is unable to interact physically and functionally with the his-
tone acetyltransferase p/CAF. Our data support an essential role
of the previously uncharacterized middle region of Smad3 for
nuclear functions, such as transcriptional activation and interac-
tion with Smad coactivators
Signaling Through Ion-channels
OP-15
Sigma1 receptor/beta1 integrin complex: a
novel target site for breast cancer cell
adhesion
R. Mahen, C. Palmer, C. Edwards, M. Djamgoz and E. Aydar
Imperial College London, Divisions of Biology, Cell & Molecular
Biology and Molecular Biosciences, Sir Alexander Fleming
Building, South Kensington Campus, London, UK.
E-mail:
The sigma receptor is a novel protein that is highly expressed
in cancer cells and tissues, and has been shown to modulate
proliferation and adhesion of breast cancer cells. The mechan-
ism of action of the sigma1 receptor in these processes; how-
ever, has not been elucidated. Using the Single Cell Adhesion
Measuring Apparatus (SCAMA; confocal microscopy, sigma1
receptor RNAi, immunoprecipitation, surface biotinylation and
lipid raft fractionation, the function of sigma1 receptor in adhe-
sion of MDA-MB-231 breast cancer cells was investigated.
Functional studies using SCAMA revealed that disruption of
lipid rafts eliminated sigma receptor modulation of adhesion.
Moreover,immunoprecipitation experiments in MDA-MB-231

cells,demonstrated that sigma1 receptor and beta1 integrin are
associated. Furthermore, both confocal microscopy experiments
and surface biotinylation experiments indicated that both appli-
cation of sigma receptor drugs and knock-down of the sigma1
receptor increased the beta1 integrin expression in the mem-
brane. Lipid raft fractionation experiments in MDA-MB-231
cells demonstrated that both application of the sigma receptor
drugs and the knock-down the sigma1 receptor levels, beta1 in-
tegrin protein in lipid rafts fraction of MDA-MB-231 cells were
altered. All these data suggest that sigma1 receptor is associated
with beta1 integrin and is likely modulate beta1 integrin levels.
Therefore, sigma1 receptor is likely to be a novel target for
breast cancer metastasis.
OP-16
HERG potassium channels and heart disease
S. Kuyucak
School of Physics, University of Sydney, NSW, Sydney, Australia.
E-mail:
The heartbeat is controlled by electrical signals mediated by the
flow of ions through specialized ion channels. Of the channels
that contribute to cardiac electrical activity, potassium channels
encoded by the Human ether-a-go-go-related gene (HERG) have
been of particular interest for many reasons. First, mutations in
HERG are the cause of one-third of cases of congenital long QT
syndrome, an inherited cause of sudden cardiac death. Secondly,
HERG is the molecular target for the vast majority of drugs that
cause drug-induced long QT syndrome, the commonest cause of
drug-induced arrhythmias and cardiac death. Thirdly, HERG
channels have very unusual biophysical properties, which suggest
that they may act as an endogenous antiarrhythmic agent. There-

fore, understanding the operation of HERG channels has become
an important goal-post in medicine, physiology and pharmacol-
ogy. While there is no crystal structure for the HERG protein
yet, homology models based on the crystal structure of bacterial
potassium channels provide a promising avenue for progress. In
this talk, a survey of advances made in the field using a com-
bined experimental/simulation approach will be given. This
involves using homology models of HERG to find the important
residues on the protein that are involved in channel dynamics
(e.g. toxin binding, fast inactivation) and then test these hypothe-
ses via mutagenesis experiments. This information is then used to
refine the model, followed by further tests.
Abstracts
46
Signaling and Apoptosis
OP-17
Mechanism of pancreatic acinar cell apoptosis
induced by crambene
S. Adhikari
1
, Y. Cao
1
, A. D. Ang
1
, M. V. Clement
2
, M. Wallig
3
and M. Bhatia
1

1
Department of Pharmacology, National University of Singapore,
Yong Loo Lin School of Medicine, Singapore,
2
Department of
Biochemistry, National University of Singapore, Yong Loo Lin
School of Medicine, Singapore,
3
Department of Pathobiology,
University of Illinois at Urbana Champaign, Urbana, IL, USA.
E-mail:
We investigated the molecular mechanisms that regulate the
apoptosis of acinar cells induced by crambene (1-cyano-2-hydrox-
y-3-butene-CHB), a plant nitrile. As evidenced by annexin
V-FITC staining, crambene treatment for 3 h induced the apop-
tosis but not necrosis of pancreatic acini. Caspase 3, 8 and 9
activity in acini treated with crambene were significantly higher
than in untreated acini. Treatment with caspase 3, 8 and 9 inhibi-
tors inhibited annexin V staining, as well as caspase 3 activity,
pointing to an important role of these caspases in crambene-
induced acinar cell apoptosis. The mitochondria membrane
potential was collapsed in crambene-treated acini than in
untreated that displayed polarized mitochondria. Also, the treat-
ment of acini with crambene induced the release of cytochrome
C by mitochondria than in untreated acini. Neither TNF-a nor
Fas ligand levels were changed in pancreatic acinar cells after
crambene treatment. These results provide evidence for the induc-
tion of pancreatic acinar cell apoptosis in vitro by crambene and
suggest the involvement of mitochondrial pathway in pancreatic
acinar cell apoptosis.

OP-18
Androgen inhibition of apoptosis in prostate
cancer cells is due to downregulation of JNK
activation
P. I. Lorenzo and F. Saatcioglu
Department of Molecular Biosciences, University of Oslo, Oslo,
Norway. E-mail:
Androgens have a significant role in normal prostate physiology,
as well as in prostate cancer. Removal of androgens during the
initial stages of prostate cancer causes tumor regression via a
combination of reduced cellular proliferation and increased apop-
tosis. We have studied the molecular mechanisms of apoptosis in
prostate cancer cells, focusing on the inhibition of apoptosis by
androgens. We show that androgen treatment protects LNCaP
prostate cancer cells from thapsigargin (TG)- and 12-o-tetradeca-
noyl-13-phorbol-acetate (TPA)-induced apoptosis through a
mechanism that involves the inhibition of c-Jun N-terminal kin-
ase (JNK). The inhibitory effect of androgens on JNK activation
is dependent on the androgen receptor (AR) since it is blocked
by the androgen antagonist bicalutamide. The inhibition of JNK
by androgens is independent of the stimulus that activates JNK,
since it occurs readily when the JNK pathway is activated in
response to TG, TPA or ultraviolet irradiation (UV). The inhibi-
tory effect of androgens on JNK activation and apoptosis
requires new gene expression, which is consistent with the time
required to observe this effect. ATP depletion experiments indi-
cate that inhibition of JNK activation by androgens is mediated,
at least in part, through an increase in phosphatase activity. This
crosstalk between AR and JNK signaling pathways may have
important implications for both normal prostate physiology, as

well as prostate cancer progression.
OP-19
Enhancement of peptidylarginine deiminase 4
enzymatic activity assists apopt osis
C Y. Lin
1
, Y F. Liao
2
, P C. Hsu
3
, H C. Hung
2
and G Y. Liu
1
1
Institute of Immunology, Chung Shan Medical University, Tai-
chung, Taiwan,
2
Department of Life Sciences, National Chung-
Hsing University, Taichung, Taiwan,
3
Department of Medicine, Da
Chien General Hospital, Taiwan. E-mail:
PAD4 post-translationally converts peptidylarginine to citrulline.
It plays an essential role in immune cell differentiation and apop-
tosis. A haplotype of single nucleotide polymorphism (SNP) in
PAD4 is functionally relevant as a rheumatoid arthritis (RA)
gene. It could increase enzyme activity leading to raised levels of
citrullinated protein and stimulating autoantibody. Inducible
PAD4 causes haematopoietic cell death (Liu et al. 2006) apopto-

sis. Herein, we further investigate whether the increase of PAD4
enzymatic activity induces apoptosis. In Tet-On Jurkat T cells,
ionomycin (Ion) only treatment did not induce apoptosis; how-
ever; it promoted inducible PAD4-decreased cell viability and
enhanced apoptosis. In vitro PAD enzyme activity assay, we dem-
onstrated PAD4 enzyme activity of SNP relative to RA was
higher than wild type (WT) relative to non-RA following Ca++
treatment. The effect of PAD4 SNP-induced apoptosis was
superior to PAD4 WT. In addition, both Ion and PAD4 SNP sy-
nergistically provoked apoptosis compared with both Ion and
PAD4 WT. Western blotting data showed apoptosome activation
during the programming cell death. Concurrently, the expression
of Bcl-xL was downregulated remarkably and Bax upregulated in
Ion treatment cells. These data demonstrated that increasing
PAD4 enzyme activity could enhance apoptosis through mitoch-
ondrial pathway and provide a conceivable explanation in the
pathogenesis of RA following the upregulation of PAD4 activity.
OP-20
Glucose-induced impairment of the
insulin-signaling path way in mouse pancreatic
beta cells
E. Tsilibary
1
, P. Venieratos
1
, A. Charonis
2
and P. Kitsiou
1
1

Institute of Biology, National Center for Scientific Research
‘Demokritos’, Greece,
2
Foundation for Biomedical Research of the
Academy of Athens, Athens, Greece.
E-mail: effi
Type 2 diabetes is characterized by progressive pancreatic b-cell
dysfunction and apoptosis. Recent reports provided evidence for
an autocrine role of insulin on the signalling cascade in b-cell
growth, function and survival are concerned. We examined the
effects of chronic glucose stress on insulin signalling. Exposure of
bTC-6 cells to high glucose resulted in impairment of insulin-sti-
mulated phosphorylation of IRS-2. These changes were accom-
panied by significant impairment of IRS-2-associated PI3-kinase
activation, and substantially decreased activation of Akt. We also
examined mTOR kinase, a downstream effector of Akt, which
stimulates protein synthesis in response to Akt phosphorylation.
High glucose abolished insulin-induced activation of mTOR
without affecting mTOR expression, thus protein synthesis
should also be impaired. A mechanism by which Akt promotes
cell survival includes phosphorylation of the pro-apoptotic
Abstracts
47
protein BAD, which keeps the pro-apoptotic protein BAX
engaged to Bcl-XL. Exposure of cells to high glucose also resul-
ted in suppression of insulin-stimulated phosphorylation of BAD
without affecting BAD expression. In conclusion, we demonstra-
ted that chronic exposure of pancreatic b-cells to increased glu-
cose concentration resulted in impaired activation of the IRS-2/
PI3-kinase/Akt signalling pathway in response to insulin. The

observed defects in insulin signalling may eventually have a neg-
ative effect on b-cell function and survival.
OP-21
Caspase-dependent and geldanamycin-
enhanced cleavage of co-chaperone p23 in
leukemic apoptosis
G. Gausdal
1
, B. T. Gjertsen
2
, K. Fladmark
1
, H. Demol
3
,
J. Vandekerckhove
3
and S. O. Døskeland
1
1
Department of Biomedicine, University of Bergen, Bergen,
Norway,
2
Department of Medicine, Haukeland University Hospital,
Bergen, Norway,
3
Department of Biochemistry, University of Ghent,
Ghent, Belgium. E-mail:
The co-chaperone p23 is a component of the Hsp90 multiprotein
complex and is an important modulator of Hsp90 activity.

Hsp90 client proteins involved in oncogenic survival signaling are
often found to be mutated in leukemia, and the integrity of the
Hsp90 complex could therefore be important for leukemic cell
survival. We demonstrate here that the Hsp90 co-chaperone p23
is cleaved in leukemic cell lines treated with commonly used che-
motherapeutic drugs. The cleavage of p23 paralleled the activa-
tion of procaspase 3 and was suppressed by z-DEVD-FMK.
Interestingly, p23 cleavage was also observed in caspase 3-defici-
ent MCF-7 cells, and in vitro translated 35S-p23 was cleaved by
both caspase 3 and 7. Two caspase target sites were identified in
the C-terminal sequence EVD142GAD145, and only Asp to Ala
mutagenesis of both sites (D142/145A) completely blocked p23
cleavage. The Hsp90 inhibitor geldanamycin, which inhibits p23
binding to Hsp90, did not induce cell death or p23 cleavage on
its own, but enhanced anthracycline-induced caspase activation,
p23 cleavage and apoptosis. This implies that Hsp90 inhibition
amplifies caspase activation. Geldanamycin also enhanced
caspase cleavage of 35S-p23 in vitro, indicating that the associ-
ation of p23 to Hsp90 protects against cleavage. These findings
underscore the importance of the Hsp90-complex in antileukemic
treatment, and suggest that p23 may have a role in survival
signaling.
OP-22
Overview how colorectal cancer cells avoid
cell elimination; mechanisms of immune- and
chemo-resistance
B. Pajak
1
, H. Engi
1

, J. Molnar
1
and A. Orzechowski
2
1
Institute of Medical Microbiology and Immunology, Faculty of
Medicine, University of Szeged, Szeged, Hungary.
E-mail: ,
2
Department of Physiological Sciences,
Faculty of Veterinary Medicine, Warsaw Agricultural University,
Warsaw, Poland
The resistance of cancer cells to deletion allows tumors to grow
and develop. In the past, several tactics were shown how colon a-
denocarcinomas avoid cell deletion and maintain cell viability. In
particular, colorectal cancer cells resist death ligands-induced
apoptosis by expressing antiapoptotic proteins, including FLIP.
By direct interaction with FADD, FLIP inhibits the signal des-
cending from death receptors in COLO 205 cells. Colorectal can-
cer cells also stimulate own survival by the cytoplasmic retention
of proapoptotic protein clusterin. In contrast, in normal cells clus-
terin translocates to the nucleus and induces cell death. We found
that apoptotic activity of clusterin is dependent on calcium ions,
and depletion of intracellular calcium caused extensive death of
COLO 205 cells. Other type of strategy of chemotherapy-induced
cell death is the activity of multidrug resistance proteins (MDR).
These cell membrane efflux pumps actively expel the drugs from
the cell interior to prevent their action on intracellular targets.
Upon phenothiazine derivatives rhodamine 123 accumulated
within cells interior, which indicates the reversal of Pgp efflux

pump in chemoresistant COLO 320 cell line. The variety of antia-
poptotic mechanisms found in colorectal cancer cells and the
knowledge how complex they are renders the anticancer therapy a
challenge but the more we know how to sensitize cancer cells to
death signals the more likely promise to eliminate them.
Embryonic Stem Cells
OP-23
Expression of the cholinergic system
components during mouse embryonic stem
cell differentiation
L. Paraoanu, G. Steinert and P. Layer
Developmental Biology, Darmstadt University of Technology,
Darmstadt, Germany. E-mail:
Expression of cholinesterases during phases of embryonic devel-
opment is a general phenomenon. However, no precise function
for cholinesterases could be described during early developmental
stages. We have examined the expression of cholinesterases and
other cholinergic components during in vitro differentiation of
CGR8 cells, an embryonic stem cell line. Undifferentiated mono-
layers of these cells can be maintained in culture, or embryoid
bodies can be generated in vitro. The embryoid bodies were
allowed to differentiate in the absence of further additives or
treated with retinoic acid, and collected at various times for fur-
ther analysis. The cholinesterase expression was analyzed by
reverse transcriptase polymerase chain reaction, histochemistry,
and activity measurement. Low levels of cholinesterase activity
and transcripts could be detected already in undifferentiated cells.
The phases of differentiation are accompanied by increased ace-
tylcholinesterase transcripts. Butyrylcholinesterase expression was
initially constant, decreasing at later differentiation stages. All

transcripts of muscarinic cholinergic receptors were present; how-
ever, their expression did not vary throughout the differentiation
steps. Choline-acetyltransferase was also detected, indicating that
the cells are possibly able to produce acetylcholine. With these
results we could show that mouse embryonic stem cells express a
primitive cholinergic system. Its function remains to be deci-
phered.
Abstracts
48
Differentiation of Stem Cells
OP-24
Human umbilical cord strom a cells
differentiate into neurons in vitro
S. Karahuseyinoglu
1
, O. Cinar
2
, F. Kara
3
and A. Can
1
1
Department of Histology-Embryology, Ankara University School
of Medicine, Ankara, Turkey,
2
Department of Infertility, Etlik
Maternity and Women’s Health Research Training Hospital,
Ankara, Turkey,
3
Department of Obstetrics, Zubeyde Hanim

Maternity Hospital, Ankara, Turkey. E-mail: alpcan@medicine.
ankara.edu.tr
A recently isolated source for stem cells is the mesenchymal cells
of human umbilical cord stroma. Although neuronal differenti-
ation has been demonstrated in human umbilical cord stroma cells
(HUSCSs), there is a still discordance among studies about the
potential of these cells to differentiate into neurons. The aim of
this study was to differentiate HUSCSs into neurons and investi-
gate the temporal development potency of newly differentiated
neuronal cells. The spatio-temporal distribution and the quantifi-
cation of various proteins during differentiation, immunofluores-
cent single and multi-immunolabeling techniques were performed
using a series of antibodies raised against major cellular markers
for neurons, such as MAP1, MAP2, GFAP, tau, NSE, NFM, b-
III tubulin, NeuN, and thyrosine hydroxylase. MAP1, a specific
structural protein in the cytoplasm, MAP2, specific for dendrites
and NFM were all positive during the course of the entire differ-
ention period whereas tau and b-III tubulin were detected at only
certain time-points of differentiation. While NeuN stained faintly,
GFAP and thyrosine hydroxylase were both negative since no dif-
ferentiation toward astrocytes and dopaminergic neurons was
accomplished. Conclusively, it appears that throughout the differ-
entiation of HUCSCs into neurons, expression of neuronal mark-
ers present a diversity which follows a similar progress as seen in
embryologic development of neurons of the body.
Gene Therapy
OP-25
Cationic nanoparticle synthesis and their use
in gene transfer
G. Guven

1
, M. Turkoglu
1
and E. Piskin
2
1
Hacettepe University Chemical Eng. Dept,
2
Hacettepe University
Chemical Eng. & TUBITAK-BIOMEDTEK. E-mail: gguven@
hacettepe.edu.tr
Polymer microspheres would be synthesized by use of an emulsi-
fier-free emulsion polymerization, which gives more pure, and
monodisperse samples. In this method the particle formation
occurs when the growing radicals reach to a certain critical chain
length depending upon the solvency of the continous phase. In
this study, monosized non-functional poly(styrene/N-[3-dimethyl-
amino)propyl]methacrylamide/poly(ethyleneglycol)ethylethermetha-
crylate) [poly(St/PEG-EEM/DMAPM)] and functional group
carrying poly(styrene/N-[3-dimethylamino)propyl]methacrylamide/
poly(ethyleneglycol) methacrylate) [poly(St/PEG-MA/DMAPM)]
nanoparticles were synthesized by emulsifier-free emulsion copo-
lymerization in the presence of a cationic initiator 2,2’-azobis(2-
methyl propionamidine) dihydrochloride (V-50). The spherical
particles with diameters in the nanometer range and cationic surface
charge were prepared. Generally, the polymeric nanoparticle dimen-
sions are below 100 nm. The smallest particle size (71 nm) with very
low polydispersity index and very high surface charge was obtained
by increasing water content. This particle size was achieved by using
both of the functional (PEG-MA) and non-functional (PEG-EEM).

Nanoparticles with such a high amount of surface charge make
these materials useful for the gene transfer especially.
OP-26
Peripheral pool of CD8+ lymphocytes contains
T cells that recognize syngeneic MHC class II
molecules
E. S. Zvezdova, T. S Grinenko, L. A Pobezinsky,
E. L. Pobezinskaya and D. B. Kazansky
Russian N.N. Blokhin Cancer Research Center.
E-mail:
A number of publications appeared, which assume that autoim-
mune cells exist in the pool of CD8+ T lymphocytes with the
potential to recognize both MHC class I and MHC class II mole-
cules. To test this, we generated autoreactive T-cell hybridomas
with the ability to recognize both allogeneic MHC class I mole-
cule and MHC class II molecules: the LN CD8+ T cells from
C57BL/6 mice were activated in the presence of allogeneic MHC
class I molecules Kbm3 and fused with BW5147 thymoma cells,
expressing CD4 coreceptor. The percentage of autoreactive hy-
bridomas in CD8+ T cells was 9.3%. One of these hybridomas
expressed two a-chains. One of these a-chains help to recognize
syngeneic MHC class II molecules, while the other allogeneic
MHC class I molecule Kbm3. To elucidate would the same situ-
ation be observed in TCRa
+/0
mice capable to express single
a-chain of TCR, we obtained hybridomas from their CD8+ T
cells. The frequency of self MHC class II reactive hybridomas
was lower in contrast with that of wild type 1.3%. We have
shown that in the peripheral pool of wild-type CD8+ T cells the

autoreactive T cells exist that recognize foreign MHC class I and
self MHC class II molecules. One part of these cells expresses the
TCR with dual specificity to MHC class I and MHC class II
molecules, while the other expresses two a-chains, one defines the
specificity to MHC class I molecule and the other to syngeneic
MHC class II molecule.
OP-27
Salmonella typhimurium aroB-encoding
murine IL-18 or CD40L: evaluation for gene
therapy
D. Aydin
1
, A. Gunel-Ozcan
2
, N. Menager
3
, G. Esendagli
1
,
M. O. Guc
4
, M. Hayran
5
, E. Kansu
1
and D. Guc
1
1
Institute of Oncology, Department of Basic Oncology, Hacettepe
University, Ankara, Turkey,

2
Faculty of Medicine, Department of
Medical Biology and Genetics, Kirikkale University, Kirikkale,
Turkey,
3
Wellcome Trust Sanger Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge, UK,
4
Faculty of Medicine,
Department of Pharmacology, Hacettepe University, Ankara,
Turkey,
5
Institute of Oncology, Department of Preventive
Oncology, Hacettepe University, Ankara, Turkey.
E-mail:
Attenuated Salmonella typhimurium (ST) strains have being
investigated as promising vectors for gene therapy. In our study,
Abstracts
49
STaroB strain was evaluated as a gene delivery vehicle for the
first time. Eukaryotic expression vectors, pTARGET and
pcDNA3, carrying murine IL-18 or CD40L genes were construc-
ted to enhance immune responses of the host for further cancer
therapy studies. mRNAs used to synthesize cDNAs to amplify
IL-18 and CD40L DNAs were obtained from lipopolysaccharide-
induced adherent peripheral blood mononuclear cells, and Phor-
bol 12-Myristate 13-Acetate and Ionomycin-induced splenocytes
respectively. A bioactive IL-18 molecule consisting of IFN-b sig-
nal and mature IL-18 sequences was constructed by overlap
extension method. The plasmids were transformed into STaroB

by electroporation. While in vitro stability of pTARGET con-
structs was very low, pcDNA3 constructs were highly stable.
HEK293 cells were transfected with pcDNA3 constructs by a cat-
ionic liposome and mRNA expressions of the molecules were
shown by RT-PCR. STaroB carrying pcDNA3 constructs were
injected to mice intravenously. Plasmids carrying insert DNAs
were found statistically more stable than vector only control in
vivo; however, in vivo stability of all was below 1% and mRNA
expressions in liver and spleen could not be detected by RT-
PCR. Our preliminary results showed that STaroB carrying
pTARGET or pcDNA3 may not be suitable to investigate the
effects of a therapeutic gene in a disease model.
OP-28
Kanamicin-induced adaptive mutation in E. coli
cells harbouring plasmids with direct repeats
S. C. Ribeiro, P. H. Oliveira, D. M. F. Prazeres and
G. A. Monteiro
Departamento de Engenharia Quı
´
mica e Biolo
´
gica, Instituto
Superior Te
´
cnico, Lisbon, Portugal.
E-mail:
This study describes a kanamycin stress-induced adaptive
mutation in Escherichia coli cells harbouring either a candidate
plasmid DNA vaccine (pGPV-PV) or its mammalian expression
vector backbone (pCI-neo) [1]. The mutation, which was shown

to occur due to the presence of a 28 bp direct repeat flanking 1.6
(pCI-neo) and 3.2 kb (pGPV-PV) intervening sequences, was
responsible for the acquisition of a kanamycin resistance pheno-
type in different E. coli strains (DH5a, Top 10F’, HB101 and
JM109). This could be explained as follows: as a result of slip-
page events, the Neo
r
gene falls under the control of a promoter-
like sequence located at the origin of replication, thus enabling
cells to strive in kanamycin. The Poisson distribution of mutants
determined by Luria-Delbru
¨
ck fluctuations tests and reconstruc-
tion experiments surprisingly, but unequivocally, indicates that
this plasmid slippage event behaves adaptively. This study high-
lights the need to carefully design plasmid vectors avoiding, for
example, repetitive sequences that are genetically unstable and
may give rise to unforeseen problems during plasmid DNA pro-
duction and subsequent utilization.
Reference:
1. Bahloul et al.Vaccine 1998; 16: 417–425.
Therapeutic Enzymes
OP-29
Engineering of nucleoside kinases to improve
the activation of nucleoside analog prodrugs
M. Konrad
1
, S. Ort
1
, C. Monnerjahn

1
and A. Lavie
2
1
Max-Planck-Institute for Biophysical Chemistry, Goettingen,
Germany,
2
Department of Biochemistry and Molecular Genetics,
University of Illinois, Chicago, IL, USA. E-mail: mkonrad@
gwdg.de
The objective of our study is to improve therapeutic enzyme-pro-
drug systems. Compounds, such as AZT for the treatment of HIV
infections, ACV and GCV used against Herpes virus, or the antican-
cer compounds AraC and gemcitabine, enter cells only in the un-
phosphorylated form (prodrug) and need to be transformed by
different kinases to their pharmacologically active triphosphate
state that interferes with DNA replication. Based on crystal struc-
ture analyses of various enzyme–nucleotide complexes we first
designed mutants of the human TMP kinase (hTMPK) that phos-
phorylate AZTMP up to 200-fold faster than wild type. Expression
of this enzyme in human cells leads to 10-fold higher intracellular
concentrations of AZTTP and to enhanced HIV inhibition. Sec-
ondly, the prodrugs ACV and GCV are not phosphorylated by
human kinases, but are converted to their monophosphates by
HSV1-TK, which has been used in enzyme/prodrug-dependent can-
cer suicide gene therapy trials. With the aim of improving this thera-
peutic system we generated enzyme variants, which show selective
and efficient phosphorylation of GCV. Thirdly, an engineered
human dCK variant catalyzes more efficiently the activation of the
prodrugs AraC and gemcitabine. Thus, the concept of a gene (or

enzyme) therapeutic treatment involving expression (or direct intra-
cellular protein transduction) of a catalytically improved human
enzyme may pave the way to the development of novel strategies in
nucleoside prodrug-dependent cancer chemotherapy.
OP-30
Effect of N2A connectin/titin on disassembly
of p94/capn3 caused by autolysis in IS2
Y. Ono
1
, F. Torii
2
, K. Ojima
3
, N. Doi
3
, K. Yoshioka
2
, D. Labeit
4
,
S. Labeit
4
, K. Suzuki
5
, K. Abe
2
, T. Maeda
6
and H. Sorimachi
1

1
Department of Enzymatic Regulation for Cell Functions, The
Tokyo Metropolitan Institute of Medical Science (Rinshoken),
Tokyo, Japan,
2
Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Tokyo, Japan,
3
CREST, Japan Science
and Technology (JST), Saitama, Japan,
4
University of Mannheim,
Mannheim, Germany,
5
New Frontiers Research Laboratories, Toray
Industries Inc., Kanagawa, Japan,
6
Institute of Molecular and
Cellular Biosciences, The University of Tokyo, Tokyo, Japan.
E-mail:
p94/calpain3 is the skeletal muscle-specific member of calpains,
Ca
2+
-regulated cytosolic cysteine protease family. Defective p94
protease activity originated from gene mutations causes muscular
Abstracts
50
dystrophy called calpainopathy, indicating p94s’ indispensability
for muscle survival. One of unique properties of p94 is its very
rapid and exhaustive autolytic activity, which is proposed to be

regulated by its binding protein connectin/titin. In vitro analysis
of p94 autolysis revealed that autolytic cleavage in each p94-spe-
cific insertion sequence, NS, IS1, and IS2, causes different impact
on molecular integrity of p94 as a protease; autolysis in IS2, but
not in IS1, causes disassembly of autolyzed fragments. Using
proteinase trapping system to semiquantitatively assay p94 auto-
lysis, where p94 was expressed in yeast as a hybrid protein
between DNA binding and activation domains of the yeast tran-
scriptional activator Gal4, N2A connectin was shown to suppress
p94 autolytic disassembly. Proximity of IS2 autolytic and connec-
tin-binding sites in p94 suggested that N2A connectin interferes
with IS2 autolysis. It was also shown that N2A connectin with
mdm mutation that is causative of dystrophy in mice and abol-
ishes connectin–p94 interaction was incapable of suppressing p94
autolytic disassembly. These data indicate that p94–connectin
interaction plays an important role in the control of p94 func-
tions by regulating autolytic decay of p94.
OP-31
Prevention of thermal aggregation of yeast
alcohol dehydrogenase by arginine and
a-cyclodextrin
A. Barzegar and A. A. Moosavi-Movahedi
Institute of Biochemistry and Biophysics, University of Tehran,
Tehran, Iran. E-mail:
Yeast alcohol dehydrogenase (YADH) is a tetrameric enzyme
and is a widely studied as a metaloenzyme for its well-known
biotechnological importance. Arginine (Arg) and a-cyclodextrin
(a-CD) are the universal reagents that are effective in assisting re-
folding of recombinant proteins from inclusion bodies. The
results showed that YADH (0.5 mg/ml) initiate to aggregation

after 15 min at 45 °C and 5 min at 50 °C in sodium phosphate
buffer (pH 7.5). In the presence of Arg (100 mM) aggregation
phenomenon completely suppressed at 50 °C until 2 h and a-CD
suppressed aggregation until 40 min (aggregation initiates after
40 min) in the same condition. The fluorescence and UV–Vis
data show that enzyme structure does not change in the presence
of these additives. The residual activity of YADH at 50 °C after
50 min was very low and negligible (£5%) and in the presence of
100 mM Arg its activity completely inhibit whereas maintained
at G30% in the presence of 100 mM a-CD. We propose Arg has
a major factor for diminishing the electrostatic forces and a-CD
has a role for controlling of the hydrophobicity due to decreasing
the aggregation of YADH.
RNA Interference
OP-32
RNA interference shows critical involvement
for NF-lkappaB p65 in the production of Wnt-1
protein
S. Tsai
1
, C. Cheng
1
, C. Sun
2
, C. Tzeng
3
and A. H. Wang
4
1
Liver Research Unit, Department of Medical Research, Chi Mei

Medical Center, Tainan, Taiwan,
2
Division of Hepatogastroentero-
logy, Department of Internal Medicine, Chi Mei Medical Center,
Tainan, Taiwan,
3
Department of Pathology, Chi Mei Medical
Center, Tainan, Taiwan,
4
Core Facilities for Proteomics Research
and Institute of Biological Chemistry, Academia Sinica, Taipei,
Taiwan. E-mail:
The link of proto-oncogenic protein Wnt-1 with NF-lkappaB
activity has been reported in PC12 cells, a rat pheochromocytom-
a cell line of neural crest lineage, showing that the Wnt-1-medi-
ated survival of these cells is dependent on NF-lkappaB
activation, and that stable expression of Wnt-1 increases NF-
lkappaB activity. In Wnt-1-mediated breast tumorigenesis, Wnt-1
is reported to be a fundamental signaling event in metastatic pro-
gression of human cancers. We have shown that enhanced NF-
lkappaB-associated Wnt-1 protein expression is detected in the
majority of hepatoma samples from both hepatitis B and C
patients. Insight into how NF-lkappaB regulates Wnt-1 protein
production may help design highly effective therapeutic agents in
treating Wnt-1-producing cancers and prevent their metastatic
progression. RNA interference (RNAi) was used in this study to
assess the role of p50 and p65 of NF-lkappaB in the regulation
of Wnt-1 protein production by Het-1A cells, a human esopha-
geal cancer cell line with Wnt-1 protein production. Cultured
Het-1A cells were transfected with sequence-specific double-stran-

ded small interfering RNAs (siRNAs) of P50, P65, and Wnt-1
respectively. All these siRNAs did not inhibit NF-lkappaB acti-
vation in Het-1A cells. The production of Wnt-1 protein was
totally abolished in cells transfected with P65 siRNAs and parti-
ally reduced in cells transfected with P50 siRNAs, indicating a
pivotal requirement for P65 in Het-1A production of Wnt-1
protein.
OP-33
Molecular mechanism of paclitaxel-induced
apoptosis in ER+/– breast cancer cell lines:
MCF-7 and MDA-MB-231
E. D. Arisan, O. Kutuk, A. Verim and H. Basaga
Biological Sciences and Bioengineering Program, Faculty of
Science and Engineering, Sabanci University, Istanbul, Turkey.
E-mail:
Taxanes such as paclitaxel (Pac) and docetaxel are cytotoxic
agents, which act by promoting deformation of stable microtu-
bules, inhibiting the normal dynamic reorganization of microtu-
bule networks required for mitosis. However, the development
intrinsic/acquired of resistance against Pac is a major obstacle to
successful cancer chemotherapy. Bcl2 is believed to have role in
drug resistance since Bcl2 overexpression is very frequent especi-
ally in ER+ breast cancer tumors through ER–responsive ele-
ment in its promoter. Bcl2 also causes prevention of Pac-induced
Abstracts
51
expression of apoptosis-related proteins. It was our aim to study
the role of Bcl2 in induced resistance to apoptosis in breast can-
cer cell lines treated with taxanes. MCF-7 (ER+) and MDA-
MB-231 (ER–) breast cancer cell lines were treated with 20 and

50 nM Pac. Cytotoxicity was determined with MTT assay after
cells were treated with the drug for 24–72 h. 20 nM Pac treat-
ment for 24 h inhibited growth of MCF-7 by 30% and MDA-
MB-231 by 44%. Cytotoxic effect was dose-dependent as 50 nM
Pac caused growth inhibition by 52% and 64% for 24 h in both
cell line. DNA fragmentation experiments showed that 20 and
50 nM Pac induced apoptosis. To understand the role of Bcl2 in
apoptotic response Bcl2 mRNA levels was checked by qPCR.
Pac at both concentrations decreased Bcl2 expression by 1.8- and
2.1-fold in ER+ and 1.2- and 1.7-fold in ER– cells compared to
control.To further clarify the role of Bcl2 we have prepared Bcl2
siRNA cells and the experiments are in progress.
Oxidative Stress
OP-34
ANKRD1 gene shows opposite atrial versus
ventricular expression kinetics at heart failure
M. Torrado
1
, B. Nespereira
1
, Y. Bouzamayor
1
, A. Centeno
2
,
E. Lo
´
pez
2
and A. Mikhailov

1
1
Institute of Health Sciences, University of La Corun
˜
a, La Corun
˜
a,
Spain,
2
Experimental Surgery Unit, University Hospital ‘Juan
Canalejo’, La Corun
˜
a, Spain. E-mail:
Background: It has been suggested that the cardiac ankyrin
repeat domain 1 (ANKRD1) gene is differentially regulated in
cardiac chambers both during development and at heart failure.
Aim: To determine a ANKRD1 gene expression fingerprint
throughout the heart, accounting for region- and disease-specific
aspects. Methods: Heart failure in neonatal piglets was produced
by intravenous administration of cardio-toxic antibiotic, Doxoru-
bicin (2 mg/kg). ANKRD1 mRNA and protein levels were deter-
mined in both atrial (A) and ventricular (V) chambers of control
and failing hearts, using quantitative PCR, Northern blot hybrid-
ization, and Western blot. Results: In the normal heart, AN-
KRD1 showed a fairly left–right (L-R) asymmetric distribution
with a twofold higher protein level in the LA when compared to
the RA, and an eightfold ANKRD1 protein enrichment in the
LV over the RV. In failing hearts, ANKRD1 transcript and pro-
tein contents were significantly augmented in each ventricle (two-
to threefold change in mRNA/protein levels), but reduced in each

atrium (twofold change in mRNA/protein levels). Owing to these
opposite expression kinetics, the normal atrio-ventricular pattern
of ANKRD1 distribution was abolished in failing hearts. Conclu-
sions: (a) ANKRD1 expression is distinctly regulated in cardiac
chambers in the postnatal heart; (b) the inverse gene expression
response in atria when compared to ventricles at advanced heart
failure may be considered a molecular sigh of severity of cardiac
disease.
OP-35
Peroxynitrite induces red blood cell membrane
reorganization
M. N. Starodubtseva
1
, T. G. Kuznetsova
1
, J. C. Ellory
2
,
T. A. Kuznetsova
3
and S. O. Abetkovskaya
3
1
Gomel State Medical University, Gomel, Belarus,
2
Department of
Physiology, Anatomy and Genetics, University of Oxford, Oxford,
UK,
3
A. V. Lykov Heat and Mass Transfer Institute of NASB,

Minsk, Belarus. E-mail:
Peroxynitrite is formed in many pathological human processes.
This study aims at studying peroxynitrite-induced rearrangements
in red blood cells (RBC) by using atomic force microscopy
(AFM; both taping and contact mode) in air. Peroxynitrite
(2.5 mM) increases RBC membrane rigidity. Young’s modulus of
RBC membrane fixed by 1% glutaraldehyde increases from
18.7 ± 7.5 P (n = 8) for control cells to 29.2 ± 2.6 P (n = 10,
P = 0.001) for peroxynitrite-treated RBCs. Peroxynitrite treat-
ment of RBCs leads to formation of macroareas with different
mechanical properties and composition in the RBC membrane.
The peak of the spatial distribution of torsion angle for peroxyni-
trite-treated RBC membranes is shifted and broadened in com-
parison with control RBC membranes. Peroxynitrite at a lower
concentration (300 lM) also causes the lipid macrodomain for-
mation. The spatial distribution density of AFM cantilever oscil-
lation phase change is bimodal with the modes at 49.30 ± 7.27°
and 333.79 ± 4.76° (n = 28). The membrane of control samples
is characterized by a unimodal distribution with mode at
46.90 ± 1.78° (P = 0.001, n = 20). The findings are evidence of
increasing heterogeneity of the mechanical properties of RBC
membrane under peroxynitrite action that seems to be one from
the most likely mechanisms of peroxynitrite-induced enhancement
of ion permeability of the RBC membrane. Acknowledgment:
We thank The Physiological Society for financial support.
OP-36
Oxidative stress fails to induce protein
misfolding in replicating cells
G. Kiss, P. Csermely and C. So
¨

ti
Department of Medical Chemistry, Semmelweis University,
Budapest, Hungary. E-mail:
Molecular chaperones or stress proteins guard the cellular con-
formational homeostasis of proteins, protecting against a num-
ber of stresses. The strongest inducer of stress proteins is the
accumulation of misfolded proteins. Proper function of chaper-
ones as well as a robust mounting of the stress response is
required for longevity. Indeed, a hallmark of ageing is the
increase of aggregated and oxidized proteins. However, little is
known about the causal relationship between protein misfold-
ing, oxidative stress and chaperone induction. In our present
studies using different cell lines we examined the relationship
between protein misfolding, aggregation and chaperone induc-
tion. As expected, heat shock, the archetype of stress as well
as geldanamycin induced the uniform elevation of the major
chaperones, Hsp70 and Hsp90. In contrast, the oxidative stres-
sor hydrogen peroxide induced only Hsp90. Investigating the
hydrophobic surface exposure to detect protein misfolding in
live cells, the fluorescence of ANS, an apolar probe was
applied. In contrast to heat shock, hydrogen peroxide was
unable to induce the ANS signal of purified model proteins,
cytosolic extracts and live cells. Moreover, did not result in
significant protein aggregation. These results suggest that short-
term oxidative stress does not induce bulk protein denaturation
and stress response, raising the question what is the depend-
ence of protein oxidation and misfolding during ageing and in
degenerative diseases.
Abstracts
52

DNA Damage Processing
OP-37
Chromosome remodel ling and DNA replication
in B. subtilis
P. Soultanas
Centre for Biomolecular Sciences, University of Nottingham,
Nottingham, UK. E-mail:
In Bacillus subtilis three essential proteins known as DnaI, DnaD
and DnaB have been implicated in a ‘primosomal cascade’ that
initiates DNA replication. We have recently discovered that both
proteins also exhibit global DNA remodelling activities. Our ini-
tial studies on DnaD and DnaB have revealed oligomeric pro-
teins that remodel DNA [1, 2]. We have suggested that the DNA
remodelling activities of DnaD and DnaB are similar to the
Escherichia coli H-NS and HU DNA remodelling activities but
whether their biological significance is analogous to these pro-
teins still remains an open question. Interestingly, there are no
H-NS and HU homologues in B. subtilis and other Gram-posit-
ive bacteria. Recently, we have proposed a model where DnaD
and DnaB act as global or local (at the vicinity of oriC) DNA-
remodelling proteins to render the chromosomal DNA replica-
tion proficient. This is compatible with previous suggestions that
DnaD recruitment to the bacterial membrane by DnaB is the
main regulatory event in the initiation of B. subtilis DNA replica-
tion. I will present our latest data on the function/structure rela-
tionships of DnaI, DnaD and DnaB proteins in relation to
initiation of DNA replication and chromosomal remodelling.
References
1. Turner IJ, Scott DJ, Allen S, Roberts CJ, Soultanas P. FEBS
Lett 2004; 577: 460–464.

2. Zhang W, Carneiro MJVM, Turner IJ, Allen S, Roberts CJ,
Soultanas P. J Mol Biol 2005; 351: 66–75.
OP-38
Coordination of DNA base excision repair
studied by photoreactive DNA probes and
functional assay
O. I. Lavrik
Institute of Chemical Biology and Fundamental Medicine,
Novosibirsk, Russia. E-mail:
Photoaffinity-labeling technique has been developed to study
coordination of base excision repair (BER) proteins. Photoreac-
tive DNA intermediates of BER pathways were created in sys-
tems reconstituted of purified proteins and in cellular/nuclear
extracts to identify proteins interacting with damaged DNA. The
main target proteins interacting with branch point BER interme-
diate were identified as Poly(ADP-ribose)polymerase1 (PARP1),
flap endonuclease1 (FEN1), DNA polymerase beta (Pol beta)
and apurinic/apyrimidinic endonuclease1 (APE1). The results
indicate that APE1 and PARP1 interact preferentially with
nicked BER intermediate carrying 3’-photoreactive dNMP resi-
due and the 5’-sugarphosphate moiety, whereas intermediate with
5’-phosphate is less favourable interaction partner. Thus, PARP1
and APE1 can discriminate intermediates of SP and LP BER
pathways to regulate BER. DNA repair synthesis catalysed by
Pol beta is modulated by the interplay between Pol beta, APE1,
PARP1 and XRCC1. APE1 can perform stimulation of strand-
displacement synthesis catalysed with Pol beta and proofreading
function while PARP1 inhibits these reactions. PARP1 inhibits
activity of FEN1 preventing formation of intact DNA structure.
The inhibition by PARP1 of these activities was not completely

reversed by poly(ADP-ribosyl)ation. Our study further estab-
lished that photoaffinity labelling combined to functional assay is
a powerful tool to explore proteomic ensembles of DNA repair.
Acknowledgement: RFBR grant 05-04-48319.
OP-39
A new family of nucleases: the uracil-DNA-
specific nuclease of pupating insects
A. Bekesi
1
, F. Felfoldi
2
, M. Pukancsik
1
, V. Muha
1
, I. Zagyva
1
,
I. Leveles
1
, E. Hunyadi-Gulyas
3
, K. Medzihradszky
3
, A. Erdei
4
and B. Vertessy
1
1
Institute of Enzymology, Budapest, Hungary,

2
MolCat Bt, Buda-
pest, Hungary,
3
Proteomics Laboratory, Biol.Res.Ctr., Szeged,
Hungary,
4
Immunology Department, Eotvos University, Budapest,
Hungary. E-mail:
Uracil in DNA may arise by cytosine deamination or thymine
replacement and is removed by members of the uracil-DNA gly-
cosylase (UDG) superfamily. The surprising lack of the major
UDG-superfamily member UNG from the fruitfly genome may
allow the fly to tolerate thymine-replacing incorporation of uracil
in its DNA. Such incorporation is usually prevented by the
enzyme dUTPase that removes dUTP from the DNA polymeriza-
tion pathway. However, fruitfly larval tissues do not contain any
detectable dUTPase (the enzyme being confined to the imaginal
disks, progenitors of adult fly organs; Be
´
ke
´
si et al. J Biol Chem
2004; 279: 22362). Here, we asked if the putative presence of ura-
cil-DNA in larval tissues, potentially accumulated due to lack of
both UNG and dUTPase, might trigger any physiological
response. Such response would require macromolecular (e.g. pro-
tein) factors specifically recognizing uracil-DNA; we therefore set
out to identify such proteins from Drosophila extracts. We show
that the most intensive hit of uracil-DNA pull-down experiments

is a uracil-DNA specific nuclease (UDE) with no detectable
homology to other proteins except a group of sequences of other
pupating insects. UDE is upregulated right before pupation by
an ecdysone-controlled mechanism potentially involving Write
bFTZ-F1. Data imply the existence of a novel DNA nuclease
family specific for uracil-DNA with a possible role in cell death
during metamorphosis.
OP-40
Zooming on metals in DNA degradation
M. Fuxreiter, L. Mones and I. Simon
Institute of Enzymology, Budapest, Hungary. E-mail: monika@
enzim.hu
Sequence-specific degradation plays pivotal role in defense
against foreign DNA. Most repair enzymes requisite bivalent
metal ions for hydrolyzing the DNA backbone, the number of
which is a long-standing puzzle in molecular biology. Despite the
wealth of structural data, the role, and contribution of the metal
ions to the different steps of the catalytic choreography remains
elusive. Recently, the mismatch repair initiating MutH enzyme
revealed similarity to PDD/ExK endonucleases, especially to the
type II restriction enzyme BamHI concerning the metal ion
arrangement. To probe whether these enzymes could employ the
same mechanism for their action that might be even further gen-
eralized for other repair enzymes we computed the activation
barrier of the potential pathways in BamHI. We found that the
general base mechanism is less likely than recruiting the attacking
OH- nucleophile from the bulk phase. Based on their contri-
bution to the total catalytic effect, Metal ion A ligating the
Abstracts
53

nucleophile was concluded to be of primary importance for the
reaction, while the removal of metal ion B was less dramatic.
Thus, we propose a unified catalytic scheme involving only one
obligatory metal ion at the active site that serves to stabilize the
attacking nucleophile, which is coming from the bulk phase and
not assisted by a general base or substrate. A second, variable
group is required to facilitate the nucleophilic attack via interac-
tions with the pentavalent transition state.
DNA Repair in Health, Disease and Aging
OP-41
Base excision repair pathway at telomeric
DNA
M. Muftuoglu, P. Opresko and V. Bohr
Laboratory of Molecular Gerontology, National Institute on Aging,
National Institutes of Health, Baltimore, MD, USA.
Telomeres consist of protein–DNA complexes that protect the
ends of linear chromosomes from inappropriate chromosome
fusions by DNA repair pathways. Telomere-repeat binding fac-
tors 1 and 2 (TRF1 and TRF2) bind specifically to duplex telo-
meric DNA and regulate telomere length. When bound to
telomeres, TRF2 allows the cell to recognize the telomeres as
chromosome ends rather than double strand breaks in DNA.
Defects in TRF2 induce telomere dysfunction that causes telo-
mere end fusions, replicative senescence, apoptosis, and genomic
instability. Oxidative damage causes loss of telomeric DNA, and
we found previously that oxidative DNA base damage disrupts
TRF1 and TRF2 binding to telomeric DNA, emphasizing the
importance of DNA repair at telomeres. We are investigating the
function of base excision repair (BER) in preserving telomeres,
and specifically roles for TRF2 in cooperation with BER proteins

for repair pathways in preventing genomic instability. We dem-
onstrated that TRF2 physically interacts with DNA polymerase
beta (pol beta), a critical enzyme in BER, and stimulates pol beta
DNA synthesis on both telomeric and nontelomeric substrates,
and in reconstituted BER reactions. Our data suggest that TRF2
can participate in BER together with pol beta during repair of
oxidative lesions at the telomeres, and/or in genomic regions out-
side of the telomeres.
OP-42
Sequence variations of fanca gene in Turkish
patients with fanconi anemia
G. Balta, F. Gumruk, H. Okur, A. Gurgey and C. Altay
Department of Ped
ı
atr
ı
cs, Section of Pediatric Hematology,
Faculty of Medicine, Hacettepe University, Ankara, Turkey.
E-mail:
Fanconi anemia (FA) is a DNA repair deficiency disorder charac-
terized by bone marrow failure, developmental defects, cancer pre-
disposition, and sensitivity to DNA cross-linking damage. To date,
12 different complementation groups have been described. FA pro-
teins function in a DNA damage-response network including
BRCA1, BRCA2, and RAD51. The molecular defects in FANCA
gene account for 65–70% of all cases. In this study, a total of 26
patients from a panel of 50 unrelated Turkish families were
assigned to group A by using linkage analysis. Genomic DNA or
cDNA of these patients were screened for the sequence variations
in the complete coding (43 exons) and flanking sequences of the

FANCA gene by SSCP/HD analysis. Ironically while sequencing
of the variant bands led to the identification of many homozygous,
non-pathogenic mutations including missense/silent mutations,
base substitutions, and deletions/insertions in introns, only a few
pathologic mutations could be detected. Despite much effort, the
molecular defects in the most of Turkish group A patients go
undetected. Given that all of the exons could be amplified by PCR
in almost all of the patients, it is unlikely that large deletions could
be cause of the disease either. This study proved that mutation
analysis of FANCA gene is quite difficult and inefficient, which
makes pre-natal diagnosis and detection of carriers in Turkish
population almost impossible. Acknowledgment: This study was
supported by Hacettepe University Research Fund (02G116).
Diabetes, Obesity and Metabolic Syndrome
OP-43
The role of TGFb1 in glycosaminoglycan
metabolism in diabetes mellitus
N. Yu. Yevdokimova
Molecular Immunology, Institute of Biochemistry, Kyiv, Ukraine.
E-mail:
The dysregulation of glycosaminoglycan (GAG) metabolism is a
typical feature of renal diabetic complications. TSP-1-dependent
TGFb1 activation is known to mediate numerous ECM disor-
ders in diabetic kidney. However, its role in the metabolism of
GAGs is studied insufficiently. We observed that high glucose
enriched the mesangial ECM with hyaluronic acid (HA) of
high-molecular weight (>2000 kDa). We detected the upregula-
tion of hyaluronan synthase 2 (HAS2) mRNA without altera-
tions of HAS3 expression. High glucose also stimulated the
production and TSP-1-dependent activation of TGFb1. The

blockage of TGFb1 action by neutralizing antibodies or by
stopping TGFb1 activation abolished the effect of high glucose
on HAS2 mRNA expression and normalized HA synthesis.
Exogenous TGFb1 revealed the same effect on HAS2 expres-
sion, as high glucose did. We consider that TSP-1/TGFb1 axis
is involved in high glucose influence on HA metabolism. In
contrast, high glucose decreased the level of heparan sulphate
(HS) in mesangial ECM and led to the undersulphation of HS
molecules. The blockage of TGFb1 intensified the pathological
changes of HS, while exogenous TGFb1 increased HS synthesis.
Therefore, the pathological alterations of HS under the action
of high glucose take place despite the upregulation and activa-
tion of TGFb1. Hence, our data underlined the ambiguity of
TGFb1 involvement in the abnormalities of GAGs metabolism
in diabetes mellitus.
Abstracts
54
OP-44
The effect of –675 4G/5G polymorphism on
PAI-1 gene expression and adipocyte
differentiation
D. Ozel
1
, M. Berberoglu
2
, H. Aktas
3
and N. Akar
1
1

Pediatric Molecular Genetics Department, Ankara University
School of Medicine, Ankara, Turkey,
2
Pediatric Endocrinology
Department, Ankara University School of Medicine, Ankara,
Turkey,
3
The Laboratory for Translational Research, Harvard
University School of Medicine, Boston, MA, USA.
E-mail:
Plasminogen Activator Inhibitor I (PAI-1), a member of the ser-
ine proteinase inhibitor family is not only inhibits fibrinolysis but
also has complex interactions with cellular matrix, further inhib-
its proteolysis and important in cardiovascular diseases. Obesity
is a metabolic disorder, associated with increased PAI-1 concen-
tration in the circulation. This increase is also related to insulin
resistance, dyslipidemia and cardiovascular diseases. In our study
we investigated the frequency of –675 4G/5G PAI-1 polymorph-
ism located at –675 in promoter of the gene in Turkish pediatric
obese patients and the effect of this polymorphism on PAI-1 gene
expression and on the adipocyte differentiation. The frequency of
PAI-1 4G/5G genotype was determined as previously described.
We constructed a dual-glo luciferase effect reporter assay to
investigate of this deletion on the PAI-1 promoter activity in pre-
adipocytes, adipocytes, and HUVEC cells. We also investigated
the effects of troglitazone and ciglitazone treatments on PAI-1
promoter activity. 4G/4G genotype was significantly higher than
the 5G/ 5G genotype in Turkish pediatric obese patients. In the
present study, we demonstrated that 4G/4G genotype increases
the PAI-1 promoter activity. PAI-1 increases the differentiation

of pre-adipocytes to adipocytes. Troglitazone and the ciglitazone
had similar effects on PAI-1 expression and decrease the level of
PAI-1 in HUVEC, undifferentiated and differentiated 3T3 L1
cells.
OP-45
Effect of modifications of Grb14 protein
expression on insulin signaling and sensitivity
N. Carre
´
, J. Girard and A. Burnol
Institut Cochin, INSERMU567 CNRS UMR8104 Endocrinology,
Metabolism, Cancer Department, Paris, France,
E-mail:
Obesity and type 2 diabetes are rapidly expanding in most coun-
tries and become a worldwide health issue. These physiopatho-
logical states are characterized by peripheral insulin resistance,
which leads to an increase in glucose and fatty acids plasmatic
concentrations. Grb14, a member of the Grb7 adaptor protein
family, directly binds to insulin receptors and inhibits their tyro-
sine kinase activity. We demonstrated that Grb14 was specifically
expressed in insulin target tissues, and that its level of expression
was inversely correlated with insulin sensitivity in insulin-resistant
rodent models and type 2 diabetics. To investigate the physiologi-
cal role of Grb14, we modified its level of expression by overex-
pressing the protein with a recombinant adenovirus or by
decreasing its expression using a siRNA approach. Grb14 overex-
pression in primary cultures of mouse hepatocytes only induced a
slight decrease in ERK1/2 activation by insulin. On the other
hand, a 70% decrease in Grb14 expression following Grb14
siRNA transfection was accompanied by a significant increase in

insulin-induced Akt and ERK1/2 phosphorylation. These results
show that modifications in Grb14 expression level can rapidly
modulate insulin signalling and action, suggesting that Grb14 can
be considered as a new target for the development of insulin-sen-
sitizing agents.
Lipid-related Disorders and Atherosclerosis
OP-46
Associations of TSH levels with bloo d lip ids,
metabolic syndrome, coronary risk factors,
coronary heart disease in Turkish adult
G. Hergenc
1
, A. Onat
2
, S. Albayrak
3
, A. Karabulut
4
,
S. Turkmen
5
, I. Sari
4
and G. Can
6
1
Yildiz Tech University, Department of Biology, Istanbul,
Turkey,
2
Turkish Society Cardiology, Istanbul, Turkey,

3
Izzet
Baysal Univ Duzce Medical Fac, Department of Cardiology,
Duzce, Turkey,
4
Siyami Ersek Cardiovascular Surgery Center,
Cardiology Department, Istanbul, Turkey,
5
Gaziantep Med Fac,
Department of Cardiology, Gaziantep, Istanbul, Turkey,
6
Istanbul
Univ Cerrahpasa Med Fac, Cardiology Institute, Istanbul, Turkey.
E-mail:
Epidemiological data related to the role of thyroid hormones in
coronary heart disease (CHD) and metabolic syndrome (MS) in
Turkish adults are lacking. Thyroid-stimulating hormone (TSH)
was measured in the 2004 follow up of the Turkish Adult Risk
Factor Study, with the aim of investigating thyroid hormone sta-
tus as a risk factor in CHD and MS in the population sample.
A subgroup of the cohort (n = 512, mean age 52 years)
was screened for TSH. Thyroid status was classified according to
cut-offs of 4.2 and 0.3 lU/ml for TSH. No distinction was made
between overt and SC thyroid states. T and LDL-Chol was low-
est, waist circumference unexpectedly highest in the hyperthyroid
group. Women (1.3 lU/ml) had significantly higher TSH levels
than men (0.95 lU/ml). Prevalence of hypo- and hyper-thyoidism
in men and women were 1.7/7% and 4.4/5% respectively. TSH
values were not significantly different among groups diagnosed
as having or not CHD, MS and DM. Multivariate linear regres-

sion analysis revealed TChol as the sole independent covariate of
TSH levels in men and both sexes combined. To conclude, TSH
was found not to contribute to CHD, hypercholesterolaemia, MS
and DM risk in logistic regression analyses. Prospective follow
ups may give a better understanding concerning the thyroid sta-
tus and CHD risk in Turkish adults.
OP-47
The carboxy-terminal region of apoA-I is
important for the biogenesis of HDL in vivo
A. Chroni
1
, A. Duka
2
, G. Koukos
2
and V. Zannis
2
1
Institute of Biology, National Center for Scientific Research
Demokritos, Athens, Greece,
2
Boston University School of
Medicine, Boston, MA, USA. E-mail:
Apolipoprotein A-I (apoA-I) promotes ABCA1-mediated lipid
efflux that results in the initial lipidation of apoA-I. This leads to
the formation of discoidal HDL and, after cholesterol esterifica-
tion by the enzyme LCAT, of spherical a-HDL particles. Follow-
ing adenovirus-mediated gene transfer in apoA-I-deficient mice
the plasma HDL levels were greatly reduced in mice expressing
the C-terminus deletion mutants apoA-I[del(185–243)] and apoA-

I[del(220–243)] and were normal in mice expressing the WT
Abstracts
55
apoA-I, the apoA-I[del(232–243)] deletion mutant or the apoA-
I[E191A/H193A/K195A] point mutant. Electron microscopy and
two-dimensional gel electrophoresis showed that the apoA-
I[del(185–243)] and apoA-I[del(220–243)] mutants formed mainly
preb1 HDL particles and few spherical particles enriched in
apoE, while WT apoA-I, apoA-I[del(232–243)] or apoA-I[E191A/
H193A/K195A] formed spherical a-HDL particles. Consistently
with these in vivo data, previous in vitro data showed that the
apoA-I[del(185–243)] and apoA-I[del(220–243)] mutants had
diminished ABCA1-mediated cholesterol efflux, while the apoA-
I[del(232–243)] mutant had normal cholesterol efflux. The apoA-
I[E191A/H193A/K195A] mutant that is normal in HDL biogen-
esis has normal ABCA1-mediated cholesterol efflux. The findings
indicate that (a) the C-terminal region 220–231 of apoA-I is
important for formation of HDL in vivo and (b) mutations in
apoA-I that diminish its functional interactions with ABCA1
diminish the formation of HDL in vivo.
OP-48
Genetic risk factors and early ischemic stroke
S. Stankovic
1
, N. Majkic-Singh
1
, Z. Jovanovic
2
and D. Alavantic
3

1
Institute for Medical Biochemistry, University School of
Pharmacy & Clinical Center of Serbia, Belgrade, Serbia &
Montenegro,
2
Institute of Neurology , Clinical Center of Serbia,
Belgrade, Serbia & Montenegro,
3
Laboratory for Radiobiology and
Molecular Genetics, VINC
ˇ
A Institute of Nuclear Sciences,
Belgrade, Serbia & Montenegro. E-mail:
The aim of this study was to determine whether the DNA poly-
morphism in apolipoprotein (apo)B, E and angiotensin-converting
enzyme (ACE) is associated with occurrence of ischemic stroke
(IS) in young adults and to find out the relationship between IS,
lipids, apolipoproteins, blood pressure (BP) levels and those poly-
morphisms. The possible association of apoB (XbaI, EcoRI,
MspI, I/D, 4311 A/G), apoE (HhaI) and ACE (I/D) polymorph-
isms were analyzed in 65 IS survivors younger than 65 and 591
age-matched healthy subjects. The occurrence of stroke was pro-
ven by computed tomography or magnetic resonance of the brain.
The results showed that patients with IS presented statistically sig-
nificant higher frequencies of X (XbaI), I (I/D), M1 (MspI), A
(4311 A/G), E4 allele, and lower E2 allele than control subjects
(P < 0.01). These results are discussed in the sense of finding
risk/protective haplotype for IS, lighting the role of circulating
lipids, BP levels in the pathogenesis of IS subtypes in different
genotypes in this study. ApoB, triglycerides and MAP were the

best predictors of IS. ApoE34 genotype/apoE4 allele and cigarette
smoking/hypertension act synergistically, increasing an indivi-
dual’s propensity to have a cerebral ischemic event. Additional
knowledge of the role of genes in IS may improve our under-
standing of the cause of IS, provide new insights in prevention
and factors that influence the outcome of IS, as new therapeutic
targets when preventive strategies have failed.
OP-49
Plasma fibrinogen level: an independent risk
factor for long-term survival in Chinese
patients with peripheral artery disease?
L. Y. Cheuk
Department of Surgery, The University of Hong Kong,
Hong Kong. E-mail:
Fibrinogen, is suggested to play a significant role in the patho-
genesis of atherosclerosis and complications of atherothrombotic
diseases. The aim of this study was to determine if plasma fibrin-
ogen is an independent risk factor for long-term survival in
patients with peripheral artery disease (PAD). Altogether, 139
Chinese patients (88 men, 51 women) with PAD were consecu-
tively recruited for the study. Atherothrombotic risk factors and
fibrinogen levels were determined at presentation, and all patients
were followed up for mortality prospectively. The mean follow
up was 6 years. All variables were first correlated with survival
rates using Kaplan–Meier analysis and compared by means of
the log-rank test. Significant risk factors were identified, and mul-
tivariate Cox regression analysis was used to evaluate the inde-
pendent contribution of the fibrinogen level to the risk of
mortality. During follow up, 95 patients (68.3%) died. The over-
all survival rate was 77.7% at 3 years, 56.8% at 5 years, and

31.2% at 10 years (SE: 0.05, 0.06, and 0.07, respectively). All-
cause mortality rate increased with an elevated fibrinogen level.
Eighty percentage of patients with a fibrinogen level >3.4 g/l
had a survival time of <3 years (P = 0.002). This relation was
also demonstrated within patients with critical ischemia. The
plasma fibrinogen level was thus identified as an independent risk
factor for mortality in PAD patients after adjusting for con-
founding factors.
Oncogenes and Tumor Suppressors
OP-50
An approach for deciphering molec ular
patterns involved in genistein effects in
human prostate
R. Laslo
1
, H. Klocker
2
, I. Rowland
3
, R. L. Hancok
4
,
R. S. Pardini
4
and A. I. Baba
5
1
Nutrition and Toxicology, University of Agricultural Sciences and
Veterinary Medicine of Cluj, Cluj-Napoca, Romania,
2

UROLAB-
IBK, Innsbruck Medical University, Innsbruck, Austria,
3
NICHE,
CMB, University of Ulster, Coleraine, UK,
4
Cancer Research
Laboratory, CABR, University of Reno, NV, USA,
5
Comparative
Oncology Department, University of Agricultural Sciences and
Veterinary Medicine of Cluj, Cluj-Napoca, Romania.
E-mail: ;
cDNA microarrays experiments revealed that Genistein caused a
differential expression of several genes. Our further question was
if deciphering gene expression data we can predict alternatively
spliced variants and protein-encoded sequences of these genes,
which could be implicated in prostate cancer prevention by set-
ting-up preventive gene sets, and transcriptionally silent genes.
To collect accurate information about alternative splicing and
protein-encoded sequences predicted associated with genes of
interest we investigated known data in the databases using two
different computational methods/software (ENSEMBL and JIG-
SAW). Three prediction criteria were measured: accuracy on
genes, on exons and on nucleotides. The goal was to provide
consistent, reproducible predictions based on statistical principles,
even n case of conflicting evidence about gene structure. Then,
we interpreted deciphering those data to find the relationship
between genes of interest, alternative splice forms and protein-
encoded sequences function and structure. We found some inter-

esting features that are presented here. We discuss the results of
genes of interest in order to temp establishing of preventive gene
Abstracts
56
set. They concern the coverage quality, in terms of consistency
and reproducible, of the protein-encoded sequences of genes of
interest, an EST analysis, the validation of splice types, and
finally the link between gene expression analysis/alternative spli-
cing and prostate cancer prevention.
OP-51
Ornithine decarboxylase interferes with tumor
necrosis factor alpha-mediated matrix
metalloproteinase- 9 productions on
macrophage-like differentiation
Y. Liao
1
, H. Hung
1
and G. Liu
2
1
Department of Life Sciences, National Chung-Hsing University,
Taichung, Taiwan,
2
Institute of Immunology, Chung-Shan Medical
University, Taichung, Taiwan. E-mail:
Proteolytic activity of matrix metalloproteinase-9 (MMP-9) is
necessary for a variety of macrophage functions such as extrava-
sation, migration, and tissue remodeling. Ornithine decarboxylase
(ODC) is a rate-limiting enzyme of polyamine biosynthesis. Poly-

amines restrain immune response in activated macrophage. The
data reported here show the critical role of ODC in the inhibition
of MMP-9. By using 12-o-tetradecanoylphorbol-13-acetate
(TPA)-differentiated human promyelocytic HL-60 and promono-
cytic U-937 cell lines, we found that polyamines blocked the
expression, secretion, and activation of MMP-9. Furthermore,
overexpression of ODC interferes with MMP-9 enzyme activity,
tumor necrosis factor alpha (TNF-a) expression and nuclear fac-
tor-kappaB (NF-kB) activation. The secretion of MMP-9 was
induced by stimulating recombinant TNF- a (rTNF- a) in ODC
overexpressed cells. Pyrrolidinedithiocarbamate (PDTC), an
inhibitor of NF-kB, suppressed TPA-induced MMP-9 enzyme
activity. Difluoromethylornithine (DFMO), an irreversible inhib-
itor of ODC, and rTNF- a could not rescue MMP-9 secretion
after PDTC treatment. Therefore, these data indicated that ODC
inhibits TNF- a expression and then outcome the NF-kB activa-
tion and that regulatory mechanism may provide the molecular
differentiation basis for ODC’s ability to suppress MMP-9
expression and phagocytosis.
OP-52
Phosphorylation by Neu directs tyrosine
phosphatase epsilon to activate Src in
mammary tumorigenesis
A. Elson, D. Berman and S. Granot-Attas
Department of Molecular Genetics, The Weizmann Institute of
Science, Rehovot, Israel. E-mail:
Aberrant tyrosine phosphorylation is a well-established cause of
cellular transformation. Protein tyrosine phosphatases (PTPs)
help regulate this process; few PTPs support, rather than inhibit,
survival of tumor cells. We present genetic and biochemical evi-

dence that the receptor-type PTP epsilon (RPTPe) supports
transformation by linking Neu with downstream events. Cells
from mammary epithelial tumors induced in vivo by Neu in mice
genetically lacking RPTPe are less transformed and proliferate
slowly. At the molecular level RPTPe activates Src, a known col-
laborator of Neu in mammary tumorigenesis, as well as the Src-
related kinases Yes and Fyn. Accordingly, absence of RPTPe
reduces Src activity and alters Src phosphorylation in tumor cells,
RPTPe dephosphorylates and activates Src in heterologous sys-
tems, and Src binds a substrate-trapping mutant of RPTPe. The
role of Neu is central, since Neu phosphorylates RPTPe specific-
ally at Y695 at its C-terminus, thereby directing RPTPe to acti-
vate Src. Finally, the altered morphology of Neu-induced
mammary tumor cells lacking RPTPe is corrected by exogenous
Src or RPTPe. We conclude that a Neu-RPTPe-Src pathway
exists in mammary tumor cells, and that phosphorylation of
RPTPe by Neu directs the phosphatase to activate Src in a man-
ner required for complete transformation. Inhibition of RPTPe
may be useful to augment direct pharmacologic inhibition of Src
in fighting mammary tumors.
OP-53
Both C and N-terminal regions of p53 are
required for its optimal Mdm2-mediated
degradation
R. Hjerpe
1
, F. Aillet
1
, T. Hay
2

and S. Rodriguez
1
1
Proteomics Unit, CIC BioGUNE, Derio, Spain,
2
University of
Dundee, Dundee, UK. E-mal:
Multiple mechanisms have been reported to regulate the activity
of the tumor-suppressor p53. Different cellular ubiquitin ligases
(E3s), such as Mdm2, COP-1, and Pirh2, have been proposed to
recognize and modify p53. These E3s are upregulated by p53 as
well as by genotoxic stress and each has been found to directly
promote p53 ubiquitination and degradation via the ubiquitin-
proteasome pathway. To face the challenge of elucidating the
molecular mechanisms providing specificity for the recognition of
p53 and the physiologic significance of various E3s, we used a
‘transfer of signals strategy’ using both b-galactosidase and GFP
fusions. This strategy allowed us to isolate the minimal p53 N-
and C-terminal regions involved in its Mdm2-mediated ubiquity-
lation and degradation. These minimal sequences are able to con-
fer the capacity of being degraded to unrelated proteins, resulting
in the degradation of the fusion protein to the same extent as the
wild-type p53 molecule. We found that, independently of the type
or size of the fusion protein, both GFP and b-galactosidase show
Mdm2-mediated degradability after having been fused to identi-
cal p53 regions. As the chimeric proteins do not contain the
DNA-binding domain of p53, we effectively dissect regions
involved in proteolysis, avoiding those problems associated with
the transcriptional capacity of p53. This strategy is ready to be
used to identify p53 regions involved on its degradation mediated

by other cellular and viral E3s.
OP-54
In vivo dynamics of MN1TEL suggest that its
immobility may be crucial in leukemi as caused
by this fusion protein
M. Ter Haar, M. Meester-Smoor, M. Janssen, A. Houtsmuller
and E. Zwarthoff
Department of Pathology, Erasmus MC, Rotterdam,
the Netherlands. E-mail:
The MN1TEL fusion gene is created by a leukemia-associated
translocation and combines the MN1 gene from chromosome 22
with the TEL (ETV6) gene from chromosome 12. TEL is a mem-
ber of the ETS family of transcription factors and contains a
DNA-binding domain (DBD). TEL represses transcription from
ETS-responsive elements. MN1 contains transcription-activating
domains. MN1 can stimulate transcription from several promot-
ers by interaction with coactivators such as p300 and members of
the p160 family and can enhance expression driven by the reti-
noic acid receptor. In the MN1TEL protein the transactivating
domains from MN1 are combined with the TEL DBD.
MN1TEL stimulates transcription but is unable to synergize with
the retinoic acid receptor and is not stimulated by p300 or p160
coactivators. To get an insight in the physical properties of the
proteins, we decided to examine their behaviour in vivo. For this
Abstracts
57
we generated inducible cell lines expressing GFP-tagged proteins.
Protein mobility was examined using the fluorescence recovery
after photobleaching (FRAP) technique. It was found that MN1
is completely mobile and that a small, but significant fraction of

TEL was immobile. We presume that this is due to DNA bind-
ing. Interestingly, a much larger fraction of MN1TEL was immo-
bile. Our current hypothesis is that MN1TEL may promote
leukemogenesis by occupying TEL responsive elements, resulting
in transcription from genes that are normally repressed by TEL.
OP-55
Ewing sarcoma oncoprotein EWS-FLI1 activity
is enhanced by RNA helicase A
J. A. Toretsky, H. V. Erkizan, O. D. Abaan and A. U
¨
ren
Georgetown University. E-mail:
RNA helicase A (RHA), a member of the DEXH box helicase
family of proteins, is an integral component of protein complexes
that regulate transcription and splicing. EWS-FLI1 oncoprotein
is expressed as a result of a chromosomal translocation that
occurs in patients with Ewing’s Sarcoma Family of Tumors
(ESFT). Although more than 95% of the tumors carry EWS-
FLI1, therapeutic applications using this target have not been
developed. Using phage display library screening, we identified
an EWS-FLI1-binding peptide containing homology to RHA
and characterized human RHA protein as a potential EWS-FLI1
interacting protein. We observed endogenous RHA and EWS-
FLI1 in the same protein complex in ESFT cell lines. GST pull-
down and ELISA assays with recombinant proteins showed that
EWS-FLI1 directly bound to RHA. Chromatin immunoprecipita-
tion experiments demonstrated both proteins bound to EWS-
FLI1 target gene promoters. RHA stimulated the transcriptional
activity of EWS-FLI1-regulated promoters, including Id2, in
ESFT cells. RHA expression in mouse embryonic fibroblasts cells

stably transfected with EWS-FLI1 enhanced the anchorage-inde-
pendent phenotype of EWS-FLI1 alone. Reduction of RHA pro-
tein levels by siRNA in ESFT cell lines decreased their growth
rate. Our results provide strong evidence for EWS-FLI1 and
RHA interaction and its oncogenic consequences. This finding
may lead to development of better therapeutic agents that may
target EWS-FLI1 and RHA interaction.
OP-56
Role of p38 MAP kinases in oncogene-induc ed
malignant transformation
A. Nebreda, I. Dolado, A. Cuadrado, V. Lafarga and A. Swat
CNIO, Melchor Ferna
´
ndez Almagro, Madrid, Spain.
E-mail:
We are investigating the role of p38 MAPKs in the processes of
cell proliferation, apoptosis and malignant transformation
induced by oncogenes. Oncogenic H-RasV12 induces a more dra-
matic transformed phenotype in p38a-deficient mouse fibroblasts
than in their wild type counterparts. The inhibitory effect of p38
MAPKs on H-RasV12-driven transformation was confirmed in
NIH3T3 fibroblasts overexpressing the p38 MAPK activator
MKK6. Interestingly, p38 MAPKs do not inhibit transformation
induced by all oncogenes but seem to rather specifically modulate
signalling pathways activated by oncogenic Ras. We have also
observed that, in fibroblasts expressing H-Ras V12, there is an
inverse correlation between p38a activity levels and the ability of
the cells to lose contact inhibition and grow in multiple layers.
Consistent with these results, p38a–/– fibroblasts grow to a
higher saturation density than wild-type fibroblasts. Moreover,

p38a-deficient cells proliferate better than wild-type cells in the
presence of low serum concentrations and are also more resistant
to apoptosis induced by oncogenes. Finally, we have found that
p38a–/– cells have impaired motility, which correlates with a less
robust actin cytoskeleton. These results indicate that p38 MAPKs
may modulate cancer cell-associated traits at multiple levels. We
have performed proteomic and microarray experiments to iden-
tify new proteins that potentially function downstream of p38
MAPKs in tumorigenesis regulation.
OP-57
Abnormal expression of gamma catenin
correlates with poor progno sis and liver
metastases of breast carcinoma
N. Erin
1
, J. Weisz
2
, D. Shearer
2
and G. Clawson
3
1
Internal Medicine and Human Gene Therapy Unit, Akdeniz
University School of Medicine, Antalya, Turkey.
E-mail: ,
2
Obstetrics and Gynecology, Penn
State University, Hershey, PA, USA,
3
Gittlen Cancer Foundation,

Penn State University, Hershey, PA, USA
We previously developed a cell line from a heart metastases of
4T1 murine breast carcinoma. This cell line named as 4THMpc
behaved more aggressively than the parental cell line (4T1) and
formed multiple macroscopic liver metastases. Expressions of
over 12 000 genes were determined in liver metastases, 4T1 and
4THMpc primary tumors. Comparison of gene array results
demonstrated that expression of adherence junctions proteins
were markedly decreased in liver metastases compared to primary
tumors formed by 4T1 and 4THMpc. Among these proteins
gamma catenin is examined at protein level. Immunoblots from
tumor lysates demonstrated the presence of gamma catenin in
both 4T1 and 4THMpc primary tumors. 4T1 primary tumors dis-
played more gamma catenin than 4THMpc. Immunohistochemi-
cally, patches of cells were stained in both groups. Interestingly,
cytoplasmic staining was observed in both groups but membrane
staining was detectable only in 4T1 tumor cells. Instead, nuclear
and perinuclear staining was observed in 4THMpc tumor.
Gamma catenin expression was undetectable in liver metastases.
Similarly, hepatocytes were mostly unstained, except around
metastases and the central vein. These findings demonstrate that
abnormal expression of gamma-catenin correlates with the more
aggressive tumor and an indicator of liver metastatic breast can-
cer. We here also demonstrated that liver metastases of breast
carcinoma do not express gamma catenin.
OP-58
Molecular and cellular characterization of
GPCR mas-induced tumour formation
W. Z. Lin, S. Y. Tsang, S. S. T. Lee and W. T. Cheung
Department of Biochemistry, The Chinese University of Hong

Kong, Hong Kong, China. E-mail:
Mas was originally isolated from a human epidermoid carcinoma
and predicted to be an orphan GPCR. Recently, we have identi-
fied a surrogate agonist for mas using phage-displayed peptide
library [1]. With dihydrofolate reductase gene as a selection mar-
ker, several stable mas-transformed CHO cell clones were isola-
ted, and mas transgene expression was further amplified by
stepwise addition of increasing doses of methotrexate. Mas-trans-
formed CHO cells induced solid tumour formation in nude mice.
Genetic analyses indicated that tumorigenicity of mas-transfected
CHO cells depends on the sites of chromosomal integration and
the levels of mas expression, suggesting that mas overexpression
is not sufficient to induce tumour formation. Histochemical ana-
lyses revealed that tumour was composed of mas-transfected cells
Abstracts
58
and mouse stromal cells. Cell-growth analyses indicated that
mas-transfected cells neither displayed an altered cell cycle pat-
tern nor grew faster than the control cells, but exhibited signifi-
cant increased anchorage-independent growth. Several differential
expressed genes were cloned from differential display studies.
Intriguingly, a novel CXC cytokine and the GRO gene were sig-
nificantly upregulated in mas-transfected cells and xenografted
tumour samples. These results suggest that cytokine genes expres-
sion and mouse stromal cells invasion might play an important
role in mas-induced tumour formation. 1. Bikkavilli RK et al.
Biochem Pharmacol 2006; 71: 319–337.
OP-59
Identification of novel genes differentially
expressed in b-catenin/TCF 4-activated

hepatocellular carcinoma cells
E. Kavak
1
,K.C¸ avus¸ og
˘
lu
1
,N.O
¨
ztu
¨
rk
2
, A. D. Duru
1
, T. Saygılı
1
,
T. Aslan
1
, E. K. Bayrakc¸ eken
1
, C. Bilgir
1
,M.C.Hız
1
, B. Ergel
1
,
D. O

¨
zer U
¨
nal
1
,M.O
¨
ztu
¨
rk
2
and A. Koman
1
1
Department of Molecular Biology and Genetics, Bog˘azic¸i
University, Istanbul, Turkey,
2
Department of Molecular Biology
and Genetics, Bilkent University, Ankara, Turkey.
E-mail:
b-catenin, a cytoplasmic component of the Wnt signaling path-
way, is observed to be aberrantly activated in several types of
tumors, including hepatocellular carcinoma. The aim of this
study is to identify novel genes that are regulated by active
b-catenin/TCF signaling in hepatocellular carcinoma. We selected
and expanded isogenic clones from hepatocellular carcinoma-
derived Huh7 cells with high and low b-catenin/TCF activities as
detected by luciferase reporter assay. In addition to high TCF
reporter activity, it was observed that Huh7 cells with active
b-catenin/TCF signaling lead to more aggressive subcutaneous

tumors in nude mice. We used Serial Analysis of Gene Expres-
sion (SAGE) to compare gene expression between low and
high TCF activity cells. We have presently identified 429
differentially expressed tags, 326 show decreased expression pro-
file in high activity cells, whereas the remaining 103 tags show
increase. We compared these tags with the SAGE data of several
different types of tumors including liver and brain tumors. We
identified several molecules whose expression profile is similar in
several different tumors and in TCF4 over-activation. Some of
these molecules have not previously been related to cancer and
may be important candidates as tumor markers.
Acknowledgments: Bog
˘
azic¸ i University Scientific Research Pro-
jects, 04B105; Scientific and Technological Research Council of
Turkey, TBAG-2417; State Planning Organization of Turkey,
DPT 01K120290.
Intracellular Trafficking in Health and Disease
OP-60
The role of nucleoside diphosphate kinase in
ADP transport across the outer mitochondrial
membrane
V. V. Voinova and T. Yu. Lipskaya
Department of Biochemistry, Faculty of Biology, Lomonosov
Moscow State University, Mosow, Russia.
E-mail:
In aerobic cells cytoplasmic ADP is transformed into ATP
mainly in mitochondria, but the outer mitochondrial membrane
has a limited permeability for ADP. In the present study, the role
of nucleoside diphosphate kinase (NDPK) in transport of ADP

across the outer mitochondrial membrane was investigated. Three
systems operating as ADP donors for oxidative phosphorylation
were analyzed. These systems employed mitochondrial NDPK
(mNDPK), yeast hexokinase (yHK), and yeast NDPK (yNDPK).
The enzymes exhibited the same activity, but yHK and yNDPK
could not bind to mitochondrial membranes. In all three systems,
muscle creatine kinase (CK) was the external agent competing
with the oxidative phosphorylation system for ADP. At large
excess of CK activity over other activities the creatine kinase
reaction reached a quasi-equilibrium state. Under these condi-
tions equilibrium concentrations of all CK substrates and Ke-
qapp of this reaction were determined for the system with yHK.
In samples containing active mNDPK the concentrations of all
CK substrates except ADP were determined and the quasi-equi-
librium concentration of ADP was calculated using the Keqapp
value. At balance of quasi-equilibrium concentrations of ADP
and ATP/ADP ratio the mitochondrial respiration rate with
mNDPK was 21% of the respiration rate assayed in the absence
of CK; in the systems with yHK and yNDPK it was 3–7%. We
concluded that mNDPK facilitate transport of ADP across the
outer mitochondrial membrane.
OP-61
Ubiquitin ligase gp78 mediates an Ufd1-
independent pathway of ER-associated
degradation
P. Ballar, Y. Shen, H. Yang and S. Fang
Medical Biotechnology Center, University of Maryland
Biotechnology Institute, Baltimore, MD, USA.
E-mail:
The misfolded and unassembled proteins are retrotranslocated

from the endoplasmic reticulum (ER) and degraded by the
proteasomes in the cytosol, a process called ER-Associated
Degradation (ERAD). Aberrant ERAD contributes to patho-
genesis of cystic fibrosis, a-1-antitrypsin deficiency, neurodegen-
erative diseases like Huntington disease and diabetes. ERAD
requires modification of misfolded proteins by chains of ubiqu-
itin. p97/VCP ATPase, which is recruited by its receptor in the
ER, then recognizes ubiquitinated misfolded proteins via its
ubiquitin-binding cofactor Ufd1-Npl4 heterodimer. gp78, ori-
ginally identified as the tumor autocrine motility factor recep-
tor promoting tumor metastasis, is also an ubiquitin ligase
(E3) involved in ERAD. We have identified a novel VCP-inter-
acting domain (VID) in gp78 and SVIP, another VCP-interact-
ing protein with unknown function. VID is transferable and
interferes with the binding of Ufd1 to VCP. Our data suggest
that VCP forms two mutually exclusive complexes, VCP-gp78
and VCP-Ufd1-Npl4. Functionally, both complexes are compet-
ent for CD3d (an ERAD substrate) binding and degradation.
Both E3 and VCP-recruitment activities of gp78 are required
for CD3d degradation. This study reveals a novel Ufd1-inde-
pendent ERAD mechanism that operates in parallel with the
previously described VCP-Ufd1-Npl4-mediated pathway.
Abstracts
59
OP-62
Ibogaine affects brain energy metabolism
R. Pas
ˇ
kulin
1

, P. Jamnik
2
,M.Z
ˇ
ivin
2
, P. Raspor
2
and B. S
ˇ
trukelj
3
1
OMI Institute, Ljubljana, Slovenia,
2
University of Ljubljana,
Biotechnical Faculty, Ljubljana, Slovenia,
3
University of Ljubljana,
Faculty of Pharmacy, Ljubljana, Slovenia.
E-mail:
Ibogaine is an indole alkaloid present in the root of the plant
Tabernanthe iboga. It is known to abandon abstinence syndrome
in animal models of opiate addiction. Since the antiaddiction
effect lasts longer than the presence of ibogaine in the body,
some profound metabolic changes are expected. The aim of this
study was to investigate the effect of ibogaine on protein expres-
sion in rat brains. Rats were treated with ibogaine at 20 mg/kg
body weight i.p. and subsequently examined at 24 and 72 h. Pro-
teins were extracted from whole brains and separated by 2-D

electrophoresis. Individual proteins were identified by MALDI-
TOF mass spectrometry. Levels of 12 proteins were observed to
increase, four of which were identified as metabolic enzymes
involved in glycolysis and the TCA cycle. The results suggest that
the vitalizing effect of ibogaine could be mediated by change in
energy availability. Since energy dissipating detoxification and
reversion of tolerance to opioids requires underlying functional
and structural changes in the cell, higher metabolic turnover
would be favourable. Understanding the pharmacodynamics of
antiaddiction drugs highlights the subcellular aspects of addiction
diseases, in addition to neurological and psychological perspec-
tives.
OP-63
Galectin-3: a lectin essential for apical protein
sorting
D. Delacour
1
, A. Koch
1
, C. I. Cramm-Behrens
1
, A. Le Bivic
2
,
H. Y. Naim
3
and R. Jacob
1
1
Department of Cytobiology, Philipps University Marburg,

Germany,
2
Institut de Biologie du Developpement de Marseille
(IBDM), Faculte des Sciences de Luminy, Marseille,
France,
3
Department of Physiological Chemistry, School of
Veterinary Medicine, Hannover, Germany.
E-mail:
Epithelial cells are characterized by their polarized organization
based on an apical membrane that is separated from the basolat-
eral membrane domain by the tight junctional complex. This
structure is maintained by highly specific sorting machinery that
separates lipids and proteins into different sets of carriers for the
apical or basolateral cell surface. Since glycans can signal apical
sorting, a lectin-based mechanism has been suggested as central
element of the apical sorting machinery. However, candidate lec-
tins identified so far did not fulfil the requirements for apical
sorting receptors. For the biochemical characterization of protein
components involved in apical transport we took advantage of
two hydrolases of the apical brush border, sucrase-isomaltase
(SI) and lactase-phlorizin hydrolase (LPH). After exit from the
trans-Golgi network (TGN), both proteins travel in distinct vesi-
cle populations to the apical cell surface, named SI-associated
vesicles (SAVs) and LPH-associated vesicles (LAVs). We have
identified galectin-3 as a major component of LAVs by 2-D gel
SDS-PAGE and MALDI-TOF-TOF analysis. Galectin-3 binds
to the apical non-raft marker proteins LPH and p75NTR in a
carbohydrate-dependent manner. Moreover, galectin-3 knock-
down results in basolateral missorting of LPH and p75NTR. As

a conclusion our data suggest that galectin-3 is the first lectin
sorting receptor identified in raft-independent apical trafficking.
OP-64
Role of ADP-ribosylation factor1 (ARF1) in
vesicular trafficking in plants
A. R. Memon and S. Usluer
TUBITAK, Institute for Genetic Engineering and Biotechnology,
Gebze, Kocaeli, Turkey. E-mail:
ADP-ribosylation factors (ARFs) are small monomeric GTP-
binding proteins, implicated in vesicle trafficking and secretion
pathway in yeast, mammalian and plant cells. ARFs have been
implicated in the formation of COPI-coated vesicles in eukaryotic
cells and ARF-GEFs are a target of Brefeldin A (BFA). We have
investigated the interaction between ARF and BFA and the
effect of a dominant inhibitory ARF [TN] mutant on Golgi-ER
transport in tobacco epidermal and BY2 cells. Our data show
that N-ST-GFP and GmMan1-RFP markers were transported to
Golgi in tobacco leaves and BY2 cells when expressed with wild-
type ARF1 or ARf1 [QL]. Most of the N-ST-GFP and
GmMan1-RFP markers were redistributed in ER when leaves or
BY2 cells were infiltrated with BFA or ARF-TN mutant. The
BFA effect was suppressed when the tobacco leaves expressed
with either ARF1 wild type or ARF1 [QL]. ARF1 wild type and
ARF1 [QL] also rescued the ARF1 [TN] phenotype in both
tobacco leaves and BY2 cells. Coexpression of either N-HDEL-
GFP or N-Sec-YFP with ARF [TN] mutant in tobacco leaves
resulted the appearance of an Endo H-resistant population of
GFP and YFP. This effect was overcome when leaves were coex-
pressed with ARF wild type or ARF [QL] mutant and was
enhanced by low concentrations of BFA. In contrast, Rab1b

(N121I), a dominant negative mutant caused accumulation of
these markers in an Endo-H-sensitive form. The role of Arf
in anterograde and retrograde traffic will be discussed in plant
cells.
OP-65
Can ChEs regulate cell signaling? Interaction
of ChEs with ERK, PKC, and PKA pathways in
r28 cell line
E. Bodur
1
and P. G. Layer
2
1
Biochemistry Department, Faculty of Medicine, Hacettepe
University, Ankara, Turkey,
2
Developmental Biology and
Neurogenetics, Darmstadt Technical University, Darmstadt,
Germany. E-mail:
During development co-regulation between acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) is observed. BChE is
related to proliferation while AChE is expressed in differentiated
cells. Antisense studies toward both ChEs revealed an interaction
between BChE expression and protein kinase C. The interaction
of cholinesterases with ERK, PKC, and PKA pathways were
investigated in the embryonic rat retinal cell line R28. Usage of
siRNAs specific for BChE displayed the presence of ChE co-
regulation. siRNA treatment caused differential activation of
ERK1/2 coupled with increases in PKC and P90rsk1 expression.
Treatment of R28 cells with a cAMP and PKC activator, forsko-

lin and DOG caused differential activation of ChEs and ERK
proteins. Forskolin treatment increased AChE expression and
caused R28 cells to develop neurites causing cells to be choline-
receptive. DOG treatment increased both BChE expression and
activity along with neurite extension. Our studies display associ-
ation of ChEs with different signaling pathways and cell cycle
events.
Abstracts
60
Neurodegenerative Disorders
OP-66
The effect of beta-boswellic acid on
microtubule polymerization
O. Karima, G. H. Riazi, A. Moosavi Movahedi, F. Mokhtari
and B. Fakurian
Biochemistry Department, Institute of Biochemistry and
Biophysics, University of Tehran, Tehran, Iran.
E-mail:
Tau protein has been shown to be essential in Alzheimer’s disease
(AD). This important protein maintains the structural integrity
of microtubule. Hyperphosphorylation of tau protein observed in
AD and several mutations of the tau genes have been considered
to result in reduced binding ability of tau to microtubule and
resulted in a lack of microtubule dynamic instability. Therefore,
disruption of its structure leads to impairment of axonal trans-
port and finally loss of synaptic function in neural cells and cell
death. Therefore the key factor, which induces the loss of mem-
ory, could be in microtubule destabilization. The gum resin of
the Boswellia species (Frankincense) has long been in use for the
prevention of the amnesia and enhancing the power of memory

in oriental medicine recommended by the most famous physician
in Middle East Avicennia. To identify the effect of beta-boswelic
acid, on the stabilization of brain microtubule proteins, tubulin
was purified from sheep’s brain and beta-Boswellic acid was
granted by Prof. Simmet. The results showed under spectroscopic
and electron microscopy methods for microtubule protein poly-
merization, those different concentrations of beta-boswellic acid
helped microtubule protein in its elongation state. By depolymeri-
zation and repolymerization of polymerized microtubule protein
in the presence of different concentration of beta-boswellic acid,
increase of the stability of microtubule protein in equilibrium
state was identified.
OP-67
c-Jun N-terminal kinase 3 deficiency protects
neonatal mice against cerebral hypoxic-
ischaemic injurury
G. Pirianov
1
, K. Brywe
2
, C. Mallard
2
, N. Kennea
1
, D. Edwards
1
,
H. Hagberg
2
and H. Mehmet

1
1
Institute of Reproductive and Developmental Biology, Clinical Sci-
ences Division, Imperial College London, Hammersmith Hospital
Campus, London, UK,
2
Department of Obstetrics and Gynaecology,
Perinatal Centre, Sahlgrenska Academy, Go
¨
teborg, Sweden.
E-mail:
c-Jun N-terminal kinase 3 (JNK3) is a member of the stress-acti-
vated group of mitogen-activated kinases (MAPK). Although
JNK3 is a potent mediator of apoptosis and neurodegenaration,
little is known about the role of JNK3 and its mechanism of
action in neonatal brain injury. The aim of the present study was
to compare the vulnerability of neonatal JNK3-KO and wild-
type mice to cerebral hypoxic-ischaemic injury (HII) using unilat-
eral acrotid occlusion combined with transient hypoxia. The
degree of neural tissue loss in JNK3-KO mice was substantially
reduced compared to wild-type mice (JNK3-KO 27.8 ± 2.8%
versus wild type 48.3 ± 2.0%, P < 0.0001) following HII. Mole-
cular analysis of JNK3 signalling revealed that HII increased
JNK phosphorylation and activation, and that JNK3 was the
major activated JNK isoform. Significantly, in JNK3-KO animals
there was no difference in the activation of either MKK4 or
MKK7. Downstream of JNK3, HII lead to increased phosphory-
lation of the transcription factors c-Jun and ATF-2. Interestingly,
although this phosphorylation was attenuated in JNK3-KO mice,
the effect on ATF-2 was significantly greater. In JNK3-KO mice

subjected to cerebral HII, decreases in caspase 3 cleavage and
Bim/PUMA protein levels, coupled with diminished Akt/
FOXO3a phosphorylation suggested that the primary mode of
JNK3 action was to promote apoptosis. In conclusion, these find-
ings strongly implicate JNK3 involvement in neural cell loss after
cerebral HII in the developing brain.
OP-68
Reduction of antioxidant capacity in the
postmortem parkinsoni an substantia nigra
E. Sofic
1
, G. P. Reynolds
2
and P. Riederer
3
1
Department of Chemistry, Faculty of Science, University of
Sarajevo, Bosnia and Herzegovina,
2
Department of Pathology, Uni-
versity of Nottingham, Nottingham, UK,
3
Clinical Neurochemistry,
Clinic and Policlinic for Psychiatry, University of Wuerzburg,
Wuerzburg, Germany. E-mail: esofi
Objectives: Parkinson’s disease (PD) is a progressive neurodegen-
erative process with neuronal loss in the neurotransmitter-syn-
thesizing areas. In PD the substantia nigra (SN) is most severely
affected. Pathogenesis of PD is unknown. The aim of this study
was to investigate the antioxidant capacity in the postmortem

motor-cortex (MC), nucleus caudatus (NC) and SN from con-
trols (C) and patients with PD. Materials and Method: The ini-
tial samples consisted of 18 subjects of PD and C. Brains were
matched with age and postmortem time. Brain tissue was homo-
genized in a phosphate buffer with pH 7.3 and separated with
two-step centrifugation at 15 000 rpm for 30\,min and
15 000 rpm for 10 min at 4 °C. Antioxidant capacity in the sup-
ernatants was measured using oxygen radical absorbance assay
(ORAC) on a Perkin-Elmer spectrometer LS 55 with a fluores-
cent filters (Ex: 485 nm; Em: 520 nm). Hydroxyl and peroxyl rad-
ical generators were used. Fluorescence was used as a target of
free radical attack. Results: No changes in the antioxidant capa-
city of postmortem MC and NC of parkinsonian’s brain in com-
parison with C were found. In the SN of parkinsonians brain,
antioxidant capacity seems to be lower in comparison with C
(P < 0.05). Conclusion: Our results showed that in the SN of
parkinsonians brain the balance between production of free radi-
cals and the neutralization by a complex antioxidant system is
disturbed.
Abstracts
61
Bioinformatics and Proteomics
OP-69
Atomic hydration potenti als using Monte
Carlo reference state advance protein
solvation modeling
S. V. Rahmanov
Bioinformatics Department, GosNIIGenetika, Moscow, Russia.
E-mail:
We present a new method for constructing knowledge-based pot-

entials for protein and DNA atomic hydration based on statistics
of their contacts with explicit water molecules in 1774 macromol-
ecule 3-D structures. The method employs a new reference state
created using Monte Carlo technique: the expected probability
densities for the hydration of atoms of different types are calcula-
ted for every structure in a direct computational experiment by
exhaustive sampling of the structure space with random probes.
The atomic potentials that are obtained are very detailed,
smooth, and also very accurate at short distances, in the range
principally not covered by the conventional statistical potentials.
The potentials are tested first for the ability to predict hydration
sites in proteins and DNA structures, and are shown to notably
outperform the existing methods for modeling protein hydration.
We used the new potentials to develop software for the analysis
of macromolecular solvation including calculation of the total
energy of macromolecular solvation, solvent expulsion or retent-
ion during ligand dock, protein–protein and protein–DNA inter-
action energy due to complex formation desolvation and complex
stability calculation. This software was successfully tested in
point mutation induced stability changes prediction and in fold
recognition experiments where the correct folds were preferred to
the misfolded decoys for 12 of 14 protein structures, based on
the hydration energy alone.
OP-70
A study of proteomics for newly isolated
halophilic micro-organisms
A. Salman Dilgimen
1
, D. Kazan
1

and B. Wittmann Liebold
2
1
Faculty of Engineering, Chemical Engineering Department,
Marmara University, Istanbul, Turkey,
2
Wita GmbH, Teltow,
Germany. E-mail:
The optimum living condition for a micro-organism is in its nor-
mal physiological environment. When any change occurs, this
change inflicts a stress on the organism. The type and the severity
of the stress factor determine the survival possibility and the con-
ditions for the micro-organism. Since the proteins are directly
responsible for maintenance of correct cellular function and con-
sequently the viability of the organism, the identification of pro-
teins and protein expression pattern under such given
physiological conditions by proteomic analysis is important for
functional studies of cellular processes. In our study, moderately
halophilic micro-organisms isolated from Anatolian saltern areas
are studied with traditional proteome tools, to determine the
characteristic proteins of the halophilic micro-organisms and to
have information of the microbial life in saltern parts of Anato-
lia. The isolates and reference strains are grown at different salt
concentrations (5% and 20% NaCl) and different temperatures
(10 and 40 °C). After growing and cell disrupture, the entire pro-
teins of the organisms are subfractionated as membrane proteins
and cytosolic proteins. An acidic character of most of the whole
cell proteins is detected both in isolates and the reference strains
(H. salina DSMZ5928 and H. halophila DSMZ4770). The 2-DE
maps of the new halophiles are compared with the reference

strains and selected spots are identified by MALDI-TOF MS
(Waters/Micromass) analysis.
OP-71
UniProtKB/Swiss-Prot: the protein sequence
knowledgebase
A. Stutz
1
, A. Bairoch
2
, Swiss-Prot Group
1
and A. Estreicher
1
1
Swiss-Prot, Swiss Institute of Bioinformatics, CMU, Geneva,
Switzerland,
2
Department of Structural Biology and Bioinformatics,
Faculty of Medicine, University of Geneva, Geneva, Switzerland.
E-mail:
UniProtKB/Swiss-Prot is a high quality manually annotated pro-
tein sequence database with minimal redundancy, maximal anno-
tation and numerous cross-references. Together with UniProtKB/
TrEMBL, its computer annotated supplement, it constitutes the
UniProt knowledgebase (UniProtKB). UniProtKB/Swiss-Prot
provides annotated protein sequences for many species to ensure
high quality annotation for representatives of all protein families.
Data integrated into UniProtKB/Swiss-Prot, including the pro-
tein sequence and current knowledge on each protein, are manu-
ally checked and continuously updated. Manual annotation

process involves sequence and literature curation. 1 In order to
achieve non-redundancy and to improve sequence reliability, all
protein sequences encoded by the same gene are merged into a
single entry. Sequence comparison allows us to find and show the
most reliable sequence. 2 Literature curation involves a critical
review of the current knowledge for each protein. Information is
extracted from scientific publications and continuously updated
thanks to external scientific expertise. A special emphasis is laid
on the annotation of biological events, which generate protein
diversity but are not predicted at the genomic level. Alternative
products, post-translational modification (PTMs) and polymor-
phisms are extensively annotated. 3 UniProtKB/Swiss-Prot is
highly cross-referenced to about 100 database.
OP-72
Fluorescent immunoprecipitation assay – a
proteomic analysis of cell surface proteins
A. Filatov
1
, G. Krotov
1
, M. Krutikova
1
and V. Zgoda
2
1
Institute of Immunology, Moscow, Russia,
2
V.N. Orechovich
Institute of Biomedical Chemistry RAMS, Moscow, Russia.
E-mail:

We proposed a new method, which is an alternative to traditional
immunoprecipitation (IP) technique based on radioactive or bio-
tin labeling of cells. According to FIPA cell proteins in situ are
covalently labeled with fluorescent dyes. Labeling was followed
by standard IP procedures including cell lysis, purification of tar-
get antigens with specific antibodies, and SDS-PAGE. Then gel
without any additional staining or fixation steps is visualized with
fluorescence imaging devices. FIPA has advantage as it does not
use potentially hazardous radioactive labelling, speeds up the
whole IP procedure and – what is most important – easily integ-
rates with other proteomic methods such as immunoblotting and
mass spectrometry. Over 20 human leukocyte surface proteins
were shown capable of labeling with covalent fluorescent dyes,
successfully immunoprecipitating and detecting in gel with fluor-
escent scanner. Modification of target proteins’ lysine residues
did not prevent their possible subsequent trypsinolysis and identi-
fication of derived peptides in electrospray ionization mass
spectrometry. In combination with tandem mass spectrometry
Abstracts
62
FIPA allowed to obtain good quality spectra and acceptable
sequence coverage for the following molecules: CD9, CD20,
CD29, CD49c, CD59, CD71, class I and II HLA.
OP-73
Ligand-specific classification of G protein-
coupled receptors using supp ort vector
machines and reduced alphabeth
O. U. Sezerman
1
, A. Akalin

1
, Z. Kasap
1
and E. Kavak
2
1
Biological Sciences and Bioengineering Program, Sabanci Univer-
sity, Istanbul, Turkey,
2
Molecular Biology and Genetics, Bogazici
University, Istanbul, Turkey. E-mail:
G-protein-coupled receptors (GPCR) are a superfamily of cell
membrane proteins that play a vital role in transmitting extracel-
lular signals into the cytosol. These signals (ligands) bind GPCRs
to control the activation of various mechanisms inside the cell.
Due to their wide range of regulatory effects, GPCRs are one of
the main targets of pharmaceutical research today. However, the
structural information on these molecules are quite scarce, and
ligand specificity for most of them are unknown. In this study,
we describe the utilization of support vector machines to predict
the ligand specificity of a given class of GPCRs, based on total
and partial primary sequence information. We used three-letter
word composition of GPCRs using reduced amino acid alpha-
beth representation and Support Vector Machines for classifica-
tion. Using these compositions we could predict whether a
protein is a GPCR with 97% accuracy. We were also able to pre-
dict the ligands of GPCRs belonging to classes A, B and C with
an accuracy of 93%, 100% and 100%, respectively. Moreover,
we could also determine structural segments that were important
for ligand binding in GPCRs.

OP-74
Predicting the size of the human interactome
M. P. H. Stumpf
1
, T. Thorner
1
, C. Wiuf
2
and M. Lappe
3
1
Molecular Biosciences, Imperial College, London, UK,
2
BIRC,
University of Aarhus, Aarhus, Denmark,
3
Max-Planck-Institute for
Molecular Genetics, Berlin, Germany. E-mail: m.stumpf@
imperial.ac.uk
Current protein interaction network data are notoriously noisy
and incomplete [1]: for most species – apart from Saccharomyces
cerevisiae – only the interactions among relatively small sets of
proteins are known. As a result present network data sets only
offer us partial insights into the structure and functional organ-
ization of molecular interaction networks in most species. Here,
we show for the first time that it is possible to estimate the size
of the true whole-organism interactome from the present partial
data sets. We present a powerful, efficient and reliable estimator
and illustrate its performance using several published S. cerevisae
data sets. We obtain consistent and similar estimates (and confid-

ence intervals) for these data sets and estimate that the complete
yeast interactome will have approximately 24 000–30 000 interac-
tions. We then apply this formalism to different data sets from
P. falciparum, D. melanogaster, C. elegans and H. sapiens to see
if predicted interactome size reflects the complexity of these
organisms. We obtain consistent interactome size estimates of
approximately 19 000, 70 000, 230 000 and 680 000 interactions,
respectively. Thus, the human protein interaction network
appears to be approximately an order of magnitude larger or
more complex than the fruitfly’s interactome. We conclude with
a discussion of the implications of these results for comparative
and functional analyses in systems biology.
1. Stumpf et al. PNAS 2005; 102: 4221–4224.
OP-75
Probable evolutionary pathways towards the
tubulo-interstitial nephritis antigen protein
family
C. Paliakasis, V. Kaltezioti, V. Sideraki, C. Zervas, A. Charonis
and S. Kossida
Foundation for Biomedical Research of the Academy of Athens,
Athens, Greece. E-mail:
Tubulo-interstitial Nephritis Antigen (TIN-ag) is involved in auto-
immune renal disease. It is a multidomain protein located at the
basement membrane of renal proximal tubule epithelium and
intestinal epithelium. Another family named ‘TIN-ag-like’ proteins
shares extensive similarity despite a different expression pattern.
More distant homologues are present in invertebrates. All mem-
bers of these two families exhibit similarity to relevant domains of
well-characterized protein families. The C-terminal half is largely
similar to the Cathepsin family of proteases; in the N-terminal

part, a Somatomedin B-like domain is detected, followed by a part
which in some cases is recognized as a ‘partial’ von Willebrand-
like domain. No similarity exists between TIN-ag and TIN-ag like
proteins in the N-terminal, rich in hydrophobic residues. The N-
terminal part has no similarity to other protein families. By a
number of criteria, TIN-ag like proteins seems older. TIN-ag ori-
ginates from them, having acquired a few extra features on the
process. Similarities were studied in order to reconstruct any
implied evolutionary pathways. The TIN-ag-like family has likely
originated through drastic rearrangements of ready-made parts
from pre-existing material. In conclusion, this analysis is of dual
value: (a) homology-driven extrapolation of potential functions
should aid deciphering TIN-ag functions and (b) it can serve as a
live example for experiments of building proteins from spare parts.
OP-76
Determination of short-range potentials for
physics-based protein-structure prediction
U. Kozlowska
1
, K. Wachucik
1
, A. Liwo
1
and H. Scheraga
2
1
Faculty of Chemistry, University of Gdansk, Sobieskiego, Gdansk,
Poland,
2
Baker Laboratory of Chemistry and Chemical Biology,

Cornell University, Ithaca, NY, USA.
E-mail:
Because of exponentially growing number of known protein
sequences, only about 30% of structure of proteins which are
potentially significant in biomedical sciences can be determined,
which calls for reliable algorithm for protein-structure prediction.
Local interactions play a very significant role in determining pro-
tein structure, because they determine the geometry of secondary
structure elements (helices and sheets), as well as that of turns
and loop regions. Therefore, accurate representation of local
interaction terms in the empirical force fields for physics-based
protein-structure prediction is of utmost importance. In this com-
munication, we present the determination of local interaction
terms for our physics-based united-residue UNRES force field [1]
for protein-structure prediction. We computed the corresponding
potentials of mean force from the energy surfaces of terminally
blocked amino acid residues calculated by the ab initio MP2/6-
31G** approach and the semiempirical AM1 method. Analytical
formulas derived based on generalized cumulant expansions were
subsequently fitted to the potentials of mean force and the result-
ing expressions were implemented in the UNRES force field. The
ability of the UNRES force field with new terms to predict pro-
tein structure by global energy minimization was tested on a
number of benchmark proteins with size from 50 to 100 residues.
1. Liwo A, Czaplewski C, Pillardy J, Scheraga HA. J Chem Phys
2001; 115: 2323–2347.
Abstracts
63
Clinical Proteomics
OP-77

Markers of aggressive behaviour in head and
neck tumors
E. H. Tajara
1
and GENCAPO Brazilian Head and Neck Gen-
ome Project
2
1
Molecular Biology Department, School of Medicine, Sa
˜
o Jose
´
do
Rio Preto, Brazil,
2
Complete authors list and addresses on: http://
ctc.fmrp.usp.br/clinicalgenomics/cp/. E-mail:
Head and neck squamous cell carcinoma (HNSCC) is a very fre-
quent neoplasia in Brazil. In order to investigate molecular mark-
ers of tumor aggressiveness in HNSCC, the Brazilian Head and
Neck Genome Project (GENCAPO) collected 826 tumor samples,
lymph nodes and surgical margins, and blood samples from
patients and 868 controls. Combining genomic and proteomic
techniques, differences in gene and protein expression profiles
from HNSCC and normal counterparts and their relations to
clinical and laboratory parameters were investigated as well as
genetic polymorphism frequencies. Samples from patients were
categorized in aggressive and non-aggressive cases based on
TNM classification. Qualitative and quantitative protein varia-
tions in both groups were consistent and eight proteins were

identified by MALDI-TOF-TOF mass spectrometry. These pro-
teins were: albumin, a-enolase, calgranulin B, galectin 7, myoglo-
bin, myosin light chain 1 and light chain 2, and tropomyosin
b-chain. Also, differences in allele frequencies of a Wnt gene fam-
ily member between aggressive and non-aggressive cases and in
alternative splicing isoform expression between normal and meta-
static lymph nodes were observed. Our results showed different
gene and protein expression pattern and polymorphism frequen-
cies between aggressive and non-aggressive tumors, which may be
used as a predictor of outcome, and rational targets in HNSCC
(FAPESP/Ludwig Institute).
DNA and Protein Microarrays
OP-78
Redox-labelled nucleotides for molecular
biology and bioelectronics
W. Wlassoff
1
, D. Di Giusto
2
and J. Gooding
3
1
Biomedical Research Centre, Sheffield Hallam University,
Sheffield, UK,
2
School of Biotechnology and Biomolecular Sciences,
The University of New South Wales, Sydney, Australia,
3
School of
Chemical Sciences, The University of New South Wales, Sydney,

Australia. E-mail:
Electrochemical DNA hybridization biosensing offers sensitivity,
selectivity and low cost for detection of selected DNA sequences
or mutated genes associated with human disease. This approach
has good potential to become an easy to use and inexpensive
miniaturized (‘lab-on-a-chip’) method for genosensing applica-
tions in the future. We have developed a new strategy for the
redox labelling of nucleic acids based on the enzymatic incorpor-
ation of electrochemically modified nucleotides into DNA and
RNA. The approach avoids cumbersome and time-consuming
chemical manipulations of DNA and exploits DNA/RNA syn-
thesizing enzymes for fast preparation of a wide range of desir-
able-labelled probes. We have demonstrated the principal
possibility of producing electrochemical sensors capable of detect-
ing individual mutations and SNPs in DNA sequence. We have
also shown for the first time the applicability of redox labelling
for detection of RNA, with promising potential applications for
differential transcriptome analysis. Further development of such
enzymatic redox labelling will help to unite technologies from
molecular biology and electrochemistry and enter the exciting
arena of bioelectronics with its potential for fast, cheap and
highly reliable analysis of an individual’s genome, especially for
medical applications.
Imaging and Non-invasive Techniques: Fluorescence
Techniques
OP-79
Dynamic binding of PcG proteins during
Drosophila development
D. J. Arndt-Jovin, J. Grendel and C. Fritsch
Molecular Biology, Max Planck Institute for Biophysical

Chemistry, Goettingen, Germany. E-mail:
Polycomb group (PcG) genes are essential genes in higher euk-
aryotes for the maintenance of the spatially distinct repression of
developmentally important regulators. Their absence, as well as
overexpression, causes transformations in the axial organization
of the body. Little is known about their stability or the exact
mechanism of repression in vivo. Using fluorescence recovery
after photobleaching (FRAP), we determined the translational
diffusion coefficients of two GFP fusion proteins of the PcG pro-
teins as well as the binding equilibrium and dissociation con-
stants and residence times for complexes of the proteins in vivo at
different developmental stages in whole living Drosophila organ-
isms and tissues. The small nuclei of Drosophila cells as well as
the movement of chromatin within the nuclear volume presented
technical difficulties. Recently a method, RICS, was developed
that utilizes the temporal information encoded in CLSM data
and time correlation analysis of the fluorescence signals (FCS) to
determine diffusion constants. We use RICS to analzye the diffu-
sion and binding equilibria of another GFP-labeled PcG protein,
ESC, which is part of the histone methylation complex that
directs PcG protein binding to chromatin. We compare the pros
and cons of FRAP and RICS as well as the results for measure-
ments of Polycomb-GFP in both systems in living embryos and
larval tissues.
1. Ficz et al. Development 2005; 132.
2. Digman et al. Biophys J 2005; 89.
Abstracts
64
OP-80
Periodic twisted structure of amyloid fibrils

revealed by differential polarization laser
scanning microscopy
G. Steinbach
1
, I. Pomozi
2
, G. Garab
1
and J. Makovitzky
3
1
Biological Research Center, Hungarian Academy of Sciences,
Szeged, Hungary,
2
PI-Vision, Budapest, Hungary,
3
University of
Rostock, Rostock, Germany. E-mail:
Amyloid fibrils, insoluble protein aggregates, are associated with
a large variety of diseases. When stained with an intercalating
dye, such as Congo Red, the fibrils exhibit strong linear birefrin-
gence due to the highly ordered molecular architecture of the
self-assembled bundles. By using a differential polarization laser
scanning microscope (DP-LSM), recently designed and construc-
ted in our laboratory, we investigated the spatial distribution of
optical anisotropy properties of isolated human amyloid fibrils
stained with Congo Red and Rivanol; we imaged the anisotropy
(r) and the degree of polarization (P) of the fluorescence
emission, and the linear dichroism detected by fluorescence
emission (FDLD). In contrast to fluorescence intensity images,

which were virtually homogenous along the fiber, the r and
FDLD images revealed a periodicity of about 1.5 lm. The
strong, periodic variations in the dichroism signals might origin-
ate from a twisted macrostructure of the amyloid fibrils. In addi-
tion, r and FDLD images yielded values for the order parameters
associated with the emission and absorption dipoles, respectively.
By also using data of P imaging, we determined the preferential
orientation of the transition dipoles and the static and dynamic
fluctuations of the dye molecules with respect to the long axis of
the fibrils.
OP-81
Capillary electrophoretic analysis of the
superoxide production by individual
mitochondria isolated from cybrid cells
D. Li
2
, M. Navratil
2
, B. G. Poe
2
, C. T. Moraes
1
and
E. A. Arriaga
2
1
Department of Neurology, University of Miami School of
Medicine, Miami, FL, USA,
2
Department of Chemistry, University

of Minnesota, Minneapolis, MN, USA.
E-mail:
The production of superoxide by mitochondria has been previ-
ously examined by fluorescence microscopy. An alternative
approach to carry out this measurement is capillary electrophor-
esis with laser-induced fluorescence detection. Using this method,
organelles are first isolated and then subject to electrophoretic
migration. When the organelles reach the detector two fluorescent
measurements are carried out: (a) detection of individual organ-
elles containing oxidized hydroethidine, a fluorogenic and mem-
brane permeable probe for superoxide; (b) detection of Mito
Tracker Green, which selectively accumulates and labels mito-
chondria. The method was validated by analyzing mitochondria
isolated from cells treated with antimycin A and rotenone. As
expected, these electron transport chain inhibitors increase super-
oxide production in individual mitochondria. The method was
used to investigate how the presence of mitochondrial DNA
mutations affects the production of superoxide released to the
mitochondrial matrix side. We monitored the distributions of
individual mitochondrial superoxide generation in the 143B cell
line and a derived cybrid cell line (DeltaH2-1) that has a long
deletion in 70% of its mitochondrial DNA. This 7522 bp dele-
tion, affects multiple genes essential for oxidative phosphoryla-
tion. The results clearly show that the cybrid cell line has
increased levels of superoxide generation.
Biocompatibility of Materials for Advanced Therapies
OP-82
Effect of use of BMP-2 and TGF-beta on
allograft survival in rat cranial critical size
defect model

H. I. Canter
1
, P. Korkusuz
2
, I. Vargel
1
, F. Oner
3
, B. Cil
4
and
Y. Erk
1
1
Faculty of Medicine, Department of Plastic and Reconstructive
Surgery, Hacettepe University, Ankara, Turkey,
2
Faculty of
Medicine, Department of Histology and Embryology, Hacettepe
University, Ankara, Turkey,
3
Faculty of Pharmacy, Department of
Pharmaceutical Technology, Hacettepe University, Ankara,
Turkey,
4
Faculty of Medicine, Department of Radiology, Hacettepe
University, Ankara, Turkey. E-mail:
Bone grafts, used to provide structural integrity of cranial vault
remodeling, could not always integrate with the remaining bone
structures. They may sequestrate forming fibrous tissue around

themselves or be totally resorbed. In this study, allografts were
covered by chitosan so that slow release of BMP-2 and TGF was
achieved. By this way, increased viability and better integration
of allografts with the cranial vault were aimed. Autograft, allo-
graft, chitosan groups were designed as control groups while allo-
graft + chitosan, TGF, BMP-2, and TGF + BMP-2 were the
study groups. Bone biopsies were obtained at 14th, 56
th
, and
90th days. Bone regeneration was evaluated by morphologic
studies detecting histologic bone healing and radiological studies
detecting bone density. In the morphologic studies, bone regener-
ation in BMP-2 group was found to be compatible with bone
regeneration in gold standard autograft group and even better
than it within 15 days. No statistical correlation between histo-
logic bone healing parameters and radiological studies could be
demonstrated. In conclusion, chitosan is a biocompatible mater-
ial. TGF alone is not effective enough in bone regeneration,
BMP-2 alone has a positive effect in every step of bone regener-
ation and combining TGF with BMP-2 does not lead to a better
bone regeneration than using BMP-2 alone. Results of histo-
morphometric studies show that a synergistic effect is not
obtained by the use of these two factors together.
Abstracts
65
Parasitic Diseases
OP-83
Chloroquine increases apoptosis and TNF-a
RNA level expression in mice infected with
P. yoelii 17xl

M. Legorreta-Herrera
1
, A. Ramos-Avila
1
,
J. L. Ventura Gallegos
2
and A. Zentella-Dehesa
2
1
Divisio
´
n de Posgrado, FES Zaragoza, Universidad Nacional
Auto
´
noma de Me
´
xico, Me
´
xico,
2
Instituto de Investigaciones
Biome
´
dicas, Universidad Nacional Auto
´
noma de Me
´
xico, Mexico.
E-mail:

Chloroquine has been used as an antimalarial drug for several dec-
ades furthermore is used successfully in patients with autoimmune
diseases, the mechanism of action of chloroquine is not completely
understood but it has been assumed that induces apoptosis in
lymphocytes. In this work we studied how the treatment with
chloroquine modifies apoptosis in mice infected with malaria, in
addition, since this cytokine is associated with pathology in cereb-
ral malaria, with the signalling for apoptosis and with Plasmodium
elimination, levels of TNF-a mRNA expression by RT-PCR and
apoptosis by TUNEL were analysed. BALB/c mice were infected
with Plasmodium yoelii 17XL, on day 7 after infection were trea-
ted with chloroquine (25 mg/kg) or received vehicle. Non-infected
mice were treated as before to be used as controls. Results showed
that chloroquine did not modify the levels of TNF-a mRNA
expression or apoptosis in normal mice. In contrast, chloroquine
increased both TNF-a level expression and apoptosis in spleen
lymphocytes of P. yoelii 17XL-infected mice. These results suggest
that chloroquine in addition of its direct antimalarial effect, could
also contribute to the parasite elimination by positively modula-
ting the TNF-a overexpression and apoptosis in mice infected with
Plasmodium, without affecting these parameters in normal mice,
which increases its therapeutic potential. Acknowledgment: This
study was supported by DGAPA IN205703-3.
OP-84
Leishmania glycosome is a store of
intracellular calcium
S. Datta, S. Gupta, B. Raychaudhury, S. Banerjee and B. Das
Indian Institute of Chemical Biology.
E-mail:
A fourth intracellular Ca

2+
pool in Leishmania donovani was iden-
tified by permeabilizing plasma membrane with digitonin. In Fura-
2 loaded cells Ca
2+
was released synergistically when mitochond-
rial function was blocked by antimycin and oligomycin. Vanadate
did not have any effect if applied before incorporation of these mit-
ochondrial poisons. However, the same inhibitor, which inhibits
Ca
2+
-ATPase activity of endoplasmic reticulum, was able to
release Ca
2+
at a slow rate when added after antimycin and oligo-
mycin. Alkalization of cytoplasmic pH allowed further release of
Ca
2+
essentially from the acidocalcisome. Purified glycosomes
could mediate Ca
2+
uptake mechanism in presence of vanadate
whereas bafilomycin, a specific and potent inhibitor of vacuolar
proton pump did not have any effect. Glycosomal Ca
2+
-ATPase
activity was optimum at pH 7.5. The apparent K
m
for calcium in
presence of vanadate was 12 nM. Taken together, it may be sug-

gested that a vanadate-insensitive Ca
2+
-ATPase is present in the
membrane of this microbody. Presence of glycosomal Ca
2+
was
further confirmed by imaging of Ca
2+
activity in the Fura-2 loaded
purified organelle using confocal laser. Results reveal that newly
localized glycosomal calcium may essentially be an effective candi-
date to play a significant role in cellular function.
OP-85
Inhibition of P. falciparum proliferation by
antisense and antisense nanoparticles against
malarial topoisomerase II
W. Noonpakdee
1
and F. Foger
2
1
Department of Biochemistry, Faculty of Science, Mahidol Univer-
sity, BKK, Thailand,
2
Department of Pharmaceutical Technology,
Institute of Pharmacy, Leopold-Franzens-U.of Innsbruck, Austria.
E-mail:
The development of new effective antimalarial agents is urgently
needed due to the ineffectiveness of current drug regimes on the
most virulent human malarial parasite Plasmodium falciparum.

Antisense (AS) oligodeoxynucleotides (ODNs) have shown prom-
ise as chemotherapeutic agents. Exogenous delivery of phos-
phorothioate AS ODNs against different regions of P. falciparum
topoisomerase II gene to P. falciparum K1 strain between 0.01
and 0.5 lM significantly inhibited parasite growth compared with
sense sequence control suggesting sequence-specific inhibition by
fluorescence-activated cell sorter assay or by microscopic assay.
This inhibition was shown to occur during maturation stages, to
improve stability and to increase intracellular penetration, ODNs
were complexed with the biodegradable polymer chitosan to form
solid nanoparticles with an initial diameter of ~55 nm. AS nano-
particles showed stronger inhibition of parasite growth compared
with free AS ODNs. These results should prove useful in future
designs of novel antimalarial agents and use of nanoparticles to
delivery ODNs.
Immune Intervention
OP-86
Induction of NK lymphocytes against cancer
cell lines by administration of Morinda
citrifolia
T. I. Toliopoulos
1
, D. B. Bougiouklis
1
, D. T. Daskalou
1
,
K. S. Karkabounas
1
, G. S. Gerou

2
and E. A. Evaggelou
1
1
Medical Faculty of Ioannina,
2
Analysi Microbiological and
Research Biomedical Labs. E-mail:
Introduction: Natural killer cells (NK cells – NKCs) are a sub-
population of lymphocytes that play an important role in immu-
notherapy. The extract from the tropical plant Morinda citrifolia
indicates some anticancer properties useful for the immune sys-
tem. Material and methods: 12 healthy volunteers participated in
the study. The methodology included four stages: (a) isolation of
peripheral blood mononuclear cells (PBMCs) from blood and
their quantification, (b) quantification of cancer cells (leiomyosar-
coma – Wistar rats, and K562 cell lines), which used as cancer
target cells (CTCs), (c) incubation of PBMCs cells with CTCs in
CO2 chamber in the ratios 12.5:1, 25:1 and 50:1, and (d) deter-
mination of cytotoxicity by flow cytometer FACScan OF BDB
corporation. The same trials were repeated after the addition of
Abstracts
66