Abstracts Young Scientist Forum April 10
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| Дата: | 2010 |
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| Формат: | Стаття |
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Інститут молекулярної біології і генетики НАН України
2010
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| Назва видання: | Вiopolymers and Cell |
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irk-123456789-1541962019-06-16T01:29:33Z Abstracts Young Scientist Forum April 10 Abstracts Young Scientist Forum April 10 2010 Article Abstracts Young Scientist Forum April 10 // Вiopolymers and Cell. — 2010. — Т. 26, № 2, доп. — С. 42-150. — англ. 0233-7657 http://dspace.nbuv.gov.ua/handle/123456789/154196 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Abstracts Young Scientist Forum April 10 // Вiopolymers and Cell. — 2010. — Т. 26, № 2, доп. — С. 42-150. — англ. |
| series |
Вiopolymers and Cell |
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2025-07-14T05:51:56Z |
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2025-07-14T05:51:56Z |
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1837600415961579520 |
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42
Abstracts
Young Scientist Forum
April 10
43
Oral Presentations
44
Cancer Research
&
Prevention
45
Anticancer potentials of a novel Streptomyces antibiotic landomycin E
Panchuk R. R., 1Matselyukh B. P., Stoika R. S.
Institute of Cell Biology, NAS of Ukraine 14/16Drahomanov Str. Lviv, 79005,
1Institute of Microbiology and Virology, NAS of Ukraine 154, Zabolotnogo Str., Kyiv, 03680
rpanchuk@ukr.net
Development of effective anticancer drugs with minimal side effects and capability of
overcoming drug resistance in tumor cells remain the crucial aims in cancer treatment.
Landomycin E (LE) is a novel antibiotic of angucycline family produced by
Streptomyces globisporus 1912 strain growing in soybean medium. It was found that
several tumor cell lines were sensitive to LE pro-apoptotic action that was mediated by
the reactive oxygen species (ROS). In this study, we addressed the molecular me-
chanisms of LE action towards human Jurkat T cell leukemia line in vitro and its anti-
tumor action towards murine NK/Ly lymphoma in vivo. Phosphatidyl serine exter-
nalization and intensive membrane blebbing (early apoptotic events) were observed as
early as 1 h after LE (2 µg/ml) treatment of Jurkat T cells, while nucleus fragmentation
(late apoptotic events revealed by DAPI staining) appeared only in 12 h after the start of
LE (2 µg/ml) action. N-acetylcysteine (NAC) was used to block ROS action and reveal
ROS-dependent signaling pathways induced by LE. It was shown that LE (2 µg/ml, 24 h)
led to splitting of anti-apoptotic PARP-1 and DFF45 proteins involved in DNA
reparation. Cleavage of these proteins is mediated by active caspase-7 and caspase-3
whose levels were increased under LE action. Pre-treatment of target cells with NAC (5
mM, 30 min) completely inhibited activation of the effector caspases-3,-6,-7 and PARP-
1/DFF45 cleavage, which indicates that these processes are ROS-dependent. Thus,
mitochondria can play an important role at late stages of LE-induced apoptosis. This was
also confirmed by a release of cytochrome c from mitochondria to cytoplasm. However,
there were no significant changes in the levels of pro-apoptotic protein Bax and anti-
apoptotic protein Bcl-xL involved in mitochondria-induced apoptosis. NAC pre-
treatment also had no effect on their levels in target cells. To check if ROS are involved
in initiator stages of apoptosis induced by LE, Western-blot analysis was performed with
antibodies to initiator caspases-2,-8,-9. It was found that LE (2 µg/ml, 24 h) induced
cleavage of all tested initiator caspases, however, caspase-8 was the only enzyme whose
activation was not blocked by NAC. We suggest that caspase-8 that is involved in
receptor-mediated apoptosis, can play a crucial role in apoptosis induced by LE in cancer
cells. Thus, LE may realize its cytotoxic effects via receptor-mediated apoptosis signaling
pathway. We found in the in vivo studies that LE (1 mg per kg) significantly increased a
lifespan of NK/Ly lymphoma-bearing mice from 14 days after tumor inoculation, in
control group of animals, to 21 days in the LE-treated animals, comparing with 30 days
under doxorubicin treatment. This correlated with an increase in number of dead tumor
cells and decrease in number of living lymphoma cells. In conclusion, we demonstrated
that landomycin E is a potent anticancer drug, which is effective, both in vitro and in
vivo, and caspase-8 may be a principal molecular target during its action towards tumor
cells.
46
Role of surface sialilation in the clearance of apoptotic cells
Tomin Andriy, Bilyy Rostyslav
Institute of Cell Biology, National Academy of Sciences of Ukraine
amtomyn@gmail.com
In our previous works we proved that exposure of subterminal galactose and mannose
residues of glycocalyx glycoproteins (GP) via their desialilation is the intrinsic feature of
apoptotic cells (Bilyy, et al., 2003, 2007; 2008) and can be effectively used for detection
of apoptotic cells (Bilyy, 2009). At the same time, exposure of desialated proteins on the
surface is one of the markers for phagocytes to eliminate the cell. Failure in the
elimination of apoptotic cells result in the leakage of apoptotic cells content to
intercellular milieu (during it conversion to secondary necrotic cells) and accompanying
inflammation. Chronic deficiency of cell clearance leads to the immune response against
self antigens and development of autoimmune disorders. In vitro, several signals have
been identified as mediators of apoptotic cell recognition and clearance. However, the
distinct mechanisms of apoptotic cell clearance are not fully deciphered to date. The aim
of current work was to study sialidase activity during apoptosis and influence this activity
with the aim to facilitate apoptotic cell clearance. In the current work we studied sialidase
activity, responsible for such desialilation. We have developed know how assay for
fluorescent vital study of enzymatic sialidase activity and proved the increase of sialidase
activity on the surface of apoptotic cells, as early as 2 h after the onset of apoptosis.
Sialidase activity was attributable to the surface of apoptotic (Anexin V positive and PI
negative) cells and was blocked by neuraminidase inhibitor DANA, as was detected by
confocal imaging. To localize the sialidase activity in the cell we performed cell
fractionation of viable and apoptotic cells and detected sialidase activity by
spectrofluorometric assay for 4-MUNA cleavage. Sialidase activity was found to be
increase at apoptosis in the plasma membrane fraction. By isolation of plasma membrane
fractions and by subsequent separation of peripheral and integral membrane proteins we
localized neuraminidase activity as characteristic feature of integral proteins’ fraction of
plasma membrane. By treatment of cellular lysates with caspases 3, 7 and 8 and by
inducing apoptosis in the presence of pan-caspase inhibitor as well as of specific
inhibitors of caspase 3, 6, 8 and 9 we have demonstrated caspase-dependent mechanism
for sialidase activation at apoptosis for both receptor-mediated (FasL) apoptosis
induction pathway or mitochondrial pathway (induced by UV-B irradiation). By artificial
desialilation of cells we were able to significantly increase their clearance by human
macrophages. While the induction of apoptosis in the presence of sialidase inhibitor led
to the inefficient clearance of apoptotic cells lacking desialilated glycotops on their
surface. Thus we claim that sialidase activity is increased in the plasma membranes of
apoptotic cells, its activation is caspase-dependent and its action lead to the formation of
desialilated glycotops, which are important markers for the clearance of apoptotic cell by
macrophages.
This work was partly supported by WUBMRC Grant, grants from NASU and German-
Ukrainian bilateral grant UKR08/035.
47
CHI3L1 stimulates proliferation and migration of glioma cells
Iershov A., Kavsan V.
Department of Biosynthesis of Nucleic Acids
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine
150, Zabolotnogo Str., Kyiv, Ukraine, 03680
a.yershov@yahoo.com
Human genome encodes six proteins of family 18 glycosyl hydrolases, two active
chitinases and four chitinase-like lectins (chi-lectins) lacking catalytic activity. CHI3L1 is
the most investigated protein among human chi-lectins. It has molecular mass of about 40
kDa and is N-glycosylated at Asn60 (two residues of β(1,4)-N-acetyl-D-glucosamine,
NAG). N-terminal amino acids of CHI3L1 mature form are tyrosine, lysine and leucine
(Y, K, L) that gives rise to its alternative name “YKL-40”. CHI3L1 protein sequence has
a cluster of basic residues (GRRDKQH, Gly143–Arg–Arg–Asp–Lys–Gln–His149), similar
to other heparin-binding proteins, while there is no conclusive evidence of its binding to
heparin. Most of growth factors contain heparin-binding sites, which as a rule serve as a
connector between two proteins, for instance, in case of FGF and FGFR. Crystallization
studies failed to reveal the CHI3L1 binding of sulfated oligosaccharide units within its
ligand-binding groove. However, it was shown that CHI3L1 could modulate the activity
of basic fibroblast growth factor (bFGF). Also, there are recent findings proposing that
CHI3L1 acts through cell membrane receptor syndecan-1. Syndecans bear most of
heparan sulfate residues on the cell surface and are involved in angiogenesis and
invasion. CHI3L1 stimulates the association of syndecan with aVb3 integrin, and
treatment with heparinase or chondroitinase removes this effect. Recombinant His-tagged
CHI3L1 protein expressed in [a] prokaryotic system was purified by affinity
chromatography using Ni-NTA agarose. Native CHI3L1 was purified by affinity
chromatography from conditioned medium of MG-63 human osteosarcoma cell line
using heparin-sepharose. We investigated CHI3L1-induced proliferation and migration
on several glioma cell lines. MTT test showed that both, recombinant and native CHI3L1
stimulate proliferation of U373 and U87 cells. Scratch test revealed CHI3L1 stimulation
effect on U87 but not on U251 migration. Glioblastoma, the most common intracranial
malignancy is the aggressive brain tumor with short median survival time (12 months),
frequent recurrence and significant drug resistance. Through four stages of progression,
tumor cells cumulate genetic abnormalities and, as a result, have aberrant signaling
networks. Glioblastomas are characterized by high invasion potential and can escape anti-
angiogenic therapy. CHI3L1 is overexpressed in glioblastomas in comparison to low-
grade gliomas and normal brain, and a correlation between high expression of CHI3L1
and short survival of patients is observed. CHI3L1 serving as a migration and
proliferation factor for glioma cells can play significant role in tumor spreading and
recurrence.
48
Cardiovascular Diseases
&
Prevention
49
N-cadherin deletion results in embryos heart malformations and
embryonic death
Piven O., Kostetskii I., Maczewich L., Lukash L., 1Radice G.
Іnstitute of Molecular Biology and Genetic, National Academy of Sciences of Ukraine
1Jefferson Medical College, Philadelphia, USA
igork@imbg.org.ua; glenn.radice@jefferson.edu
The structural integrity of the heart is necessary for its function and is maintained by the
end-to-end connection between the myocytes called the intercalated disc. Cell adhesion
molecules are critically involved in these interactions, in particular cadherins, the Ca 2+ -
dependent proteins family with homophiles type of interaction. In cardomyocytes
adherent junction is presented only by N-Cadherin. Classical cadherins are
transmembrane proteins. Extracellularly, they mediate specific cell-cell adhesion, while
intracellularly they interact with catenins and through them with actin cytoskeleton. The
catenins family includes α-, β- and γ- Catenin. Cadherins and catenins are structural
compounds of adherent junction. In this study we focused on the analysis of N-Cadherin
and α- and β-Catenin function during heart development using conditional knock-out
approach. For this, we provided morphological and immunological analyses of heart
tissue after N-Cadherin, αЕ-Catenin and β- Catenin missing at different terms of
gestation. For determination of N-cadherin function at cardiogenesis we used 10- and 5-
day embryos. The morphology of embryos was studied by whole mount embryo
examination at E9.5 or E10.5. Mutant embryos were delayed and exhibited delays are in
development comparable with wild type embryos and severe heart malformation.
Neuronal and somatic defects evident for N-cadherin constitutive knockout (Radice
1997) have not been observed suggesting normal N-cadherin function in those tissues.
The heart looked ballooning, looping morphogenesis was not completed, and
extraembryonic circulatory system was poorly developed in the N-cadherin CKO
embryos. Analysis of embryos at term of gestations more than 10, 5 days revealed
complete maceration of mutant embryo. Transverse sections through the heart region of
mutant embryos and control embryos at term of gestations E 10,5 demonstrated the
expressive violations of cardiogenesis after N-Cadherin ablation, namely: thinner wall of
myocardium compared to control, violation of heart tissue forming (intercellular
connections). N-cadherin mutant embryos at E9.5 were immunostained with N-cadherin
and β-catenin antibody. N-cadherin was missing from the heart tissue; there was also no
detectable β-catenin expression in heart myocardium. These data suggest that N-Catherin
plays a critical role in the regulation of β-catenin expression during early cardiogenesis. It
is also possible, that N-cadherin affects the distribution of β-catenin, especially during
the formation of intercalated disks. In our investigation we have shown that ablation of
N-cadherin in the developing heart results in severe cardiac malformations and has
lethality effect. Our data suggest a critical role of N-cadherin in the regulation of early
cardiogenesis. Overall, our findings have extended the understanding of the role of cell
adhesion components in early heart development and in adult heart.
50
Twins can help to prevent atherosclerosis disease. Findings of International
twin study 2009
1Tarnoki A. D., 1Tarnoki D. L., 2Stazi A., 2Medda E., 2Cotichini R., 2Fagnani C., 2Nisticò L.,
3Lucatelli P., 3Boatta E., 3Zini C., 3Fanelli F., 4Baracchini C., 4Meneghetti G., 5Osztovits J.,
5Jermendy G., 6Littvay L., 7Metneki J., 8Horvath T., 1Karlinger K., 9Lannert A., 10Molnar A. A,
11Garami Z., 1Berczi V.
1Department of Radiology and Oncotherapy, Semmelweis University, Budapest, Hungary
2Genetic Epidemiology Unit, National Centre of Epidemiology, Istituto Superiore di Sanità, Rome, Italy
3Department of Radiological Sciences, La Sapienza University of Rome, Rome, Italy
4Department of Neurosciences, School of Medicine, University of Padua, Padua, Italy
5Bajcsy Zsilinszky Hospital, III. Semmelweis University, Budapest, Hungary
6Central European University, Budapest, Hungary
7National Centre for Healthcare Audit and Inspection, Budapest, Hungary
8Institute of Human Phisiology and Clinical Experimental Research, Semmelweis University, Hungary
9Semmelweis University, Faculty of Pharmacy
10Semmelweis University, Budapest, Hungary
11The Methodist Hospital DeBakey Heart and Vascular Center, Houston, TX, USA
tarnoki4@gmail.com
Objective: Atherosclerosis process is a pathophysiological condition in which the artery wall thickness is
the result of a chronic inflammatory response in the walls of arteries which tends to be slowly progressive
over decades and usually remains asymptomatic. Twin studies by comparing identical with non-identical
twins produce information on the relative contribution of genes and environment, and how the two
interact. Purpose: To estimate heritability and environmental effects on arterial stiffness using a twin
sample. Methods and Materials: 126 Italian (66 MZ, 60 DZ), 48 American (45 MZ, 3 DZ) and 82
Hungarian (60 MZ, 22 DZ) twin pairs were included in the study as part of International twin study 2009.
TensioMed Arteriograph was used to measure the arterial stiffness parameters. Results: Based on 512
samples (342 MZ, 170 DZ; mean age 50.9±15.2 years), mean Aixbra and PWVao indicated -16,4±32,3%
and 9,008±2,4 m/s. Age-adjusted intraclass correlation of Aixbra and PWVao were 0.65 (95% CI, 0.55 to
0.72) and 0.46 (95% CI, 0.33 to 0.57) in MZ, 0.42 (95% CI, 0.24 to 0.57) and 0.28 (95% CI, 0.08 to 0.47)
in DZ pairs; heritability 0.45 (95% CI, 0.12 to 0.71) and 0.42 (95% CI, 0.02 to 0.57) (Figures 1 and 2).
Shared environmental effect of AIxbra and PWVao indicated 0.20 (95% CI, 0.0 to 0.49) and 0.04 (95%
CI, 0.0 to 0.38), unshared environmental effects indicated 0.35 (95% CI, 0.28 to 0.45) and 0.54 (95% CI,
0.43 to 0.67) adjusted by age, respectively.
Conclusions: Arterial stiffness parameters are moderately heritable. Due to the long-lasting process
of atherosclerosis it can be detected in early stage by pulse wave analysis in order to be treated by
appropriate therapy to prevent consequences of atherosclerosis like stroke, heart attack or peripheral
vascular disease.
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51
Mother and Child Health
Reproductive Health
52
Mathematical model enabling to differentiate between various
modes of perinatal PCB exposure
Lancz Kinga, 1Trnovec Tomáš, 2Dedik Ladislav, Palkovičová Ľubica,
1Wimmerová Soňa, 1Kočan Anton
Department of Environmental Medicine, Slovak Medical University, Bratislava, Slovakia
1Department of Toxic Organic Pollutants, Slovak Medical University, Bratislava, Slovakia
2Faculty of Mechanical Engineering, Slovak University of Technology, Bratislava, Slovakia
kinga.lancz@szu.sk
Introduction: The adverse effects of polychlorinated biphenyls (PCBs) on the developing
organism may result from exposure prior to conception (either parent), during prenatal
development or postnatally to the time of sexual maturation. The perinatal exposure consists
of prenatal exposure due to passage of PCB through placenta and postnatal exposure through
its uptake by breast or formula feeding. Materials: Blood samples were collected from
newborns during years 2002-2004 at delivery from umbilical cord and at 6, 16 and 45
months of their age by venepuncture. The lipid adjusted serum concentration of the most
abundant PCB congener #153 was used for modelling of PCB exposure. Methods: The time
course of the measured PCB 153 concentration after birth of the child C(t) was described by
the relationship C(t) = C0 + ∆C(t) where t is time, C0 is PCB 153 concentration (ng/g serum
lipids) in cord blood serum and ∆C(t) is the concentration increase due to breast feeding until
time tbf and normal food intake after weaning. As a best fit a linear one compartment model
has been chosen on the basis of Akaike's Information Criterion with unit amplification.
Results: The correlation between Cbf, inf and AUC was very high. The usefulness of the
model can be demonstrated by computing for each child its PCB 153 serum concentration
adjusted to cord serum PCB 153 concentration (C/C0) at the time of weaning.
Conclusions: Kinetics of PCB 153 in the serum of 103 infants was approximated using a
„system model“ representing a novel approach compared to compartmental or
physiologically based pharmacokinetic models. Duration of breast feeding and mean time
were found to be correlated with PCB 153 concentrations in steady-state without normal
food intake (Cbf, inf) and without breastfeeding (Cf, inf) and with both parameters adjusted
to concentration at delivery (Cbf, inf /C0 and Cf, inf/C0).
The usefulness of the model was demonstrated by predicting of PCB 153 serum
concentration at weaning adjusted to cord serum PCB 153 concentration (C/C0) from breast
feeding duration. For approximation a modified Weibull function was used.
Dedík, L., Ďurišová, M. System approach in technical, environmental, and bio-medical studies. STU,
Bratislava, 1999.
Hertz-Picciotto I., Trnovec T, Kočan A., et al. PCBs and early childhood development in Slovakia: Study
design and background Fresen Environ Bull 2003, 12, 208-214.
53
Kynurenic acid concentration in the perinatal period
Flach Edina, 1Vámos Enikő, 1Klivényi Péter, Gyarmati Judit, 1Zádori Dénes,
1Vécsei László, Bódis József, Ertl Tibor
Department of Obstetrics and Gynaecology, Medical School, University of Pécs, Pécs
1Department of Neurology, Albert Szent-Györgyi Clinical Centre
University of Szeged, Szeged, Hungary
flachedina@freemail.hu
Background and aim: The essential amino acid tryptophan is metabolized in the brain
by two major pathways, through either the kynurenine or methoxyindole pathways.
Kynurenic acid (KYNA), synthesized from L-kynurenine in reactions mediated by
kynurenine aminotransferases in astrocytes, glial cells and neurons, is a metabolite of the
kynurenine pathway. KYNA is one of the few known endogenous NMDA (N-methyl-D-
aspartate) receptor antagonists with neuroprotective properties. Our aim was to determine
the concentration of KYNA in the umbilical vein and arteries of term neonates. Methods:
Umbilical venous and arterial blood samples were taken from twenty-four healthy term
infants immediately after birth as well as by peripheral venipuncture on the fourth
postnatal day (D4). Mean (+/-SD) birth weight and gestational age were 3445.0 (+/-
499.3) g and 39.0 (+/-0.9) weeks, respectively. Serum concentrations of KYNA were
measured by high performance liquid chromatography. Results: KYNA level in the
umbilical vein was significantly (p=0.022) higher than in the umbilical arteries and in D4
venous blood, whereas the arterial and D4 samples did not differ significantly. There was
no significant difference for KYNA neither in the umbilical arterial and venous samples
nor in D4 samples between newborns delivered spontaneously and by cesaerean section.
The KYNA concentration in the umbilical vein was significantly (p=0.044) higher in
female newborns than in males. Conclusions: The higher KYNA concentration in the
umbilical vein suggests that in normal term newborns KYNA level is not elevated during
delivery and on the fourth postnatal day. The increased umbilical venous KYNA level
can be of placental or maternal origin. Our findings may help to understand the
neuroprotective and neurotoxic mechanisms in the infant brain during the perinatal
period.
54
Inflammatory
&
Immune Response
55
Melatonin – novel cytoprotective and healing modulator of upper
gastrointestinal tract by angiogenesis impact
Savytska M., Yaciv S., Zayachkivska O., Gzhegotsky M.
Physiology Department, Lviv National Medical University,
69 Pekarska Str., Lviv, Ukraine, 79010
merymed11@gmail.com
Recent data indicated that risk for premalignant Barrett’s esophagus (BE) increases by 30
to 50% per decade of life in patients with gastroesophageal reflux disease (GERD).
Modern day data revealed that widespread use of prolonged acid suppression is
associated with side effects, as well known dismotility, dysbiosis in the upper
gastrointestinal (GI) tract, hypergastrinemia etc. It is well known that melatonin (MT)
possesses GI resistance to luminal damaging agents, improved motor dysfunction and
reveled antineoplastic effect. Previously we have shown that disturbances of oral and
esophageal mucosa (OM and EM, respectively) cytoprotection were followed by
disorders in endogenous defence signalling pathways for inflammation, re-
epithelialization, and formation of granulation tissue, interaction between the various
cells and matrix, and apoptic cell recognition. However, the role of MT on angiogenesis
signalling on mechanisms of maintaining OM and EM integrity and healing has not yet
demonstrated. Materials: several series were used on rats after sialoadenectomy (SAE) vs
rats with intact salivary glands (ISG) without/with MT (20 mg/kg/ip) treatment by water-
immersion restraint stress (WRS) by Takagi, 1964; the acute injury and healing process of
OM and EM were monitored at various time points: after WRS induction immediately
and after 24 and 48 hr of WRS induction; estimation of injury, inflammation and
hyperplasia via histological score index (HSI); VEGF, EGF, IL-1β, TNFα by ELISA.
Results: WRS-induced non-erosive OM lesions and stress-associated esophagitis,
constantly increased HSI in 150% in compare to MT treated rats; in SAE rats HIS was
2,5-folds time more than in ISG rats. MT decreased expression of VEGF and
inflammatory mediators including IL-1β, TNFα, and increased EGF synthesis. At 24
hr&48 hr after WRS EM healing was delayed and defective response of EM basal layer
indicated vs MT-treated ISG rats. In SAE rats pro-inflammatory interleukins were
increased twice than in ISG rats; was shown MT treatment accompanied decreased
synthesis of IL-1β, TNFα during hyposalivation and accelerated OM and EM healing vs
SAE rats without MT. We concluded that MT increased resistance of epithelial barrier of
upper GI tract and accelerated the healing of OM and EM lesions via increased
expression of EGF and decreased of VEGF and inflammatory mediators, suggesting that
it exhibit mucosal repair through the activation of angiogenesis signaling to induce
proliferation activity and accompanied anti-inflammatory effects.
56
C60 fullerenes modify protein tyrosine phosphorylation patterns in
normal and transformed T cells treated with apoptosis-inducing
agents
Palyvoda K., Samoylenko A., Drobot L., 1Matyshevska O.
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine,
9 Leontovycha Str., Kyiv, Ukraine, 01601
2Taras Shevchenko Kyiv National University, Kyiv, Ukraine
zuav@ukr.net
In recent years there has been increasing interest in the possible application of carbon
nanospheres, known as fullerenes, in biomedicine. С60 fullerenes are nanodimensional
molecules, which due to their small size and hydrophobicity can incorporate into cell
membranes and specifically interact with cellular proteins, thereby exerting a variety of
biological effects at relatively low concentrations. Nevertheless, effects of C60 fullerenes
on normal and transformed cells as well as mechanisms of C60 fullerenes interaction with
the cell structural components have been poorly studied. The aim of the present work was
to study the effects of pristine С60 fullerenes on viability of normal and transformed T-
lymphocytes treated with different apoptosis-inducing agents. Since phosphorylation of
proteins on tyrosine residues plays a crucial role in the control of cellular signaling
networks, the phosphotyrosine status of these cells was also investigated. Primary
cultures of rat thymocytes and human T lymphoma Jurkat cells were used in the
experiments. Cell viability was assessed by the MTT reduction assay. Protein
phosphotyrosine patterns were analyzed by Western-blot analysis using monoclonal anti-
phosphotyrosine antibodies. Colloidal solutions of С60 fullerenes were prepared in
Technical University of Ilmenau (Germany). Preincubation of thymocytes with fullerenes
(10-5М) for 1 h significantly reduced cytotoxic effects of such agents as staurosporine
(0.01 µМ), cytosine arabinoside (10 µМ) and hydrogen peroxide (50 µМ). After 24 h
incubation viability of thymocytes was increased by 25±9%, 16±7% and 19±6%,
respectively. By contrast, C60 fullerenes did not protect human Jurkat T lymphoma cells
from death induced by these agents. Next, investigation of phosphotyrosine status of T
cells demonstrated, that the main immunoreactive bands detected with anti-
phosphotyrosine antibodies correspond to proteins with Mr 17, 30, 50, 72 and 100 kDa in
thymocytes and 17, 26, 30, 50, 55, 70, 90 kDa in Jurkat cells. In both cell types the
intensity of phosphorylation of almost all phosphotyrosine-containing proteins was
decreased after incubation with all the apoptosis-inducing agents studied. The lowest
level of protein tyrosine phosphorylation was detected after 12 and 24 h incubation with
investigated agents in both cell types. The effects of these cell death inducers on protein
tyrosine phosphorylation were modifided in the presence of C60 fullerenes. In conclusion,
the selectivity of C60 fullerenes effects in normal and transformed T-cells might be
helpful for the development of the complex approaches to therapy of T-lymphomas.
57
Nanotechnology
58
Identification of novel tumor-associated antigens of medullary
breast carcinoma by modified SEREX-approach
1, 2Kostianets О. I., 1Maliuchik S. S., 2Demidov S. V., 1Filonenko V. V.,
1, 3Gout I. Т., 1Kiyamova R. G.
1Institute of Molecular Biology and Genetics NAS Ukraine, Kyiv, Ukraine
2National Taras Shevchenko University of Kyiv, Kyiv, Ukraine
3University College London, London, UK
kostjanez@ukr.net; r.g.kiyamova@imbg.org.ua
Tumors elicit humoral and cellular immune response in the host organism.
Autoantibodies specific to proteins that are mutated, misfolded, improperly glycosylated,
overexpressed, truncated, or aberrantly localized in tumor cells have been detected in
cancer patient sera. These autoantibodies or their aberrant targets can be utilized as
molecular signatures of tumor genesis, thus being excellent candidates for cancer
immunotherapy and diagnostics. Our aim was to identify and characterize novel tumor-
associated antigens (TAA) of medullary breast carcinoma (MBC) which is heavily
infiltrated by lymphocytes, indicating the possible presence of specific antigens on the
surface of tumor cells, applying SEREX methodology (Serological expression of
recombinantly expressed clones). For this purpose we modified SEREX approach to
create a cDNA library depleted of immunoglobulin (IgG) clones, because heavy
infiltration of this tumor by lymphocytes impedes the use of standard SEREX
methodology for the search of TAA, due to very high percentage of IgG positive clones
in generated library. Screening of depleted cDNA library from MBC tumor with
autologous sera allowed us to identify 59 positive clones corresponding to 41 different
genes, 9 of which were represented by several clones – up to 6. According to
involvement of protein products of these genes in different biological processes we have
categorized them in following groups: transcription, translation, cell signaling, cell
adhesion, protein folding and others. It is noteworthy that 14 from identified clones have
been already identified by SEREX techniques during screening of cDNA libraries from
different tumors types, including breast carcinomas. Based on literature data analysis we
found that protein products of 5 genes have immunomodulatory properties such as
modulation of functional activity of macrophages, cell migration, activation of T-cells by
regulation of the IL-1 pathway and regulation of cytokines expression. We cannot
exclude that expression of these proteins in medullary breast carcinoma can cause such
heavy lymphocytic infiltration of this type of tumor and relatively good prognosis for
MBC patients. Further investigation of identified genes and protein products can help to
understand the molecular mechanisms of breast cancerogenesis and to select antigens
which can be used for immunotherapy as targets for creation of anti-tumor vaccines or
the panel of TAAs to devise diagnostic tests that provide better sensitivity and specificity
than single biomarkers alone.
59
Novel synthetic vehicles and nanoscale drug delivery systems
overcoming cell membrane
Moskvin M., Skorokhoda T., 1Panchuk R., 1Boiko N., Mitina N.,
Zaichenko A , 1Stoika R.
1Lviv Polytechnic National University,
2Lviv Institute of Cell Biology of National Academy of Science of Ukraine
s04143609@polynet.lviv.ua; stoika@cellbiol.lviv.ua
Novel telechelic, comb-like and branched oligoelectrolytes of tailored molecular weight,
narrow molecular weight distribution, and functionality possessing controlled solubility
and surface activity were developed in the lab of Lviv Polytechnic National University.
The originality of the developed approaches is based on the synthesis of novel functional
oligoperoxides with end or side ditertiary peroxide fragments and their subsequent using
for initiation of block or graft copolymerization. That provides controlling the copolymer
characteristics and behavior in water media of various polarities, namely: conformational
state and size of the micelle-like zones, pH and temperature responsivity, untoxicity and
biological compatibility or genuine physiological activity. As a result of the development
of theoretical and experimental principles of the controlled synthesis of such
oligoelectrolytes – surfactant novel telechelic, block and branched copolymers of vinyl
acetate, vinyl alcohol, unsaturated acids, amines, and nonionic PEGylated monomers of
desired macro- and microstructure and chain length as well as functional polymeric and
polymer-mineral nanoparticles were synthesized and studied. There was shown by light
scattering and SAXS techniques that size of the nanoscale drug delivery systems is in the
range 20 – 100nm depending on oligoelectrolyte nature and content. Such novel
functional oligoelectrolytes and nanoparticles possess ability to immobilize low
molecular weight physiologically active substances (biocides, antibiotics including
anticancer ones) via mechanisms of solubilization, intermolecular complex formation,
covalent binding etc and to form nanoscale water drug delivery systems. They can be
labeled with luminescent, MRI and X-ray detectable markers. The availability of reactive
functional fragments in their structures provides irreversible binding antibodies, lectins or
saccharide-containing fragments possessing specific interaction with cell membranes.
The toxicity and genuine biological activity studied in the lab of Institute of Cell Biology
witness their strong dependence on the molecular weight and molecular weight
distribution as well as functionality and surface activity. Testing of oligoelectrolytes and
nanoparticles in vitro and in vivo showed very low toxicity some of them and allowed to
select the most promising ones as carriers for antimicrobial and anticancer drug delivery
systems. Water based systems consisting of novel oligoelectrolytes carriers and
immobilized chloramphenicol or ampicillin were successfully tested on microbial and
fungi cultures. Study of anticancer drug (doxorubicin) delivery systems testified to their
overcoming cell membranes and natural biological barriers and as a result high efficiency
of the action on some tumor cells in vitro and in vivo and their low toxicity at the same
time. This provides significant lowering the amount of anticancer drug. The study of the
novel developed drug delivery systems are on the stage of the patenting.
60
Cell Biology
61
PPAR-α agonist BAY PP1 attenuates renal fibrosis in rats
1,2,3Boor Peter, 4Celec Peter, 5Villa Luigi, 4Pálffy Roland, 1Klenovicsová Kristína,
5Esposito Ciro, 6Schäfer Stefan, 6Albrecht-Küpper Barbara, 2Ostendorf Tammo,
7Heidland August, 1Šebeková Katarína
1Department of Clinical and Experimental Pharmacotherapy, Research Base of Slovak Medical
University, Bratislava, Slovakia
2Division of Nephrology, RWTH University of Aachen, Aachen, Germany
3Institute of Pathology, RWTH University of Aachen, Aachen, Germany
4Institute of Molecular Biomedicine & Institute of Pathophysiology & Department of Molecular Biology,
Comenius University, Bratislava, Slovakia
5Unit of Nephrology, Dialysis and Transplantation, University of Pavia, Pavia, Italy
6Bayer Schering Pharma AG, Cardiology Research, Wuppertal, Germany
7Department of Internal Medicine, University of Würzburg, Würzburg, Germany
boor@email.cz
Renal fibrosis is the final common pathway of most progressive renal diseases. Effective
treatment options are limited. There are no data on the role of peroxisome proliferator-
activated receptor-α (PPAR-α) in the development and progression of kidney fibrosis.
Therefore, we have examined the effect of two PPAR-α agonists, fenofibrate and BAY
PP1, in a rat model of tubulointerstitial fibrosis: the unilateral urethral obstruction
(UUO). Compared to unobstructed kidneys, five days after obstruction renal PPAR-α
mRNA expression was reduced by 94%. Compared to vehicle treated rats with UUO,
BAY PP1 significantly ameliorated the renal cortical expression of collagen type I, III,
IV and fibronectin. The number of proliferating tubulointerstitial cells (mitotic figures,
PCNA positive cells) and of PDGFR-β/PCNA double-positive cells was significantly
lower in BAY PP1 treated rats. Treatment with fenofibrate had no effect on these
parameters. The infiltration with monocytes/macrophages was not reduced by either
treatment. In vitro, BAY PP1 had no direct effect on the proliferation or expression of
fibrosis markers in rat renal fibroblasts. Conversely, rat tubular cells treated with BAY
PP1 produced less collagen, fibronectin, TGF-β1 and the conditioned media of these cells
reduced proliferation of fibroblasts. In conclusion, the PPAR-α agonist BAY PP1, but not
fenofibrate, reduced renal fibrosis in rats with UUO by affecting the cross-talk between
tubular cells and fibroblasts. These data suggest that novel PPAR-α agonists could be an
important treatment option in the early stages of renal fibrosis.
62
Kinetic model of the effects of extracellular pH and [K+] on the
inactivation of the Kv1.3 potassium channel
Vincze Janos
Department of Biophysics and Cell Biology, Medical and Health Science Centre, University of
Debrecen
98, Nagyerdei krt., Debrecen, Hungary, H-4012
janos.vincze@unideb.hu
Kv1.3 is a voltage-gated potassium channel, which plays an important role in the
membrane potential regulation of human T cells. During prolonged depolarization Kv1.3
enters a non-conducting state by the slow P/C type inactivation, whose exact mechanism
is yet to be determined. Previous studies of our lab show that both the pH and the
potassium concentration of the extracellular space ([K+]e) influence the kinetics of
inactivation. According to these previous results acidic pH accelerates the inactivation in
the presence of low [K+]e, while decelerates it in the presence of high [K+]e.
The aim of the present study was to asses whether the proposed kinetic models were able
to explain the experimental results and therefore determine the model which is the most
suitable to describe the inactivation gating of the Kv1.3 channel. To examine the models
the author's own computer programme was used, which is able to generate simulated
recordings from a given kinetic model. In this study whole cell patch-clamp
measurements were modelled, 1500 channels per run were simulated. 10 runs were
analyzed for each parameter set. Inactivation kinetics of the simulated traces was
determined using exponential curve fits performed from 90% of the amplitude to the
onset of the steady-state part of the curve. The results of the simulations show that a
model containing only two open microstates and one inactivation pathway from both
open microstates is not sufficient to describe the experimental results accurately. By
contrast, the introduction of a new binary parameter and with this the duplication of the
inactivation pathway leads to a good approximation of the experimental results. Based on
these results it is likely that a kinetic model in which the speed of inactivation is the
function of the binding of a sole potassium ion into the cavity of the channel, as proposed
by earlier, does not fit the experiments. The probable structure of microstates corresponds
to a model in which the conducting pore can have two different conformations with
different conduction and inactivation properties depending on the [K+]e. The simulation
provides the basis for future experiments verifying the model and leading to better
understanding of the P/C type inactivation mechanism. The software that was developed
for this study can be used to stochastically simulate the kinetics of any ion channel. Since
ion channels are necessary regulatory elements of the almost all cells of a human body
and play an important role in the pathogenesis of several diseases, e.g. long QT
syndrome, the better understanding of their gating kinetics, which can be achieved by this
simulation, could lead to results with clear clinical significance.
63
Stat5 deficiency influences survival and plasticity of hippocampal
neurons affecting erythropoietin and growth hormone signaling
Byts N., 1Rost C., 1Klingelhöffer C., 2Ehrenreich H, 1Sirén A. L.
Palladin Institute of Biochemisry, National Academy of Sciences of Ukraine
9, Leontovycha Str., Kyiv, Ukraine, 01601
1Department of Neurosurgery, University of Würzburg, Würzburg, Germany
2Max Planck Institute of Experimental Medicine, Göttingen, Germany
nabyts@yahoo.de
Signal transducers and activators of transcription Stat5 represent an important
downstream signaling pathway activated by cytokine type I receptors. Upon binding to
their membrane receptors, growth factors such as erythropoietin (EPO) and growth
hormone (GH) activate Janus kinase 2 (JAK2) and cause activation of Stat5 inducing
expression of genes that regulate cell survival, proliferation and differentiation. Stat5
deficiency is characterized by a dwarf phenotype, anemia, and high perinatal mortality
due to ineffective hematopoiesis. Recently Stat5 has been suggested to play a role in
neuronal migration and axon guidance in the developing brain. Stat5 is activated by the
neuroprotective and neurotrophic cytokines, including erythropoietin (EPO) and growth
hormone (GH). To directly test whether activation of Stat5 in neurons is essential for
EPO actions, we examined the neuroprotective and neurotrophic effects of EPO in
hippocampal neuronal cultures isolated from Stat5-/- and Stat5+/+ mouse fetuses after
acute abrogation of JAK2/Stat phosphorylation by the newly available pathway-specific
inhibitor, cucurbitacin I. Since Stat5 has been shown to be crucial for intracellular
signalling and the somatotrophic effects of GH, we also studied its effects in the Stat5-/-
and Stat5+/+ neurons. Our data obtained in hippocampal neuronal cultures from Stat5a/b-
knockout mouse fetuses suggest a dissociation of the intracellular pathway mediating the
protective effect of EPO against glutamate toxicity from that needed for its neurotrophic
activity. Both pretreatment and post-treatment with EPO counteracted glutamate-induced
cell death in Stat5+/+ and Stat5-/- neurons. Acute pharmacological inhibition of
JAK2/Stat signaling had no effect on EPO neuroprotection, whereas inhibition of
phosphatidylinositol-3' kinase (PI3K)/Akt pathway abolished the protective effect of EPO
in both Stat5+/+ and Stat5-/- neurons. GH effectively protected Stat5+/+ cells against
glutamate toxicity but had no effect in Stat5-/- neurons or in Stat5+/+ neurons treated
with JAK2/Stat or PI3K inhibitor. EPO and GH stimulated neurite outgrowth and
branching of Stat5+/+ neurons through activation of PI3K/Akt signaling but had no
trophic effect in Stat5-/- cells. We conclude that in hippocampal neurons Stat5 is not
required for neuroprotection by EPO but along with Akt is essential for its neurotrophic
activity. Both Stat5 and Akt are needed for neuroprotective and neurotrophic signaling
induced by GH in neurons.
64
Drug Development
&
Pharmaceutical Research
65
New one-pot synthetic method of biologically active 2-substituted-4-
thiazolidinones
Kaminskyy Danylo, Khyluk Dmyto, Lesyk Roman
Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
dankaminskyy@gmail.com; dr_r_lesyk@org.lviv.net
Design of “small molecules” as innovative biological active compound base on well-known
scaffolds is one of most commonly employed approach in drug discovery. Among 4-
azolidinone derivatives 2-aryl(heteryl) substituted 4-thiazolidinone attract attention of
scientists as sources of antimicrobial, antimycobacterial, antiviral and anticancer compounds.
Nowadays “one-pot reactions” of heterocyclic systems synthesis are effective method in
medicinal che-mistry. Synthesis of 2,3-disubstituted derivatives of 4-thiazolidinone involves
a one-pot three-component condensation of primary amine, oxo-compound and
mercaptoacetic acid (scheme a) and describe for different condition/medium and catalysts.
The row of new 4-thiazolidinone derivatives (compounds 1) base on amino-acids or primary
aromatic amines; aromatic aldehydes/cyclic ketones or isatine was synthesized according
mentioned reaction. For synthesis of compounds with carboxylic acids moieties in position 3
of core heterocycle the MAOS (microwave assisted organic synthesis) method involved.
R
NH2
SH
OH
O
R 1 R 2
O
S
N
HO
S
A
O
OH
SH
A
S
N
O R
R 2
R 1
O
A
H
S
N
O R
R 2
R 1
A
O
OH
SH
A
R 1 R 2
O
R
NH2
++
1. NaOH
2. HCl
+ +
R= substituted-C6H4;
(CH2)nCOOH
R1=H; R2= substituted-C6H4
R1+R2=(CH2)n; isatine moiety
1
2
a)
b)
A= substituted-C6H4;
C6H4CHCH
a,b,c
a
a - a/h C6H6, ZnCl2; b - THF, DCC; c - EtOH, MW
C6H6
tert-BuONa Increase of biologycal
activity level
Optimization of obtaining substances was carrying out via condensation reaction with aromatic
aldehydes that allowed us obtained 5-ilydene derivatives (compounds 2). Direction of chemical
modification was substantiate by our previously investigation about critical role of presence or
nature of moiety in position 5 for mentioned compound biological activity level. However, the
general applications of the described processes are limited as the reactions are requiring the
expenditure and have low yield according to low reactivity of CH group. We focused on
developing new variant of one-pot three-component reaction (scheme b) which allowed us to
obtain 5-ylidene derivatives of compounds 1 in one stage. In this case instead of mercaptoacetic
acid we used 2-mercapto-3-arylacrylic acids obtained with high yields via alkali hydrolysis of
appropriate 5-arylidene rhodanines.The all chemical reactions, purity and structure of new
compound were determined by TLC and spectral data. Biological activity screening was realized
according international programs of NIH (Bethesda, USA): DTP of NCI for anticancer activity,
TAACF for anti-tuberculosis activity and AACF for antiviral activity of NIAID. The screening
data allowed us to identify the hit-compound and show the increase of activity level after
structure modification of compounds 1.
66
Search for thiazolidon derivates means with immunomodulating
activity in experimental immunodeficiency
1Poshyvak Olesya, 1Pinyazhko Oleh, 2Lesyk Roman
Danylo Halytsky Lviv National Medical University
69, Pekarska str., Lviv, Ukraine, 79010
1Pharmacology department,
2Department of Pharmaceutical, Organic and Bioorganic Chemistry
olesya.poshyvak@gmail.com; dr_r_lesyk@org.lviv.net
Thiazolidones and its’ derivatives have been a great success in the field of chemistry and
pharmacology over the last decades. The row of the thiazolidones act on the different sta-
ges of the clinical research as anticancer, antithyroid, anti-inflammatory, cardiovascular,
antiviral drugs. Objective: to identify among thiazolidon derivates means with
immunomodulating activities on the base of pharmacological screening and on the model
of cyclophosphan immunodeficiency to identify leading compound as potential mean of
immune status correction. Materials and Methods: pharmacological, toxicological,
immunological, morphological and statistical methods were used. The experiment has
been carried out on white rats with the weight of 70-80 g. Experimental
immunodeficiency modeling was made by subcutaneous introduction of cyclophosphan
in a doze of 10 mg/kg of weight during 10 days. The immune system functional status
was conducted on the 12th day of experimental therapy with the use of morphological,
laboratory and immunological methods. Five groups of animals underwent the
experiment: control group, animals with placebo, animals with immunodeficiency
(cyclophosphan model), animals with immunodeficiency, which were treated by
polyoxidonium in a dose of 10 mg/kg 10 days, animals with immunodeficiency, treated
by experimental substances in the doses of 1/10 LD50 during 10 days. Results: On the
base of pharmacological studies, grounded by virtual screening, a group of non-toxic
lead-compounds which are characterized by high immunomodulating activity was
chosen. Studied thiazolidon derivates possessed high activity in treating of
cyclophosphan immunodeficiency comparing to the standard therapy (using
polyoxidonium). It was confirmed, that there using leads to increasing of immune system
basic morphological parameters (body weight, thymus and spleen mass and mass index),
activity of phagocytosis, CD3, CD4, CD16, CD22 cells levels in animals with
immunodeficiency. Also, increasing of IgA, IgM and IgG quantity was observed. Highly
active lead-compound (LES 5849) among thiazolidon derivatives based on immunotropic
activity was identified. The data obtained let us to enhance present spectrum of means –
potential drugs with immunomodulating properties.
67
Combinatorial biosynthesis as a strategy of new biologically active
compounds creation
1,2Klymyshin D., 1Makitrynskyy R., 1Gren T., 1,2Nimets O., 2Honchar M.,
1Hrubskyy Y., 1Fedorenko V.
1Ivan Franko National University of L’viv, Hrushevskuy str.4, L’viv, Ukraine; 79005
2Institute of Animal Biology, Stoosa str. 38, L’viv, Ukraine, 79034
dedima@rambler.ru
Actinomycetes are producers of variety antibiotics and other secondary metabolites.
Biosynthetic gene clusters from several antitumor pathways in actinomycetes are presently
being characterized and expressed in order to generate novel drugs. Several methylated and
glycosylated antitumor-drug derivatives have been produced that show a relaxed substrate
specificity for secondary-metabolic enzymes, which opens up the possibility of generating
novel drugs by genetic manipulation. Streptomyces nogalater Lv65 is a producer of a
cytotoxic antibiotic nogalamycin. This antibiotic was reported to be markedly effective
against a variety of gram-positive bacteria and also found to be active against several rodent
tumor models. To understand the regulation of nogalamycin biosynthesis and to facilitate
design of novel anthraciclines we have cloned and characterized several genes that code for
enzymes involved in nogalamycin biosynthesis (snogD, snogZ, snogE , snogY, snogL and
snogM) and regulation (snorA). In our experiments the transcriptional regulator of
nogalamycin biosynthesis encoded by snorA gene has been inactivated within the
chromosome of S. nogalater Lv65. The snorA gene was replaced with the mutated allele that
was generated by insertion of spectinomycin resistance gene cassette aadA at a position
adjacent to snorA start codon. The obtained mutant strain did not produce nogalamycin and
its intermediates. Loss of nogalamycin production caused significant changes in morphology
of the snorA deficient strain. Introduction of snorA gene, encoding a putative antibiotic
regulatory protein, restored nogalamycin production in the mutant strain. We also decided to
express snorA gene in the wild type S. nogalater strain in a multicopy plasmid. Finally, it
was shown that the presence of extra copies of snorA caused increasing in nogalamycin
production compared to that by the wild type. To elucidate the role of snogD, snogZ, snogE
(a putative glycosyltransferases), snogL (a putative O-methyltransferase) genes in
nogalamycin biosynthesis, there disruption was performed within the chromosome of S.
nogalater Lv65 strain. We succeeded in generation of snogD, snogZ, snogE and snogL
disruption mutants and verified them by Southern-blot hybridization. Using the TLC
chromatography new hybrid compounds, probably amethylated and aglycosylated
nogalamycins were observed. In our studies on O-methylation in nogalamycin biosyntheses,
we were interested in the expression of snogY, snogL and snogM genes in heterologous
hosts. This strategy is based on the relaxed substrate specificity of secondary-metabolic
enzymes and the fact that structurally related polyketide drugs share part of their biosynthetic
pathways. In order to caring out such experiments the putative O-methyltransferases were
cloned into pKC1218E plasmid under the control of the erythromycin promoter. The
obtained vectors were transferred into S. echinatus strain (aranciamycin producer) by
conjugation from Escherichia coli. Using the TLC chromatography it was shown that the
expression of snogM gene in S. echinatus leads to the production of a new hybrid compound,
probably O-methylated aranciamycin.
68
Genomics
69
Hepatic gene expression in Prague Hereditary Hypercholesterolemic
(PHHC) rat – a model of polygenic hypercholesterolemia
1,2Zimolová M, 1,2Bohuslavová R, 3Stránecký V, 4Ivánek R, 1Jirsa M, 1,2Poledne R,
1,2Kovář J
1Institute for Clinical and Experimental Medicine, Prague, Czech Republic
2Centre of Cardiovascular Research, Prague, Czech Republic
3Institute of Inherited Metabolic Disorders, 1st Faculty of Medicine
Charles University, Prague, Czech Republic
4Institute of Molecular Genetics, Academy of Sciences, Prague, Czech Republic
mizi@ikem.cz
Most of the hypercholesterolemic patients have a hypercholesterolemia of polygenic
origin and the genes involved in are not well characterized yet. Unfortunately, there are
almost no animal models that could be used for study of polygenic hypercholesterolemia.
Prague Hereditary Hypercholesterolemic (PHHC) rat may be an exception – PHHC rats
develop polygenic hypercholesterolemia (characterized by accumulation of cholesterol in
nonHDL lipoproteins) after feeding cholesterol. Objectives: To determine differences in
hepatic gene expression in the response of PHHC rats and control Wistar rats to the
cholesterol in the diet. Methods: Male PHHC and Wistar rats were fed chow (C) and 1%
cholesterol (CHOL) diet for three weeks. Hepatic gene expression was evaluated using
Affymetrix GeneChip arrays. Results: On CHOL diet, cholesterol and triglycerides
accumulated in the liver of both PHHC and Wistar rats; cholesterolemia rose
significantly only in PHHC rats. Cholesterol feeding resulted in down regulation of
several genes of lipid metabolism – most of the affected genes were genes involved in
cholesterol biosynthesis. Surprisingly, no significant differences in the response of gene
expression to dietary cholesterol were found between both strains (in spite of the fact that
using microarrays we were able to detect expression of more than 6 500 genes in the
liver). On the other hand, we identified several genes that were expressed differently
between PHHC and Wistar rats independently of the diet. Among those genes, Aldh1a7,
Yc2, and Apof genes were markedly up regulated and Ugt2b, Cdh17, Ltc4s, and Slc6a6
genes distinctly down regulated in PHHC rats. Conclusions: The response of gene
expression to dietary cholesterol was affected in PHHC and Wistar rats in the same way.
The development of hypercholesterolemia in PHHC rats involves genes whose exact role
in lipid metabolism remains to be determined.
70
Over expression of eEF1 subunits in human lung carcinomas.
Veremieva M., 1Zakharychev V., Negrutskii B.
Institute of Molecular Biology and Genetics National Academy of Sciences, Kyiv, Ukraine
1P. Schupik Kyiv National Medical Academy of Postgraduate Education UMH, Kyiv, Ukraine
vermarina@list.ru
The impressive list of different translational components whose mRNA or protein levels
appear higher in cancerous tissues as compared with non-malignant ones clearly
demonstrates a link between the translational apparatus and cancer progression. In
particular, the increase in expression of one or another subunit of the translation
elongation factor 1, comprising eEF1A1/2, eEF1Bα, eEF1Bβ and eEF1Bγ components,
in different types of cancer was reported. Unfortunately, almost all previous data
concerning the expression level of a single eEF1H subunit were based on examination of
the mRNA expression. Very narrow information about the cancer-related changes in
corresponding proteins amount is available so any functional consequences of the known
elevation of the level of one or another eEF1B mRNAs in cancerogenesis remained
unidentified. We have investigated the expression of mRNAs coding for eEF1A1,
eEF1A2, eEF1Bα, eEF1Bβ and eEF1Bγ components of eEF1H, and the level of
corresponding proteins in 25 human lung cancer specimens by Northern blot and Western
blot analysis. The increase in expression of one or another subunit of the eEF1H complex
in lung cancer specimens was observed in 44% cases at the mRNA level and in 52%
cases at protein level. Surprisingly, a significant increase of the protein components of
eEF1H and elevated level of mRNAs coding for these components did not coincide in the
majority of tumor specimens. We have concluded that the cancer-related increase in the
level of mRNA coding for translation components is not coupled with the increase in the
level of corresponding proteins in lung cancer. Thus, the elevation of the eF1H subunits
level seems to be controlled by translational or post-translational regulation while the
mRNA rise has no direct effect on the eF1H proteins level and may have additional
regulatory consequences remained to be studied. Moreover, the increases in the amount
of different protein components of the eEF1H complex were found to be unbalanced,
suggesting a specific cancer-related role of individual eEF1H subunits rather than of the
eEF1H complex as a whole. Thus, the stability and regulation of eEF1H complex may be
one of the factors playing an important role in lung carcinogenesis.
The adaptor protein Ruk/CIN85 is involved in the regulation of
hypoxia-induced gene expression
Samoylenko A., 1Dimova E., Marchenko S., 1Kietzmann T., Drobot L.
Palladin Institute of Biochemisry, National Academy of Sciences of Ukraine,
9, Leontovycha Str., Kyiv, Ukraine, 01601
1University of Kaiserslautern, Kaiserslautern, Germany
toljas@yahoo.com
Adaptor proteins, which link protein-binding partners together and stimulate formation of
signalling complexes, play an important role in the regulation of intracellular signalling
pathways. The adaptor/scaffold protein Ruk (also known as CIN85, SETA and CD2BP3)
is a member of a distinct, evolutionary conserved family of SH3-containing proteins.
Ruk/CIN85 was implicated to play a role during carcinogenesis by influencing a number
of processes such as cell adhesion, motility and apoptosis. Though intratumoral hypoxia
is one of the driving forces in cancer progression and a lot of genes involved in
carcinogenesis are induced by hypoxia, there are no data concerning the involvement of
Ruk/CIN85 in the regulation of hypoxia-dependent gene expression during
carcinogenesis. The present study is focused on determining the role of Ruk/CIN85 in
hypoxia-dependent gene regulation using MCF-7 breast adenocarcinoma cells as a
model.
The effects of hypoxia on Ruk/CIN85 expression as well as effects of Ruk/CIN85 over
expression on the hypoxia-dependent gene regulation were examined. By using Western
blot analysis with both monoclonal and polyclonal antibodies against Ruk/CIN85, no
significant changes in its expression under different oxygen concentrations were detected.
However, it was found that in MCF-7 cells, which either stably or transiently over
express Ruk/CIN85, hypoxia-inducible factor-1α (HIF-1α) induction by hypoxia was
significantly higher as compared both to mock-transfected cells. Next, MCF-7 cells were
cotransfected with a luciferase reporter gene construct containing three copies of the
hypoxia-responsive element (HRE) from erythropoietin gene in front of SV40 promoter
and Ruk/CIN85 expression vector. It was shown that in the presence of Ruk/CIN85
luciferase activity was induced both under mild hypoxia and normoxia. It was also shown
that Ruk/CIN85 interfered with the proline hydroxylation-dependent HIF-1α protein
destabilization. The Ruk/CIN85-dependent stimulation of HIF-1α was partially blocked
by the PI3K/Akt inhibitor LY294002 but not the MEK inhibitor U0126. Further,
upregulation of Ruk/CIN85 increased cellular sensitivity to IGF-1-induced and HIF-1
mediated proliferation.
In conclusion, our data show that Ruk/CIN85 is involved in modulation of the effects of
hypoxia in breast adenocarcinoma cells. This work was supported by grants from the
Ministry of Education and Research of Ukraine (№170-2009 and № Ф 28.4/041-2009).
71
72
Proteomics
73
Cysteinyl peptide enrichment in combination with iTRAQ as a tool
for discovering low abundance protein biomarkers
Tambor Vojtech, Lenco Juraj, 1Kacerovsky Marian, Stulik Jiri
Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence
Hradec Kralove, Czech Republic
1Charles University in Prague, Faculty of Medicine in Hradec Kralove, Czech Republic
tambor@pmfhk.cz
Since proteomics was proven to be capable of characterizing a large number of
differences in both protein quality and quantity, it has been applied in various areas of
biomedicine, including the discovery of potential diagnostic biomarkers. However, the
enormous complexity of clinical samples is one of the major stumbling blocks in
biomarker discovery efforts. Numerous earlier projects aimed at biomarker discovery
reported altered concentrations mostly in high abundance proteins. Unfortunately, these
changes are often ambiguous and insignificant, whereas the truly interesting low
abundance proteins remain undetected. In order to provide a deeper insight into the
proteome of clinical samples, several fractionation and separation strategies emerged.
Here we describe a method that combines the fractionation power based on cysteinyl
peptide enrichment (CPE) with the robustness of iTRAQ quantification. First, we used
liquid scintigraphy to optimize the protocol by employing radioactive 35S cysteine-
labeled iTRAQ peptides. This allowed us to fine-tune individual parameters during the
workflow in order to ensure maximum efficacy of the capturing process. The optimized
protocol was then applied onto an iTRAQ-labeled BSA digest to assess the effect of CPE
fractionation on iTRAQ quantitation, which was proven not to be influenced by the
fractionation process. The final phase of the experiment involved application of the
protocol on a complex clinical sample. We analyzed amniotic fluid samples obtained
from patients with preterm premature rupture of membranes (PPROM) with
intraamniotic infection (n=2) as well as from patients with PPROM with ruled out
intraamniotic infection (n=2). Respective samples were combined into two pooled
samples and subjected to immunoaffinity depletion in order to remove ballast proteins
with no diagnostic potential and unmask lower abundance proteins. These were digested
with trypsin and the resulting peptides were subjected to the optimized iTRAQ/CPE
protocol. Peptides from individual fractions were then analyzed using LC-MALDI-
TOF/TOF. First, MS spectra of intact peptides were acquired and based on these data;
suitable peptides were selected, fragmented, and analyzed in MSMS mode. The resulting
MSMS spectra allowed both protein identification and quantification. In total, we
identified 242 proteins at 95% confidence. We proved this method to be able to uncover
interesting differences among the samples. Therefore, a clinical proteomics study
incorporating this method is currently under way.
74
ITSN2 adaptor protein: functional comparison and impact on early
development
Novokhatska O., Tsyba L., Skrypkina I., Nikolaienko O., Dergai O., 1Moreau J.,
Rynditch A.
Institute of Molecular Biology and Genetics NAS of Ukraine, Kyiv, Ukraine
1Institute Jacques Monod, Paris, France
olga.novokhatska@gmail.com
Endocytosis is a vital cellular process that is mediated by machinery of proteins
organized into functional complexes on multimodular scaffolds. One of such
undermembrane scaffolds are proteins of intersectin (ITSN) family. Whereas most
analyses are focused on ITSN1, participating also in cytoskeleton rearrangements, cell
signaling and survival, little is known about ITSN2 high expression level of which
predicts recurrence of breast cancer after chemotherapy. For better understanding of
intersectin function we focused our research on ITSN2. The results on colocalization of
intersectins and endocytic markers evidenced that both intersectins function in the same
cellular compartments. The possibility of intersectins oligomerization was examined and
not detected. Furthermore, investigation of ITSN2 interactions revealed that majority of
protein partners of ITSN2 and ITSN1 are common – dynamin 1, SOS1, Sj, N-WASP, c-
CBL – but the specificities in binding could be observed. Interaction with adaptor
CIN85/Ruk was shown for ITSN1 but not for ITSN2. We found a new protein ITSN
partner, Sema6A, implicated in axon guidance and endocytic adaptors of Reps family.
Thus, ITSN1 and ITSN2 operate in common cell compartments providing similar but
also meaningfully different interfaces for their protein partners from endocytosis,
signaling and cytoskeleton rearrangements processes. Using Xenopus in vivo model and
microinjection technique to study ITSN2 properties we observed the strongest effect of
hyperpigmentation and gastrulation failure in case of over expression of ITSN2 long
isoform C-terminal part (DH/PH-C2 domains). We showed that this isoform is present in
Xenopus oocytes and embryos starting from 2-cell stage and could be involved in early
development. DH/PH tandem is responsible for the observed phenotype as overexpressed
alone it was sufficient to give a similar phenotype. Also we found that membrane-
targeting by CAAX is not necessary for phenotype development but CAAX-tagged form
showed more severe defects presumably due to targeting it to Cdc42. The distribution of
DH/PH was more uniform while DH/PH-CAAX associated mainly with F-actin and cells
formed extensive filopodia. This could explain the effect of hyperpigmentation as the
pigment is located under the membrane and is connected with actin integrity. We
demonstrated that the mechanism of DH/PH action involved activation of Cdc42. Cdc42
constitutively active produced strong hyperpigmentation resembling the effect of DH/PH-
CAAX overexpression. Dominant negative Cdc42 rescued loss of cell-to-cell contacts in
case of DH/PH overexpression and reduced the hyperpigmentation effect of DH/PH-
CAAX. The results obtained suggest a possible role of ITSN2 as a participant (through
Cdc42) of the coordinated changes in actin cytoskeleton during early embryonic
development of vertebrates.
75
Metabolomics
76
Phosphatidylcholine-enriched diet ameliorates the endotoxin-
induced inflammation in the hippocampus and the colon
1,2ErősGábor, 1Tőkés Tünde, 3Várszegi Szilvia, 1Hartmann Petra, 2Bebes Attila,
1Kaszaki József, 3Gulya Károly, 1Boros Mihály
1Institute of Surgical Research, University of Szeged, Szeged, Hungary
2Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary
3Department of Cell Biology and Molecular Medicine, University of Szeged, Szeged, Hungary
egabor@expsur.szote.u‐szeged.hu
Background: Our laboratory has repeatedly shown that orally-administered
phosphatidylcholine (PC) pretreatment exerts significant anti-inflammatory effects in
experimental models of different pathologies. Peripheral endotoxin (ETX) release
contributes to cytokine production and leukocyte accumulation, and induces generalized
inflammation which affects the central nervous system also. Besides, it has been
demonstrated that this reaction is leading to decreased hippocampal neurogenesis via an
interleukin 1ß-dependent signal. Preventive approaches play increasing roles in modern
medicine and functional foods offer potential means for the effective prevention of
different pathological conditions. Therefore, the goal of our study was to examine the
neuroprotective effect of PC-enriched diet in the hippocampus and the colon in a rodent
model of ETX-induced systemic inflammation. Materials and methods: The
experiments were performed on male CD rats (180-230g bw). Inflammation was induced
by 2 mg/kg i.p. ETX; control animals received sterile saline in the same volume. The
control group and one group of ETX-treated animals were nourished with standard
laboratory chow, while another ETX-treated group received a special diet enriched with
1% PC for 5 days prior to the administration of ETX and thereafter during the 7-days
observation period. The subgroups were sacrificed 3 hr, 1 day, 3 days or 7 days after the
administration of ETX, respectively. The rats were treated with bromo-deoxyuridine
(BrdU, 50 mg/kg/day) i.p. during the 7 days of observation. Tissue biopsies were taken
from the hippocampus and the ascendant colon and immunohistochemistry was used to
visualize the BrdU- and doublecortin-positive neuroprogenitor cells and the Iba1-positive
microglia. Biopsies from the colon were examined after hematoxylin-eosin staining as
well. Furthermore, the activities of pro-inflammatory myeloperoxidase (MPO) and
xanthine-oxidoreductase (XOR) enzymes were also determined. Results: The ETX-
caused inflammatory challenge decreased the neurogenesis in the hippocampus and led to
an accumulation of microglia. PC pretreatment prevented these changes and also reduced
the colonic activities of MPO and XOR which are considered as markers of inflammatory
process. Conclusion: Oral PC pretreatment provided protection against ETX-induced
peripheral inflammation and seems to be able to prevent the inflammation-linked
decrease of neurogenesis in the central nervous system.
77
Poster Presentations
78
Cancer Research
&
Prevention
79
Hazardous second hand smoke exposure in hospitals
Tarnoki D. L., Tarnoki A. D., 1Travers M. J., 1Hyland A., 1Dobson K., 2Laszlo T.,
3Mechtler Laszlo L., 1Cummings K. Michael
Department of Radiology and Oncotherapy, Semmelweis University, Budapest, Hungary
1Roswell Park Cancer Institute, Department of Health Behavior, Buffalo, NY, USA
2Department of Otorhinolaryngology, Head and Neck Surgery, Jahn Ferenc Teaching Hospital,
Semmelweis University of Medicine Affiliate, Budapest, Hungary
3Roswell Park Cancer Institute, Department of Neurology, Buffalo, NY, USA
tarnoki4@gmail.com
Objective: Exposure to secondhand smoke (SHS) is a serious threat to public health, and a
significant cause of lung cancer and heart disease among non-smokers. Hungarian hospitals
have been declared smoke free since 2005. However, compliance with this law may not be
100 percent. The purpose of this study was to assess the level of compliance with the smoke-
free hospital law by measuring levels of indoor air pollution in different indoor areas of a
large public hospital in Hungary [1, 2]. Methods: The TSI SidePak AM510 Personal
Aerosol Monitor was used to measure the concentration of particulate matter less than 2.5
microns in diameter (PM2.5) observed in the ambient air of 117 locations sampled, containing
16 health care and 6 non-health care (other) departments, in a large public hospital in
Hungary. The air monitoring was done between January and April 2009. Results: We
observed evidence of smoking such as cigarette butts and ashtrays s in several indoor areas
of the hospital. Air monitory revealed
concentrations of respirable suspended particulates
(RSPs) ranging from 1 to 336 µg/m3 depending on
the area sampled. Clinical departments averaged 83
µg/m3 by average smoker density of 1,73 and the
non-clinical departments averaged 37 µg/m3 with
average active smoker density of 1,71, respectively.
In the rooms where evidence of smoking was not
seen the mean PM2.5 level was 6 µg/m3. In areas
where evidence of smoking was observed the mean
PM2.5 level was 138 µg/m3. The highest levels of
indoor air pollution were found in the Radiology
(336 µg/m3), Intensive Care Unit (230 µg/m3) and
Neonatology (163 µg/m3), respectively (Fig.).
Fig. Average particle pollution by clinical departments
Conclusions: Conclusively, at least in one large public hospital it appears that compliance
with the smoke-free law is incomplete resulting in high levels of indoor air pollution which
pose a health risk to both patients, visitors and staff. It is clear that proper implementation
and enforcement of the legislation that bans smoking in hospitals is imperative to protect the
health of patients and staff alike.
1. World Health Organization. 2008. Report on the Global Tobacco Epidemic. In the MPOWER
Package, 2008. Geneva, Switzerland: World Health Organization.
2. Tárnoki Á, Tárnoki D, Travers M, et al Tobacco smoke is a major source of indoor air pollution
in Hungary’s bars, restaurants and transportation venues. Clin. Exp. Med.J.2009;3(1):131-138.
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80
Primary systemic therapy of breast cancer - our experiences
1,2Székely B, 1Szász A.M, 1Tőkés A.M, 1Szentmártoni Gy, 2Dank M, 1Kulka J
12nd Department of Pathology, Semmelweis University, Budapest
2Department of Diagnostic Radiology and Oncotherapy, Semmelweis University, Budapest
szekely@radi.sote.hu
Purpose: To identify breast cancer subtypes by immunohistochemistry likely to respond
to neoadjuvant chemotherapy and to analyse the used chemotherapy regimen and the
range of response rates. Compare the accuracies of physical examination (PE), breast
ultrasound (US), and pathologic evaluation in tumor and lymph node assessment.
Methods: Analysis of a prospectively collected clinical database was performed. Eighty-
one patients were identified in our files who received neoadjuvant chemotherapy between
1998 and 2009. Prior to neoadjuvant therapy the diagnosis of carcinoma was established
through core biopsy (NCB) and/or fine-needle aspiration biopsy (FNAB) of the primary
tumor and palpable lymph node, if present. We used immunohistochemical profiles (ER,
PgR, and HER2) of FNAB and additionally Ki67 and p53 of NCB and surgical breast
specimens to subclassify the tumors. Pathological response rates were assessed following
surgical removal of tumors by using the Chevallier classification. Tumor and lymph node
measurements by PE and US were obtained before and after neoadjuvant chemotherapy.
Concordance among different clinical measurements was assessed and compared with the
tumor and lymph node staging by pathology. DFS and OS was measured in 68 cases
from the date of definitive surgery to the date of last follow-up or death. Results:
Pathological complete or near-complete remission (pCR = Chevallier I and II) was
observed in 12 of 81 cases (14, 8 %). According to the preoperative characteristics of the
12 tumors achieving pCR 8 of the cases were triple negative, one of 12 was ER-/HER2+
and three of 12 ER+/HER2+. 20 of 81 patients received taxane based neoadjuvant
chemotherapy, 27 of 81 anthracyclin based neoadjuvant chemotherapy, 32 of 81 taxane +
anthracyclin regimen and 2 of 92 CMF regimen. In the taxane treated group of patients
the pCR rate was 30 %, in the anthracyclin group 7% and in the taxane + anthracyclin
treated group 12,5 %. After neoadjuvant chemotherapy, PE correlated better with
pathology than US (p = 0,017 and p = 0,037). The decrease of tumor size after the second
cycle of the chemotherapy was able to predict the pathological response diagnosed after
the final cycle (p = 0,001). Concerning DFS, significant difference was observed between
the Chevallier III and IV group (p = 0,016), and less events were observed in the pCR
group (not significant). pCR was associated with significantly better OS (p=0,033).
Conclusions: It seems that even limited, routinely used immunohistochemical profiling
of tumors is able to predict the likelihood of pCR to neoadjuvant chemotherapy: Patients
with triple negative and HER2-positive cancers are more likely to achieve pCR after
neoadjuvant chemotherapy. Breast US is an accurate imaging study at baseline but PE
correlates better with the pathology finding. US after the 2nd cycle of chemotherapy
predicts the pathological response diagnosed after the final cycle.
81
Metastasis-regulatory microRNAs: one step further
Szasz A. M., Szekely B., Lukacs L. V., Szendroi M., Fillinger J., Soltesz I.,
Hanzely Z., Richardson A. L., Kulka J.
Semmelweis University, 2nd Department of Pathology and Department of Orthopaedics;
Korányi National Institute for Tuberculosis and Pulmonology;
National Institute of Neurosurgery;
Harvard Medical School, Brigham and Women's Hospital
cac@korb2.sote.hu
Despite the advancements, understanding of genetic programs and molecular
mechanisms required for cancer metastasis are still incomplete. Genes that specifically
regulate the process of metastasis are useful tools to elucidate molecular mechanisms and
may become markers for anti-metastatic therapy. Recently, several non-coding regulatory
RNA genes were identified, which play roles in various steps of metastasis. MicroRNAs
are ideally suited to regulate tumor metastasis due to their capacity to coordinately
repress numerous target genes, thereby potentially enabling their intervention at multiple
steps of the invasion-metastasis cascade. In breast cancer, there tend to exist pro- (mir-
10b, -21, -373, -518d, -520c) and anti-metastatic (mir-31, -34, -126, -200b, -205, -206, -
335) microRNAs. We previously identified a microRNA, miR-31, whose expression
correlates inversely with metastatic recurrence in human breast carcinomas. In this study
we aimed to analyze the expression of these molecules: we compared their expression in
primary breast carcinomas (without relapse: 23 cases, and with recurrence: 23 patients)
and their corresponding distant metastatic sites (applicable: 40 samples). As the studies
investigating this topic usually lack this data, because of sample unavailabilty, we are
taking the identified microRNAs under inquisitory inspection.
82
Endocytic adaptors in traffic of latent membrane 2A of Epstein-
Barr virus.
Dergai O., Dergai M., Skrypkina I., 1Gosta W., 1Matskova L., Tsyba L, Rynditch A.
Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine, Kyiv, Ukraine
1Karolinska Institute, Stockholm, Sweden
rynditch@imbg.org.ua
Endocytosis is a fundamental process of membrane vesicle-dependent substrates uptake
and intracellular trafficking. Large number of nutrient, growth factors, messengers and
pathogens enter cells utilizing different types of endocytosis.
Intersectin 1 is an endocytic adaptor protein known to be crucial at the earliest stages of
endocytosis. Moreover, it was shown to control RTK-signaling in concert with c-CBL,
promote actin cytoskeleton nucleation in Cdc42- and NWASP-dependent manner. Often,
different pathogens manipulate hosts mechanisms of endocytosis and signaling to enter
cells and affect cell differentiation and proliferation. Epstein-Barr virus infects epithelial
cells and B-lymphocytes. Viral latent membrane protein 2A (LMP2A) mRNA is
frequently detected in peripheral blood B lymphocytes from healthy individuals and the
protein is often present in tumor biopsies from EBV-associated malignancies. Here we
report about interaction between viral protein LMP2A and endocytic adaptor intersectin1.
The immunoprecipitation data evidence for the complex formation between LMP2A and
ITSN1 in vivo in HEK 293, MCF-7 cells and in B-lymphocyte cell line CBMI. SH3-
domains of ITSN1 are sufficient to precipitate LMP2A in vitro, thus it was supposed that
ITSN1 binds -PXXP- motives of LMP2A. Moreover, another endocytic adaptor -
amphyphisin1 was found to bind -PXXP- of LMP2A through its SH3 domain. Mutational
analysis of LMP2A sequence evidences for at least two binding sites for ITSN1 and
three for Amphiphysin. According to our data, intersectin1 interacts with both isoforms:
LMP2A and -2B, while amphiphysin 1 binds only LMP2A. Amphyphisin1 binds two
sites at the N-terminus of LMP2A that were known to mediate interaction of this viral
protein with ubiquitin-ligases Nedd4-2 and Aip4. Thus, it is tempting to speculate that
amphyphisin 1 competes with mentioned ubiqutin-ligases inhibiting LMP2A-mediated
down regulation of Syk and Lyn. Analysis of subcellular distribution of LMP2A and
ITSN1 in MCF7 cell line supported our data about these proteins interaction in vivo.
Overlapping of the signals of LMP2A and ITSN1 is rather a rare event, LMP2A is co
localized with ITSN1 only in clathrin-coated pits. According to immunofluorescence
analysis we suggest that ITSN1 and LMP2A interact initially during LMP2A
internalization on plasma membrane. Summarizing these data we can propose a model of
clathin-mediated internalization of LMP2A which could be ITSN1-dependent.
Investigation of ITSN1 – LMP2A interaction could provide important clues for
understanding of LMP2A role in EBV cycle in epithelial cells during viral infection and
EBV-dependent cancerogenesis.
83
Immunocytochemical analysis of subcellular localization and content of S6
kinase during cell cycle progression
1,2Kukharchuk V, 1Khoruzhenko A., 1Cherdnyk O., 1Tykhonkova I., 1Filonenko V.
1Institute of Molecular Biology and Genetics, NAS of Ukraine
2Biological Faculty, Taras Shevchenko National University of Kyiv
a.i.khoruzhenko@imbg.org.ua; v.v.filonenko@imbg.org.ua
Introduction. The ribosomal protein S6 kinase is an important component of PI3K signal
transduction pathway. Members of this signaling pathway, including S6K, have been
shown to be either mutated or overexpressed in malignant tumors, therefore inhibition of
some of them is supposed as one of approaches to suppress cancer growth. S6K plays a
key role in the regulation of cell growth by stimulating protein synthesis when growth
factors and nutrients are available. Its activation is initiated by phosphatidylinositide-3-
OH kinase (PI3K)-mediated activation of downstream effector molecules, such as
mTOR, PDK, AKT. There are several substrates of S6K, including rpS6, some
translation and transcription factors and apoptosis related proteins. In mammalian cells
two highly homologous forms of S6Ks are expressed, namely S6K1 and S6K2 that play
both redundant and distinct functional roles. S6K1 and S6K2 have cytoplasmic and
nuclear isoforms. It was shown, that activity of S6Ks increased during cell cycle.
Selective inhibition of S6Ks with neutralizing antibodies led to proliferation delay.
Besides, CCT (Chaperonin containing TCP1 complex) providing folding of important
participants of mitosis (tubulin, cdc 20, PLK1 etc.) is the substrate of S6K. The goal of
our work was to study subcellular localization and content of S6K1 and S6K2 during all
stages of the cell cycle. Materials and methods. Human breast cancer cells MCF-7 were
cultured on the cover glass slides until the cells reached 70% monolayer. Anti-S6K1
rabbit antibodies/anti-Ki-67 mouse antibodies and anti-S6K2 rabbit antibodies/anti-Ki-67
mouse antibodies mixtures were used to detect S6K1 and S6K2 content in proliferating
cells. Furthermore, proliferating cells were detected using Hoechst 33258 that binds to
DNA. The patterns were examined using fluorescent microscopy. The correlation of
qualitative data was detected by tetrachoric test. Results. Our data show, that S6K1 and
S6K2 are localized predominantly in the cytoplasm during interphase, however weak
positive reaction was revealed in cell nuclei as well. It may be explained by a high level
of protein synthesis at this stage of cell cycle. The population of studied cells
demonstrated the same level of intracellular content of S6Ks. Immunocytochemical and
immunofluorescent analyses revealed that the content of S6K1 and S6K2 increased
during mitosis. Some increase of S6K1 and S6K2 content was observed in prophase. In
metaphase, anaphase and telophase the immunofluorescent reaction becomes much more
bright and prominent. The statistical analysis confirmed the correlation between
increased S6K1, S6K2 content and activation of proliferation process. So, the increased
level of S6K1 and S6K2 content has been revealed in proliferating cells that give
additional grounds to study the influence of PI3K inhibitors on the growth of malignant
cells
84
Synthesis and pharmacological screening of novel 4-thiazolidinones
with heterocyclic moieties in molecules
Havrylyuk Dmytro, Mosula Ludmyla, Zimenkovsky Borys, Lesyk Roman
Danylo Halytsky Lviv National Medical University, Pekarska 69, Lviv, Ukraine, 79010
d‐gavrylyuk@ukr.net; dr_r_lesyk@org.lviv.net
Systematic study of various 4-thiazolidinones allowed us to identify series of compounds as
potential anticancer, antiviral and anti-tuberculosis agents. As we established the
combination of thiazolidone and diazole or benzothiazole moieties in molecule is perspective
direction for new "drug-like" structures design, considering significant biological potential
of 4-thiazolidones and the affinity of diazole derivatives (including pyrazolines) to the
following antitumor targets, such as cyclin-dependent kinase, heat shock protein (HSP90),
vascular endothelial growth factor (VEGF) and P-glycoprotein, etc. We obtained the broad
group of 4-thiazolidone derivatives based on chemical modification of thiazolidone cycle in
positions 2, 3, 4 and 5. For realization of synthetic schemes the various types of reactions,
such as Knoevenagel condensation, [2+3]-cyclocondensation, diazotization, reaction of N-
alkylation, acylation, etc were used. Biological activity screening is conducted within the
framework of scientific programs of National Institutes of Health (Bethesda, USA): DTP of
NCI for anticancer activity, TAACF for anti-tuberculosis activity and AACF for antiviral
activity of NIAID. Anticancer activity assays of more then 110 compounds allowed us to
identify 3 lead-compounds, which currently are under in-depth in vivo studies according to
decision of NCI Biological Evaluation Committee. As a result of anti-tuberculosis assays we
identified one lead-compound which was selected for in-depth in vivo study. Antiviral
activity screening allowed us to identify some “hit-compounds” which showed moderate or
high activity against influenza virus, corona viruses and Tacaribe.
S
N
O
R1
R2
O
N
O
N
Ar
Ph
S
N
H
O
X
N
N
Ar S
N O
Ph
R1
R2
N
N
Ar
S
N O
Ph
R2
R1
S
N
H
O
ON
O
N
Ar
Ph
S
N
O
R1
R2
S
N
H
N
N
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R3
O
N
S
N
SO
O
S
N
O
R1
R2
N
H
S
N
CH3
X = S, O
63 compounds 78 compounds
13 compounds
15 compounds 15 compounds
70 compounds
Main directions of design of 4-thiazolidones with related moieties
NCI DTP Program
(anticancer activity)
Tested 118 compounds
Selected 12 "hits" in vitro
Selected 3 compounds for
in-depth study
as drug-candidates
Tested 52 compounds
Selected 4 "hits" in vitro
Selected 1 "lead"
compound
Tested 21 compounds
Selected 5 "hits"
NIAID TAACF Program
(anti-tuberculosis activity)
NIAID AACF Program
(antiviral activity)
Pharmacological research
Docking studies have shown that the most probable biotarget for anticancer compounds is
Bcl-XL-BH3 protein complex. QSAR modeling resulted into the row of predictive linear
regression models of “anticancer activity” / “molecular descriptors” relationships, which
could be used for preliminary prediction of antitumor activity for structurally related
compounds.
85
Lack of pttg-1 gene can cause myelodysplasia and autoimmune
disorders
Filyak Yevhen, Kanyuka Olena, Afanasyef Serhiy, Stoika Rostyslav
Institute of Cell Biology
14/16, Drahomanov Str., Lviv, Ukraine, 79005
yevhenfilyak@cellbiol.lviv.ua
Pituitary tumor transforming gene-1 is known to induce malignant transformation of
pituitary cells and found to be over expressed in many types of tumors. Pttg-1 is also
believed to be involved in activation of anaphase-checkpoint during the mitosis.
Knockout of pttg-1 gene caused thrombocytopenia and disproportion of CD4/CD8
lymphocytes in blood. In human, pttg-1 gene is located in 5q35.1 (Entrez Gene, HGNC)
or 5q33.3 (Ensembl) locus. Deletion of q arm of 5th chromosome, in human leads to
myelodysplasia 5q syndrome. This syndrome is characterized by myelodysplastic
changing in blood cells concentration and development of autoimmune disorders.
Patients with 5q syndrome often die from cancer. It is known that 5q-syndrome develops
if deleted 5q31-33 locus, but the molecular explanation of the importance of 5q31-33
locus in development of 5q syndrome was not revealed. We showed that deletion of pttg-
1 gene can induce myelodysplastic changes and autoimmune disorders in mice similar to
5q syndrome in human. In particular, we found that pttg-1 knockout in mice leads to
decrease in erythrocytes, thrombocytes and neutrophils number in the blood. However,
lack of this gene caused increase in erythrocytes degradation and elevation of bilirubin
concentration in the blood, accompanied with compensatory 100% increase in
erythrocytes production and doubling number of young erythrocytes in the blood. We
also found that blood of pttg-1-knockout mice contain elevated level of anti-dsDNA
antibodies, while amount of anti-ssDNA and antibodies against simple negatively
charged compounds were not elevated. We discovered, that in pttg-1 knockout mice anti-
dsDNA antibodies are increased owning to 200% elevation of low-affinity anti-dsDNA
antibodies, while the level of high affinity anti-DNA antibodies was comparable to that in
the blood of wild type mice. Investigation of cancer receptivity of pttg-1 knockout mice
is in progress.
Summarizing, obtained by us results suggest the deletion of pttg-1 gene to cause
autoimmune disorders and myelodysplasia symptoms in mice, similar to 5q syndrome in
human. The obtained data drives at functional connection of pttg-1 deletion and
development of 5q syndrome in human.
86
Novel expression platform of human arginase I as anticancer
enzyme.
Krasovska O.S., Mayevska O. M., Vynnytska B.O., Stasyk O.V.
Department of Cell signaling
Institute of Cell biology, National Academy of Sciences of Ukraine
krasovska@cellbiol.lviv.ua
To meet the expected practical needs, many foreign proteins of medical and
biotechnological importance have to be expressed in a heterologous host. Often this is not
a straightforward task. One of such examples is recombinant human liver arginase I
(rhARGI). This enzyme is of great interest for several practical applications but is not
currently commercially available due to the absence of efficient producers and
purification protocols. First of all, rhARGI is potentially the best enzyme for the
development of non-toxic, selective and efficient anticancer enzymotherapy based on
single amino acid, arginine, deprivation. We recently developed a novel yeast-based
expression system for secreted rhARGI and started the testing of different protocols for
its purification to homogenous state. As a heterologous host for rhARGI expression we
have chosen the methylotrophic yeast Hansenula polymorpha, which is known as an
efficient expression platform for human and other eukaryotic proteins. As a eukaryotic
host, H. polymorpha provides correct folding, little unwanted posttranslational
recombinant protein modifications, like hyperglycosylation, little secretion of own
proteins and produces virus-free products. We also designed a set of constitutive and
regulatable promoters and vectors for stable, including multicopy, integration into H.
polymorpha genome that do not rely on antibiotic-resistance markers (this is important
for bio-safety reasons). Cultivation conditions for yeast producers were optimized that
provide stable accumulation of the secreted rhARGI in the culture medium.
Enzymatically active purified rhARGI preparations have been obtained on a laboratory
scale. Testing of our rhARGI preparations in vitro on the models of cultured human
cancerous cell lines (as hepatocarcinoma HepG2 sensitive to arginine deprivation)
revealed that the enzyme produces strong antiproliferative and proapoptotic effect.
Interestingly, rhARG1 appeared to be an efficient antitumor enzyme also in combination
with canavanine, arginine analogue of plant origin known as antineoplastic agent.
Summarizing, our data suggest that rhARGI heterologously produced in yeast is an
effective antitumor agent and can be further developed as an agent of novel
combinational enzymotherapies.
87
Arginine deprivation induces autophagic process in SKOV3 human
ovarian carcinoma cells
Shuvayeva G.Y., Bobak Y.P., Igumentseva N.I., Stasyk O.V.
Department of Cell signaling, Institute of Cell biology, NAS of Ukraine, Lviv, Ukraine
shuvayeva@cellbiol.lviv.ua
It is known that some tumor cells have an elevated requirement for exogenous arginine (ARG) in
vitro and in vivo, the reason for which is not clearly understood. This feature has been exploited
in the development of anticancer ezymotherapy based on ARG deprivation. We hypothesized
that upon amino acid restriction, the process of autophagic protein turnover may affect cells
response and fate. To address this question, we developed a model of cultured SKOV3 human
ovarian carcinoma cells. Using monodancylcadaverine (MDC), a classical fluorescent dye that
selectively labels autophagosomes and autolysosomes, we revealed that ARG deprivation
induces process of autophagic degradation in SKOV3 cell already after 30 min of starvation.
Treatment with classic inhibitors of autophagy, 3-methyl adenine (3MA) and asparagine (Asn),
abolished the fluorescent signal. We also monitored the appearance of autophagolysosomes,
characteristic of the last stage of autophagic process, using immunofluorescent staining of the
autophagosome proteins MAP LC3 and Beclin1 as well as lysosome membrane proteins LAMP1
and Golgin97. The colocalization of MAP LC3/LAMP1 and Beclin 1/Golgin 97 signals was
observed already at 30 min of ARG deprivation, what is the strong evidence for induction of
autophagy (Fig. 1A). Colocalization of these proteins was not detected upon treatment with
different autophagy inhibitors (3MA, Asn, and chloroquinone (CQ)). We also observed
accumulation of MAP LC3 protein upon treatment with autophagy inhibitors (Fig. 1B). SCOV3
cells were resistant to classical apoptosis upon ARG deprivation as suggested by the absence of
the cleavage of caspase-3 into its activated 17 kDa fragment, even upon treatment with
autophagy inhibitors (Fig. 1B).
Fig.1. ARG depletion rapidly induces autophagy in SKOV3 cells:
A - Immunofluorescence staining of MAP LC3 and Beclin 1 (green fluorescence) as well as
LAMP1 and Golgin 97(red fluorescence) in SKOV3 cells upon ARG starvation. Nuclei labeled
with DAPI. B – Immunoblot of the lysates of SKOV3 cells incubated in ARG-free medium
(AFM) using polyclonal MAP LC3 and caspase3 antibodies.
Our data suggest that single amino acid, ARG, deprivation strongly induces autophagic response
in the ovarian carcinoma SKOV3 cells. Autophagy may serve as a protective mechanism upon
such conditions providing the additional recycling of ARG from preexisting proteins. It remains
to be elucidated whether there is a positive correlation between autophagy induction and survival
of different tumor cells upon ARG deprivation. We suggest that ovarian carcinoma SKOV3 may
serve as a suitable model for studying the role of autophagy modulation in anticancer therapy.
A
0,5 4 8 control
IF
:
LC
3/
LA
M
P1
IF
:
B
ec
lin
1/
G
97
Arginine deprivation, h
88
Over expression of adaptor protein Ruk/CIN85 decreases estrogen
sensitivity and promotes resistance to TNF-α-induced apoptosis of
breast adenocarcinoma MCF-7 cells
Fedoseienko A., Samoylenko A., Byts N., Kozlova N., Drobot L.
1Palladin Institute of Biochemisry, National Academy of Sciences of Ukraine
9, Leontovycha Str., Kyiv, Ukraine, 01601
alifed@ukr.net
Ruk/CIN85 is a SH3-containing adapter/scaffold protein that plays important roles in the
regulation of homeostasis in normal cells and also can be involved in carcinogenesis by
influencing a number of processes such as cell adhesion, motility and apoptosis. Our
previous findings suggest that high levels of Ruk/CIN85 modulate EGF-dependent
signalling and contribute to the conversion of breast adenocarcinoma MCF-7 cells into a
more malignant phenotype. Since estrogens are critically involved in the development of
breast cancer, it was the aim of the current study to compare sensitivity of MCF-7 cells
with different levels of stable Ruk/CIN85 expression to β-estradiol treatment. Mock-
transfected MCF-7 cells as well as sublines that stably over express either low (D4) or
high (G4) levels of Ruk/CIN85 were treated with β-estradiol (20 and 100 pM). MCF-7
cell viability was analyzed by MTT-test after three days of β-estradiol stimulation. In
concordance with previous data, β-estradiol in our study increased proliferative activity
of MCF-7 cells in a concentration-dependent manner. However, β-estradiol sensitivity of
cell sublines that stably over express Ruk/CIN85 was lower as compared to control MCF-
7 cells. There are data that Ruk/CIN85 plays a role in control of apoptosis induced by
TNF-α treatment. Therefore, another aim of the study was to investigate whether over
expression of Ruk/CIN85 affected MCF-7 sensitivity to TNF-α-induced apoptosis.
Control MCF-7 cells and three MCF-7 sublines with different levels of Ruk/CIN85 over
expression (D4, G4, and G10) were cultured for 3 days in presence of TNF-α (5, 10, 50
ng/ml). Cell viability was analyzed by MTT-test. It was shown that TNF-α decreased
viability of control as well as D4, G4, and G10 subline cells in a concentration-dependent
manner but viability of Ruk/CIN85 over expressing cells was higher in comparison with
control cells. Apoptotic cell death was determined by increase of cleaved PARP levels.
For this purpose, control MCF-7 cells and D4, G4, G10 sublines were cultured in the
presence of TNF-α (10 ng/ml) for three days. The cell lysates were analyzed by Western
blotting using antibodies against cleaved and intact PARP. The results of Western blot
analysis correlated with MTT test results: the highest accumulation of cleaved PARP was
detected in control cells and the lowest - in G10 subline cells. Taken together, our data
show that over expression of Ruk/CIN85 in MCF-7 cells is accompanied by attenuation
of estrogen sensitivity and increased resistance to TNF-α-induced apoptosis.
89
The role of inositol requiring enzyme-1α signaling in brain tumor
growth and gene expression
Minchenko O. O., Hubenya O.V., Terletsky B.M., Minchenko D. O., 1Moenner M.
Department of Molecular Biology, Palladin Institute of Biochemistry
National Academy of Science of Ukraine
9, Leontovich Str., Kyiv, Ukraine, 01601
1INSERM U920 Molecular Mechanisms of Angiogenesis Laboratory
University Bordeaux 1 (Talence, France);
ominchenko@yahoo.com
Astrocytes represent the most abundant cell type in mammalian brain, and play an
important role in the maintenance and regeneration of neuronal functions. Inadequate
responses of astrocytes to ischemic conditions may contribute to the development of
various pathologies of the nervous system. Hypoxia and glucose deprivation, which are
essential features of ischemia and chemicals have been shown to induce a set of complex
intracellular signaling events known as the Unfolded Protein Response (UPR). This
adaptive response is activated upon accumulation of misfolded proteins in the
endoplasmic reticulum (ER) and is mediated by three ER-resident sensors named PERK
(PRK-like ER kinase), IRE1/ERN1 (Inositol Requiring Enzyme-1) and ATF6 (Activating
Transcription Factor 6). Activation of the UPR tends to limit the de novo entry of
proteins in the ER and facilitate both ER protein folding and degradation. Cells’ ability to
handle these stresses may therefore condition their intrinsic capacity to adapt for cell
survival or, alternatively, to enter cell death programs through ER-associated
machineries. IRE1 activation and signaling is a common molecular determinant linking
hypoxia- and hypoglycemia- dependent responses to the up-regulation of several pro-
angiogenic and neurotrophic factors, including vascular endothelial growth factor,
fibroblast growth factor 2, hepatocyte growth factor, CSF1R, tissue factor, death receptor
6 and DATF1, in various cell types including glioma-derived cells. IRE1 is a single-
spanning ER transmembrane protein that expresses both intrinsic Ser/Thr protein kinase
and endoribonuclease activities in its cytosolic domain. Astrocytes subjected to ischemia
trigger distinct signaling pathways contributing to angiogenic stimulation, neuronal
stabilization and survival. The blockade of IRE1 ser/thr kinase and RNase activities is
thought to either lead to a decrease in survival signals between astrocytes and neuronal
cell, as suggested by transcriptomic analyses, or/and triggered cytotoxic signaling
towards neuron-type cells. The blockade of IRE1 RNase activities precipitates U87 tumor
cells to death after only a few passages in culture. We determined a set of genes which
expression depends from IRE1 RNase activity and possibly involved in death processes.
Thus, the evaluation of respective roles of the two intrinsic catalytic activities (kinase and
endoribonuclease) of IRE1 in its signaling is very important in order to get chemical
inhibitors that block IRE1 as a whole or only on its RNase activity.
90
Circadian and 6-phosphofructo-2-kinase/fructose-2,6-
bisphosphatase genes expression in glioma cells: effect of hypoxia
and nutrient starvation
1Terletsky B. M., 1Minchenko D. O., 1Lypova N. M., 1Bozhok I. V., 1Marunych R.Y.,
2Moenner M., 1,2Minchenko O. H.
1Department of Molecular Biology, Palladin Institute of Biochemistry NAS of Ukraine
9, Leontovycha Str, Kyiv, Ukraine, 01601
2INSERM U920 Molecular Mechanisms of Angiogenesis Laboratory, University Bordeaux 1
Talence, France
terletskiy@max‐well.com; ominchenko@yahoo.com
Hypoxia and glucose deprivation, which are essential features of ischemia, have been
shown to induce a set of complex intracellular signaling events known as the Unfolded
Protein Response (UPR). This adaptive response is activated upon accumulation of
misfolded proteins in the endoplasmic reticulum and is mediated by Inositol Requiring
Enzyme-1 (IRE1) as well as by two others endoplasmic reticulum-stress sensors: PRK-
like ER kinase (PERK) and Activating Transcription Factor 6 (ATF6). We studied effect
of hypoxia and nutrient (glucose or glutamine) starvation on the expression of several
circadian and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase genes in U87 glioma
cells and its subline without IRE1 ser/thr kinase and ribonuclease activities using real
time polymerase chain reaction. We have shown that the expression of Clock, BMal2 and
Per3 was decreased in hypoxic conditions in glioma U87 cell line. However, expression
of BMal1b, BMal1e, Per1 and casein kinase-1 epsilon circadian genes as well as 6-
phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and 6-phosphofructo-2-
kinase/fructose-2,6-bisphosphatase-4 was increased in this line of glioma cells in hypoxic
conditions. The blockade of IRE1 ser/thr kinase and ribonuclease activities is lead to an
increase of most circadian genes (Clock, BMal1b, BMal1e, Per1, Per2, Per3 and casein
kinase-1 epsilon) and PFKFB genes (PFKFB-3, PFKFB-4 and PFKFB-2), but to a
decrease of BMal2 and casein kinase-1 delta. Glutamine or glucose deprivation
conditions are lead to suppression of the expression of 6-phosphofructo-2-
kinase/fructose-2,6-bisphosphatase-3 and 6-phosphofructo-2-kinase/fructose-2,6-
bisphosphatase-4 in glioma cells without IRE1 kinase and ribonuclease activities only.
Expression of circadian genes casein kinase-1 epsilon, BMal1b and BMal1e was
increased both in U87 glioma cells and in its IRE1-negative subline but expression of
BMal2 was decreased in these cells. However, the expression of circadian genes Clock,
BMal1b, BMal1e and Per1was increased only in the cells without kinase and
ribonuclease activities in glutamine deprivation conditions. The blockade of IRE1 kinase
and ribonuclease activities in U87 glioma cells was induced expression of Clock, Per1
and BMal1b under glucose deprivation conditions. Thus, the expression of different
circadian genes is depended from inositol requiring enzyme-1 signaling as well as from
nutrient (glucose or glutamine) starvation.
91
Cardiovascular Diseases
&
Prevention
92
F2 G20210A, F5 G1691A, MTHFR C677T polymorphisms –
involvement in stroke
1,2Tatarskyy P., 1,2Kucherenko A., 1Kravchenko S., 3Shulzenko D., 3Kuznetsova S.,
1Livshits L.
1Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine
Department of Human Genomics
2Taras Shevchenko Kyiv National University, Faculty of Biology
3Gerontology Institute of the Academy of Medical Sciences of Ukraine
livshits@imbg.org.ua
Stroke is considered to be a complex polygenic disorder arising from a wide number of
gene–gene and gene–environment interactions. The F2 G20210A and F5 G1691A
mutations are the most common genetic risk factors of venous thrombosis and ischemic
stroke. F2 G20210A is associated with higher plasma prothrombin concentrations and
augmented thrombin generation. F5 G1691A provokes a structural change of a factor V
molecule and shifts the balance toward thrombosis in the clotting cascade. The MTHFR
C677T gene variation is associated with 30-50% reduction of the enzyme activity and is
the most common inherited cause of hyperhomocysteinemia, which increases the risk of
ischemic stroke. Aim of the study was to establish possible involvement of the F2
G20210A, F5 G1691A, MTHFR C677T genes alleles into stroke development. Unrelated
individuals from different regions of Ukraine were included in this study: case group -
patients with ischemic stroke (n=183, men-95, women-88), average age – 64.6±9.1,
control group I - individuals from the general population (n=100, men-50, women-50),
average age – 30.2±7.6, control group II – healthy individuals elder than 65 years (n=88,
men-35, women-53), average age – 73.9±6.4. No significant difference between the age
of men and women within each group was observed. Blood samples for DNA analysis
were obtained after informed consent. Genotyping was performed by PCR followed by
RFLP analysis. The differences were considered significant at P<0.05 value of Fisher
exact test. F2 20210A carriers frequency in the case group (4.6%) was significantly
higher than in control group II (0%) and was higher in men (7.4%) comparing to women
(1.1%) in the case group. Frequency of genotype with at least one risk allele of MTHFR
677T was higher in women from the case group (54%) than in women from control group
II (41%). Frequency of combined genotype with at least one of the risk alleles of F2 or
F5 or bearing two copies of the MTHFR 677T frequency in men from case group (20%)
was higher than in men from control group II (5%). In women from case group (57%)
frequency of the combined genotype with at least one of F2, F5, MTHFR genes risk
alleles was higher than in women from control group II (41%). Our findings suggest a
relevant role of F2 20210A, F5 1691A, MTHFR 677T alleles in stroke development.
This study was supported by the NASU and the RFFR grants.
93
Vasculometry of upper and lower extremities in correlation with
development of pathologic conditions like the diabetic foot
Nikolić Vasilije, Radić Radivoje, Selthofer Robert, 1Mišević Tonči,
2Dmitrović Branko, Šnajder Darija
School of Medicine, University J. J. Strossmayer Osijek, Croatia
Department of Anatomy and Neurosciences,
1Department of Radiology, Clinical Hospital Center Osijek, Croatia
2Department of Pathology, School of Medicine, University J. J. Strossmayer Osijek, Croatia
dsnajder@mefos.hr
We assume that the vascular apparatus of the lower limb did not evolve to adapt to leg
mass and volume. The lower limb is greater in length and volume than the upper limb,
and therefore the arteries should have a bigger diameter and cross-sectional area. During
pathoanatomic autopsies on the Department of Pathology of Clinical Hospital Center
Osijek we have taken segments of 1 cm of length from the subclavian, femoral, radial and
tibial artery. Our sample contained segments from 51 bodies, 24 female and 27 male. We
have measured leg and arm length and circumference. From these data the idealized
limbs volume was calculated by geometric approximations to a cone fragment. The
relation between idealized leg and arm volume and arterial cross-sectional area were
calculated. For statistical analysis, Student’s t-test was used. There is a slight difference
between the diameter and cross-sectional area of subclavian and iliac (femoral) artery.
Leg length was for 48.5% bigger than arm length and the difference in volume between
upper and lower limb is significantly different. The foot has four to five times greater
volume than the arm, and is vascularised by an arterial tree of similar diameter. This fact
proves our hypothesis that the blood supply to the lower limbs compared to the mass of
tissue is smaller.
94
Remote monitoring of single lead implantable defibrillator holders
Németh M., 1Miklós Z. S., 2Abrahám H., Szabados S., Melczer L.
Heart Clinic, Faculty of Medicine, University of Pécs, Hungary
1Department of Surgical Research and Techniques, University of Pécs, Hungary
2Central Electron Microscope Laboratory, University of Pécs, Hungary
nemethmarianna85@gmail.com
Lack of direct discrimination of atrial and ventricular rhythm disturbances is the major
disadvantage of VVI-ICD. Remote monitoring is a goal of modern ICD follow-up.
The aim of this study is to analyze the usefulness and reliability of single lead
implantable cardioverter defibrillators (VDD-CD) with home monitoring system (HM).
Patients and methods: 18 patients - 12 males, mean age: 58,9 ± 14,9 years, ejection
fraction: 36,33±12% - underwent single-lead VDD-CD (Biotronik Lexos A+T)
implantation between June 2007 and December 2009 in our clinic. All patients were
monitored for 14,7 ± 8,3 months, mean clinical follow-up was 3,11 ± 2,29. HM events
(supraventricular tachycardia (SVT), ventricular tachycardia (VT), ventricular fibrillation
(VF)) were analyzed. Results: in about 44, 4% (n=8) of the patients 911 HM events (747
SVT, 116 VT, 48 VF) were detected. Antitachycardia pacing (ATP) was used in 195 and
cardioversion (CV) in 83 cases. 93 ATPs were successful, 102 were inadequate. Out of
34 started CV with low energy following failed ATPs, 24 were successful, 9 were failed
and 1 was aborted. The 9 failed CV occurred in 1 patient, and the reinitiation of the
arrhythmia was stopped due to the repeated ATP or CV. 49 high energy shocks were
initiated due to VF, and 13 of them were successful. 35 detected VF were terminated
spontaneously, 1 shock was ineffective. Conclusion: The method of implantation of
VDD-CD is equal with that of VVI-CD. Atrial sensing ensures a direct discrimination of
atrial arrhythmias, decreases the number of false detection and therapy. In patients with
atrio-ventricular block either AV conduction with long PR interval, or P wave triggered
ventricular stimulation is possible. Remote monitoring allows early detection of
arrhythmias and shortens physicians’ reaction time from their development to therapy.
The VDD-CD with HM option is a milestone in the follow-up of patients living with
ICD.
95
Effects of postconditioning on kidney ischemia/reperfusion injury in
hypercholesterolemic rats
Miklós Zsanett, Kürthy Maria, 1Jancsó Gábor, 2Degrell Péter, Ranczinger Eszter ,
Lantos János, 1Sinay László, 1Arató Endre, Horváth Szabolcs, Németh Marianna,
Ferencz Sándor, Wéber György, Rőth Erzsébet
University of Pécs Medical School, Department of Surgical Research and Techniques
1Baranya County Hospital, Department of Surgery
2University of Pécs Medical School, 2nd Department of Internal Medicine and Nephrology Center
miklosz23@yahoo.com
Background: Ischemia/reperfusion injury frequently threats the integrity of the organs
during surgery. The protective effect of postconditioning (PK), the short repetitive
ischemia/reperfusion cycles, applied in the beginning of reperfusion, has been improved
the outcome in vital organs. Signaling cascades are induced by PK interfere in several
points to preconditioning, which is blocked by metabolic diseases, such as insulin
resistance and type 2 diabetes. The aim of our study was to compare the efficacy of PK
after reperfusion injury of both kidneys in metabolically healthy and
hypercholesterolemic rats. Methods: Male Wistar rats (n=30) were fed by either normal
or high cholesterol (1.5%) containing chow for 8 weeks. Following pretreatment period,
both groups were divided into two subgroups, where under general anesthesia both
kidneys were exposed either to 45 min ischemia followed by 2 hours of reperfusion, or to
additional 4x15 sec postconditioning cycles. Serum creatinine, carbamide, oxLDL levels,
PMA-induced free radical production was determined before and after surgery. After
euthanasia both kidneys were removed for TNF- alfa immunohistochemistry, and for
PAS and HE staining. Results: Cholesterol feeding resulted in a significant elevation in
serum cholesterol and triglyceride levels (p<0.05). In the control rats significant elevation
was observed in free radical production (p<0.01), lipid peroxidation (p<0.01), and serum
TNF-alfa levels (p<0.05), following ischaemia and reperfusion, which were markedly
reduced by post conditioning. However, we did not reveal any beneficial effect of post
conditioning in the cholesterol fed rats. Tissue TNF- alfa level was significantly higher in
cholesterol fed, than in control animals, and this high level did not change in response to
post conditioning. In healthy animals post conditioning caused a significant reduction
(p<0.05) in tissue TNF- alfa level. Conclusions: PK proved to be a very effective
defense against I/R in healthy animals, but it was ineffective in hypercholesterolemic
ones.
96
Comparison of red wine, beer and vodka effects on oxidative stress
and increase in arterial stiffness after normobaric oxygen breathing
in healthy humans
Modun Darko, 1Krnic Mladen, Budimir Danijela, Gunjaca Grgo, Jajic Ivan,
Vukovic Jonatan, 2Kozina Bernard, Boban Mladen
Department of Pharmacology, University of Split School of Medicine
Soltanska 2, 21000 Split, Croatia
1Department of Endocrinology, Internal Medicine Clinic, University Hospital Center
Split, Croatia
2Department of Viticulture and Enology, Faculty of Agriculture
Svetosimunska 25, University of Zagreb, 10000, Zagreb, Croatia
modun@mefst.hr
Introduction: We determined and compared acute effects of different alcoholic
beverages on oxygen-induced increase in oxidative stress plasma marker and arterial
stiffness in healthy humans. Materials: Ten males randomly consumed one of four tested
beverages: red wine (RW), beer, vodka (0.32 g ethanol / kg body wt) and water as
control. Every beverage was consumed once, a week apart, in a cross-over design. The
volunteers breathed 100% normobaric O2 between 60th and 90th min of 3h study
protocol. Plasma lipid hydro-peroxides (LOOH) and uric acid (UA) concentration, blood
alcohol concentration (BAC) and arterial stiffness (evaluated as augmentation index,
AIX) were measured before and 30, 60, 90, 120 and 180 min after beverage
consumption. Results: Intake of all alcoholic beverages caused a similar increase of
BAC. In contrast to that, only RW caused significant increase in plasma UA (34±4 vs.
15±3, -6±2 and -8±2 µmol/L for, beer, vodka and control, respectively). Exposure to
oxygen resulted in increased plasma LOOH in all groups. However, in RW group this
increase was lowest (1.1±0.5) in comparison to the control (2.5±0.4), beer (1.6±0.3) and
vodka (2.1±0.5 µM/L H2O2). The oxygen-induced elevation in AIX was similarly
reduced in all three alcoholic beverage groups relative to the control (13.7±2.6 vs.
3.4±1.3, 0.2±1.6 and 5.4±2.2 % for red wine, beer, and vodka, respectively). Conclusion:
RW provided protection against oxygen-induced oxidative stress, in contrast to beer and
vodka. This beneficial effect was mainly mediated by corresponding increases in plasma
UA levels. All three alcoholic beverages provided similar protection against oxygen-
induced increase in arterial stiffness, probably due to central vasodilatatory effect of
alcohol itself.
97
Mother and Child Health
Reproductive Health
98
PAPP-A as an early marker for Fetal Growth
Ceauşu Iuliana, Toderici Ina, Lazăr Virginia, Chelu Roxana, Poalelungi C.,
Bacalbaşa N., Hudiţă D
“CAROL DAVILA”, University of Medicine and Pharmacy
Department of Obstetrics and Gynecology,
“DR. I. CANTACUZINO” Hospital, Bucharest, Romania
iulianaceausu2004@yahoo.com
Objective: Determination of pregnancy associated plasma protein (PAPP-A) and beta-
hCG for bi-test evaluation in week 11w0d-13w6d may help in predicting an accelerate
growth later in pregnancy and a Large for Gestational Age (LGA) or macrosomia at birth.
Method: Prospective study on 193 patients who made the bistest between 11w0d-13w6d
and weight at delivery. LGA was defined as a weight over 90 percentile for gestational
age and macrosomia as a weight over 4000 grams at term (over 37 gestational weeks).
Statistical correlation was made for Mom and values in MoM (calculated by PRICA
software) and in mUI/mL, and the statistical power was evaluated by Mann-Whitney test
is SPPS 12 ROC curves. Results: PAPP-A values were significantly increased in women
who delivered both macrosmic and LGA babies (n=23/131, 1.12 MoM; p=0.036). The
cutoff value for an increase for fetal accelerate growth was 5.41 mUI/mL. Association
with other pregnancy complications as intrauterine growth retardation, small for
gestational age, pretermbirth and gestational diabetes are under analasys.
Conclusion: Level of PAPP-A in maternal serum can be an early predictive factor of
fetal growth.
99
Complex role of ultrasound and biochemical markers for prenatal
diagnosis of chromosomal abnormalities
Lototska-Savchak O, 1Huleyuk N., 2Beskorovaina H.
Lviv National Medical University named after D.Halytsky, Ukraine
1State institute “Institute of Hereditary Pathology of AMS Ukraine”, Lviv, Ukraine
2Interregional medical genetic center, Lviv, Ukraine
doctor‐lototska@mail.ru
Introduction: Prenatal study is used to identify major genetic and congenital
abnormalities in a developing fetus. Prenatal karyotyping was performed in women with
high risk of children’s birth with chromosomal abnormalities. Actual problem for today
remains to determine the most specific clinical and laboratory markers to the
chromosome abnormalities. Aim: Establish communication between the deviations of
indicators of ultrasound and biochemical examinations of pregnant women and
established prenatal chromosomal abnormalities. Materials and methods: Cytogenetic
analyses of the chromosomes from amniocyte in vitro culture were performed according
to standard protocols. Ethidium bromide (10mcg/mL) was added simultaneously with
colchicines in order to obtain high-quality chromosomes of early and middle mitotic
stages. GTG and CBG banded chromosomes were analyzed at the 550 bands resolution
level. Analyzed the frequency of detected chromosomal abnormalities depending on
indications to the amniocentesis. Results: Prenatal study have been performed in
pregnancies after genetic counselling in the next cases: changes in maternal serum
markers during first/second-trimester; ultrasonic markers of congenital malformations;
family history of stillbirth, recurrent spontaneous abortions, affected child with
congenital birth abnormalities; advanced maternal age >35 year; previous child with a de
novo chromosomal abnormality; presence of structural chromosome abnormality in one
of the parents. 74 prenatal cytogenetic studies have been performed. In 14 amniocyte
cultures (18,9%) numeral (9 cases) and structural (5 cases) changes of the karyotype were
detected. In 11 cases unbalanced chromosomal abnormalities was observed: trisomy of
21-st and 18-th chromosome; simple and mosaic forms of gonosomal monosomy;
additional marker chromosomes. Non-balanced derivative chromosomes were detected in
two fetuses — 46,XY,der(4)(?::p16 q35)de novo and 46,XY,der(10), t(10;7)(q26,13;
p21.2)pat. High level of unbalanced chromosomal abnormalities was confirmed in
fetuses from pregnancies with ultrasound markers of congenital malformations – 16.6%
and maternal serum markers – 13.3%. The highest percentage established prenatal
unbalanced chromosomal abnormalities – 33.3%, observed deviation of ultrasound and
biochemical markers simultaneously. Conclusion: Therefore, the combination of
ultrasound and biochemical studies of pregnant women is most effective for establishing
appropriate prenatal cytogenetic analysis.
100
Analysis of the potential role of Apolipoprotein E polymorphism in
genetic predisposition to spontaneous abortion.
Rynekrova Jitka, 1Fait Tomas, Hubacek Jaroslav A.
Institute for Clinical and Experimental Medicine, Prague
1General Faculty Hospital, Prague
rynji@seznam.cz
Introduction: According to estimates, up to 20 % of pregnancies end in the first
trimester by spontaneous abortion. There are many causes (negative influences of the
environment, genetic determination, etc.) but a significant number still remains
unexplained. The aim of this study is to investigate the role of variants in the gene for
Apolipoprotein E in the genetic determination of spontaneous abortions. Material and
methods: We collected DNA from 171 samples of spontaneous abortions and genotyped
Apolipoprotein E by PCR-RFLP method. The frequencies were compared with known
population sample of adults (N=653). Results and discussion: The frequencies of the
Apolipoprotein E genotypes did not significantly differ from the frequencies in analyzed
population study (Table). Out pilot study suggests that apolipoprotein E is not a major
determinant of spontaneous abortions in Caucasians.
Table
Numbers of carriers according the Apolipoprotein E
genotype
Genotype Controls Aborts
n % n %
22 3 0,5 4 2,3
32 75 11,5 16 9,4
33 444 68,0 109 63,7
43 108 16,5 33 19,3
42 12 1,8 5 2,9
44 11 1,7 4 2,3
101
Pregnancy loss – "Split protocol" for successful next pregnancy
Mijaljica G., Čulić V., Žegarac Ž., Lozic B., 1Resic J., 1Karelovic D., 2Pavelić J.
Department of Pediatrics, Division of Medical Genetics; University Hospital Center Split and University
of Split School of Medicine; Split, Croatia
1Department of Obstetrics and Gynaecology; University Hospital Center Split and University of Split
School of Medicine; Split, Croatia
2Department of Molecular Medicine, Institute Ruđer Bošković; Zagreb, Croatia
goran.mijaljica@mefst.hr
Chromosome aberrations and infections come first as potential causes of pregnancy loss.
The two hits theory is established acknowledging the role of infections in pregnancy loss.
The first hit could have been triggered by patient's prior genitourinary system infection,
while the second hit is triggered by the reactivation of latent EBV infection.
The presented results support this hypothesis, which encouraged us to propose the "Split
protocol" for planning a successful next pregnancy. Couples (all together 442) having
one, two, three, four or more pregnancy losses (PL) were analyzed in this retrospective
case study. The majority of couples (265) had two (530) PL. From 312 samples of the
aborted material, aberrant karyotype (trisomy, monosomy, aneuploidy, tetraploidy and
others) was found in 21.8% of cases. Pathological analysis revealed hydropic
degeneration in the majority of samples. Genitourinary infections prior to pregnancy
were reported in 58% female and 41% male participants. These infections were most
frequently caused by E. coli, Enterococcus, Streptococcus agalactie, Ureaplasma
urealyticum and/or Mycoplasma and Chlamydia. Serological analysis on samples of 309
female and 293 male participants revealed the presence of EBV EA in 67 female and 22
male participants. Presence of IgM and/or IgA against CMV, HSV1/2, Rubella,
Toxoplasmosis, HHV6, Parvo B19, RSV, Adenovirus and Varicella Zoster, as a result of
reactivation of infection in pregnancy, was also noted. Forty-three percent of couples
with aneuploidy in the aborted material (54) had positive serological results. Serum iron
values and feritine values for 65 and 74 women respectively, indicate that they have
suffered from sideropenia and low iron reserve. Urinary and/or genital infection and low
iron reserve must be regulated before the next pregnancy. The effect of the reactivated
infection must be excluded in next pregnancy.
Research of the genes involved in this process is the goal of the future “Split protocol
project".
102
Evaluation of cervical, pharyngeal and anal HPV status of female
sex-workers in Hungary
Marek E., Cseh A., Gőcze K., 2Várszegi D., 1Benczik M., Gőcze P., Bózsa Sz.
Department of Obstetrics and Gynaecology, Medical Faculty, University of Pécs, Hungary,
1GenoID, The HPV Diagnostics Company, Budapest, Hungary,
2Department of Dermatology, Venereology and Oncodermatology, University of Pécs, Hungary
erika.marek@freemail.hu
Human papilloma virus (HPV) is the most common sexually transmitted infective agent
known today. Sexually active women are the most affected, approximately half of them is
infected at least once during their lifetime. Chances of being infected and transmitting the
virus correlates with the number of sexual partners. Sexual intercourse is not even
mandatory in terms of development of HPV infection, genital contact (skin to skin) is fair
enough. The use of condom decreases the risk, but doesn’t give full protection. When
talking about sexually transmitted diseases (STDs), including HPV, we must focus our
attention on prostitutes, who are mostly endangered. Switching partners is not without
risk nowadays. Every fifth switch goes hand in hand with an HPV infection, which
means that prostitutes and their partners are in greater jeopardy every day.
In malignant transformations of chronic HPV infections high risk HPV types are
involved. Earlier HPV was only connected to cervical cancer, but latest data show, that
HPV is the primary cause of more than 95% of cervical, 50% of genital (vagina, vulva,
penis), 70% of anal and 20% of oro-pharyngeal cancers. A survey based prospective
study was carried out screening the sexual habits of women prostitutes and their
knowledge of HPV. Thorough STD screening was supplemented with HPV testing using
cervical, anal and pharyngeal samples along with Pap smear screening. The questioned
population had bare knowledge about HPV, the infection and its long distance
consequences. It can be stated that almost every one of them uses condoms on a regular
basis as protection against sexually transmitted diseases. We found HPV DNA positivity
in 82,4% (14/17) of the cases. 11 HPV genotypes were identified, mostly HPV 16 and 33.
53% of HPV positive women (9/17) tested positive for high risk HPV, 23,5% (4/17) was
positive for more than one type of HPV. High risk HPV positivity was proven as follows:
41,1% (7/17) of cervical, 17.7% of anal (3/17) and 11,8% (2/17) of pharyngeal samples.
Screening, sampling, testing and evaluation are still under way.
HPV infection is in most cases symptomless and remains latent for months or even years,
carrying the potential of malignant transformation. Since prostitutes are sources of
infection and their partners are in danger of getting infected thus becoming a significant
health hazard, routine STD screening among prostitutes (Chlamydia, Hepatitis-B, HIV,
Syphilis, Gonorrhea – once in every 3 months) should include HPV testing as well.
103
Inflammatory
&
Immune Response
104
Anti-inflammatory and antioxidant effects of thiazolidin derivatives
possessing dual cox/lox inhibition upon the heart tissue and gastric
mucosa of rats
Fomenko I. S., Bondarchuk T. I., Sklyarov A. Ya.
Danylo Halytskyi National Medical University, Lviv, Ukraine
biochemistry@meta.ua
Compounds that combine COX/LOX inhibition are potential new drugs to treat
inflammation. Dual inhibitors, by acting on the two major arachidonic acid metabolic
pathways – cyclooxygenase (COX) and lipooxygenase (LOX), possess a wide range of
anti-inflammatory activity. Besides that, dual inhibitors appear to be almost exempt from
gastric and cardiovascular toxicity, which is the most troublesome side effect of COX
inhibitors.
The purpose of the research was to determine PGE2, LTB4 concentration and activity of
the antioxidant protection system in heart tissue and gastric mucosa under prolonged
application of thiazolidin derivatives possessing dual COX/LOX inhibition. {2,5-Dioxo-
3-[4-oxo-5-(3-phenyl-allylidene)-2-thioxo-thiazolidin-3-yl]-pyrrolidin-1-yl}-acetic acid –
(agent 1) (10 mg/kg) and {2,5-Dioxo-3-[4-oxo-5-(3-phenyl-allylidene)-2-thioxo-
thiazolidin-3-yl]-pyrrolidin-1-yl}-benzene-sulfonamide – (agent 2) (10 mg/kg) were
introduced per os for 14 days. The concentrations of PGE2 and LTB4 were determined in
homogenates of healthy rats’ tissues using ELISA method after blockage of COX/LOX.
It was shown that the content of both metabolites of arachidonic acid had the tendency to
decrease, proving the inhibitory action of both agents. Injection of agents 1 and 2
considerably increased activity of the antioxidant protection system enzymes (catalase,
SOD, GP, GR) in both investigated tissues. After dual inhibition by agents 1 and 2 MDA
content in heart tissue was increased by 28% and 30% subsequently. MDA concentration
remained unchanged gastric mucosa after action of these 2 types of inhibitors. COX/LOX
dual inhibition led to considerable rise in NO concentration in gastric mucosa (by 40%
and 22%). Morphological changes of gastric mucosa due to injection of agent 1 that are
evidence of a higher degree of integrity of the mucous barrier components in comparison
to the action of COX-2 inhibitor celecoxib, increased density of epithelial cells of the
surface of mucosa, reduced degree of edema. Due to injection of agent 2, protective
effect upon the status of mucous membrane was less manifested.
105
Enhancement of cytoprotective mechanisms under combined action
of amaranth oil with blockage of inos in experimental colitis
Panasyuk N. B., Sklyarov O. Ya.
Danylo Halytskyi national medical university of Lviv
45/13, Pl. Rynok, Lviv, Ukraine
sklyarov@meduniv.lviv.ua
In ulcerogenic colitis lipoperoxidation processes and activity of NO-synthases intensify,
and content of nitric oxide and proinflammatory cytokines in the mucous membrane of
large intestine (MMLI) increases. Ulcerogenic colitis is treated with the drugs blocking
iNOS, cyclooxigenase-2 and 5-lipooxigenase, and different oils containing considerable
amounts of ω-3, ω-6 and ω-9 of unsaturated fatty acids. Purpose of the research was to
study the effect of blockage of iNOS activity with aminoguanidine combined with
injection of amaranth oil on the cytoprotective and metabolic processes under
experimental ulcerogenic colitis. Methods. Ulcerogenic colitis in rats (n = 60) was
modeled with 4% acetic acid. In the MMLI homogenates were determined: content of
TBA products, activity of SOD, catalase, eNOS, iNOS, NO and concentration of L-
arginine in the plasma of blood. Results. Injection of 4% acetic acid induced
development of ulcerogenic defects, erosions, massive hemorrhages of the area of 77.2 ±
25.1 mm2. Content of TBA products increased by 2.2-fold (p < 0.001), activity of SOD
enhanced by 71 %, of catalase – by 54 % (p < 0.01), of general NOS – by 2.5-fold (p <
0.001), of eNOS – by 21 %, of iNOS – by 6.9-fold, content of NO in the MMLI increased
by 64 % (p < 0.05). Concentration of L-arginine in the blood decreased by 51%
(p<0.001). Due to iNOS blockage with aminoguanidine, the area of ulcerogenic lesions
decreased to 69.6±19.7mm2. Activity of general NOS reduced by 41 % (P < 0.01), of
iNOS – by 46 % (P < 0.0!). MDA content decreased by 28 % 9p < 0.05) and catalase
activity diminished by 20%. Injection of amaranth oil caused reduction of the area of
ulcerogenic lesions to 45.4 ± 20.83mm2. Activity of general NOS reduced by 52 % (p <
0.01), of iNOS – by 69 % (p < 0.001), NO content decreased by 29 %, and concentration
of L-arginine in the blood increased by 23 %, contents of TBA products decreased by 35
% and catalase activity reduced by 27 %. In combined action of amaranth oil with the
blockage of iNOS by aminoguanidine, area of MMLI lesions considerably decreased and
was 38.8 ± 30.1mm2 (p < 0.01). Activity of general NOS diminished to the normal and
activity of iNOS – by 73 % (p < 0.001). Content of MDA and catalase was at the level of
intact animals. Thus, combined action of aminoguanidine with amaranth oil manifests
pronounced cytoprotective and anti-inflammatory effects in ulcerogenic colitis that is
associated with a steep decline in the activity of NO-synthases and indices of oxidative
stress. Obtained results substantiate application of combined action of oils with high
concentrations of unsaturated fatty acids and selective blockers of iNOS in treatment of
inflammatory processes.
106
Peculiarities of apoptosis-induced DNA degradation against
experimental esophagitis and its correction by melatonin
Pinyazhko R., Grzegotsky M.
Department of physiology, Danylo Halytsky Lviv National Medical University
69, Pekarska, Lviv, Ukraine, 79010
pinyazhko.roman@gmail.com
Gastroesophageal reflux disease is one of the topical issues in medicine. Great interest in
this issue is caused not only to increase of prevalence rate of the said disease, requiring
thorough investigation of terminal exchange components, functional failure of which
reduces resisting power of epithelial barrier of mucous coat of esophagus. We conducted
the research of apoptosis-specific internucleosomal DNA degradation of cells of mucous
coat of esophagus in rats against esophagitis simulation and its correction by melatonin.
The research was conducted on sexually mature male rats. Experimental esophagitis was
induced by means of dosed administration (using an external perfusion technique) of
acidic and pepsic mix for days (an experimental group). Melatonin was administered
(intra-abdominal administration in the dose of 20 mg/kg per day) for days in a separate
group of experimental animals against perfusion of the acidic and pepsic mix. A fluid
rich in cells of mucous coat of esophagus was used, after pre-treatment, for separation
and cleaning of fragmented DNA using traditional DNA ladder technique (Herrmann,
1994). Fragmented DNA was visualized, after separation, on a transilluminator, and
photographed. Conducted research is indicative of the fact that significant apoptosis-
specific internucleosomal DNA degradation can be observed against experimental
esophagitis model used by us. With the view to peculiarities of change of other
parameters explored by us (ultrastructural changes, production profile of anti-
inflammatory cytokines), and reference data, such pattern of changes in mucous coat of
esophagus may be rated as a moderately grave condition. Death of epithelial esophageal
cells occurred in individual animals among analyzed general population with
experimental esophagitis by means of apoptosis, as well as necrosis. According to
reference data, apoptosis is treated as a highly regulated active process, mostly depending
on efficiency of oxidative energy-producing metabolism. Findings prove reasonability of
use, under the present conditions, of melatonin that can universally reduce, according to
reference data, level of tissue hypoxia, improve usage of lipid peroxidation products,
stimulate production of NOS with further vasodilatation and improvement of local blood
flow. Degree of manifestation of ultrastructural pro-apoptotic changes appeared certainly
lower in the group of experimental animals for whom melatonin was used as a corrective
medium. Positive effect of melatonin use against experimental gastroesophageal reflux
disease model is apparently due to the ability of melatonin to provide for cell oxidation
homeostasis and optimize local blood supply of epithelial cells of esophagus by means of
attraction of metabolites of nitrogen oxide cycle.
107
The effect of rabeprazole on gastric acid and mucoid-electrolyte
secretion in patients with duodenal ulcer
Sklyarova Helena
Outpatient and Family Medicine Department, Lviv National Medical University, Lviv, Ukraine
sklyarov@meduniv.lviv.ua
Background and aim. Proton pump inhibitor (PPI) rabeprazole has been widely used in
clinical practice due rapid suppression of gastric acid secretion. However, it is still little
known about the mucoid-protecting action of rabeprazole. The aim of the study was to
evaluate the effects of rabeprazole on gastric acidity and mucoid-electrolyte secretion in
patients with duodenal ulcer after two weeks therapy with this PPI. Methods. 15 patients
with duodenal ulcer (Helicobacter pylori-positive) were investigated. The size of ulcerous
defect ranged from 0,5 to 1,5 cm. All patients received triple therapy that included the
standard dose of rabeprazole (20 mg/day), clarythromycin and metronidazole for two
weeks. The volume of gastric aspirate, hydrogen ion concentration, gastric acid output,
pepsin concentration, N-acetylneuraminic acids (NANA) and Na ions concentrations in
gastric juice and insoluble mucus were measured and compared before and after
treatment with rabeprazole. Results. After two weeks, complete healing of ulcer was
documented in 60 % of patients. Rabeprazole demonstrated inhibitory effect on gastric
acidity by 72 %, volume secretion by 50 %, and pepsin concentration by 82 %. At the
same time level of NANA and Na-ions increased by two times in gastric juice compared
in insoluble mucus. Conclusion. Healing of the ulcerous defect after rabeprazole
admission is due to effective lowering of acidity and peptic secretion. At the same time
increased the frequency of duodenogastral reflux and mucous secretion. Future
investigations are needed to determine influence of switching from function of high
specialized parietal cells to the phylogenetically older mucous secretion in consequence
of epithelial cells rebuilding.
108
Role of hemodynamic forces in the regulation of vasomotor tone of
skeletal muscle venules
1Marki A., 1Gara E., 1Debreceni B., 1Racz A., 1,2,3Koller A.
1Department of Pathophysiology, Semmelweis University, Budapest, Hungary
2Department of Pathophysiology and Gerontology, University of Pécs, Pécs, Hungary
3Department of Physiology, New York Medical College, Valhalla, NY, USA
Alex.marki@yahoo.com
Background: The roles of hemodynamic forces, such as pressure and wall shear stress
have been well characterized in arterioles. It has been shown that smooth muscle and
endothelial mechanisms (nitric oxide (NO) and dilator prostaglandins) contributes to the
development of vasomotor responses and in certain conditions reactive oxygen species,
as well. Much less is known regarding the importance of hemodynamic forces-induced
regulation of vascular resistance in venules. Methods and results: Isolated rat gracilis
muscle venules developed substantial tone in response to increases in intraluminal
pressure (active diameter: ~260 µm, whereas passive diameter: ~360 µm at 10 mmHg).
Presence of catalase or indomethacin reduced significantly the pressure-induced
development of myogenic tone (control: 40 +/- 5 % vs. control+CAT: 60 +/- 11 % of PD,
at 5 mmHg pressure). In addition, venules dilated as a function of intraluminal flow,
which was augmented in the presence of the thromboxane A2 (TxA2) receptor antagonist
SQ 29,548 or the TxA2 synthase inhibitor ozagrel. The selective cyclooxygenase (COX)-
1 inhibitor SC 560 reduced, whereas the selective COX-2 inhibitor NS 398 enhanced
flow-induced dilations. We have also found that H2O2 elicited concentration dependent
constrictions (max.: 137 +/- 8 to 61 +/- 18 µm), which were inhibited by the presence of
indomethacin or SQ 29,548. Conclusion: Skeletal muscle venules exhibit myogenic
response, which is - in part - mediated by H2O2, which elicits the release of constrictor
TxA2. In addition, flow/shear stress dependent regulation of diameter is present in
venules, which is mediated by simultaneous release of constrictor TxA2, the dilator NO
and PGs, with an overall effect of limited dilation. These responses and mediators are
likely to have important roles in the multiple feedback regulation of wall shear stress and
resistance in skeletal muscle venules.
Supported: Hungarian Sci. Res. Funds/OTKA – T48376, K67984; AHA Founders Aff.
0855910D, AHA NE Aff. 0555897T; Health Sci. Council/ETT 364/2006
109
SNAC – The last step???
Szakács Noémi, 1Kovács Rita
Semmelweis University, Budapest, Hungary
1National Institute for Sports Medicine, Budapest, Hungary
noemi.szakacs@gmail.com
Aim: We would like to present the biomechanical evaluation of the “scaphoid nonunion
advanced collapse” – a not very well known, but severe arthrotic disease of the wrist –
regarding each stage with its adequate treatment. In a case presentation we will discuss a
patient with stage II SNAC deformity where we performed proximal row carpectomy.
Background: Long-standing untreated scaphoid nonunion results in carpal collapse and
subsequent arthrosis, which is termed “scaphoid nonunion advanced collapse”. However,
this is a progressive disease, the stage depends on which joint surface is involved, and
regarding the stage we can choose the adequate therapy. At stage I SNAC deformity the
proximal pole of the scaphoid is free of degenerative changes, the distal fragment of the
scaphoid flexes and arthrosis arises only between this fragment and radial styloid. In
stage II arthrosis occurs furthermore between the proximal fragment of the scaphoid and
the head of the capitate, the severity depends on the amount of carpal instability. Further
shift and collapse of the scaphoid results in an increasing load on the capitolunate joint
with shear loading of the capitolunate cartilage resulting in arthrosis in the midcarpal
joint which is termed stage III SNAC deformity. Regarding the different joint
involvations, in stage I styloidectomy of the radius is recommended with scaphoid
reconstruction – interpositional bone graft and screw fixation; in stage II proximal row
carpectomy or four-corner fusion with scaphoid excision should be considered, and in
stage III the procedure of choice is four-corner fusion with scaphoid excision or
radiocarpal arthrodesis. Materials and methods: At the Department of Sports Surgery in
the National Institute for Sports Medicine in our practice the most frequent stage of
SNAC deformity was stage II, which correlates with the literature. Between 2005 and
2009 we performed radial styloidectomy in 7 cases, proximal row carpectomy in 9 cases
and radiocarpal arthrodesis in 3 cases. We would like to present one case operated in
august of 2008, emphasizing the clinical and radiological results after one and a half year.
Collected data: range of motion, grip strength, VAS, DASH, satisfaction, radiological
evaluation. Conclusion: The “scaphoid nonunion advanced collapse” can be divided in
different stages and the treatment is based on the involved joint surfaces; the diagnosis of
this disease itself is not an indication of the radiocarpal arthrosis – as we can see it
unfortunately in the everyday practice.
110
Analysis of the K+ current in human T cells in
hypercholesterinaemic state
Balajthy Andras, Pethoe Zoltan Denes, Somodi Sandor
Department of Biophysics and Cell Biology, Debrecen Medical and Health Science Centre
University of Debrecen
98, Nagyerdei krt., Debrecen, Hungary, H-4012
endy09@vipmail.hu
It is well documented in the literature that the changing the cholesterol content of the cell
membrane may influence the functioning of the membrane proteins, including that of the
ion channels. In most of the relevant studies the cholesterol content of the membrane is
altered artificially, using various forms of cyclodextrins. Our laboratory has reported
previously that the increase of the cholesterol content of the cell membrane (in vitro,
using cholesterol/cyclodextrin inclusion complex treatment) modified the biophysical
parameters of the gating of Kvl. 3 K+ ion channels in human T-lymphocytes.
As Kv1.3 channels have a pivotal role in the regulation T cell proliferation and function
we addressed whether the changes described in the model experiments could be observed
for Kvl.3 channels of T cells isolated from patients with hypercholesterinaemia.
T-lymphocytes were isolated from the peripheral blood of patients with cholesterol level
considered normal (< 5.2 mmol/l) according to the laboratory standards (control group)
and patients with hypercholesterinaemia („hc”, the cholesterol level is the 1.5 – 2-fold of
the normal one, n = 5). Patient samples (’hc’ samples) were obtained form untreated
(first-visit) outpatients of the Department of Medicine and they did not suffering from
any additional other diseases.
The biophysical characteristics of the Kv1.3 ion channel were studied using the whole-
cell configuration of the patch-clamp technique. We determined the kinetic (activation
and inactivation kinetics, time constants for recovery from inactivation) and equilibrium
parameters (voltage-dependence of steady-state activation and inactivation)
characterizing the gating of Kvl. 3 channels. The expression level of the channels was
characterized by current density (pA/pF) calculated as the peak current normalized to the
membrane capacitance, the latter being proportional to the membrane surface area.
Our results indicate that the biophysical parameters of Kv1.3 gating are similar in the
control group and in the ’hc’ samples (p= 0.05). Thus, the increase of the serum
cholesterol level does not result in such biophysical changes in Kv1.3 gating as those that
we measured in model experiments using cholesterin/cyclodextrin inclusion complex
treatments. On the other hand the current density measured in T cells isolated from the
’hc’ sample (CDhc =610 ± 98 pA/pF) was significantly higher than in the case of the
control cells (CDc = 398 ±10.8 pA/pF). This increase in the current density may reflect
chronic inflammatory processes coupled with hypercholesterinaemia and the cytokines
produced.
111
Nanotechnology
112
Inhibition of phosphate transporter NaPi2b function with specific
antibodies
Gryshkova V., 1Gout I., Filonenko V., Kiyamova R.
Department of Cell Signaling, Institute of Molecular Biology and Genetics, NAS of Ukraine,
150, Zabolotnogo Str., Kyiv, Ukraine, 03680
1Department of Structural and Molecular Biology, University College London,
London WC1E6BT, United Kingdom
v.s.gryskova@imbg.org.ua
Ovarian cancer is the most common gynecologic malignancy that usually becomes far
advanced before it is diagnosed. So far, only few markers and antigens specific for
ovarian cancer are known. MX35 antigen is one of them and it has been recently
identified as sodium-dependent phosphate transporter NaPi2b (SLC34A2, NaPi3b, Npt2).
NaPi2b belongs to the SLC34 family of sodium-dependent phosphate transporters which
are involved in the transport of inorganic phosphate and the maintenance of phosphate
homeostasis in human body.
Since the clinical trials with labeled MX35 antibody and its Fab2 fragments suggest their
therapeutic potential in patients with ovarian cancer it is important to know how they
affect NaPi2b phosphate transporter. The main objective of this work was to investigate
the effect of MX35 monoclonal antibodies on NaPi2b protein expression and function.
For this purpose the expression level of NaPi2b protein was compared in stable cell lines
expressing NaPi2b protein after incubation with MX35 MAbs in Western-blot analysis
whereas phosphate uptake assay was applied to study NaPi2b phosphate transporting
function. We used stable cell lines HEK293 expressing wild type WT_NaPi2b and
mutant form of transporter T330V_NaPi2b. This mutant form is not recognized by MX35
MAb because of single amino acid substitution (T330V) in the major extracellular loop
of NaPi2b phosphate transporter. WB analysis showed that incubation of cells expressing
WT_NaPi2b with MX35 MAbs led to a significant reduction of NaPi2b protein while
application of MX35 MAbs to cells expressing mutant T330V_NaPi2b had no effect on
the NaPi2b protein level. The results of phosphate uptake assay indicated approximately
40% inhibition of inorganic phosphate transport in cells expressing WT_NaPi2b after
incubation with MX35MAb but this antibody did not affect NaPi2b function in cells
expressing mutant T330V_NaPi2b. The data obtained allowed us to suppose possible
internalisation and further degradation of NaPi2b transporter initiated by MX35 MAb.
To summarize, we have shown specific inhibition of phosphate transporter NaPi2b
function by MX35 monoclonal antibodies. This knowledge could be useful for the
investigation of therapeutic potential of MX35 MAbs in the treatment of ovarian cancer
patients and in the developing of new specific inhibitors for NaPi2b-mediated phosphate
transport.
113
Effect of oligoperoxide coating of magnetic nanoparticles on the
efficiency of stem cell labeling
Šponarová Daniela, 1Jendelová Pavla, Horák Daniel, 2Zaichenko Alexander,
3Stoika Rostyslav
Institute of Macromolecular Chemistry AS CR, Czech Republic
1Institute of Experimental Medicine AS CR, Czech Republic
2 Lviv National Polytechnic University, Ukraine
3 Institute of Cell Biology National Academy of Sciences of Ukraine, Lviv, Ukraine
sponarova@imc.cas.cz
With the progress in regenerative medicine, magnetic resonance imaging (MRI) is a
suitable noninvasive method for in vivo transplanted cell tracking in the host organism.
Dextran-coated superparamagnetic iron oxide nanoparticles (e.g., Endorem®) are widely
used for cell labeling in combination with transfection agents or at high iron
concentrations, both of which can be cytotoxic. To overcome these drawbacks, the search
for better coatings of superparamagnetic iron oxide nanoparticles is required.
Maghemite (γ-Fe2O3) nanoparticles (~ 12 nm) were obtained by the coprecipitation of
Fe(II) and Fe(III) salts in alkaline medium which was followed by the controlled
oxidation of magnetite to maghemite. Resulting nanoparticles were coated with
oligoperoxide in an aqueous solution. Advantage of oligoperoxides consists in the
possibility to initiate polymerization of another monomer grafting thus various functional
biocompatible chains. In this report, poly(vinyl acetate-co-5-tert-(butylperoxy)-5-
methylhex-1-en-3-yne-co-butyl acrylate-co-maleic anhydride) of low molecular weight
(Mw =4,500) was selected as a coating. Carboxyl groups of the oligoperoxide formed by
hydrolysis were of crucial importance for subsequent attachment of the oligomer on iron
oxide surface. Oligoperoxide-coated γ-Fe2O3 nanoparticles were thoroughly
characterized by ATR FT-IR spectroscopy, SAXS, QELS, AAS and elemental analysis.
Coating had a marginal effect on the size and the morphology of the particles, however, it
strongly affected efficiency of labeling of mesenchymal stem cells (MSCs) with the
nanoparticles. Oligoperoxide-coated nanoparticles were compared with Endorem®
(control) in terms of MSCs cytotoxicity, labeling efficiency and MRI detection limit.
Financial support of the Academy of Sciences of the Czech Republic (project
KAN401220801) is gratefully acknowledged.
114
Enhanced antisense oligodeoxynucleotides delivery for prevention
prion infections
Izyumova L., Stadnyk V., 1Skorohoda T.
Institute of Animal Biology UAAS
1Lviv Polytechnik National University
izyumova_lyudmyla@yahoo.com
Prospects of disorders correction of humans and animals using antisense oligonucleotides
have attracted much attention of scientific community as antisense technology is a
powerful tool against many diseases which were incurable yet. One of those cases, for
which there are no means of treatment and prevention are prion infections. The
characteristic feature of prion infections is an accumulation of pathological form of prion
in a brain and some other tissues, which is derived from the host-encoded cellular prion
(PrPC). It was ascertained that presence of PrPC is absolutely necessary for illness
development. Thus, targeting PrPC has the potential to remove the substrate for the
pathogenesis. Based on this, we decided to reduce the PrPC expression level through the
inhibition its mRNA translation using antisense oligonucleotides (asODNs).
For effective inhibition of PrPC gene mRNA translation it was decided to select asODNs
for 5’-nontranslating region, start codon and middle of open reading frame of the gene.
Efficiency of application of asODNs substantially increases by using drug delivery
systems, which protect therapeutic agent from degradation, continue his half-life time and
prolong his action. We used cationic liposomes and synthetic poly dimethylamino-
ethylmetacrylate (polyDMAEM) as carriers. PolyDMAEM is water-soluble cationic
oligoelectrolite and it can efficiently bind negatively charged DNA through electrostatic
interactions as it was shown by turbidimetric analysis. The potential cytostatic and
cytotoxic effects of polyDMAEM were verified on the rat fibroblasts cell culture. It was
shown that the most optimal doses are 0.1 and 1 mkmole/ml culture medium. After 4
hours of incubation L1210 cells with asODNs incorporated into liposomes all used
asODNS decrease the PrPC expression level approximately on 80 % (r < 0,05).
Immunoblot analysis demonstrate that asODN complementary to the area of start codon
of prion mRNA was less effective and decreased expression of PrPC on 60-70 % (r <
0,01) by comparison to two other oligonucleotides that inhibited PrPC expression on 75-
90% (r < 0,01). If on 4th h. of incubation L1210 cells with complex asODN and
polyDMAEM the most effective was asODN complementary to the start codon then all
three asODNs showed 95-98% (r < 0,01) efficiency in decreasing PrPC expression level
on 24th h. Notably, it was found that polyDMAEM is able to decrease PrPC content by
itself.
This work demonstrates potential possibility of using asODN for prophylaxis and therapy
of prion infections. As a result of carried out research it was synthesized nanocarrier of
polymeric nature for delivery of nucleic acids. It was found that polyDMAEM is able to
reduce expression level of cellular prion in L1210 cells independently.
115
Flavocytochrome b2 and recombinant Hansenula polymorpha cells,
overproducing this enzyme, as perspective tools for chromate
bioremediation
1Smutok Oleh, 2Broda Daniel, 1Dmytruk Kostyantyn, 1,2Gonchar Mykhailo
1Institute of Cell Biology, National Academy of Sciences of Ukraine
14/16, Drahomanov Street Lviv, Ukraine, 79005
2Branch Campus of the Faculty of Biotechnology, Rzeszów University
26, ul. Sokołowska, PL-36-100 Kolbuszowa, Poland
smutok@cellbiol.lviv.ua
In spite of the great interest to the study of biological role of chromium, as well as of
toxic influence of Cr (VI)-species on living organisms, the molecular mechanisms of
chromate bioremediation remain vague. Among possible mechanisms for chromate
biodetoxification, a reductive pathway resulted in formation of the less toxic Cr (III)-
species is suggested to be the most important. The yeast L-lactate: cytochrome c
oxidoreductase (flavocytochrome b2, FC b2) has absolute specificity for L-lactate,
although is non-selective with respect to the nature of electron acceptors. Such properties
allow consider this enzyme as a potential candidate for reduction of chromate by living
cells.
To elucidate this hypothesis, recombinant strain of thermotolerant methylotrophic yeast
Hansenula polymorpha was used as a model organism possessing a six fold increased FC
b2 enzymatic activity (up to 3 µmol min-1 mg-1 protein in cell-free extract) compared to
the initial strain. The lyophilized recombinant cells, stored in dried state, as well as living
yeast cells were tested in respect of their chromate reducing activity in vitro in the
presence of L-lactate (as an electron donor for chromate reduction) and different low-
molecular redox-active dyes: dichlorophenolindophenol (DCPIP), Methylene blue,
Meldola blue, Nile blue facilitating electron transfer from the reduced form of the
enzyme to chromate (as a final electron acceptor). It was shown that the highest
chromate-reducing activity of the cells was achieved in the presence of DCPIP.
The ability to catch electrons from the reduced flavocytochrome b2 by chromate was also
demonstrated on the model of purified enzyme immobilized on the surface of platinum
electrode. It was clearly shown that with increasing concentration of Cr(VI) the peak of
enzyme-mediated L-lactate oxidation is decreased, indicating Cr(VI)-dependent
competition between reaction of chromate with reduced FC b2 and direct electron
transfer from the enzyme to the electrode surface.
The perspectives of the application of the observed chromate-reducing ability of FC b2-
overproducing recombinant cells of H. polymorpha for chromate bioremediation, as well
as for construction of cells-based biosensor for chromate monitoring in environment are
discussed.
116
Cell Biology
117
The effect of sildenafil versus erdostein on the monocrotaline
induced pulmonary disease in the Wistar rat a morphopathological
study
Bogdan S. N., 1Dumitrache-Rujinski S., 2Ardeleanu C., Dorobantu M.,
1Bogdan M. A.
Clinical Emergency Hospital, Bucharest, Romania
1"Marius Nasta" Institute of Pulmunology, Bucharest, Romania
2Victor Babes" National Institute, Bucharest, Romania
maria.dorobantu@gmail.com;
Introduction. Monocrotaline (MCT) a pyrrolizidine alkaloid is a phytotoxin used
experimentally to cause a pulmonary vascular syndrome in rats characterized by
proliferative pulmonary vasculitis, pulmonary hypertension and cor pulmonale. Sildenafil
(SIL), the phosphodiesterase 5 inhibitor, is an effective pulmonary vasodilator. Erdostein
(ERD) is a mucolytic agent with antioxydative effects.
Aims. Compare the benefits of administering Sildenafil versus Erdostein to the MCT
inoculated Wistar rats. Methods. Three groups of 15 rats each received subcutaneously 1
dose of MCT 60 mg/kg.The first group received SIL orally for 28 days, the second
received ERD orally for 28 days, the third remained untreated. Sacrifices were made on
the 14th and 28th day.Pulmonary tissue samples were processed using hematoxylin-eosin
stain and immunohistochemistry techniques with active antibodies. Results. The same
type of pulmonary lesions inflammatory interstitial and alveolar infiltrates, interstitial
edema, remodeled pulmonary arterial vessels, was found in all three groups. Lesions
were found to be less severe in the two treated groups vs non-treated group (p<0.05).The
SIL treated group had less severe lesions compared to the ERD treated group (p<0.05).
Mortality during the first 14 days reached: 1 in the SIL treated group, 5 in the ERD
treated group and 4 in the non-treated group.
Conclusion. SIL and ERD are both effective in diminishing the severity and extent of the
MCT induced pulmonary disease, with a direct positive effect on the vascular
remodeling. SIL was more efficient than ERD and was the only one to positively
influence mortality.
118
K+ current expression and proliferation of human T-lymphocytes
induced by various stimuli
Pethoe Zoltan Denes, Balajthy Andras
Department of Biophysics and Cell Biology, Debrecen Medical and Health Science Centre,
University of Debrecen
98, Nagyerdei krt., Debrecen, Hungary, H-4012
pethozo@freemail.hu
Ion currents of human T-lymphocytes and specifically Kv1.3 potassium channel currents
are crucially important for the triggering of an immune response. Resting T cells also
express this channel, but as a result of stimulation (e.g. an immune reaction), the
expression of the channel increases. In contrast to the in vivo activation of T cells, where
the interaction with specialized antigen presenting cells triggers T cell proliferation, in
vitro stimulation of the cells uses molecules that can act on specific signal pathways. Our
aim was to investigate whether Kv1.3 channel expression in T-cells depends on the
nature of the signaling pathway activated in vitro.
We used healthy human peripheral blood T-cells in our experiments. We monitored the
division of the cells in a flow cytometer using CFSE (carboxyfluorescein succinimidyl
ester) dilution assay. The cells were stimulated with a mitogenic lectin
(phytohaemagglutinin: PHA); a combination of a diacylglycerol analog (phorbol 12-
myristate 13-acetate: PMA) and a calcium ionophore (ionomycin); furthermore with
antibodies affecting the TCR-CD3 complex (anti-CD3 by itself; anti-CD3 and anti-CD28
used in combination). Five days after application of the stimulus we measured the
proportion of dividing cells, change in cell size and cellular granulation. Subsequently we
measured the Kv1.3 ion currents through the T-cell membrane as well as biophysical
parameters of this channel (e.g. activation and inactivation kinetics and voltage
dependence of conductance), using the whole-cell patch-clamp technique.
Our flow cytometric measurements indicated that as the cells divided, cell size and
amount of granulation increased after all treatments. Our patch-clamp experiments
showed that there are significant changes in Kv1.3 ion currents at +50 mV depolarization
after both PHA and anti-CD3+anti-CD28 stimulation. Inactivation kinetics changed only
as a result of anti-CD3+anti-CD28, which could be a sign of posttranslational modulation
of the channel. Similarly, increase in the current density was only significant in case of
anti-CD3+anti-CD28 treatment. We can conclude that for measurement of Kv1.3 currents
(e.g. for ion channel pharmacology), PHA treatment is sufficient but for the simulation of
in vivo T-cell activation the most effective method is stimulation with anti-CD3+anti-
CD28.
119
Formaldehyde dehydrogenase from the methylotrophic yeast
Hansenula polymorpha as bioanalytical instrument for assay of toxic
formaldehyde
Demkiv O. M., Gayda G. Z., Gonchar М. G.
Institute of Cell Biology, National Academy of Sciences of Ukraine
14/16, Drahomanov Str., Lviv, Ukraine, 79005
demkiv@yahoo.com
Formaldehyde (FA) is a highly reactive compound that has a toxic effect on all organisms
due to a non-specific reactivity with proteins and nucleic acids. FA reacts as an electrophile
with the side chains of arginine and lysine and the amino groups of RNA and DNA and
causes protein–protein, protein–DNA, and DNA–DNA cross-links. This substance is a
hazardous air pollutant and prolonged exposure to formaldehyde FA can cause serious health
effects. FA has been connected to cancer deaths; recent observations show that factory
workers who had been exposed to high FA levels are at increased risk for leukaemia. At
home, off-gassing of FA over time from pressed wood products can also result in health
hazards. Indoor, non-industrial exposure to chemical hazards can occur continuously at low
levels, contributing to symptoms such as headaches, fatigue, and upper respiratory and eye
irritation. Effective detection of chemicals in the environment requires simple, rapid,
sensitive and selective analytical methods. Such devices could continuously monitor
surrounding media and give warnings about the level of toxic chemicals in workplaces,
factories, and homes, even when they are present in extremely low concentrations. All
organisms employ certain metabolic pathways of FA detoxification, involving glutathione-
dependent FA oxidation by Formaldehyde dehydrogenase (FdDH). FdDH, a key enzyme of
FA metabolism in microorganisms, is proposed for bioanalytical purposes. We suggest using
FdDH, isolated from the gene-engineered recombinant strain Tf 11-6 of methylotrophic yeast
H. polymorpha for the enzymatic assay of FA. The method is based on the photometric
detection of a colored product, formazan, formed from nitrotetrazolium blue in a reaction
coupled with FdDH-catalyzed oxidation of FA. The reliability of the developed analytical
approach was tested on real samples of waste waters, pharmaceuticals, FA-containing
industrial products, and vaccines. The comparison of formaldehyde FA content values
obtained by biosensors (enzyme and cells-based), enzymatic methods and two routinely used
chemical ones (chromotropic acid and 3-methyl-2-benzothiazolinone hydrazone) showed a
good correlation between these approaches. Using nanosized matrix as a carrier for FdDH
immobilization will allow increase a local enzymes concentration, enhance a stability of the
protein and, probably, increase catalytic activity of the enzyme. We suppose to apply FdDH
bound with nanoparticles in biosensors on FA in air. We carried out immobilization of FdDH
on magnetic nanoparticles (γ-Fe2O3) and biocompatible poly(2-hydroxyethylmethacrylate-
co-40%ethylene dimethacrylate)/P(HEMA-EDMA)/microspheres using carboxylic groups
for covalent biofunctionlization of matrix. The bioanalytical properties of such FdDH–
modified materials were studied.
Acknowledgements. This work was supported by Joint Ukraine-Israel research grant MES of
Ukraine “Control of formaldehyde content in air by formaldehyde dehydrogenase-based
biosensors and its elimination by bioreactors with immobilized alcohol oxidase” M-197-2009.
120
Enhanced antioxidant formula based on selenium-enriched biomass
of the yeast Phaffia rhodozyma
1,3Nechay H. I., 2Deneha I. O., 3Kolisnyk H.V., 1Gonchar M.V.
1Institute of Cell Biology, NAS of Ukraine, 14/16 Drahomanov Str., Lviv, Ukraine, 79005
2Institute of Animal Biology, UAAS, 38 Stus Str, Lviv, Ukraine, 79034
3I. Franko National University of Lviv, Biology Faculty, 4 Hryshevskyi Str., Lviv, 79005 Ukraine
nechai_g@ukr.net
The red yeast P. rhodozyma is able to produce astaxanthin as a final product of carotenoid
biosynthesis. The properties of astaxanthin as antioxidant, anticancer agent, and coloring
agent are well-known. Aim of this work was to combine this powerful antioxidant with
selenium in P. rhodozyma biomass. We suggest that this formula will play a potential role in
human and animal's health by controlling free radical and antioxidant balance, thus
protecting cells from damage caused by ROS. Moreover, yeast biomass can efficiently
accumulate selenium from growth medium and transfer inorganic form of Se to organic, this
is considered to be more bioavailable and suitable for dietary application. Wild type strain
Xanthophyllomyces dendrorhous (P. rhodozyma) NRRL Y-10921 was cultivated in sucrose
medium, supplemented (if necessary) by sodium selenite to the final concentration 5 µg/L.
On 6th day of cultivation, astaxanthin content was measured after treatment of the cells by
DMSO and extraction by a mixture of hexane:ethylacetate (1:1), and, finally, measuring the
absorbance of an organic phase at 480 nm. The determination of total selenium content in the
cells was performed using atomic absorption spectrometer. Cells for analysis were prepared
using acid-hydrogen peroxide mineralization. Male Wistar rats with an initial body weight of
approximately 120 g were divided into 2 groups. The first group was intact animals and for
another we used tetrachloromethane (TCM) for intoxication. Groups were divided to 4
subgroups which differ by the feeding: 1 - got standard combined feeding (SCF); 2 - SCF
with 4% of P. rhodozyma biomass which consisted astaxanthin 370 µg/g of dry weight; 3 -
SCF with 4% of yeast biomass which contained 2 µg of organic selenium; 4 - SCF with 5 µg
of sodium selenite. Animals were killed on 16th day of the experiments and their livers and
blood were taken to assay enzymes activities and oxidative modification of lipids. Injection
of TCM resulted in increase of activity of alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) in serum and liver in comparison with intact animals. Activity of
ALT and AST were decreased to 11 and 14 % for animals treated by TCM, which were fed
by SCF - containing yeast biomass. The activity of enzymes for the subgroup, which also
were treated by TCM and were fed by selenium-enriched biomass was decreased in 47 and 9
%. Activity of catalase in liver was not changed in subgroups influenced by TCM, while
activity of glutathione peroxidase was decreased to 44, 33 and 55 % for 2, 3, 4 subgroups,
respectively. TCM also activated peroxidative modification of lipids. The addition of the
yeast biomass to SCF resulted in the decrease of conjugated dienes (CDs) content to 16 %
and malondialdehyde (MDA) to 20 %, while selenium-enriched biomass provoked
decreasing CDs to 4-fold and MDA - to 24 %, respectively. The addition of sodium selenite
did not result in positive influence against oxidative stress. We have demonstrated that the
formula, containing selenium-enriched biomass of astaxanthin-synthesizing yeast P.
rhodozyma can be used as an effective biologically active additive with a highly extensive
antioxidant potential and can be applied in various human and animal diets.
121
Auto-antibodies in human milk and blood serum: characteristics of
biological activity
Starykovych M., Mahorivs’ka I., Stoika R., Kit Y.
Institute of Cell Biology, National Academy of Science of Ukraine
14/16, Drahomanov St., Lviv, Ukraine, 79005
stoika@cellbiol.lviv.ua
Antibodies against DNA, nucleoprotein complexes, as well as enzymes participating in
metabolism of nucleic acids, have been described in patients with a variety of autoimmune
diseases and certain infections. Increasing evidence exists that the same auto-antibodies can
also hydrolyze their specific antigens. Antibodies possessing catalytic activity have been
called catalytically active antibodies or abzymes. They belong to a new group of
physiologically active substances with dual characteristics: they represent a pool of canonical
autoantibodies and possess catalytic activity. Proteolytic and DNA-hydrolyzing auto-
antibodies are of special value among them. An increase in their activity correlates with
clinical manifestations of the autoimmune disorders, disease severity, and the rate of
progressing disability. Abzymes are crucial for immune homeostasis regulation. They can be
of practical value for the development of modern immunodiagnostic tools and
immunotherapy schedules. With a few exceptions, blood serum of normal human donors
(men and women) usually does not contain catalytic antibodies. During pregnancy and
immediately after delivery (i.e. at the beginning of lactation), female organism is frequently
characterized by an immune status similar to that in patients with autoimmune diseases. In
addition, lactation is associated with an appearance of catalytically active antibodies with
DNAse, RNase, ATPase, amylolytic, protein kinase and lipid kinase activities present in
breast milk. These data suggest that abzymes play an important role in the innate and
adaptive immunity. Our study addressed the characterization of antigen specificity and
catalytic activity of antibodies of blood serum and milk of healthy human donors and
patients with some autoimmune pathology. Besides, we estimated the effect of these
antibodies towards growth and survival of cultured mammalian cells. It was shown that
different samples of immunoglobulins isolated from colostrum of healthy women and blood
serum of some patients with multiple sclerosis, induced cell death in vitro. However, in some
cases they stimulated proliferation of the studied target cells. Functional activity of
antibodies toward mammalian cells could be linked to their antigen specificity and catalytic
activity. Anti-DNA sIgA isolated from human colostrum with nuclease activity, possessed
different extent of cytotoxic activity towards Namalwa, Jurkat, L1210 and L929 cell lines.
We firstly found anti-histone sIgA- and IgG-antibodies capable of catalyzing a hydrolysis of
linker histone H1 in colostrum of healthy woman and blood serum of some patients with
autoimmune diseases - multiple sclerosis or systemic lupus erythematosus. Anti-histone IgGs
were internalized into Jurkat T cells and L929 transformed fibroblasts, and also stimulated
proliferation of human Jurkat T-cells.
Summarizing, we found that functional characteristics of immunoglobulins isolated from
human colostrum and blood serum depend on individual peculiarities of humoral immunity.
These characteristics can be of diagnostic and prognostic value, namely for complex
detection of early autoimmune disorders linked to pregnancy and delivery.
122
Metabolic enzyme CoA synthase is tyrosine phosphorylated and is a
part of signaling network in the cell
1Breus Oksana, 1Panasyuk Ganna, 1Gudkova Daria, 1,2Gout Ivan,
1Nemazanyy Ivan, 1Filonenko Valeriy
1Department of Cell Signaling, Institute of Molecular Biology and Genetics NAS of Ukraine
150, Zabootnogo Str, Kyiv, Ukraine, 03680
2Research Department of Structural and Molecular Biology, University College London, London, UK
oksanabreus@gmail.com; v.v.filonenko@imbg.org.ua
In the recent years the mechanisms which underlie complex interplay between metabolic
pathways and fundamental processes in multicellular organisms, such as proliferation,
differentiation and others, are starting to emerge. Some of the metabolic molecules and
enzymes are shown to play an active role in the regulation of cell behavior by acting on
different regulatory cellular systems including signal transduction. Objectives: we
studied relationships between an enzyme involved in de novo Coenzyme A biosynthesis
– CoA Synthase and the signaling proteins in mammalian cells. Methods: Metabolic
enzyme CoA synthase (CoASy) mediates two final stages of de novo biosynthesis of
coenzyme A. This protein possesses multiple proline-rich and phosphorylated tyrosine-
based motifs which according to in silico predictions could mediate its interactions with
SH2/SH3 domains of different signaling proteins. Based on these data we hypothesize
that CoASy has some unknown functional relationships with components of cellular
signaling. We tested this prediction of bioinformatics experimentally in pull down
analysis of tissue extracts with a number of recombinant GST-SH2 and GST-SH3
domains of different signaling molecules and confirmed several protein-protein
interactions by further co-immunoprecipitation. Namely, existence of CoASy complexes
with signaling proteins – protein tyrosine phosphatase Shp2, p85α regulatory subunit of
PI3K and Shc protein was shown in mammalian cells. Importantly, formation of
complexes between CoASy and the listed proteins is sensitive to availability of growth
factors indicating the regulatory nature of observed interactions. Furthermore, the
tyrosine phosphorylation of CoASy in vivo which regulates CoASy interaction with SH2
domains of these proteins was revealed. Involvement of c-Src kinase in CoASy
phosphorylation in vivo and its dephosphorylation by Shp2PTP in vitro were
demonstrated. Further studies of physiological relevance of CoASy interactions with
signaling proteins using siRNA technology revealed that CoASy knockdown leads to
decreased phosphorylation of substrates of PDK and Akt protein kinases. Altogether our
data indicate the existence of an unexplored physiological link between CoASy as a part
of CoA biosynthetic pathway and signal transduction pathways in mammalian cells. The
mechanisms and physiological significance of the observed phenomena are under
investigation.
123
Effects of fluid resuscitation methods on the pro- and anti-
inflammatory cytokines and expression of adhesion molecules after
burn injury
Foldi Viktor, Lantos Janos, Bogar Lajos, Roth Elizabeth, Weber Gyorgy,
Csontos Csaba
University of Pécs, Department of Anesthesiology and Intensive Care
noemi.szakacs@gmail.com
Objective: Fluid resuscitation management can influence inflammatory response after
burn injury. The aim of this study was to analyze the effects of two fluid resuscitation
methods on the cytokine production and on the expression of the leukocyte surface
markers.
Methods: Thirty patients were included in this prospective randomized study with burn
injury affecting more than 20 % of the body surface area. Fluid resuscitation was guided
by hourly urine output (HUO, n = 15) or by intrathoracic blood volume index (ITBVI, n
= 15). Blood samples were taken on admission and on the next five consecutive
mornings. Concentrations of IL-1ß, IL-6, IL-8, IL-10, IL-12p70 and TNF-a were
measured in phorbol myristate-acetate stimulated and non-stimulated samples. Leukocyte
surface marker expressions (CD11a, CD11b, CD14, CD18, CD49d, CD97) were also
determined.
Results: In the ITBVI group IL-6 levels on days 2-3 and IL-6/IL-10 ratios on days 2-3,
and the IL-8/IL-10 ratios on days 3-5 were significantly higher than in HUO group (p <
0.05). In the HUO group IL-10 levels were significantly higher (p < 0.05) on days 4 and
5. Granulocyte CD11a levels on day 2, CD11b levels days 4-6, lymphocyte CD11a on
days 5-6, CD11b on days 3-6, CD49d on days 2-6 and CD97 on day 6 and monocyte
CD11a, CD11b, CD18 levels on days 4-6, CD14 levels on days 3-5 were significantly
higher in the HUO group (p < 0.05).
Conclusions: Our study suggests that ITBVI guided fluid resuscitation of burned patients
suppresses the shift towards anti-inflammatory imbalance and the expression of
leukocyte surface markers more than HUO guided resuscitation.
124
Expression of endothelial selectin ligands on leukocytes following
repeated dive in SCUBA divers
Čulić Vedrana Čikeš, 1Martinić Roko, Anita Markotić, 2Ljubković Marko,
2Brešković Toni, 2Ljubković Jasna Marinović, 2Dujić Željko
Department of Medical Chemistry and Biochemistry, University of Split School of Medicine,
Split, Croatia
1Department of Pathophysiology, Laboratory for Tissue Typing, University of Split Hospital Center,
Split, Croatia
2Department of Physiology, University of Split School of Medicine,
Split, Croatia
vedrana.cikes.culic@mefst.hr
Leukocyte cell surface adhesion molecule CD11b, decorated with CD15s, plays a critical
role in regulation of β2 integrin functions during neutrophil endothelial transmigration.
Hyperbaric oxygenation reduces neutrophil-endothelial cell adhesion which is mediated
by Mac-1 (CD11b/CD18) β2-integrin. This study investigated expression of CD15 and
CD15s on leukocytes, following repeated trimix (a mixture of oxygen, helium and
nitrogen) dives in two series: in study I, 7 divers performed 6 consecutive dives from 55-
80 m (total dive time varied from 65-75 min), while in study II, 7 divers performed 3
consecutive dives from 63-65 m (total dive time varied from 59-83 min). More intense
dive profile was used in study II as can be seen from longer total dive time. 5 divers took
part in both studies. CD15 and CD15s were determined before and after the 1st and the
last dive in study I and before and after the 1st and the last dive in study II. Leukocyte
subpopulations were not elevated after both dives in study I. Only CD15+CD15s+
granulocytes were significantly decreased after the 1st dive (p = 0.006). In study II,
monocyte proportion was increased (p = 0.014) and lymphocyte decreased (p = 0.020)
within total leukocyte population, and CD15s+ monocytes and CD14+CD15s+
granulocytes were elevated (p = 0.019, and p=0.018, respectively) after the 1st dive in the
study II. CD15+CD14+ granulocytes were decreased after the 1st and the last dive (p =
0.048 and 0.017, respectively), while CD15s+ granulocytes were decreased only after the
last dive of study II (p = 0.006). The current findings of decreased endothelial selectin
ligand CD15s expression on CD15+ granulocytes after certain dives point the role of this
subpopulation in the endothelial damage prevention.
125
Drug Development
&
Pharmaceutical Research
126
Development of an in vitro-in vivo correlation for poorly soluble
drugs
Anuta Valentina
University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania
cmirc@gg.unibuc.ro
For poorly soluble substances, both the dissolution and the diffusion kinetics are limited
by the reduced solubility, which is the reason for which both the in vivo and in vitro
assaying use surface active agents (saa) which amplify and accelerate both phenomena.
The surface-active agents have fundamentally different behavior at levels before and after
the critical micelle concentration, and a chaotic behavior around that concentration.
Furthermore, due to accumulation at interfaces, there is a superficial excess of surface-
active agents and the critical micelle concentration is reached faster than in the case of
solutions. For pharmaceutical products containing poorly soluble active substances, the
dissolution methods stipulated in pharmacopoeias and used by the manufacturers are
mandatory tests for quality control. The aim looked for by producers is to fully and
rapidly dissolve the active ingredients. Following this unnecessary “quality”
pharmaceutical companies are frequently using non-physiological pHs and very large
amounts of surface-active agents Such tests are not predicting the way in which the given
product will behave in vivo. Therefore, it is possible for products which in compendial
conditions yield similar in vitro release profiles to behave essentially different in vivo.
The present study concerned development of in vitro dissolution methods, using optimal
concentrations of surfactants able to obtain reliable results concerning in vivo dissolution
and bioavailability. Ketoconazole and nimesulide were chosen as test products. The
dissolution studies were performed by using USP apparatus 2 (paddles). Polysorbate 80
(Tween 80) in the concentration range 0-2,5% was used in combination with a phosphate
buffer solution to increase the solubility of the tested drugs. Pharmacokinetic data were
obtained from as a single-dose, randomized, two-treatments, two-periods, two-sequence
cross-over bioequivalence study, performed in accordance with Good Clinical Practice
regulations. Venous blood samples were collected through a catheter up to 24 hours after
drug administration for both compounds. The active substances were extracted from
plasma samples by means of a liquid-liquid extraction method, and analyzed using a fully
validated HPLC method, with UV detection. The in vitro dissolution profiles were
compared with the in-vivo pharmacokinetic data, by using the FDA Level A correlation
recommended method (fraction of drug dissolved versus fraction absorbed calculated
using Wagner Nelson formula). The surface-active agent concentration 0.5 %, i.e. two-
three fold lesser than that used in official methods was found as optimum. A new
correlation method was tested, by substituting the absorbed drug fraction with the
eliminated one. This alternative method proved to provide a better correlation between in
vitro dissolution and in vivo pharmacokinetic data.
127
In vitro studies regarding the interactions of some ruthenium (II)
fluoroquinolones complexes and some plasmatic proteins
Arsene Andreea L., 1Uivarosi Valentina, Dragoi Cristina M., Mitrea Niculina,
Nicolae Alina
Department of Biochemistry
University of Medicine and Pharmacy ”Carol Davila”, Bucharest, Romania
1Department of Inorganic Chemistry
University of Medicine and Pharmacy ”Carol Davila”, Bucharest, Romania
andreeanitulescu@hotmail.com
The study on the interaction of small molecules (usually called ligands) with DNA and
plasmatic proteins has been the focus of many recent works. Investigating the interaction
of drugs with these endogenous biomolecules has a great significance for disease defense
and drug development.
Numerous biological experiments have demonstrated that DNA is the primary
intracellular target of anticancer drugs, due to the interaction between small molecules
and DNA, which cause DNA damage in tumor cells, blocking their division and resulting
in cancer cell death.
Of these studies, the interaction of transition metal complexes with DNA and plasmatic
proteins has gained much attention.
The present paper presents the DNA-binding properties and the plasmatic proteins
interactions (namely human serum albumin-HSA and transferrin) of some ruthenium (II)
complexes with ofloxacin and norfloxacin.
In this regard we investigated in vitro the interactions of the ligands studied with double
stranded calf thymus DNA through fluorescence emission spectrophotometry.
We also performed fluorescence studies for evaluating the HSA-binding properties of our
complexes, while the transferrin interactions were assessed through UV absorption
spectroscopy.
Our results showed that the studied complexes developed concentration-dependent
interactions with the DNA, HSA and transferrin.
128
Docking studies of 4-thiazolidone derivatives as potential COX-2-
pathway blockers in oncogenesis
Atamanyuk Vasyl’, Lesyk Roman
Danylo Halytsky Lviv National Medical University,
AtamanyukV.pharmchem@gmail.com
Last years COX-2 and arachidonic acid metabolites expression was found in human colon
cancer cells, human breast cancer cells, mouse melanoma, human fibrosarcoma, invasive lung
adenocarcinoma cases, human colorectal carcinoma, gastric carcinoma and human skin
epidermal cancer cells. 4-Thiazolidinone derivatives are well-known class of substances that is
related with a wide spectrum of biological activity and usage as pharmacological agents. This
class of compounds possesses hypoglycaemic, anti-inflammatory, choleretic, antitumor,
diuretic, immunostimulant, and other activities. Recently, attention has been paid to the
antitumor activity of thiazolidinone derivatives. It has been known that 4-thiazolidinones can
behave as COX-2-inhibitors by showing affinity to COX-2 enzyme. This determines novel
direction in search of new anticancer agents among molecules containing 4-thiazolidinone
moiety. Our aim was to conduct molecular docking of some 4-thiazolidinone and related
heterocyclic derivatives into COX-2 active site for purposeful search of COX-2 inhibitors as
potential anticancer agents. Docking studies were conducted with OpenEye Scientific Software
program package that include Fred Receptor, Vida, Flipper, Babel3, Omega2 and Fred2.
Chrystallographic models of COX-2 were obtained from Protein Data Bank (www.rcsb.org),
particularly medels 6COX (COX-2 in complex with selective inhibitor SC-558 at a resolution
of 2,8 Å) and 1СХ2 (COX-2 in complex with selective inhibitor SC-558 at a resolution of 3,0
Å). 145 compounds among 4-thiazolidinone and related heterocyclic derivatives, which
possessed in vitro anticancer activity, were selected for docking studies from in-house library,
as well as few known selective COX-2 inhibitors such as Celecoxib, Etoricoxib, Rofecoxib,
Valdecoxib, Meloxicam and non-selective COX inhibitor Aspirin. The molecular docking
included the following stages: a) generation R-, S- and cys-, trans-isomers of ligands using
program Flipper (obtained 461 isomers of studied compounds), b) 3D optimization of isomers
using program HyperChem 7.5 (www.hyper.com) (molecular mechanics method ММ+ with
following semi-empirical quantum-mechanical method РМ3), c) conformers generation
(Omega2) and d) 3D molecular docking (Fred2). Obtained seven scoring functions values
(Chemgauss2, Chemscore, PLP, Screenscore, Shapegauss, Zapbind and Consensus) were used
for in silico estimation of COX-2-compound binding. Consensus (cumulative) scoring function
ranking allowed us to select 20 compounds, which can prospectively be selective COX-2
inhibitors at the level of celecoxib, for in-depth pharmacological studies and templates for
synthesis of various related analogues. Compounds Les-942 and Les-1009, as the first in
compound ranking, to our opinion could be the most promising structures for the optimization
of the COX-2 inhibitors search with anticancer profile.
N
S
SO
O
O
OEt
N
S
N
CH2
OH
O
N
H
O
Les-1009 (6COX)Les-942 (1CX2)
Preliminary docking studies of in-house library could be considered as a first stage of long-term
project dedicated to rational design of selective COX-2-inhibitors.
129
A radial distribution function (RDF) approach to the QSAR study
of thiazolidine analogs
Klenina Olena
Lviv National Medical University by Danylo Halytskiy
oklenina11@rambler.ru
The prediction of anticancer activity of newly synthesized compounds provides a novel lead
for the drug-development process. In this study, RDF descriptors were used to predict the
antiproliferative activity for thiazolidine analogs. A series of 28 compounds was studied with
their activity IC50 values (concentration which inhibited cell growth by 50%) being examined
in a SK-MEL-188 human melanoma cell line [1].
N
H
S
NR1R2
OR N
S
NR1R2
OR N
S
NR1R2
O
CH3
R
I II III
R = H, 3,4,5-OCH3,
3,4-OCH3,
3,4-OCH2O-,
NHCOCH3, NH2
R1 = Alkyl, Enyl, OCH3
R2 = H, CH3
Firstly we carried out geometry optimization of each compound, using the quantum chemical
semi-empirical method PM3 included in HyperChem 7.5 [2]. The DRAGON computer
software, web version 3.0 was used to calculate 150 RDF molecular descriptors for each
compound. The atomic masses, van der Waals volumes, Sanderson electronegativities and
polarizabilities were used as bond weightings. All statistical analyses were carried out using
BuildQSAR program [3]. GA-MLR algorithm was used for the QSAR models construction.
Two-, three-, four- and five-variables linear regressions were built. They were validated by
calculating Q2 values from “leave-one-out” (LOO) testiness, known as cross–validation and
can be considered a measure of the predictive power of a regression. Correlation coefficients
r, the Fischer ratio (F) and the standard deviations (s) are of higher quality for the models.
The most important variables in the two-variables equations are the atomic masses and van
der Waals volumes. The negative contribution to the IC50 values (meaning the increasing of
activity) RDF020m descriptors give while the positive contributions are supplied with
RDF120m and RDF120v descriptors. This corresponds to a radius of 2.0 to 12.0 Å. In this
sense a spherical molecular volume could have certain restrictions to the addition of bulky
substituents. The r values are in the range of 0.82-0.76, the values of Q2 are 0.529-0.498
while the Fischer ratio lies between 25.786 and 17.590.
Three-, four- and five-variables models include also weighted by atomic polarizabilities and
electronegativities being of a significant contribution into the IC50 value. Most correlation
coefficients were higher than 0.85. One of the compounds presents large residual and should
be considered as an outlier. The outlier number represented only a 3.57% of the whole data.
The RDF descriptors can be used for predicting the anticancer activity of new chemicals,
thus contributing to the design and development of safe drugs. The linear regression models
developed are easily calculated and suitable for the rapid prediction of activity, and cross-
validations of the final models support this claim.
1.Wei Li, Yan Lu, Zhao Wang et all. Bioorg. & Med. Chem. Lett. 17 (2007), 4113-4117.
2.HyperChem 7.5 (HyperCube, Inc.)/http:www.hyper.com
3.de Olivera D. B., Gaudio A. C. Quant. Struct. – Act. Relat. 19, № 6 (2000), 599-601.
130
Synthesis of novel 4-thiazolidinones with (3,5-diaryl-4,5-dihydropyrazol-1-yl)-
ethanone moieties in molecules and evaluation of their antitumor activity
Kovach Nataliya, Havrylyuk Dmytro, Zimenkovsky Borys, Lesyk Roman
Department of Pharmaceutical, Organic and Bioorganic Chemistry,
Danylo Halytsky Lviv National Medical University
d‐gavrylyuk@ukr.net; dr_r_lesyk@org.lviv.net
Investigation of 4-thiazolidinones antitumor activity is actual and perspective tendency in
medicinal chemistry. The mechanisms of antitumor activity of 4-thiazolidinones and
related heterocycles can be associated to their affinity to anticancer biotargets such as
phosphatase of regenerating liver (PRL-3), nonmembrane protein tyrosine phosphatase
(SHP-2), JNK-stimulating phosphatase-1 (JSP-1), tumor necrosis factor TNFα, and anti-
apoptotic biocomplex Bcl-XL-BH3 etc. Among 4-thiazolidones the integrin αvβ3
antagonists and inhibitors of necroptosis have been established. Combination of
thiazolidine template with pyrazoline moiety is a perspective approach for drug-like
molecules design (Havrylyuk, 2009), considering that pyrazoline derivatives have a wide
spectrum of pharmacological activities.
The purpose of our work was the synthesis of new 4-thiazolidinones with pyrazoline
fragments for pharmacological screening. 3,5-Diaryl-4,5-dihydropyrazoles 1 easily react
with chloroacetyl chloride yielding derivatives 2. Compounds 2 were tested as alkylating
agent in the reaction with of 5-arylidene-2,4-azolidinones potassium salts, thus the corresponding
derivatives 3 have been obtained. It is known, that nature of arylidene moiety in position 5 of
azolidinone cycle has a critical influence on the antitumor activity (Lesyk, 2004). Therefore
another focus of our research was the synthesis of [2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-2-
oxoethoxy]benzaldehydes 4 and 1-[2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-2-oxoethyl]-1H-
indole-2,3-diones 6. The Knoevenagel reaction of 4, 6 with several 4-thiazolidinones yielded
the group of new 5-arylidenederivatives 5, 7. The structures of compounds were confirmed by 1H
NMR spectra.
O
H
N
NAr'
Ar
O
O
S
N
HX
Y
S
N
HX
Y
N
H
N
Ar2
Ar1
N
N
Ar'
Ar
O
Cl
NK
X
O
O
Ar'' N
X
O
O
Ar''
N
O
N
Ar
Ar'
N
N
Ar'
Ar N
O
O
O
R
OH
N
H
O
O
R
NN
Ar' Ar
N
O O
R
S
N
HX
Y
S
N
HX
Y
O
H
N
N
Ar
O
OAr'
X=O, NH,
Y=O, S
4
ClCH2COCl
1
2
Et3N
3
X = S, NH
6
R = H, Br
7
AcOH,
AcONa
AcOH,
AcONa
5
R. B. Lesyk, and B. S. Zimenkovsky. 2004. 4-Thiazolidones: Centenarian History, Current Status and Perspectives
for Modern Organic and Medicinal Chemistry. Curr. Organic Chem. 8: 1547–1577.
D. Havrylyuk, B. Zimenkovsky, O. Vasylenko et al. 2009. Synthesis of novel thiazolone-based compounds
containing pyrazoline moiety and evaluation of their anticancer activity. Europ. J. Med. Chem. 44(4): 1396–1404.
Antitumor activity scre-
ening of the synthesized
derivatives has shown
their moderate activity
with high selectivity to
individual cell-lines of
lung, renal, ovarian and
CNS cancers.
131
Phytochemical research of Hedera helix leaves
Lutsenko Yu.O.
Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
jullu@list.ru
Hedera helix L. (English ivy, Common ivy) is an evergreen dioecious woody liana, one
of 15 species of the genus Hedera, Araliaceae family. Whole or cut, dried leaves of
Hedera helix L. from vegetative and reproductive forms are used as medical plant
material. The major types of biologically active compounds, that are responsible for the
medicinal use of H.helix, are triterpene saponins with hederagenine or oleanolic acid as
aglycones (α- and β-hederin, hederacosides A-J, glycosides L-1, L-2a, L-2b, L-3, L-4a,
L-4b, L-6a, L-6b, L-6c, L-7a, L-7b, L-8a and hederoside B).
H.helix is traditionally used in the folk and official medicine, homeopathy and
cosmetology. The German Comission E confirms Hedera leaves use as the treatment for
catarrhs of the respiratory passages and for symptoms of chronic inflammatory bronchial
conditions.
The aim of our research was detailed phytochemical investigation of H.helix leaves. We
separated and identified rutin, caffeic, chlorogenic, isochlorogenic and rosmarinic acids
by several methods of chromatography (CC, PC, TLC). The structures of pure substances
were determined by the methods of spectral analyses (UV, MASS, Н1- NMR (COSY,
HSQC, HMBC) and С13-NMR).
Some elements and amino acids were determined in the plant material by
spectrophotometric method. Hedera may be considered a perspective material for
manufacturing of phytosubstances with potential immunological and antioxidant activity,
due to high concentrations of Ca, Mg, Mn, Fe, Zn, Cu. High concentrations of proline,
asparagine, γ-aminobutyric аnd glutaminic acids seem to become perspective for use of
Hedera leaves as reparative, detoxicant, antioxidant remedies and ones with positive
influence on central nervous system.
The results of phytochemical researches prove the perspective and the possibility of wide
use of Hedera phytopharmaceuticals in medicine and cosmetology and stimulate
pharmaceutical science for further investigations for development of new qualitative,
effective and competitive medicines.
132
RDF and 3D-MORSE descriptors in thiazolidine derivatives
anticancer activity prediction
Myrko Iryna
Danylo Halytsky Lviv National Medical University
irynaoliinyk@gmail.com
Thiazolidine derivatives represent a well-known class of patented drugs and substances at
different stages of research, which possess various biological activities. Recently,
attention has been paid to the antitumor activity of thiazolidine derivatives as novel
potential anticancer agents. Quantitative structure–activity relationships (QSAR) have
been broadly used for some years mainly in medical research. This methodology makes
use of the molecular descriptors offering valuable and simple information about the
structure of the molecules which is used later in the elaboration of the predictive models.
The employment of this methodology allows cost savings by reducing the laboratory
resources needed, and the time required to create and investigate new drugs with certain
desired biological activity. A data set of 17 thiazolidine derivatives, which possess the
most significant antitumor activity on two renal tumour cell lines 780-0 and UO-31, was
used. Primary anticancer assays were performed according to the US NCI protocol. The
Radial Distribution Function and 3D-MORSE descriptors for the given compounds were
calculated using DRAGON software on the (x,y,z)-atomic coordinates of the minimal
energy conformations determined by the AM1 method in Hyperchem 7.01 Evaluation
Package. Descriptors with constant or near constant values inside each group were
discarded. Mathematical QSAR models were obtained by means of Multiply Linear
Regression technique as implemented in BuildQsar software. Quality of the obtained
models was determined by examining the following data: correlation coefficient (R) and
Fisher’s statistic (F). Robustness of the obtained models was examined by Leave-One-
Out (LOO) cross-validation technique, which characterises by cross-validation
coefficient (Q2). The best three-dimensional QSAR models for two different tumour cell
lines are given below together with the statistical parameters of the regression:
UO-31 = -36.79RDF115m - 3.61RDF045e +51.78RDF145p +151.31
R = 0.97, F = 69.10, Q2 = 0.888;
UO-31 = +381.23Mor30u +17.48Mor03m -191.88Mor25p +194.92
R=0.797, F=7.553, Q2=0.325;
786-O = +30.49RDF055m +22.33RDF105m -21.23RDF100e -87.63
R=0.923, F=24.848, Q2=0.787;
786-O = -71.33Mor16u +230.46Mor24u -607.92Mor28v +161.24
R=0.945, F=36.278, Q2=0.821.
Variables in the obtained models encode specific 3D structural information about the size
and arrangement of particular atoms or atom groups which presence induces anticancer
effect. To posses significant antitumor activity on renal tumour cell line UO-31 optimal
size of thiazolidine derivatives must be equal to 2.5-14.5 А° and for line 786-O – 4-15.5
А°. Also we can make an assumption about different mechanisms of action on different
tumor cell lines. Obtained QSAR models possess high predictive ability and quality and
may be used for virtual screening of potential anticancer drug candidates.
133
The possibility of modern evidential pharmaceutical care
elaboration in Ukraine
Ryvak Tatyana, Nastyucha Yulia, Zimenkovsky Andriy
Department of Clinical Pharmacy, Pharmacotherapy and Medical Standardization
Danylo Halytsky Lviv National Medical University
azimenkovsky@ukr.net
WHO strategy in relation to antimicrobial drugs argues their rational use as: cost-
effective, with a maximum clinical effect, minimal toxicity and with account of
resistance. GCP offers accounting of according group of these drugs to infectious agents
and diseases, sensibility to certain antimicrobial drugs. Until today in Ukraine, with the
exception of the State formulary of drugs, first release (2009), level of clinical effect
evidence, particularly antimicrobial drugs, was not practically considered. We consider
that modern pharmaceutical care that conduces to the improvement of pharmacotherapy
quality must be based on the priority – high evidence of the given information about
drugs. In a similar aspect a problem is examined for the first time in Ukraine.
Aim of this stage of research was to prove the possibility of elaboration the modern
evidence-based pharmaceutical care in Ukraine. For realization of research’s tasks the
following methods were used: modern informative search, statistical, analytical;
principles of evidence (to evaluate the quality of information). On the basis of using the
high-quality world databases (SIGN, NICE, Cochrane Collaboration), and also most
authoritative pharmaceuticals formulary (BNF, WMF), we analyzed 22 antimicrobial
drugs (9 different pharmacotherapeutical groups) with the best clinical efficiency
(evidence level A, B) at gynecological pathology (sexual Chlamydia infection, gonorrhea
during pregnancy, post-natal endocentric). Instructions for medicines contain a number of
indications, including for use in gynecology. However, the evidence of clinical efficacy
of antimicrobial drugs (level A, B), especially in gynecology, available today, only for
single illnesses. Instead, responsibility for use of certain antimicrobial drug, in particular,
during pregnancy, fully, by the instruction for medicines, relies on a doctor who hasn’t,
usually, access to the high-quality information, and sometimes doesn’t know about its
existence. Therefore, he makes clinical decision which is based only on his experience,
considerably lowering the level of evidence.
Modern pharmaceutical care requires the use of evidence-based information about drug’s
clinical efficiency, which is possible only with high-quality computer database using. The
evidence of high truth level clinical efficacy, in the moment of research, has only specific
indications or illnesses, instead of all marked in instruction for medicines. The
elaboration of modern evidential pharmaceutical care is possible in Ukraine and can
reduce pharmacotherapy medical errors because of its influence on the clinician’s
decision.
134
Traditional honey food product abbamele – chemical
characterisation and antioxidant activities
Jerković I., Kasum A., 1Marijanović Z., 2Tuberoso C. I. G.
Faculty of Chemistry and Technology, University of Split, Croatia
1Department of Food Technology, Marko Marulić Polytechnic in Knin, Croatia
2Department of Toxicology, Faculty of Pharmacy, University of Cagliari, Cagliari, Italy
igor@ktf‐split.hr
Sardinian abbamele is a typical product originally obtained from the recuperation of honey
from the combs, but without information about its useful properties as well as used honey
types in the production. On the other hand, a honey in general is an excellent nutritional food
with health benefits. It has been used for the treatment of flu and common cold, healing of
wounds and burns, as anti-microbial agent as well as the source of natural antioxidants.
Therefore, five abbamele samples were obtained. The long thermal treatment applied in
abbamele production caused very high (1007.0 - 4405.8 mg/kg) HMF content (HPLC-DAD),
while glucose and fructose amounts were quite similar to the honey ones (HPLC-RI).
Thermally derived furan derivatives and terpenes were abundant among the headspace
volatiles (HS-SPME), particularly limonene (0.5 - 76.0%) that probably originated from citrus
rinds addition during abbamele production. GC and GC-MS analyses of ultrasonic solvent
extracts revealed HMF predominance as well as the compounds originated from the honey
(if/when existing) such as methyl syringate (up to 49.2%), marker of Asphodelus microcarpus
honey previously determined by HPLC-DAD (chemical traceability of A. microcarpus honey
is of great importance since melissopalynology does not allow the unambiguous determination
of its botanical origin). However, it was necessary to confirm its predominance in volatile
compounds of Asphodelus honeys. Therefore, GC and GC-MS analyses were performed on
Asphodel honey ultrasonic extracts (USE). Methyl syringate was the major compound ranging
from 50.8 to 87.0%. High isophorone percentage (up to 30.9%) determined by HS-SPME
followed by minor percentage of 4-ketoisophorone and norisoprenoides in one abbamele
sample indicated Arbutus unedo L. honey use in the production. HPLC-DAD analysis
confirmed the presence of specific honey markers: two samples showed high methyl syringate
concentrations (150.4 - 120.1 mg/kg) while homogentisic acid and other specific markers of A.
unedo honey were found in one sample. The compared GC-MS and HPLC-DAD data proved
to be useful to obtain information about the use of specific honeys in abbamele production and
to verify citrus addition. Total antioxidant activity (FRAP assay) of the samples ranged
between 13.3 and 71.2 mmol Fe2+/kg, while antiradical activity (DPPH assay) ranged between
3.8 and 23.3 mmol TEAC/kg. Antioxidant values were linearly correlated with total phenols
amount (1297.8 - 4469.5 GAE mg/kg) determined by Folin-Ciocalteau method. However,
because of the strong correlation between total polyphenols content and abbamele antioxidant
activity, the total phenol amount is an interesting aspect, even affected from the contribution of
Maillard reaction products (antioxidant activity of the honey is greatly influenced by its
botanical origin as well as heat treatment). Such high antioxidant values indicate a good
potential in the scope of food pharmacy in comparison to the honey. Particularly, Asphodelus
honey showed average antiradical activity value of 0.7 mmol of TEAC/kg, whereas the
antioxidant activity ranged between 3.0 and 5.7 mmol of Fe2+/kg.
135
Genomics
136
Results of molecular genetic analysis of SMN1 and NAIP gene
deletions in the high risk of SMA families
Tretyak Bohdan, Zastavna Danuta, Makukh Halyna
State Institution “Institute of Hereditary Pathology of Academy of Medical Sciences of Ukraine”
Lviv, Ukraine
irynej@ukr.net
The Spinal muscular atrophy (SMA) (I-Werdning-Hoffmann disease; II-intermediate
form; III-Kugelberg-Welander disease) vary according to the age of onset and the rate of
degenerative changes of spinal cord motor neurons progression, are connected with the
Survival Motor Neuron gene (SMN1) mutation. Two highly homologous copies of
Survival Motor Neuron gene: telomeric gene (SMN1) and centromeric gene (SMN2) are
defined in this genome areal. Deletions of the SMN1 gene appear to be directly involved
in SMA and are detected in over 95 % of SMA patients. The differences in exons 7 and 8
sequences are used to detect the genes in DNA analysis.
The NAIP gene lies in the region adjacent to the SMN1 gene and is present in the SMA
region with multiple pseudogenes, which apparently arise independently of the inverted
duplication. The neuronal apoptosis inhibitory protein gene has been postulated to have
neuroprotective effect and acts as a negative regulator of motor neuron apoptosis.
DNA of the patients with the clinical diagnosis of SMA was extracted from blood
leucocytes and PCR was conducted. The amplified products were analysed by RFLP with
endonuclease DraI and DdeI for exon 7 and exon 8 of SMN1 correspondingly. Deletion
analysis of exon 5 of NAIP gene was conducted (detection of exon 5 of NAIP has been
taken as evidence for the existence of at least one active copy of the gene).
The DNA bank of 63 probands with SMA clinical features and 82 family members’ DNA
samples has been collected. Among the probands homozygous deletion of SMN1 exons 7
and 8 has been found in 23 (36,5 %) cases. Homozygous deletion of SMN1 exon 7 only
has been identified in 2 (3,2 %) probands. The detection of homozygous deletion of
SMN1 exons 7 and 8 enables to verify the diagnosis of SNA in 39,7 % of affected
individuals. The absence of these genetic defects does not rule out the diagnosis of SMA.
The homozygous deletion of NAIP gene was detected in 11 patients with deletions
SMN1 exons 7 and 8 and the deletion rate was higher in SMA type I patients than that in
SMA type II or III.
These findings could be of relevance concerning a potential role of the NAIP gene as a
modifying factor in the pathogenesis of SMA.
The further research should be aimed at the detection of heterozygous deletion carriage
and the possible gene conversion which can be conducted using quantitative molecular
genetic analysis.
Supported by: West-Ukrainian BioMedical Research Centre (WUBMRC).
137
The essence of the spontaneous point mutations: quantum-chemical
seeking for means of the general structural principles
1Brovarets O.О., 2Hovorun D.M.
Faculty of Radiophysics, Taras Shevchenko National University of Kyiv
2Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine,
brovarets@list.ru; hovorun@imbg.org.ual
Nowadays the occurrence of spontaneous transitions is explained by two physico-chemical
mechanisms – the first is a formation of mismatched (wobble) base pairs in the center of
recognition of highly replicative DNA-polymerases ; the second is considered in the framework
of tautomeric hypothesis that suggests the formation of the base pairs involving rare (imino and
enol) tautomers in the recognition centre [1]. Thus, the advantage of the first mechanism lies in
the fact that the wobble pairs Gua·Thy and Ade·Cyt are registered experimentally and
incorporated satisfactorily into the double helix of DNA. The advantage of the second
mechanism is quasiisomorphism of the complementary base pairs which invoke imino and enol
tautomers (or protonated species) to the Watson-Crick base pairs but its shortcoming is that such
[however, these] mispairs invoking rare tautomers are not experimentally registered. Analyzing
the literature we come to a conclusion that these two mechanisms are regarded as alternative [2].
In the present work we attempt for the first time to prove that the aforementioned approaches
to the basic nature of spontaneous transitions actually are interrelated, so we proposed a new
mechanism of spontaneous transitions appearance. We hypothesize that they emerge due to the
transformation of the wobble DNA base pairs Gua·Thy and Ade·Cyt into the pairs invoking the
mutagenic tautomers Gua*·Thy and Ade·Cyt* accordingly (the mutagenic tautomers unlike the
canonical are designated with an asterisk).
Moreover we proved the existence of a new, unknown before, molecular mechanism of the
spontaneous transitions during DNA biosynthesis, namely – the tautomerisation of the bases
induced by the center of DNA base pairs recognition of the replicative DNA-polymerases
resulting in the formation of the mispairs Gua*·Thy and Ade·Cyt*.
The object of our research are molecular structures – wobble DNA base pairs, DNA base
pairs invoking the rare tautomers and the transition states of their reciprocal transformation. The
subject of our investigation is physico-chemical mechanism of the transformation of the wobble
DNA base pairs into the pairs invoking rare tautomers of the isolated bases or, hypothetically , in
the center of recognition of Watson-Crick DNA base pairs by the replicative DNA-polymerases.
The method of research – quantum-chemical modeling on the MP2/6-
311++G(2df,pd)//B3LYP/6-311++G(d,p) level of theory.
For the first time a new physicochemical mechanism has been proposed and grounded in
order to understand the origin of the spontaneous transitions. It is based on the tautomerization,
induced by the interaction of the DNA-polymerase recognition center with the canonic
nucleotide bases, of the pyrimidine bases in wobble base pairs Gua·Thy and Ade·Cyt which
transit into the pairs Gua*·Thy and Ade·Cyt* accordingly.
1. . Brovarets O.O., Bulavin L.A., Hovorun D.M. The physical model of Watson-Crick base pairs DNA
recognition by the proteins of replicative complex // Reports of NASU. – 2009. – №10.-P.194-200.
2. Brovarets O.O., Bulavin L.A., Hovorun D.M. How do the proteins of replicative complex block the
synthesis of DNA base pairs at participation of mutational tautomeres? Simple physical explanation // Reports of
NASU. – 2009. – №11.-P.175-182
138
The expression of cell cycle and several other genes in embryonic
kidney cell line HEK293 is controlled by ZXDC transcription factor
signaling
1,2Galkin O.V., 1Minchenko D. O., 1Ratushna O.O., 1Minchenko O. H.
1Department of Molecular Biology,
Palladin Institute of Biochemistry National Academy of Sciences of Ukraine
9, Leontovicha Str., Kyiv, Ukraine, 01601
2University of Kansas Medical Center, Kansas City, Kansas, USA
galkino@gmail.com; ominchenko@yahoo.com
The zinc finger X-linked duplicated type C (ZXDC) protein is the founding member of
ZXD family proteins. The other two members, ZXDA and ZXDB, are retrogenes derived
from ZXDC and are located on the short arm of the X-chromosome. The transcriptional
factor ZXDC contains ten zinc fingers and a strong transcriptional activation domain.
Previously was shown that transcription factor ZXDC in complex with ZXDA is
participating in the regulation of major histocompatibility complex class II genes
expression as the class II trans-activator. In this work we created subline of embryonic
kidney cell line HEK293 which over express transcription factor ZXDC for identification
of genes, transcription of which is depended from ZXDC signaling. Overexpression of
transcription factor ZXDC was confirmed by polymerase chain reaction and Western blot
analysis. Cells stable transfected with beta-gal was used as control. Using microarray
analysis we have shown that overexpression of transcription factor ZXDC significantly
changes the level of expression of large group of genes which control different processes
in the cells. Thus, we identified several genes which participate in the control of cell
cycle and which expression significantly increases in subline of embryonic kidney cell
line HEK293 which over express transcription factor ZXDC: CDC14 homolog A
(CDC14A) and R-spondin 3 (RSPO3) – in eight fold, fibroblast growth factor 18
(FGF18) and cyclin A1 (CCNA1) – in six and four fold, respectively, fibroblast growth
factor receptor 3 (FGFR3) and cyclin-dependent kinase inhibitor 1C (CDKN1C) – in
three fold. However, expression of mitogen-activated protein kinase 1 (MAPK1) and
cyclin-dependent kinase 10 (CDK10) is decreased (in four and five fold, respectively).
Significant induction of gene expression (from four to nine fold) was also observed for
many other genes: early growth response 2 (EGR2), interleukin 5 receptor, alpha
(IL5RA), interleukin 9 receptor (IL9R), intercellular adhesion molecule 4 (ICAM4),
matrix metalloproteinase 15 (MMP15), brain-derived neurotrophic factor (BDNF),
gamma-aminobutyric acid (GABA) A receptor, gamma 3 (GABRG3) and cholinergic
receptor, nicotinic, alpha polypeptide 3 (CHRNA3). Induction of the expression of
IL5RA, BDNF, EGR2 and CDKN1C mRNA was confirmed by polymerase chain
reaction using GAPDH mRNA expression as control. These results clearly demonstrated
that transcription factor ZXDC participates in the regulation of expression of large group
of genes, which control very important cell processes.
139
Anticancer antibiotic kigamicin D induces of cell apoptosis via a
Bax-initiated mitochondria-dependent pathway.
Minchenko D. O., Hubenya O. V., 1Moenner M., Katsuya T., 2Esumi H.,
Minchenko O. H.
Department of Molecular Biology, Palladin Institute of Biochemistry NAS of Ukraine
9, Leontovicha Str., Kyiv, Ukraine, 01601
1INSERM U920 Molecular Mechanisms of Angiogenesis Laboratory, University Bordeaux
1 (Talence, France);
2Research Center for Innovative Oncology, National Cancer Center Hospital,
Kashiwa, Chiba 277-8577, Japan;
ominchenko@yahoo.com
Kigamicin D is a member of novel anticancer antibiotics which were discovered from the
culture broth of Amycolatopsis by their selective killing activity against pancreatic cancer
cell line Panc-1 using a new screening strategy that targets the tolerance of cancer cells to
nutrient starvation. Pancreatic cancers are known to be among the most aggressive
malignancies despite their poor blood supply. We examined the possible molecular
mechanisms of cell death caused by kigamicin D using pancreatic cancer cell line KP-3
and PSN-1. Cell lines were cultured in glucose-deprived media with or without kigamicin
D (0.1 µg/ml during 1 or 2 hours). Kigamicin D was added to the cell cultures in one
hour after pre growing with glucose-deprived media. Results of this investigation shown
that kigamicin D induces the accumulation of BCL2-associated X protein (Bax) protein
in the mitochondria of KP-3 and PSN-1 cancer cells. Moreover, kigamicin D initiates
cytochrome c release from the mitochondria to the cytosol. Effect of kigamicin D was
observed in one hour and kept at the same level in two hours. Kigamicin D suppresses the
expression of mRNA Bax induced by glucose starvation in PSN-1 cancer cells. Because
Clock and activating transcription factor 4 (ATF4) transcription system regulates drug
resistance in human cancer cell lines we have also studied effect of glucose-deprivation
and kigamicin D on the expression of Clock and ATF4 mRNA in pancreatic cancer cell
line PSN-1. Glucose starvation significantly induces expression of Clock mRNA and
slightly – ATF4. Kigamicin D was observed to block completely the expression of both
Clock and ATF4 induced by glucose starvation in these cancer cells.
We have also shown that human glioma cancer cell line U87 is sensitive to kigamicin D
without glucose starvation. Moreover, subline of these cancer cells without IRE-1α
signaling are more sensitive to killing by kigamicin D. Blockade of IRE-1α signaling in
U87 glioma cells leads to significant increase in Clock and especially death receptor 6
mRNA expression as well as suppress ATF4 and clusterin mRNA expression.
Thus, our results showed an induction of Bax translocation to the mitochondria and
cytochrome c release from the mitochondria to the cytosol in two different pancreatic
cancer cell lines, demonstrating that this compound induces apoptosis through a Bax-
initiated mitochondria-dependent pathway.
140
Proteomics
141
Metformin reduces production of reactive oxygen species in renal
glomerular podocytes through an AMPK-dependent mechanism.
1Piwkowska Agnieszka, 2Rogacka Dorota, 1,3JankowskiMaciej, Stępiński Jan K.,
1,4Angielski Stefan
1Mossakowski Medical Research Centre Polish Academy of Sciences
Laboratory of Molecular and Cellular Nephrology, Gdańsk, Poland
2Medical University of Gdańsk, Department of Clinical Immunology and Transplantology, Poland
3Medical University of Gdańsk, Department of Therapy Monitoring and Pharmacogenetics, Poland
4Powiślański College in Kwidzyń, Poland
angielsk@gumed.edu.pl
Nephropathy is one of the most serious complications in diabetes and a major cause of
chronic renal failure worldwide. Recent data suggest that malfunction of glomerular
epithelial cells, podocytes, plays an essential role in the development of diabetic
nephropathy, although the mechanism of this is unknown. Hyperglycemia increases the
production of reactive oxygen species (ROS). NAD(P)H oxidase, producing superoxide
anion, is the main source of ROS in diabetic podocytes and their production may lead to
oxidative damage. Metformin is one of the major antidiabetic drug with stimulates
activity of AMP-dependent kinase (AMPK). We have investigated the effect of
metformin on the production of superoxide anion in cultured podocytes and attempted to
elucidate underlying mechanisms.
The experiments were performed in normal (NG, 5.6 mM) and high (HG, 30 mM)
glucose concentration. Overall ROS production was measured by fluorescence of a DCF
probe. Activity of NAD(P)H oxidase was measured by chemiluminescence method. The
AMP-dependent kinase (AMPK) activity was determined by immunobloting, measuring
the ratio of phosphorylated AMPK to total AMPK. Glucose accumulation was measured
using 2-deoxy-[1,2-3H]-glucose.
ROS production increased by about 27% (187±8 vs. 238±9 arbitrary units AU, P<0.01)
in HG. Metformin (2 mM, 2h) markedly reduced ROS production by 45% in NG and
60% in HG. Metformin decreased NAD(P)H oxidase activity in NG and HG (from
3.16±0.11 to 2.08±0.07 and from 6.86±0.32 to 1.15±0.13 nmol O2
−/mg protein/min,
respectively, P<0.05). AMPK activity was increased by metformin in NG and HG (from
0.58±0.07 to. 0.99±0.06, and from 0.53±0.03 to 0.64±0.03; P<0.05). We have also shown
that most of the antioxidative activity of metformin is determined by its ability to activate
AMPK. This is confirmed by the decreased generation of O2
− by metformin and by no
effect of metformin on NAD(P)H oxidase activity when AMPK was inhibited by
compound C (100 µM). In fact, our data underscores that AMPK is an important
regulator of NAD(P)H oxidase.
These observations provide evidence that, in podocytes, metformin exerts has
intracellular antioxidant properties. It decreases ROS production by activating the
AMPK, which in turn inhibits the NAD(P)H oxidase activity.
142
Functional characterization of novel mammalian isoform of adaptor
protein ITSN1.
Dergai M., Dergai O., Skrypkina I., Tsyba L., Rynditch A.
Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine
150, Zabolotnogo Str., Kyiv, Ukraine, 03680
m.dergai@gmail.com
Investigation of cellular signaling during last decade could be considered as a robust
breakthrough in the understanding of the role of signaling molecules such as kinases and
small GTPases. Despite this progress, relatively poor knowledge was gained about
proteins without catalytic functions that provide frameworks for the signaling. There is a
great number of components of signalosomes that are not mentioned as signal transducers
or messengers or even are not considered as direct participants. Nevertheless, their role is
difficult to overestimate. Adaptor proteins function to bring together components of
signaling complexes, to serve framework for the complexes assembly and
rearrangements. Our understanding of adaptor proteins becomes more intricate due to
existence of multiple splicing events affecting expression, stability, domain structure,
regulation of the encoded proteins, etc.
Our research is focused on investigation of adaptor protein intersectin 1 (ITSN1) and
plethora of its isoforms in cells of vertebrates. Here we report functional characterization
of the novel isoform (ITSN1-22a) of adaptor protein intersectin 1 (ITSN1) with
alternative C-terminus. ITSN1 is engaged in clathrin-mediated endocytosis, mitogenic
signaling and actin cytoskeleton rearrangements. The most explored role of intersectin
deals with internalization events of activated receptor complexes and other cargos. It also
participates in cell survival, cell polarity, neurons outgrowth. Expression of ITSN1-22a
was observed in all tissues tested, levels of expression were significantly lower in
comparison to ITSN1-s. ITSN1-22a is also engaged in clathrin-mediated endocytosis,
forms complex in vivo and is codistributed with ITSN1-s isoform. Some differences were
observed in isoform localization at the plasma membrane. Alternative C-terminus of
ITSN1-22a is engaged in homodimerization via disulphide bonds. Moreover, it provides
specific interactions with SH3 domains of amphiphysin 1 and ITSN1. The direct
interaction is not needed for engagement of the ITSN1-22a and ITSN1-s in mutual
complexes; presumably this interaction is mediated via common protein partners. Both
isoforms undergo monoubiqutination; this modification does not depend on serum
starvation/stimulation. We have shown that tandem SH3A-22a does not affect dynamin1-
amphiphysin1 complex assembly/stability in vitro thus, ITSN1-22a and amphiphysin 1
do not compete for dynamin 1. Our results suggest that novel isoform ITSN1-22a
complements expands ITSN1 function in mammalian cells linking additional endocytic
protein and providing alternative assembly platform consisting of SH3A-22a in
comparison to SH3A-E block of ITSN1-s.
143
Characteristics of ribosomal protein S6 kinase interactions with
novel partner - TDRD7
Skorokhod Oleksandr, Panasyuk Ganna, Breus Oksana, Nemazanyy Ivan,
Filonenko Valeriy
Department of Cell Signaling,
Institute of Molecular Biology and Genetics National Academy of Sciences od Ukraine
150, Zabolotnogo, Kyiv, Ukraine, 03680
v.v.filonenko@imbg.org.ua
Ribosomal S6 kinase 1 (S6K1) is an important player in cellular PI3K/mTOR signaling
network involved in regulation of cell growth and differentiation. Our recent two hybrid
yeast screening, using S6K1 as bait, allowed us to identify tudor domain containing
protein 7 (TDRD7) – a protein with unknown function as a novel binding partner of
S6K1. TDRD7 is a scaffold protein identified in complexes with proteins which regulate
cytoskeleton dynamics, mRNA transport and protein translation apparatus.
To confirm and investigate a role of S6K1-TDRD7 interaction we conducted more
detailed studies of these proteins interplay. First, bioinformational analysis of TDRD7
primary structure was carried out. It allowed us to determine several functional domains
within TDRD7 and possible S6K1 sites of phosphorylation on this protein. At the next
step six different fragments of TDRD7 were cloned, over expressed and purified from
bacteria cells. These recombinant proteins were used in a set of pull-down experiments
with full-length S6K1. Direct interaction between C-terminal tudor domain of TDRD7
and S6K1 has been shown. This interaction was further confirmed in Far-Western blot on
recombinant S6K1 and TDRD7 fragments.
Also, purified domains of TDRD7 were used as antigens for mouse immunizations and
generation of monoclonal antibodies (Hybridoma, 2008). The generated antibodies were
used for studying S6K1/TDRD7 interaction in mammalian cells in vivo. We have
detected the interaction between TDRD7 and S6K1 in reaction of co-
immunoprecipitation in HEK293 and some rat tissues extracts.
144
Adaptor protein Ruk/CIN85 is involved in regulation of EGF/ uPA -
dependent signaling in MCF-7 cells
Samoylenko A., Kozlova N., Marchenko S., 1Kietzmann T., Drobot L.
Palladin Institute of Biochemisry, National Academy of Sciences of Ukraine
9, Leontovycha Str., Kyiv, Ukraine, 01601,
1University of Kaiserslautern, Kaiserslautern, Germany
kozlovanina@gmail.com
Cell migration, adhesion and invasiveness play a key role in tumor progression and
metastasis. Urokinase-type plasminogen activator (urokinase, uPA), its receptor (uPAR)
and inhibitor (PAI-1) are the major regulators of these processes. However, the
intracellular mechanisms that mediate uPА/uPAR/PAI action remain incompletely
understood. Because cell-surface receptor of uPA lacks transmembrane and cytoplasmic
domains, it is assumed that uPA signaling is transduced via growth factor receptors, such
as EGFR, associated with uPAR.
Ruk/CIN85 is an adaptor protein implicated to play a role in carcinogenesis by
influencing ligand-induced endocytosis of growth factor receptors, cell adhesion, motility
and apoptosis. We aimed to explore the dynamics of Akt and ERK1/2 activation by EGF
in the presence of uPA using breast adenocarcinoma MCF-7 sublines with different
levels of stable Ruk/CIN85 expression. It was shown that in the cells over expressing
Ruk/CIN85 both Akt and ERK1/2 were significantly activated already at 1st min after
EGF addition and maintained at high levels up to 30-th min after growth factor treatment,
as compared to control. After treatment of cells, which over express Ruk/CIN85 with
both uPA and EGF together, ERK1/2 activation was much stronger compared to EGF-
treatment only.
In order to analyze whether Ruk/CIN85 over expression affects uPА/uPAR/PAI system
in MCF-7 cells we developed polyclonal antibodies by immunizing rabbits with PAI-1
protein. As an antigen we used recombinant PAI-1, obtained from lysates of induced E.
coli BL-21 cells, which were transformed with pT7-PL-PAI-1 vector containing N-
terminal His6-tag sequence. His6-PAI-1 was purified using Talon Sepharose. High
homogeneity of obtained recombinant PAI-1 protein was demonstrated by SDS-PAGE.
The specificity of generated antibodies was examined by ELISA and Western blotting.
It was shown that PAI-1 expression was significantly up regulated in MCF-7 sublines
with high levels of Ruk/CIN85. In these experiments hypoxia treatment and Ruk/CIN85
over expression had an additive effect on gene expression. Next, MCF-7 cells were
cotransfected with a luciferase reporter gene construct containing 806 base pairs of PAI-1
promoter and Ruk/CIN85 expression vector. It was shown that in the presence of
Ruk/CIN85 luciferase activity was induced both under normoxia and mild hypoxia.
Ruk/CIN85-dependent stimulation of PAI-1 expression was partially blocked by
PI3K/Akt pathway inhibitor LY 294002.
In conclusion, our data show that over expression of Ruk/CIN85 affects EGF-induced
signaling and expression of uPА/uPAR/PAI system components in MCF-7 breast
adenocarcinoma cells.
145
Metabolomics
146
Evaluation of the corneal endothelium in patients with diabetes
mellitus type I and II
Szalai Eszter, Kemény-Beke Ádám, Berta András, Módis László
Department of Ophthalmology
University of Debrecen, Medical and Health Science Center
esztisz@gmail.com
Aim: To determine corneal physiology and endothelial morphology after proper image
analysis technique in type I and II diabetic patients. The HbA1c level and the grade of
retinopathy were also recorded and correlated with the endothelial parameters.
Methods: 41 eyes of 21 patients with type I and 59 eyes of 30 patients with type II
diabetes mellitus (mean age was 40.97±15.46 and 64.36±10.47 years) were examined and
compared to age-matched controls. Endothelial cell density (ECD), mean cell area,
coefficient of variation of cell area, central corneal thickness, intraocular pressure, and
grade of retinopathy were recorded.
Results: There was a statistically significant decreased endothelial cell density in type I
disease (2428±219 cell/mm2) in comparison with healthy subjects (2495±191 cell/mm2, P
= 0.02). The diabetic corneas were thicker than normal (P = 0.001). The HbA1c level was
inversely correlated with the ECD (r = -0.60; P < 0.0001) and correlated with the mean
endothelial cell area (r = 0.60, P < 0.0001). Significant correlation was observed between
the endothelial morphology and grade of diabetic retinopathy (r = -0.40, ECD; r = 0.38,
mean cell area; P = 0.01 for both). In type II diabetes mellitus no significant difference
was found in the evaluated values.
Conclusions: The present study disclosed the alteration of the corneal endothelial
morphology in type I diabetes mellitus as compared to normal subjects. The results
indicated that the type I diabetic corneas are more susceptible to environmental changes
than type II corneas.
147
Expediency of HFE gene mutation screening among patients with
idiopathic conditions related to impaired iron metabolism
Bilevych O., Makukh H., Akopyan H.
State Institution “Institute of Hereditary Pathology of Academy of Medical Sciences of Ukraine”
Lviv, Ukraine
bilevych@gmail.com
It remains controversial whether general screening for HH (hereditary hemochromatosis)
mutations should be performed or whether genetic tests should only be done in subjects
found to have a disturbed iron metabolism. Individuals with hemochromatosis gene
(HFE) mutations and iron overload develop hepatocellular and some extra hepatic
malignancies at increased rates. Some authors hypothesized that due to the pro-oxidant
properties of iron, altered iron metabolism in HFE carriers may promote breast
carcinogenesis. Associations have suggested that the HFE mutations may also be
involved in the development and clinical modification of other non-malignant diseases
(cardiovascular diseases, cystic fibrosis, diabetes, arthritis, neurodegenerative disorders,
and alcoholic liver disease). The molecular – genetic analysis of C282Y and H63D HFE
gene mutations have been performed among 155 persons living at Western Ukraine area.
Among them 42 children with idiopathic hepatobiliar disturbances, 63 Cystic Fibrosis
patients with identified CFTR gene mutations (34 homozygous for F508del), 20 women
with incidents of breast cancer in family and 30 persons from control group. For
molecular-genetic testing of C282Y and H63D HFE gene mutations the PCR based
procedures, which required DNA isolation, PCR and RFLP - analysis have been used. 12
heterozygous carriers of C282Y mutation, 35 heterozygous carriers of H63D mutation
and one compound heterozygous C282Y/H63D were identified among analyzed group.
General frequency of C282Y mutation carriers among studied groups of patients was
8,39%, H63D – 23,2%. Molecular-genetic research showed reliably higher C282Y
mutation frequency among patients with hepatobiliar pathology (11,9%) and among CF
patients (9,5%) as compared to control group (3,3%) that suggests a relationship between
the development of hepatobiliar conditions in CF and HFE C282Y mutation. The
differences in H63D mutation frequency among studied groups weren’t reliable. Among
patients with incidents of familial breast cancer 5% individuals were carriers of C282Y
and 20% - H63D mutation, that don’t differ from general population data. Accordingly
to obtained data every 15 – 30 person is heterozygous carrier of C282Y mutation and
every forth is heterozygous carrier of H63D mutation that point at necessity of increasing
watchfulness concerning HH diagnosis. Molecular – genetic testing of HFE gene
mutations enable to detect the disease in pre-symptomatic stage and under proper low-
cost therapy prevent the complications development, among which hepatocellular
carcinoma is the most severe.
Supported by: West-Ukrainian BioMedical Research Centre (WUBMRC).
148
Subcutaneous fat and metabolic risk factors in pre- and
postmenopausal women.
Žecová Silvia, 1Lejsková Magdaléna, Poledne Rudolf, Stávek Petr, Piťha Jan
Laboratory for Atherosclerosis Research, Institute of Clinical and Experimental Medicine,
1Institute for Postgraduate Medical Education, Prague, Czech Republic
silvique@post.sk
Background: Subcutaneous fat is discussed as a potential risk factor for insulin
resistance and cardiovascular disease. We analyzed association between subcutaneous fat
and metabolic risk factors with regard to reproductive status in middle-aged women.
Methods: Women aged 45-54 years (n = 602) from general population were evaluated
for the presence of cardiovascular risk factors including plasma lipids and markers of
insulin resistance, calculated as fasting plasma glycemia (mmol/l) multiplied by fasting
insulin (IU/l)/22.5 (HOMA-IR). In addition, subcutaneous fat was measured by
ultrasound in all participants. Association between subcutaneous fat and plasma
metabolic risk factors (triglycerides, HDL cholesterol, LDL cholesterol, HOMA-IR and
C-reactive protein, measured by highly sensitive method) was analyzed, separately for
premenopausal (n = 375) and postmenopausal (n = 227) women. The (independent)
association between subcutaneous fat and risk factors under study was evaluated by
multiple regression analysis (STATA), inculding also age and waist circumference.
Results: In the whole group subcutaneous fat was significantly associated with all
metabolic risk factors under study. Nevertheless, after waist circumference was included
into the statistical model, no significant association between subcutaneous fat and any
risk factor was found. Separate analysis according to the reproductive status revealed
significant and independent association between subcutaneous fat and HOMA-IR in
premenopausal (p = 0.014), but not in postmenopausal (p = 0.507) women.
Conclusion: Subcutaneous fat was independently associated with markers of insulin
resistance in premenopausal but not in postmenopausal women. This finding may have
implications for diagnostic and preventive strategies focused on metabolic syndrome and
on the unfavorable fat distribution in women.
149
The role of aromatic compounds in food on dietetic behaviour of
healthy rats
Klenovicsová K., Boor P., 1Celec, 1Behuliak M., 2Ráczová K., 3Somoza V , Šebeková K.
Department of Clinical and Experimental Pharmacotherapy, Slovak Medical University,
Bratislava, Slovakia
1Institute of Molecular Biomedicine and Department of Molecular Biology, Comenius University,
Bratislava, Slovakia,
2St. Elisabeth Hospital, Bratislava, Slovakia;
3German Institute for Food Chemistry, Garching, Germany
kristina.klenovicsova@szu.sk
Maillard reaction products (MRPs) are formed in high amounts in foods during thermal
processing. MRPs occur from non-enzymatic glycation, reaction between free amino-
groups of proteins and reducing sugars. MRPs render foods aroma, taste and colour.
Consumption of diets rich in MRPs was shown to exert deleterious health effects, among
others weight gain and diabetogenic effects, both in experimental animals and humans
(1). Herein, we addressed the question whether the mechanisms of satiety and behaviour
in the adult healthy rats are affected by extractable volatile compounds of bread crusts, or
MRPs. Male Wistar rats were randomized into 3 dietary intervention groups (n=8 each):
control (CTRL, standard rat chow), bread crust (BC, standard diet supplemented with
25% w/w bread crust), and extracted bread crust (EX, standard diet supplemented with
25% w/w ether-extracted bread crust). During 3 weeks intervention food and water
intake, body weight gain and urine volume excretion was recorded. Behavioural tests
(openfield, light-dark box, forced swimming and anhedonic test) were performed. Plasma
insulin, leptin and adiponectin (rat specific ELISA tests), and urinary excretion of 5-
hydroxyindoleacetic acid (5HIAA, HPLC method) was determined. Food intake was
slightly higher in the BC and EX groups than in the controls, but CTRL rats gain more
weight. Compared to CTRL rats BC and EX groups had higher plasma AGEs levels,
reflecting the higher amount of MRPs in their diet (p<0.001). Diet enriched with bread
crusts was associated with decreased insulin sensitivity (HOMA: p<0.05) and increased
plasma leptin (p=0.001) and adiponectin levels (p=0.001). Urinary excretion of 5HIAA
was significantly higher in both, EX and BC groups than in the controls. Rats consuming
aroma-rich BC diet showed less explorative behaviour and more anxiety. In conclusion,
the satiety regulating hormones insulin, leptin, adiponectin and serotonin metabolite 5-
HIAA were all most affected by administration of the bread crust diet, whereas no
significant effect was observed for the aroma extracted diet compared to the control diet,
with exception of adiponectin. These results suggest the effect of both, MRPs and
extractable volatile aroma compounds on satiety regulation and behaviour of healthy rats.
Study was supported by the Slovak medical university internal grant, No. 19-90-05.
1. Šebeková K, Hofmann T, Boor P, et al. Ann N Y Acad Sci 2005; 1043:482-491.
150
Effects of pituitary adenylate cyclase activating polypeptide in
diabetic retinopathy induced by streptozotocin in rats
3Banki Eszter, 3Csanaky Katalin, 1Atlasz Tamas, 2Szabadfi Krisztina, 3Kiss Peter,
3Reglodi Dora, 2Gonczi Pete, 4Szabo Aliz, 4Kovacs Krisztin, 5Setalo Gyorgy Jr.,
6Jakab Feren, 2Gabriel Robert
1Department of Sportbiology, University of Pecs, Pecs, Hungary
2Department of Experimental Zoology and Neurobiology, University of Pecs, Pecs, Hungary
3Department of Anatomy, University of Pecs, Pecs, Hungary
4Department of Biochemistry and Medical Chemistry, University of Pecs, Pecs, Hungary
5Department of Medical Biology, Medical School; University of Pecs, Pecs, Hungary
6Department of Genetics and Molecular Biology, Faculty of Sciences;
University of Pecs, Pecs, Hungary
bankieszti@gmail.com
Introduction: Pituitary adenylate cyclase activating polypeptide (PACAP) is a
neurotrophic and neuroprotective peptide that has been shown to exert protective effects
in different neuronal injuries, models of neurodegenerative diseases and cerebral
ischemia. About 200 million people are estimated to have diabetes in the world. Diabetic
retinopathy, a vascular disorder affecting the microvasculature of the retina is a leading
cause of adult blindness and is the most common complication of diabetes. Although
neurotrophins, such as PACAP, have been assessed as therapeutic agents for neural
complications, their involvement in diabetic retinopathy has not been characterized yet.
Methods: Diabetes was induced by 70mg/kg streptozotocin in Wistar rats. We
administered 3 times intravitreal PACAP (100pmol) injection into the right eye by
Hamilton-syringe. We used histological, different kind of immunohistochemical (vertical
cryosections and whole mount preparations) and molecular biology (Western blot, RT-
PCR) methods. Results: In streprozotocin-induced diabetic rats dopaminergic amacrine
cells appeared to be degenerated, as revealed by tyrosine hydroxylase (TH)
immunoreactivity. Diabetic retinopathy resulted in severe degeneration of dopaminergic
amacrine cells in the inner nuclear layer, as shown by the shape of their soma and their
connection. The quantification of the dopaminergic amacrine cells showed significant
reduction. Neuroprotective effects of PACAP were observed in streptozotocin-induced
retinal degeneration. Intraocular PACAP treatment led to a nearly intact appearance of
the soma, connections and also cell number. According to RT-PCR and Western blot
analyses utilizing TH primary antibody, intensity of immunostaining was increased by
PACAP treatment compared to diabetic retinas. Conclusion: Intravitreal administration
of PACAP protected dopaminergic amacrine cells, demonstrating its therapeutic potential
in streptozotocin-induced diabetic retinopathy.
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