Poster Sessions RECOOP HST Young Scietists Abstract Review
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Інститут молекулярної біології і генетики НАН України
2012
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irk-123456789-1568592019-06-21T01:26:03Z Poster Sessions RECOOP HST Young Scietists Abstract Review Poster Sessions - RECOOP HST Young Scietists Abstract Review 2012 Article Poster Sessions RECOOP HST Young Scietists Abstract Review // Вiopolymers and Cell. — 2012. — Т. 28, № 2, доп. — С. 85-217. — англ. 0233-7657 http://dspace.nbuv.gov.ua/handle/123456789/156859 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Інститут молекулярної біології і генетики НАН України |
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Poster Sessions
RECOOP HST Young Scietists
Abstract Review // Вiopolymers and Cell. — 2012. — Т. 28, № 2, доп. — С. 85-217. — англ. |
| series |
Вiopolymers and Cell |
| first_indexed |
2025-07-14T09:10:46Z |
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2025-07-14T09:10:46Z |
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1837612922012958720 |
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85
Poster Sessions
RECOOP HST Young Scietists
Abstract Review
86
CROATIA
87
Women’s Health and Cardiovascular
Diseases
88
Female stroke
1Rostohar Bijelic B., 2Kadojic D.
1.Scientific Research Unit, University Hospital Center Osijek,
Huttlerova 4, Osijek, Croatia
2.Clinic of Neurology, University Hospital Center Osijek,
Huttlerova 4, Osijek, Croatia
nbijelic@mefos.hr
Aim: Stroke has mostly been considered a male disease, and male gender as a risk factor
for cerebrovascular diseases. However, recent research shows that women suffer from
stroke more often, and that it is more lethal for female than male patients. The aim of this
pilot study was to investigate the situation in Osijek area.
Methods: A group of 100 patients, average age 70.5 years; 45 females and 55 males with
stroke treated in Department of neurology, Clinical hospital center Osijek was included.
Patients were grouped according to gender and the following were investigated in each
group: distribution of stroke according to groups of age, frequency of hemorrhagic and
ischemic stroke, frequency of ischemic stroke subtypes, presence of certain risk factors,
occurrence of complications during hospitalization and final outcome at discharge.
Results: In average, women were older than men at the time of first stroke. Differences
in occurrence of risk factors were also noticed. The frequency of complications was
almost equal, except for uroinfections, which were, as expected, more frequent in women
due to specific structure of female urinary tract. Women had poorer outcome of stroke
and higher case–fatality rate than men.
Conclusions: Biological differences like hormonal status (effect of estrogen and
testosterone on endothelium and vascular system), influence of risk factors present only
in women (oral contraceptives usage, hormone replacement therapy, and pregnancy),
specific life-styles, comorbidity (migraine, thrombophilia), and longer life-span of
women can explain some of the gender differences, however, a lot of details still remain
unclear.
Key Words: women, stroke, risk factors, complications, outcome
89
Anxiety, depression and stress in acute myocardial infarction: a
pilot study
1Gašpar T., 2Topić J., 3Makarović Z., 3Steiner R., 1Heffer M. , 4Ilakovac V.
1 Department of Medical Biology, School of Medicine, J.J.Strossmayer University of Osijek, Osijek,
Croatia
2 Department of Psychiatry, General Hospital Vukovar, Vukovar, Croatia
3 Department of Cardiology, University Hospital Center Osijek, Osijek, Croatia
4 Department of Biophysics, Medical Statistics and Medical Informatics, School of Medicine, J.J.
Strossmayer University of Osijek, Osijek, Croatia
tgaspar@mefos.hr
Aim: The aim of our study was to detect the connection between depression, anxiety and
stress level with the course of acute coronary syndrome. A comparison of psychological
test results obtained from cardiology ward patients was conducted to determine
differences between patients with acute myocardial infarction and angina pectoris.
Methods: The sample included 25 patients hospitalized for acute coronary syndrome in
2012 in Osijek.The general questionnaire, Beck Depression Inventory, Beck Anxiety
Inventory, Type D Personality Test and Life Event Stress Scale were used to assess levels
of depression, anxiety and stress. The patients were divided into two groups – the one
with acute myocardial infarction (MI) and the other with angina pectoris (AP), and were
further compared and contrasted by psychological parameters.
Results: Study included 14 male and 11 female patients, average age 61 yrs (SD=9.5).
There were 12 patients in MI group and 13 in AP group. Certain level of depression was
reported by 11 patients, while 5 achieved middle score on anxiety. In comparison to AP
patients larger portion of MI patient were both – middle score on anxiety and showing
characteristic of social inhibition. The results of anxiety and depression tests suggest
differences between two groups of patients, which are not significant due to small
number of examinees. Two fifths of patients in AP group had a middle score on Life
Event Stress Scale and none in MI group. Type D Personality Test and Life Event Stress
Scale showed difference between MI and AP patients.
Conclusions: Beside already known risk factors for cardiac diseases such as
hypertension, dyslipidemia and diabetes, patients who show also symptoms of
depression, anxiety and stress have greater risk of developing acute myocardial infarction
rather than angina pectoris.
Keywords: depression, anxiety, stress, acute myocardial infarction
90
Coronary artery disease risk factors in coastal and continental
Croatia
1,§Mlinarević D., 2,§Lukin A., 3Makarović Z., 3Boban D., 4Vari S., 1Heffer M.,
3Steiner R.
1 Department of Medical Biology, School of Medicine, J.J.Strossmayer University of Osijek, Osijek,
Croatia
2 Department of Cardiology, University Hospital Centre Split, Split, Croatia
3 Department of Cardiology, University Hospital Center Osijek, Osijek, Croatia
4International Research and Innovation Management Program, Cedars - Sinai Medical Center, Los Angeles,
CA, USA
§ Both authors contributed equally
mheffer@mefos.hr; marija.heffer@gmail.com
Aim: A comparison of clinical data obtained from cardiology ward patients in two major
regional hospital centers (UHC Osijek and UHC Split) was conducted to determine
differences between patients with CAD in continental and coastal Croatia.
Methods: Data was obtained from patients hospitalized for CAD in 2007 in Osijek and
Split, respectively. The variables included age, gender, BMI, diagnosis, duration of
hospitalization, history of arterial hypertension and diabetes, laboratory findings, ECG
and coronarography data. The sample included 295 patients from UHC Osijek and 195
patients from UHC Split.
Results: Patients from the coastal region (UHC Split) were found to be older than
patients in continental Croatia (UHC Osijek; p<0.001), mainly due to the difference in
the age of male patients in the two regions. The patients in Osijek had a higher BMI than
patients in Split – 28.1±4.9 versus 23.91±2.02 (p<0.001). Arterial hypertension was
found more often in UHC Split male patients, compared to UHC Osijek male patients
(p=0.011), but a difference was not found when comparing all patients’ data or female
patients’ data. Dyslipidemia was found more often in Osijek in all patients and separately
in males and females (p<0.001, respectively). The laboratory data analysis revealed that
UHC Split patients had higher HDL (p=0.005), but also higher LDL (p=0.012) and
triglyceride levels (p<0.001) than UHC Osijek patients. Gender-related differences were
observed in both centers. In Osijek, females were older (68.62±10.77 versus 62.25±11.25
years, p<0.001) and had higher HDL levels (1.27±0.36 versus 1.13±0.34 mmol/L,
p<0.001) than men, but in Split the differences were not observed. Unlike in Osijek, men
in Split had higher BMI than females (24.78±1.69 versus 22.83±1.89, p<0.001).
Conclusions: Patients from coastal Croatia have less risk regarding some traditional
cardiovascular risk factors than those from the continental region, delaying the onset of
clinically apparent CAD. Furthermore, dissimilar patterns of gender-related differences
between clinical data from Split and Osijek also suggest a discrepancy between CAD
patients in two Croatian regions.
Keywords: Coronary artery disease, obesity, dyslipidemia, BMI
91
Peripheral expression of adipocytokines and role of sympathetic
nervous system in function of perivascular adipose tissue and
vascular reactivity
Nenad Nešković, Željka Perić, Radivoje Radić
Josip Juraj Strossmayer University in Osijek, Faculty of Medicine, Department of anatomy and
neuroscience,
J. Huttlera 4, 31000 Osijek
neskovic@mefos.hr
Aim: Visceral (VAT) and perivascular adipose tissue (PAT) are active endocrine and
paracrine organs. Two opposite functions are assigned to PAT. It has antiatherogenic and
proatherogenic effects. In this research, changes of PAT and sympathetic nervous system
will be measured also influence of mother`s nutrition during gestation on function of
PAT and VAT in offspring will be determined.
Methods: 10 female rats will be randomly divided in 2 groups. One group will be fed
with high content of saturated fatty acid – FD group (n=5), and the other will be fed with
standard laboratory chow – CD group (n=5). After coupling and lactation offspring will
be randomly divided in 2 groups – one will be fed with high content of saturated fatty
acid (FD-FD2 and CD-FD2), and other with standard laboratory chow (FD-CD2 and CD-
CD2). We will have 4 groups of offspring genetically similar, but exposed to different
intrauterine and postnatal nutritional environments. Also, blood concentrations of
adiponectin, insulin, glucose, LDL, HDL, triglycerides, TNF-α and IL-6 in four separate
groups of animals will be measured. After decapitation, expression of ADIPOQ and β3-
AR gene will be measured in samples of VAT and PAT, and expression of SLC6A2 and
DβH will be measured in coeliac ganglion. Role of PAT will be tested by measuring
vascular reactivity after norepinephrine perfusion through isolated branches of superior
mesenteric artery.
Results: By measuring expression of ADIPOQ gene and β3-AR gene in adipose cells of
PAT in regard to different environment of offspring we expect to confirm that lipolysis
mediated by sympathetic nervous system in predisposed rats is decreased. By analyzing
offspring of fat diet mothers which are on control diet we will attempt to determine role
of postnatal diet on sympathetic nervous system activity in PAT and synthesis of
adiponectin in PAT and VAT. We will try to link the changes in ADIPOQ gene
expression in PAT with changes in expression of SLC6A2 gene and DβH gene in coeliac
ganglion. Also, we expect to comfirm protective role of PAT in control diet rats and lack
of that protection in fat diet rats by measuring vascular reactivity of superior mesenteric
artery with and without PAT.
Conclusions: Understanding relationship between PAT and sympathetic nervous system
will enable us to create more rational pharmacotherapy for obesity and metabolic
syndrome. In general, it could enable great social, economic and health benefits for
individuals and society.
92
Cyp450 Ω-hydroxilase expression in hyperbaric oxygenation
Paradžiković I., Mihaljević Z., Ćosić A., Novak S., Drenjančević I.
Faculty of Medicine University J. J. Strossmayer Osijek, Department of Physiology and Immunology,
J. Huttlera 4, Osijek, Croatia
astralsource@gmail.com
Aim: Our previous studies showed that hyperbaric oxygenation could affect vascular
sensitivity and reactivity on vasodilators and vasoconstrictors in healthy and diabetic rats.
CYP450- Ω- hydroxilaze (CYP450-4A) is one of the oxygen sensors in blood vessels,
producing increased amounts of vasocontrictor metabolite 20-HETE in positive
correlation with increase in pO2. The aim of this study was to evaluate the effects of
intermitent hyperbaric oxygenation (HBO) on expression of CYP450-4A in aortic and
cerebral vessels of rats.
Methods: Type I diabetes mellitus (T1DM) was induced in Sprague-Dawley (SD) rats by
a single intravenous injection of streptozocin (60 mg/kg). Groups: SD control healthy
rats, control healthy rats that underwent HBO (2 h for 4 days), T1DM group and T1DM
rats that underwent HBO. The expression of CYP450 -4A was determined by Western
blot in aortic and cerebral vessels samples.
Results: Densitometrical analysis of the protein bands demonstrated that the CYP450-4A
aortic/cerebral vessel expression has increased in the control group of rats treated with
HBOT, as well as in diabetic rats group treated with HBO, compared to their respective
controls.
Conclusions: This study, for the first time demonstrated the increased expression of
CYP450-4A enzyme in control and T1DM rats after hyperbaric oxygenation, thus
confirming previous functional studies on the role of CYP450-4A metabolites in vascular
responses to various physiological stimuli.
Keywords: hyperbaric oxygenation, cyp450 Ω-hydroxilaze, Western blot, diabetes
mellitus
Supported by grant of Croatian Ministry of Science, Education and Sport No. 219-
2160133-2034
93
Epicardial adipose tissue is associated with central adiposity but not
with CAD
Vrselja Z.; 1Šram M.; Radić R.
Department of Anatomy and Neuroscience, Faculty of Medicine Osijek,
J. Huttlera 4 31000 Osijek;
1Departmnet for the cardiovasular diseases, Clinical Hospital Center Osijek,
J. Huttlera 4 31000 Osijek
vrselja@gmail.com
Aim: Increased amount of visceral adipose tissue (VAT) and epicardial adipose tissue
(EAT) have been linked to coronary artery disease (CAD). Clinically central adiposity is
used as proxy for higher amounts of VAT. The aim of this study is to determine if central
distribution of adipose tissue and CAD are associated with higher amounts of EAT.
Methods: In this study 55 patients were selected into the peripheral and central group
using waist circumference. All 55 patients were subjected to coronarography and
echocardiography. According to the results patients were selected into the CAD group or
control group. EAT thickness was determined by ultrasound.
Results: In regional distribution groups, selected by standard borderline values of waist
circumference, no significant difference was found in thickness of EAT (p=0.847). When
tested in regional distribution groups selected by adjusted criteria for Caucasians a
significant difference was found (p<0.001) in thickness of EAT between peripheral and
central group. There was no significant difference (p=0.330) in thickness of EAT
between CAD and control group. Weak correlation is found between the waist
circumference and the EAT (r=0.238; N=55; p=0.080).
Conclusions: Central distribution of the adipose tissue has higher amount of EAT which
is a known risk factor for CAD. Waist circumference did not show a strong correlation
with thickness of the EAT which was expected due to its undiscriminating nature
between subcutaneous and visceral adipose tissue.
Keywords: epicardial adipose tissue, cardiovascular disease, ultrasound, waist
circumference
94
Mother and Child Health
95
Bone loss among premenopausal women on long-term suppressive
therapy with thyroid hormone
Kokot A.., Šijanović S., 1Vidosavljević D.
JJ Strossmayer University Osijek, School of Medicine, Department Obstetrics and Gynaecology,
Huttlerova 4, HR31000 Osijek, Croatia;
1Vukovar General Hospital, Bolnička
5, HR 32000 Vukovar, Croatia
sinisa.sijanovic@os.htnet.hr
Introduction: It is controversial whether the long-term treatment with thyroid hormone
given at suppressive doses has a negative effect on bone metabolism. Aim of this
prospective study was to determine whether the long-term thyroxin therapy in the
premenopausal period is a risk factor for the development of secondary osteoporosis, and
whether those women have increased bone loss during the premenopausal period.
Patients and methods: There were a selected group of women in premenopausal period
(N=19) aging 39,08,0 suffering from differentiated thyroid gland carcinoma. To all of
them, the total thyreoidectomy was done and the thyroxin suppressive therapy was
introduced. The duration of the suppressive therapy from the beginning of the research
was 9,46,4 years. Laboratory results have excluded other possible factors for secondary
osteoporosis. The prospective study of bone densitometry was done during four years to
all examinees using the method of dual photon x-ray absorptiometry (DXA) of the spine
and the femoral neck, and also by the method of single-photon absorptiometry (SPA) of
the distal radius.
Results: In the beginning of this study, while the patients were treated for 10 years
osteopenia was found in the spine and femoral neck in two examinees for every region.
On the distal radius osteopenia was found in 4 examinees. The total was 8 out of 19 with
osteopenia. One year later, after the second measurement no statistically significant loss
of bone mass was in any region of skeleton. However, looking at the individual scores,
osteopenya was found in 6 examinees on the distal radius but on the spine and the
femoral neck there were bone loss in several women not to the point that would create
osteopenia. But, after the 4 years of measurement t-test had shown some significant bone
loss.
Conclusion: after careful examination, our conclusion is that women who begin with a
long-term thyroxin therapy (about 10 years) in the premenopausal period can develop
osteopenia at the beginning of menopause. In that case, those women should complete
mammography testing and bone densitometry before hormone replacement therapy is
used.
Keywords: menopause, osteopenia, thyroxin, absorptiometry
96
Impact of high-fat diet on fertility in Sprague Dawley rats
1,2Šnajder D., 1Nešković N., 1Perić Ž., 1Radić R.
1Department of Anatomy and Neurosciences, School of Medicine, J.J.Strossmayer University of
Osijek,
J. Huttlera 4, 31000 Osijek, Croatia
2Clinical Institute of Nuclear Medicine and Radiation Protection, University Hospital Centre Osijek,
J. Huttlera 4, 31000 Osijek, Croatia
dsnajder@mefos.hr
Aim: Pilot study examined the impact of high-fat diet on fertility.
Methods: 10 female Sprague Dawley rats from the same litter were at puberty (age of six
weeks) randomly divided into two groups (N=5 for both groups) and fed with standard
chow or high-fat-diet (20% lard, enriched with vitamin and mineral-mix, mixed with
standard laboratory chow) ad libidum. At the age of twelve weeks the female rats were
placed in separate cages with one male per cage for mating. The male Sprague Dawley
rats were also all from the same litter to minimize genetic-dependent diversity in future
brood size and quality. The number of pregnancies and the size of litter were recorded for
each group of females.
Results: In the control group fed with standard chow the birth dates were spaced between
23rd and 39th day with a mean size of litter of 14, and a standard deviation of 1.414. In
experimental group all females also gave a birth between 23rd and 40th day, but
conception was delayed for two weeks in the case of 3 females. Although mean size of
litter was also 14, standard deviation was higher, 2.646.
Conclusions: All female control group-rats besides one gave birth within a week time
from the date of expected birth. In comparison, only two HFD rats had their litter in that
week. Although the mean litter size was the same, the HFD rat-litter showed a greater
diversity in number of pups. The prolonged study with a bigger number of animals
should further investigate influence of high-fat diet on fertility.
Keywords: High-fat diet, obesity, fertility
97
Metal contamination and its effect on reproductive system
1Vidosavljević D., 2Puntarić D., 3Šijanović S., 4Gvozdić V., 5Jergović M.
1Vukovar General Hospital,
Bolnička 5, HR 32000 Vukovar, Croatia
2 JJ Strossmayer University Osijek, School of Medicine, Department of Public Health,
Huttlerova 4, HR31000 Osijek, Croatia
3 JJ Strossmayer University Osijek, School of Medicine, Department Obstetrics and Gynecology,
Huttlerova 4, HR31000 Osijek, Croatia
4 JJ Strossmayer University Osijek, Department of Chemistry,
Kuhačeva 20,HR 31000 Osijek, Croatia
5 Zagreb Public Health Institute, Department of Health Ecology,
Mirogojska cesta 16, HR10000 Zagreb, Croatia
sinisa.sijanovic@os.htnet.hr;
The War in1991 in Croatia resulted with a significant release of contaminants into the
environment as a result of the use of combat assets.
Aim of the study was to investigate the concentrations of metals and metalloids in serum,
hair, urine, soil, water and vegetables on 11 locations (8 exposed to heavy and 3 exposed
to mild combat activities) of eastern Croatia, in order to create biobank for further
research.
Methods: Total of 391 participants (204 male, 187 female) gave samples of urine , hair
and serum in order to determine levels of metal for each settlement and answered an
questionnaire in order to establish level of their professional exposure, war or
environmental exposure.
Water was collected form 14 different sites, soil from 28 sites, vegetables from 41 sites.
Inductively coupled plasma mass spectrometry was made to measure 66 elements.
Results: All results suggest an association between the intensity of war activities and the
degree of contamination by metals used in ammunition. Significantly higher
concentrations of Al, As, Ba, P and V in the blood, significantly higher concentrations of
As and Cd in urine and Al, Fe, Cd, Pb and V in hair of all subjects exposed to war
operations were found.
Furthermore, environmental biomonitoring has shown high levels of As, Fe and Cd in
drinking water of some settlements and also higher levels of Cd, As, Hg , Sb and Mg in
soils and vegetables collected from number of sampled locations (even exceeding
maximum allowed concentrations) .
Conclusion: By making full environmental and population biomonitoring for selected
settlements a necessity for further research emerged. Many of elevated metals have toxic
effects, either on male or female reproductive system or on pregnancy outcomes.
Cadmium is one of the most interesting in this study , since is known “metallohormone”
activity on uterus and on impaired sperm production. Further extended research is
planned .
Key words: war, contamination, metals, metalloids, Croatia
98
Cardiovascular medications during pregnancy
1Boskovic Jelena, 2Leppée Marcel, 3Eric Mirela
1School of Pharmacy and Biochemistry, University of Zagreb,
A. Kovacica 1, 10000 Zagreb, Croatia.
2Department of Pharmacoepidemiology, Andrija Stampar Institute of Public Health,
Mirogojska 16, 10000 Zagreb, Croatia
3School of Medicine Novi Sad, University of Novi Sad,
Hajduk Veljkova 1, 21000 Novi Sad, Serbia
jelboskovic@gmail.com
Aim: The course of pregnancy is associated with a number of changes in the woman's
body. Literature data indicate that 1%-3% of pregnant women develop some cardiac
disorder. The aim was to assess the prevalence of cardiovascular drug use and the rate of
congenital malformations in neonates. One arm of the study (one-month study) was
performed at four maternity hospitals in Zagreb, Croatia and the other arm of the study
(one-year study) was performed in Novi Sad, Serbia.
Methods: The study was designed as cross-sectional study, and included 893 pregnant
women from Zagreb and 6099 pregnant women from Novi Sad.
Results: According to data collected in the Zagreb arm, the prevalence of cardiac
disorders in pregnancy was 3.6% (n=32), whereas data from the Novi Sad arm indicated
a prevalence of 1.7% (n=102), yielding a predominant use of -blockers in Zagreb (n=32;
3.6%) and of antihypertensive agents in Novi Sad (n=64; 1.0%). In Zagreb,
malformations were detected in 26 (2.9%) fetuses and newborns in the general population
and in 3 (9.4%) fetuses and newborns in utero exposed to cardiovascular agents. The
agents taken during pregnancy were almost exclusively from FDA C and D classes. The
prevalence of congenital malformations in fetuses at in utero exposure to FDA class D
drugs was 3.1% (n=1). In Novi Sad, malformations were detected in 326 (5.35%) fetuses
and newborns in the general population and in 5 (4.9%) fetuses and newborns in utero
exposed to drugs for cardiovascular disorders. Pregnant women most frequently used
FDA class C drugs. Class C and D drugs were most commonly taken in first trimester,
when the fetus is most sensitive to the action of teratogenic factors. A severalfold
increase in the use of methyldopa and slight increase in the sporadic use of furosemide
was recorded by the end of pregnancy, whereas the use of other drugs was reduced or
completely discontinued. One case of a major malformation, palatoschisis, was found in a
woman having taken a C class drug.
Conclusions: For most cardiac disorders, the risk posed by the disease itself for both the
mother and the fetus generally exceeds the postulated risk of medications used to treat the
disease. If a pregnant woman requires such therapy, a respective agent with the best
safety profile should definitely be prescribed. The exact cause of these malformations
cannot be positively identified because the fetus is generally not exposed to a single
teratogenic factor but to a combination of such effects.
Keywords: pregnancy; cardiovascular medication; Zagreb, Novi Sad
99
Nanobiotechnology and Cancer
Research
100
Expression of TFF3 in epithelia of mouse fetus
1Bijelić N., 1Belovari T., 1Tolušić Levak M., 2Mišković K., 2,3Baus Lončar M.
1.Department of Histology and Embryology, School of Medicine,
J.Huttlera 4, 31000 Osijek, Croatia
2.Department of Clinical Chemistry and Biochemistry, School of Medicine,
J.Huttlera 4, 31000 Osijek, Croatia
3.Department of molecular medicine, Institute Ruđer Bošković,
Bijenička 54, 10000 Zagreb, Croatia
nbijelic@mefos.hr
Aim: TFF3 protein has an important role in maintenance of epithelial integrity by acting
through various mechanisms. In this work we wanted to get insight in TFF3 expression in
mouse fetus
Methods: Mouse fetuses, day 15 to 17 were isolated, fixed in 4% paraformaldehyde,
paraffin embedded, cut (6µm), and processed for immunohistochemical staining. Primary
polyclonal rabbit anti-TFF3 antibody was used, and PBS as a negative control.
Results: TFF3 expression was found in keratinocytes that constitute basal and middle
layers of stratified epithelia in developing skin of fetuses and in keratinocytes at the
vestibulum of the oral cavity. Goblet cells of the nasal cavity, bronchi and gut also
stained positively. Mild signal was found in the epithelium of kidney collecting ducts.
Negative controls showed no signal.
Conclusions: TFF3 has been shown to promote migration of epithelial cells in vitro and
in vivo, partly by reducing cell-to-cell adhesion. It participates in immune response, and
activates various signaling cascades. TFF3 is also known to affect apoptosis. Since it is
expressed in various epithelial tissues during fetal development, it could play an
important role in these actively proliferating tissues. Cancers which express or
overexpress TFF3 probably only exploit the physiological properties of TFF3 in growth
and metastasing, which has diagnostic and potentially therapeutic implications.
Keywords: epithelial cells, fetal development, mouse, neoplasms, TFF3 protein
101
Translational Research and Drug
Development
102
Novel monomethine derivatives as detection tools
1Mišković K., 1,2Baus Lončar M., 2Piantanida I., 1,3Glavaš-Obrovac L. J.
1. School of Medicine,
J.Huttlera 4, 31000 Osijek, Croatia
2. Institute Ruđer Bošković,
Bijenička 54, 10000 Zagreb, Croatia
3. University Hospital Centre Osijek,
J.Huttlera 4, 31000 Osijek, Croatia
kmiskovic@mefos.hr
Aim: Development of new monomethine derivatives based on cyanine dyes and their
application in molecular biology as detection tool is highly prospected and promising
thanks to their fluorescent ability. Depending on their model of interaction with nuclear
acids and preference for AT or GC region they can be used for nuclear acids visualization
and as marker for cell staining.
Methods: Fluorescent microscopy were used for evaluation and detection of cell
localization of monomethine cyanine derivatives no.1,2,3 on live HeLa cells with
Hoechst 33258 as control. Agarose gels post staining was applied for DNA/RNA
visualization with SybrSafe as positive control.
Results: Evaluated cyanine derivatives no. 1,2,3 are spread all over the cell expressing
green fluorescent signal. 30 minutes after incubation signal from derivatives no.1 and 2 is
strong and easily detected. Signal is stable during the next 2h. Derivative no.3 need 90 –
120 minutes to enters in to the cell and localize. Signal is weaker then for other two
mentioned before. 15 and 45 minutes gel post staining showed that derivatives no.2 and 3
gives good visualization of DNA/RNA under the UV and trans white light. Results are
comparable to SybrSafe. Derivative no.1 gives no visual results of DNA/RNA on gel.
Conclusions: Monomethine derivative no.1 is the best candidate for fluorescent
microscopy since it enters quickly giving strong signal. It can be used in combination
with other dyes for multiple detection. Derivatives no. 2 and 3 are promising candidates
for further development as visualization tool.
Keywords: monomethine cyanine derivatives, visualization, cell localization,
DNA/RNA, SybrSafe
103
Association of ACE DD-genotype in women and renal disease
1Avdičević M., 4Krajina-Andričević M., 3Zibar L., 2Štefanić M., 2Karner I.,
2Glavaš-Obrovac Lj.
University Hospital Centre Osijek:
1Scientific Unit for Clinical-Medical Research,
2Clinical Institute of Nuclear Medicine and Radiation Protection,
3Internal Clinic, J. Huttlera 4, 31000 Osijek
4General Hospital Vinkovci, Zvonarska 57, 32100 Vinkovci
monika.avdicevic@gmail.com
Aim: To investigate the significance of insertion/deletion polymorphism of angiotensin-
converting enzyme (ACE) as a possible contributing factor in the development of
diabetic nephropathy.
Methods: Genomic DNA was extracted from whole blood of 100 patients with diabetic
nephropathy and 102 diabetic patients with normal renal function (urinary protein
excretion rate less than 300 mg/day and creatinin clearance level ≥ 80 ml/min).
Genotyping was carried out using primers and fluorescent probes in a LightCycler
System (Roche). Statistical analysis was performed using the SPSS 19.0 software (SPSS
Inc, Chicago, IL, USA).
Results: Genotype frequencies of the ACE I/D polymorphism were in accordance with
the Hardy-Weinberg equilibrium. Females with end stage renal disease showed higher
frequency of DD genotype than those with preserved renal function (41% vs. 28%;
p=0,038). In addition, the D allel frequency was significantly higher in women with
diabetic nephropathy compared to controls (χ²=6,345, p=0,011), respectivly, only one of
ten women does not posses the D allel.
Conclusions: Based on obtained results could be concluded that the ACE DD genotype
has an impact for the development of renal disease in females with type 2 diabetes,
indicating a possible DD genotype-associated gender effect in renal disease.
Keywords: diabetes mellitus type 2; diabetic nephropathy; angiotensine converting
enzyme, insertion deletion polymorphism
104
CZECH REPUBLIC
105
Women’s Health and Cardiovascular
Diseases
106
Influence of SNP in gene for prepro-orexin on prevalence of obesity
in the Czech population
1Jurčíková L., 1Hubáček J. A., 1Adámková V., 2Zlatohlávek L.
1 Institute for Clinical and Experimental Medicine;
2 3rd Department of Internal Medicine, 1st Faculty of Medicine, Charles University; Prague, Czech
Republic
jurcikova.l@gmail.com
The obesity is current world problem that reaches pandemic proportions. OrexinA and
orexinB are produced by neuron cells of lateral hypothalamic area, also known as
“hunger center”. Involvement of these neuropeptides in control of feeding behavior was
previously confirmed.
We investigate a role of single nucleotide polymorphism in gene for prepro-orexin
(rs760282). SNP was monitored in population study Czech postMONICA (N=2500).
This first study provided information on the frequency of mutations in this SNP in the
general population. Second study was realized on the obese children group – patients
(N=349) before and after spa intervention. Investigated genotype (-909T/C) is
substitution T (wild-type allele) for C (mutant allele).
Distribution of genotype in population TT, TC and CC: 53.93, 39.44 and 6.63%
respectively. Database of patients exhibited 55.30, 39.54 and 5.16% respectively.
Genotype distribution between databases is not statistically differ (P=0.57).
Data in male and female postMONICA groups were tested for BMI, waistline (WL), hips
(H) and cholesterolemia (CHOL). In female group was found significant increasing trend
in investigated parameters. The trend is most markedly in WL (85.99±13.36,
85.81±13.32 and 84.43±13.34cm) and H (105.69±10.49, 105.49±10.61 and
105.18±10.91cm) for TT, TC and CC allele respectively. In male group significant
difference between monitored parameters was not found.
Results in patients for BMI before spa intervention was 30.52±4.35, 31.02±4.66 and
30.57±4.78kg/m2 for TT, TC and CC allele respectively and after spa intervention
28.17±4.09, 28.77±4.38 and 28.29±4.39kg/m2. Change of BMI before and after
intervention was – 2.31±0.73, – 2.30±0.76 and – 2.28±0.78kg/m2 for TT, TC and CC
allele respectively.
Supported by grant 00023001 (IKEM, CR)
107
Mother and Child Health
108
Role of MTHFR gene in spontaneous abortions
1,2Rynekrova J., 4Kasparova D., 1,3Adamkova V., 4Fait T., 1,2,3Hubacek J. A.
1Institute for Clinical and Experimental Medicine,
Prague, Czech Republic
2Centre for Cardiovascular Research,
Prague, Czech Republic
3South Bohemia University, Faculty for Public Health and Social Studies,
Ceske Budejovice, Czech Republic
4Department of Obstetrics and Gynecology, 1st Faculty of Medicine and GeneralFaculty Hospital,
Prague, Czech Republic
rynji@seznam.cz
Introduction: According to estimates, up to 20% of pregnancies end in the first trimester
by spontaneous abortion (SA). Apart from a negative impact of some environmental
factors on the embryonal development, it is evident that genetic factors also play an
important role. One of the candidates is methylenetetrahydrofolate reductase gene
(MTHFR) which is associated with disorders in folate dependent homocystein
metabolism and DNA methylation.
Material and methods: We collected DNA from 538 samples of spontaneous abortions
and genotyped MTHFR variant C667T by PCR-RFLP method. The frequencies of
genotypes were compared with known population sample of adults (N = 2 555). For the
statistical analysis we used χ2 test.
Results: Genotype frequencies which are within the HW equilibrium are similar to the
neighbouring populations. There was a significantly lower frequency of the TT genotype
in SA in comparison with the controls (8.7% vs. 12.1%) when we compared CC genotype
carriers (44.4% in SA and 42.7% in controls) with t-allele carriers (32.2% in SA and
34.7% in controls, P=0.028, OR=1.071, CI=0.89-1.29).
Conclusions: Our results suggest that TT genotype in MTHFR may have a protective
effect on embryo development.
Keywords: spontaneous abortions, methylenetetrahydrofolate reductase gene
Supported by project No 00023001 (IKEM, CR)
109
Translational Research and Drug
Development
110
Verification of novel potential biomarkers of intraamniotic infection
and inflammation in preterm birth patients
1Tambor V., 2Vajrychova M., 1,2Lenco J., 3Menon R., 2Link M., 4Kacerovsky M.
1 Biomedical Research Center, University Hospital,
500 05 Hradec Kralove, CZ
2 Institute of Molecular Pathology, University of Defence,
500 05 Hradec Kralove, CZ
3 OB/GYN Department, University of Texas Medical Branch,
775 55 Galveston, TX, USA
4 OB/GYN Department, University Hospital,
500 05 Hradec Kralove, CZ
tambor@pmfhk.cz
Aim: We have recently performed global quantitative proteomic analyses of amniotic
fluid from preterm birth patients to identify novel potential biomarkers of intraamniotic
infection and inflammation (IAI). This phase of the project, the discovery phase, pointed
on several amniotic fluid proteins with altered levels, which could be thus associated with
the presence of IAI. Our mail goal was to verify these initial findings using
complementary techniques.
Method: Except the antibody-based approach ELISA, which is the golden standard in
protein quantitation, we used a novel perspective proteomic tool for targeted protein
detection and quantitation – multiple reaction monitoring mass spectrometry (MRM MS).
Without the need for a specific antibody, this approach can detect and quantify multiple
proteins in complex matrices with high sensitivity, accuracy and precision.
Results: Using ELISA and MRM, we targeted and quantified selected proteins, which
showed statistically highly significant dysregulation in our initial discovery phase
experiments. We were able to confirm altered levels of all of these selected candidates.
Most of these proteins showed correlation with the presence of IAI.
Conclusions: Our findings show, that protemics is indeed capable of providing novel
insights into various pathologies, with one of the goals being the identification of novel
biomarkers. We sucessfully demonstrated this using a two stage strategy, where we
idetified and verified several novel potential IAI biomarkers.
Keywords: proteomics, biomarkers, intraamniotic infection and inflamation
111
HUNGARY
112
Women’s Health and Cardiovascular
Diseases
113
Oxidative contractile dysfunction in human cardiomyocyte
Kalász J., Pásztorné E. T., Balogh A., Fagyas M., Pahlavan S., Tóth A., Édes I.,
Papp Z., Borbély A.
University of Debrecen, MHSC, Institute of Cardiology, Division of Clinical Physiology
kalaszjudit@hotmail.com
Aim: Oxidative myofilament protein alterations have been shown to contribute to
myocardial contractile dysfunction. We aimed to characterize the effects of
myeloperoxidase (MPO) on cardiomyocyte contractile function and to identify the related
myofilamentary protein modifications in left ventricular human myocardium.
Methods: Ca2+ dependent (Factive) and independent (Fpassive) forces were measured in
permeabilized human cadiomyocytes before and after applications of hydrogen peroxide
(H2O2), MPO and in the presence or absence of an MPO-inhibitor (MPO-I) and the
reducing agent dithiotreitol (DTT). Ellman’s and sulfhydryl (SH) group biotinylation
assays were used to quantify the extent of the SH oxidation of myofilament proteins.
Results: Application of H2O2 significantly decreased cardiomyocyte Factive (from
23.1±3.7 kN/m2 to 16.0±2.8 kN/m2, n=7, P<0.01) and increased Fpassive (from 3.5±0.9
kN/m2 to 4.0±0.9 kN/m2, n=7, P<0.01). When H2O2 and MPO were applied together, a
reduction in Factive and an additional increase in Fpassive were observed. The MPO-I could
partially prevent the effects on Factive and Fpassive (from 16.3±3.4 kN/m2 to 11.1±1.6
kN/m2; and from 1.8±0.4 kN/m2 to 2.3±0.5 kN/m2 (n=5), respectively). Combined
application of H2O2 and MPO significantly decreased the relative SH content of
myofilament proteins (to 87.04±1.2% from 100%, P<0.05), which effect was reversed by
the reducing agent DTT. DTT also reversed the MPO induced increase in Fpassive (from
2.4±0.3 kN/m2 to 1.4±0.2 kN/m2, n=6). H2O2 and MPO significantly decreased the
number of SH groups of the actin and a recently unidentified ~60 kDa molecular weight
protein.
Conclusions: MPO-derived oxidants may contribute to myocardial contractile
dysfunction via decreasing cardiomyocyte force production and increasing Fpassive of
human cardiomyocytes. These effects are mainly attributed to myofilament protein
oxidation and could be partially prevented by MPO-inhibition.
Keywords: myeloperoxidase, hydrogen-peroxide, SH oxidation
114
Adverse effects of statin on skeletal muscle cells
Vincze J., Fuzi M., Jenes A., Cseri J., Szentesi P., Csernoch L.
Department of Physiology, MHSC, University of Debrecen, Debrecen,
Hungary; 98 Nagyerdei krt., Debrecen, Hungary, H-4012
janos.vincze@unideb.hu
Aim: Statins are the most widely used drugs in the treatment of hyperlipidaemia. Their
side-effects on skeletal muscle, however, have been reported with increasing frequency.
Although the pathomechanism of the statin associated myopathy has been studied in
detail and several theories do exist, there is still no real consensus.
Methods: To the study of the effects of statin on skeletal muscle rats were fed with a
special diet to achieve high blood cholesterol levels and fluvastatin was administered to
them orally. Fluvastatin and rosuvastatin were also applied on rat primary cell cultures.
Morphometric analysis was used to detect effects on cell proliferation and differentiation.
Effects on the cytoskeletal structure of muscle cells were visualized using
immunofluorescence.
Results: Cholesterol levels rose more than seven fold (from 1.5±0.1 to 10.7±2.0 mmol/L;
n=15 and 16) with a dramatic increase in LDL/HDL ratio (from 0.29±0.02 to 1.56±0.17).
While the latter was reversed by statin treatment, an elevation in blood creatine kinase
level remained. In the control cell cultures the number of myogenic nuclei increased, as
compared to the end of the 1st culturing day, by 58% after 24 hours and by further 54%
after an additional day. In contrast, the number of myogenic nuclei in the fluvastatin-
treated group rose by only 3% after 24 hours and fell by 18% (decrease not significant)
after another 24 hours. Cytoskeletal components of the cells were visibly degraded; most
profound effects were seen on the distribution of fibrillar actin.
Conclusions: Our results clearly demonstrate that statins elicit skeletal muscle related
adverse effects in rats therefore such an animal model could be used to test their side-
effects. The effects of statins on the proliferation, differentiation and structure of
developing muscle cells may explain certain adverse effects seen in clinical use.
Keywords: skeletal muscle, cholesterol, statin, cell proliferation
115
Twins and veins
1Tarnoki A. D., 2Molnar A. A., 1Tarnoki D. L., 2Kulcsar Z., 3Littvay L., 4Garami Z.,
2Preda I., 2Kiss R. G., 1Berczi V., 5Lannert A., 6Monos E., 6Nádasy G. L.
1Department of Radiology and Oncotherapy, Semmelweis University, Budapest, Hungary
78/a Ulloi street, Budapest 1082, Hungary
2Research Group for Inflammation Biology and Immunogenomics of Hungarian Academy of Sciences
and Semmelweis University, Budapest, Hungary
4 Nagyvarad ter, Budapest 1089, Hungary
3Central European University, Budapest, Hungary
Nador u. 9, 1051 Budapest, Hungary
4The Methodist Hospital DeBakey Heart and Vascular Center, Houston, TX, USA
6565 Fannin St Houston, TX 77030, USA
5Semmelweis University, Faculty of Pharmacy, Budapest, Hungary
26 Ulloi street, 1085, Budapest, Hungary
6Experimental Research Department and Department of Human Physiology, Semmelweis University,
Budapest, Hungary
37-47 Tuzolto street, Budapest 1094, Hungary
tarnoki4@gmail.com
Aim: To estimate the heritability of venous biomechanics on a twin sample.
Methods: 79 monozygotic twin pairs (age 43.0±1.3 years, mean±standard deviation/SD/)
and 38 dizygotic twin pairs (age 46.7±2.0 years, mean±SD) were involved.
Anteroposterior and mediolateral diameters of the common femoral vein at both sides
were measured by B-mode ultrasound. Measurements were made both in supine and
standing body positions, with or without controlled forced expiration (Valsalva test).
Results: Genetic factors play a decisive role in determining the geometrical properties in
its distended state, that is, in the erect body position and with substantial Valsalva
pressures (Clark’s heritability indices between 0.41-0.57 for capacity in the erect
position). Environmental factors (shared and unshared) determine geometry at lower
pressures. Both environmental and genetic factors shape distensibility. Correlations of
capacity between twin pairs are high in all age groups, especially between monozygotes
in the aged group.
Conclusions: Venous biomechanical behavior is strongly influenced by genetic factors at
high pressure, while environmental factors shape the venous biomechanics at low
pressure. Elucidation of the genes that influence venous biomechanics at different
pressures may provide important new insight in the pathophysiology of venous diseases
that are associated with altered venous biomechanics.
Keywords: venous biomechanics, heritability, genetics, Valsalva manouver
116
Do you see double?
1Rácz Adél, 1Tóth Georgina Zsófia, 1,2Székelyhídi Zita, 3Tárnoki Ádám Domonkos,
3Tárnoki Dávid László, 4,5Littvay Levente, 6Garami Zsolt, 7Lannert Ágnes, 3Bérczi
Viktor, 1Süveges Ildikó, 1Németh János
1 Semmelweis University, Department of Ophthalomology, Budapest, Hungary
2 Szent György Hospital, Székesfehérvár, Hungary
3 Semmelweis University, Department of Radiology and Oncotherapy, Budapest, Hungary
4 Department of Political Science, Central European University, Hungary
5 Visiting Scholar, Department of Political Science, Washington State University, USA
6 The Methodist Hospital DeBakey Heart and Vascular Center, Houston, TX, USA
7 Faculty of Pharmacy, Semmelweis University, Budapest, Hungary
adel.racz@t‐online.hu
Aim: To estimate the relationships between intraocular pressure, arterial stiffness and
central blood pressure.
Methods: As part of the International Twin Study 2009, intraocular pressure (Goldmann
applanation tonometer), arterial stiffness (aortic augmentation index, Aixao, aortic pulse
wave velocity, PWVao) and central systolic blood pressure (SBPao) of 155 twin subjects
(mean age 43±16,2 years, 26% men) were investigated. Regression coefficients (β)
between intraocular pressure, arterial stiffness and central blood pressure were calculated.
Possible statistical biases originating from familial relationship were corrected.
Results: Age- and sex-adjusted β and significance (p) values are shown in the table.
Right ocular pressure Left ocular pressure
β p value β p value
AIxao -0,093 <0.001 -0,097 <0.001
PWVao 0,163 0,237 0,212 0,05
SBPao 0,035 0,037 0,046 0,01
Conclusions: Significant relationships between the intraocular pressure, Aixao and
SBPao were estimated bilaterally, but only in one side concerning the PWVao, however
the direction of β was the same. Ophtalmological follow-up of patients with
hypertension, and detailed cardiovascular check-up in case of ocular hypertension is
recommended.
Keywords: Arterial stiffness, central blood pressure, intraocular pressure, hypertension
117
Genetic relation between arterial haemodinamics and exhaled nitric
oxide
1Tarnoki D. L., 1Tarnoki A. D., 2Medda E., 3Littvay L., 4Lazar Z., 2Cotichini R.,
2Fagnani C., 2Stazi M. A., 2Nisticó L., 5Lucatelli P., 5Boatta E., 5Zini C., 5Fanelli
F., 6Baracchini C., 6Meneghetti G., 7Koller A., 8Osztovits J., 8Jermendy G.,
9,10Préda I., 9,10Kiss R. G., 1Karlinger K., 11Lannert A., 12Schillaci G.,
9,10Molnar A. A., 13Garami Z., 1Berczi V., 4Horvath I.
1 Department of Radiology and Oncotherapy, Semmelweis University, Budapest, Hungary
2 Istituto Superiore Di Sanitá, Italian Twin Registry, Rome, Italy
3 Central European University, Budapest, Hungary
4 Department of Pulmonology, Semmelweis University, Budapest, Hungary
5 Vascular and Interventional Radiology Unit, Department of Radiological Sciences, La Sapienza
University of Rome, Rome, Italy
6 Department of Neurosciences, School of Medicine, University of Padua, Padua, Italy
7 Department of Pathophysiology and Gerontology, University of Pécs, Hungary
8 Bajcsy Zsilinszky Hospital, 3rd Department of Internal Medicine, Semmelweis University, Budapest,
Hungary
9 Research Group for Inflammation Biology and Immunogenomics of Hungarian Academy of
Sciences and Semmelweis University, Budapest, Hungary
10 Department of Cardiology, State Health Center, Budapest, Hungary
11 Semmelweis University, School of Pharmacy, Budapest, Hungary
12 Unit of Internal Medicine, Angiology and Arteriosclerosis Disease, Department of Clinical and
Experimental Medicine, University of Perugia, Perugia, Italy
13 The Methodist Hospital, DeBakey Heart and Vascular Center, Houston, TX, USA
tarnoki4@gmail.com
Aim: Nitric oxide, measured by fractional exhaled nitric oxide (FENO) has an important
role in the airways and vessel walls. Several factors are known to influence its level and
arterial stiffness in the general population, but the association between them is unknown.
Methods: 117 adult twin pairs (48 American, 35 Hungarian and 34 Italian; 83
monozygotic and 34 dizygotic pairs; 48±16 years /mean±standard deviation/) were
investigated. FENO was measured by an electrochemical sensor-based device and the
markers of arterial stiffness (brachial and aortic augmentation index, Aixbra, Aixao; aortic
pulse wave velocity, PWVao) by the oscillometric TensioMed Arteriograph. Cholesky
decomposition models were applied.
Results: Genetic effects accounted for 59% (95% confidence interval[CI]: 43%, 71%) of
the variation in FENO. Shared environment accounted for 0%, while non-shared
environmental influences explained 41% of the variation (95% CI: 29%, 57%).
Significant negative within-twin correlation was observed between FENO and Aixbra (r=-
0.19, p<0.05), Aixao (r=-0.16, p<0.05) but no significant correlation was found between
FENO and PWVao (r=-0.08, p=0.27). The genetic covariances of FENO and Aixbra, FENO
and Aixao were 100%.
Conclusions: Variations in FENO are explained by genetic and non-shared environmental
effects. There is a strong genetic covariance between FENO and augmentation index
providing a basis for further studies on the cardiovascular effects of airway diseases.
118
Study of postischaemic glucose uptake and glucose metabolism in
isolated rat hearts by various NMR spectroscopy methods
1Kriszta G., 1Kiss G.N., 2Kalai T., 2Hideg K., 1Sumegi B., 1Berente Z.
1Department of Biochemistry and Medical Chemistry,
2Department of Organic and Pharmacological Chemistry, University of Pecs, Medical School.
H-7624 Pecs, Szigeti u. 12. Pecs, Hungary
zoltan.berente@aok.pte.hu
Aim: The glucose uptake of heart increases after ischaemia. The extent of this increase is
modified by experimental and cardioprotective agents. The study was aimed to trace the
metabolic fate of the glucose taken up in excess.
Methods: The rate of glucose uptake was studied in situ in Langendorff-perfused rat
hearts by 31P NMR spectroscopy after the administration of 2-deoxyglucose.
The metabolic fate of the glucose was traced by 1H-13C heteronuclear single quantum
coherence 2D NMR measurements of tissue extracts of perfused rat hearts after the
administration of universally 13C-labelled glucose to the perfusion liquid.
These studies were performed for normoxic and postischemic hearts both in the presence
and in the absence of poly(ADP-ribose) polymerase (PARP) inhibitors as known
cardioprotective agents.
Results: The postischaemic increase of glucose uptake is moderated by most of the
studied PARP inhibitors. HO-3089, in contrast, further boosts the increase.
When comparing the metabolite maps of the heart extracts of different groups, HO-3089
treated hearts showed among others (a) protection against lactate accumulation caused by
ischaemia (b) further increase of aspartate accumulation caused by ischaemia (c) a
glucose concentration between the normoxic and postischaemic values.
Conclusions: HO-3089 appears to improve mitochondrial function in postischaemic rat
hearts as reflected by increased amounts of high-energy phosphate metabolites, decreased
amounts of lactate and increased amounts of aspartate.
Keywords: ischaemia-reperfusion, perfused rat heart, glucose metabolism, NMR
spectroscopy.
119
Mother and Child Health
120
The impact of maternal hypertension on neonatal adaptation
Stalzer A., Szalay Zs., Bekő B., Berényi K.
Department of Neonatology, Obstetrics and Gynecology, Public Health,
Medical Scool,University of Pécs,
7624 Pécs, Édesanyák útja 17., Hungary
stalzer.anna@gmail.com
Aim: To evaluate the relationship between the hypertension of the mother during
pregnancy and the respiratory and metabolic adaptation of their infants after birth during
their hospital stay.
Methods: We analyzed the data of neonates who were born between the 1st of January,
2009 and the 31st of December, 2011 at the Department of Obstetrics and Gynecology,
Medical School, University of Pécs, Hungary. The data were collected data from medical
records of e-MedSol platform and we used t-test, Mann-Whitney-test, Kolmogorov-test,
relative risk calculation with 95% confidence interval to compare neonatal outcome.
Results: In this period 257 out of 3748 women suffered from hypertension during
pregnancy. It leads to preterm birth 1.34 (RR:1.34 CI:1.219-1.471) times higher than in
the healthy group. We found that the need of oxygen therapy (RR:1.18 CI:1.108-1.260)
and transfusions (RR:1.06 CI:1.019-1.103), the incidence of hypoglycemia (RR:1.2
CI:1.115-1.300), hyperbilirubinemia (RR:1.06 CI:1.010-1.103), apnea (RR:1.05
CI:1.007-1.084) were significantly higher as compared to those without the maternal
disease. The neonatal infection does not have significant connection with maternal
hypertension.
Conclusions: Women who suffer from hypertension during pregnancy have higher risk
to preterm delivery and maternal hypertension is a risk factor to respiratory and metabolic
adaptation of their newborns.
Keywords: maternal hypertension, preterm delivery, neonatal adaptation
121
Morbidity data of early-term birth
Bekő B., Szalay Zs., Stalzer A., Berényi K.
Department of Neonatology, Obstetrics and Gynecology, Public Health, Medical School, University of
Pécs, 7624 Pécs, Édesanyák útja 17., Hungary
bglrk@yahoo.com
Aim: Timing of deliveries is an important issue, especially just before (34-36 weeks, late-
preterm) and after (37-38 weeks, early-term) the 36th gestational week. We compared the
morbidity data of early-term neonates to those who born on the 40th week of gestation.
Methods: We examined the data of neonates who were born between January 1, 2009 and
December 31, 2010 at the Department of Obstetrics and Gynecology, Medical School,
University of Pécs, Hungary. We compared the morbidity data of early-term (n=963) to
term infants who were born in the 40th gestational week (n=1235) using t-test, Mann-
Whitney-test, Kolmogorov-test, relative risk calculation with 95% confidence interval.
We analyzed the birth weight, rate of respiratory difficulties, oxygen therapy, infection,
apnea, hypoglycemia and hyperbilirubinemia.
Results: The risk is 2 times higher for mechanical ventilation in the group of early-term
infants (RR:2,00 CI:1,06-3,80) compared to those who were born at the 40th week of
gestation. There is no significant difference between the two groups with regard to apnea
and respiratory problems, but the risk of respiratory difficulties is 2.6-fold if we take only
the babies born on the 37th week into account (RR:2,57 CI:1,40-4,73). To be an early-
term baby means 2.0-fold risk for hyperbilirubinemia (RR:1,97 CI:1,39-2,79), 2.3-fold
risk for hypoglycemia (RR:2,28 CI:1,74-3,00), 1.4-fold for infections (RR:1,43 CI:1,26-
1,62). We examined the infants who conceived by in vitro fertilization and found 1.5-fold
risk for being delivered on the 37-38th weeks (RR:1,55 CI:1,31-1,83) and 2.8-fold risk
for late-preterm delivery versus late-terms (RR:2,81 CI:2,31-3,41).
Conclusions: Early term infants have more difficulties with regard to respiratory and
metabolic adaptation compared to late-term infants. Our results draw attention to the
magnitude of the monitoring of the early-term neonates’ adaptation.
Keywords: early-term delivery, neonatal adaptation
122
Screening of pregnant women for Streptococcus agalactiae at
University of Debrecen
1Schreil A., 1Dombrádi Zs., 2Major T., 2Krasznai Z., 1Szabó J.
1 Department of Medical Microbiology, University of Debrecen, Medical and Health Science Center,
Nagyerdei krt.98. H-4032. Debrecen, Hungary
2 Department of Obstetrics and Gynecology, University of Debrecen, Medical and Health Science
Center, Nagyerdei krt.98. H-4032. Debrecen, Hungary
szabjud@dote.hu
Background: Streptococcus agalactiae or group B streptococcus (GBS) is a Gram-
positive bacterium which may cause invasive diseases in newborns or in pregnant and
postpartum women. It is considered to be the leading cause of neonatal sepsis and
meningitis in the United Sates and Western Europe. Vaginal or cervical GBS
colonization is detected by a culture-based screening strategy, which should be carried
out in 35-37 week of gestation to indicate intrapartum antibiotic prophylaxis.
Aim: The aim of the present study was to analyse the results of GBS screening among
pregnant women at University of Debrecen between 01.January 2011 and 31. December
2011.
Methods and patients: Screening for GBS colonizaton and susceptibility testing were
performed according to the recommendations of CDC 2010. During the examined period,
we tested the cervical samples of 293 pregnant women for S. agalactiae. The bacteria
were isolated on blood agar (Oxoid). The beta-heamolytic, catalase negative strains were
identified to species level by slide agglutination. The antibiotic susceptibility was
performed by disk diffusion test according to the Clinical Laboratory Standards Institute
(CLSI, 2010) recommendations.
Results: The prevalence of S. agalactiae among the pregnant women was 13.7 %
(40/293). Heavy colonization was detected in 29 cases (72.5 %), while in 11 cases a low
level of colonization was recorded (27.5%). All isolated strains proved to be susceptible
to penicillin, and beta-lactam. 73 % and 75 % of the strains were susceptible to
clindamycin and erythromycin, respectively.
Conclusion: As a conclusion it is noted that detection of the colonization status for S.
agalactiae and the antibiotic susceptibility of the isolated strains are important data for
determination of the appropriate intrapartum antibiotic prophylaxis.
Keywords: GBS, prevalence, pregnant women, colonization
123
Peripheral Th1/Th2/Th17/regulatory T-cell balance in asthmatic
pregnancy
1Toldi G, 2Molvarec A, 2Stenczer B, 3Müller V, 3Eszes N, 3Bohács A, 3Bikov A,
R2igó J Jr, 4,5Vásárhelyi B, 3Losonczy G, 3Tamási L
11st Department of Pediatrics,
21st Department of Obstetrics and Gynecology,
3Department of Pulmonology and
4Department of Laboratory Medicine, Semmelweis University, H-1085 Budapest, Hungary
5Research Group of Pediatrics and Nephrology, Hungarian Academy of Sciences, H-1083 Budapest,
Hungary
toldigergely@yahoo.com
Aim: Asthma is a common chronic disease that may complicate pregnancy and a risk
factor for complications; however, immunological mechanisms of the bilateral
interactions between asthma and pregnancy are not fully understood. Healthy gestation is
characterized by a sensitive balance of Th1/Th2/Th17/regulatory T (Treg) cells that may
be altered in asthmatic pregnancy. The aim of this study was to describe the prevalence
of these cell subsets in asthmatic compared with healthy pregnancy.
Methods: The prevalence of Th1, Th2, Th17 and Treg lymphocytes was identified by
cell surface and intracellular marker staining in blood samples of 24 healthy non-
pregnant (HNP), 23 healthy pregnant (HP), 15 asthmatic non-pregnant (ANP) and 15
asthmatic pregnant (AP) women using flow cytometry.
Results: The Th1/Th2 cell ratio was decreased in both HP and ANP compared with HNP
women; however, no further decrease was observed in the AP group. The Th17/Treg
ratio was decreased in HP, but not in AP women, compared with HNP data. Healthy
pregnancy increased Treg cell prevalence compared with HNP data (4.64% versus
2.98%; P < 0.05), and this pregnancy-induced elevation was absent in AP women (2.52%
versus 4.64%; P < 0.05). Th17 cell prevalence was similar in the HP and HNP groups
(2.78% versus 3.17%; P > 0.05). Asthma increased Th17 prevalence in non-pregnant
patients (3.81% versus 3.17%; P < 0.05), and this asthma-specific increase of Th17 cell
prevalence was also observed in AP patients (AP versus HP: 3.44% versus 2.78%; P <
0.05).
Conclusions: The abnormal asthma-dependent Th17 elevation together with blunted
Treg increase may play a role in the compromised immune tolerance characterizing
asthmatic pregnancy.
Keywords: asthma, pregnancy, Tc17, Th17, Treg
124
Nanobiotechnology and Cancer
Research
125
Primary systemic therapy of breast cancer and monitoring the
tumour response
1Molnosi Zs., 1Szentmártoni Gy, 2Tőkés A. M., 1,2 SzékelyB, 1Dank M
1Semmelweis University Department of Oncology of the Clinic of Diagnostic Radiology and
Oncotherapy,
1082 Budapest, Üllői street 78/a
2Semmelweis University 2nd Department of Pathology,
1091 Budapest, Üllői street 93.
fuzsi1@gmail.com
Introduction: Neoadjuvant or primary systemic therapy (PST) is a commonly used
therapy for treating locally advanced breast cancers nevertheless its application has
increased in the therapy of primary operable breast tumors as well. PST has many
advantages: increasing the chances of breast-conserving surgeries, the tumour response
for the chemotherapy drug can be monitored in vivo, in case of complete pathological
response (PCR) the length of disease free survival period is longer.
Aim: To compare the tumour response monitored by ultrasonography (US) and physical
examination (PE) and examine breast cancer subtypes likely to respond to PST.
Methods: We have processed the data of 81 patients of Semmelweis University
Department of Diagnostic Radiology and Oncotherapy who received PST between 1997
and 2009. The database was analyzed retrospectively. The statistical analysis was
performed by a SPSS Stat 17 program.
Results: Most of the patients discovered their tumors by self-examination (67%), X-ray
mammography was performed at 60.3%, US was performed at 71.8% of all the patients.
The tumours were typically IDCs. Grade 3, high proliferation rated and HER2 and
oestrogen receptor positive lesions were dominant. The patients received antracycline and
taxane based treatment, typically 6 cycles. The decrease in the size of the tumours was
significant by both US and PE (p=0.003 and p=0.001), mammographical monitoring data
were not available at the majority of the patients. PCR could be reached at 8 % of the
patients, which was more frequent at triple negative and high proliferating lesions.
Mastectomy was avoidable at 40.5% of the patients. During the monitoring period
(average 5.5 year) distant metastasis appeared at 21.8 % of the patients, and we lost only
1 patient because of breast cancer.
Conclusions: PST is efficiently applicable on patients with breast cancer. Both PE and
US can be used in monitoring of response to therapy.
Keywords: PST, ultrasonography, physical examination.
126
Examination of primary breast cancers and their distant metastasis
*Nagy Zs., *Faragó Zs,. Tőkés A. M., Szász A. M., Székely B., Kulka J., 1Dank M.
2nd Department of Pathology, Semmelweis University
1Radiology and Oncotherapy Cinic Semmelweis Univ. Üllői út 93. 1091 Budapest, Hungary
*authors contributed equally for this work
nagy.zsofiailona@gmail.com, zsofia.farago@gmail.com
Aim: Breast cancer is the most common malignant tumorous disease among women.
Nowadays it can be treated succesfully but after the appearance of distant metastases it is
considered to be incurable. Our aim is to examine primary breast cancers compared to
their distant metastasis and also to monitor the patients’ survival rate related to
oncological treatment.
Methods: We reviewed the autopsies done in the 2nd Department of Pathology
(Semmelweis Univ.) of patients who died in metastatic breast cancer between 2001 and
2011. We examined 18 out of the 82 cases we found who had primary breast cancer and
their complete clinical documentary was available. Beside the primary tumors we
examined 61 distant mets. For analysing the datas we used Statistica for Windows and
SPSS Stat 17.
Results: The average age of the patients at the time of diagnosis was 59. The overall
survival was on the average 5 years. The elapsed time until the appearance of the first
distant metastasis was on the av. 3 years, the survival time after this was on the av. 1.33
years. 66.7% of the primary tumors were IDC, 27.8% were ILC., medullar carcinoma
occured only in one case. We could only determine the immunohistochemical subtypes
of the tumors in 15 cases. 2 were HER2 positive, 5 were triple negative, 4 were luminal
A and 4 were luminal B. Poorly differentiated grade III tumors prevailed (53.34%)
among the cases. Distant mets were mostly found in liver (61.1%), bones (50%) and lung
(38.9%). The expression of Ki67 in the primary tumors were significantly higher than in
distant mets (15.23 % vs. 3.31% p= 0.001). The hormone receptor status in the primary
tumor and the distant mets were in occurence of ER 56.25% and PR 60% concordant. In
the other cases the receptors were identified in the primary tumors but the distant mets
were proved to be negative. The patients were likely treated with multiple lines of
chemotherapy.
Conclusions: According to our research metastatic breast cancers are principally
invasive ductal carcinomas. Nearly half of the tumors don’t express estrogen receptor and
a phenotype change can be occured in the distant metastasis. After the hematogenous
spread of the cancer, the survival rate is expressly bad. To learn more about the biological
characteristics of the examined tumors, we are planning to do their deeper molecular
pathological examination.
127
Translational Research and Drug
Development
128
PP1 dephosphorylates the retinoblastoma protein
Gaál Zs., Kiss A., Dedinszki D., Erdődi F.
Department of Medical Chemistry, Medical and Health Science Center, University of Debrecen
zsuzsanna.gaal.46@gmail.com
Aim: The phosphorylation level of retinoblastoma protein (pRb) plays an important role
in the regulation of cell proliferation and cell cycle progression, and therefore has a major
impact on the survival of malignant cells. While the phosphorylation of pRb by kinases is
well characterized, our knowledge on the dephosphorylating phosphatases is limited.
Protein phosphatase-1 (PP1) has been considered as one of the pRb phosphatases,
however its direct role in the dephosphorylation has not been fully established. The goal
of our study was to investigate the role of PP1 and possible PP1 inhibitory proteins on
pRb phosphorylation.
Methods: The catalytic subunit of PP1 (PP1c) was silenced by transfection of siRNA in
HeLa cells. Vectors coding for Flag-tagged KEPI (kinase enhanced phosphatase
inhibitor) and LIM-kinase-2 (LIMK-2) were transfected into MCF-7 and HeLa cells,
respectively. The efficacy of the transfections was checked by Western blot experiments
and immunofluorescence using anti-KEPI, anti-LIMK-2 and anti-Flag antibodies. The
viability of cells was determined by MTT assay.
Results: Silencing of PP1c by pan-siRNA reduced markedly the amount of PP1c and in
turn it resulted in increased phosphorylation level of pRb. Both KEPI and LIMK-2
include a phosphorylatable peptide sequence implicated in the inhibition of PP1.
Expression of KEPI in MCF-7 cells led to its phosphorylation by endogenous kinases and
inhibition of PP1 reflected in enhanced pRb phosphorylation. Similarly, expression of
LIMK-2 in HeLa cells decreased protein phosphatase activity leading to increased
phosphorylation of pRb.
Conclusions: Our results indicate the essential role of PP1c in determining the
phosphorylation level of pRb and suggest that PP1 is implicated in the regulation of cell
cycle progression and survival of malignant cells, thereby PP1 could be considered as a
drug target with therapeutic means.
Keywords: Protein phosphatase-1; retinoblastoma protein (pRb); pRb
dephosphorylation; LIM-kinase-2; kinase enhanced phosphatase inhibitor (KEPI).
129
Inhibiting PARP prevents experimental demyelination
1Veto S., 2Acs P., 3Bauer J., 3Lassmann H., 1Berente Z., 1Sumegi B., 2Komoly S.,
1Gallyas Jr. F., 2Illes Z.
1 Department of Biochemistry and Medical Chemistry, University of Pecs,
Szigeti str 12, 7624 Pecs, Hungary
2 Department of Neurology, University of Pecs,
Ret str 2, 7623 Pecs, Hungary
3 Center for Brain Research, Medical University of Vienna,
Spitalgasse 4, 1090 Wien, Austria
zoltan.berente@aok.pte.hu
Aim: Mitochondrial dysfunction has been indicated to play a role in loss of
oligodendrocytes in multiple sclerosis (MS). A nuclear-mitochondrial crosstalk
dependent on poly(ADP-ribose) polymerase (PARP) activation is critical in determining
the fate of injured cells. Here, we investigated activation of PARP in MS lesions, and the
effect of PARP inhibition on experimental demyelination.
Methods: MS lesions were studied by poly (ADP-ribose) (PAR) and apoptosis inducing
factor (AIF) immunohistochemistry in 13 MS patients. In a primary demyelinating mouse
model induced by cuprizone myelination of the corpus callosum (CC) was investigated
by in vivo serial 9.4 T MRI and in vitro quantitative MBP
immunoblotting/immunohistochemistry. On CC of mice also electron microscopy, PAR
and AIF immunohistochemistry, MAP and Akt kinase and caspase-3 immunoblotting
was performed.
Results: Strong PAR reactivity reflecting PARP activation was observed in apoptotic
oligodendrocytes in pattern III MS lesions. AIF co-localized with anti-PAR staining in
condensed nuclei. The same pathology was observed in the cuprizone model: PARP
activation in CC; apoptotic morphology with enlarged mitochondria in oligodendrocytes;
caspase-independent apoptosis with the nuclear translocation of AIF. 4HQ, a potent
inhibitor of PARP blocked cuprizone induced poly(ADP-ribosyl)ation. Inhibition of
PARP attenuated oligodendrocyte depletion and decreased demyelination, suppressed
JNK and p38 MAP kinase phosphorylation, increased the activation of the cytoprotective
PI-3/Akt pathway and prevented caspase-independent AIF-mediated apoptosis.
Conclusions: In summary, PARP activation may play a crucial role in the pathogenesis
of pattern III MS lesions. Since PARP inhibition also reduces inflammation by
attenuating activation of NF-κB, it may target all subtypes of MS: either by preventing
oligodendrocyte death or attenuating autoimmune inflammation.
Keywords: multiple sclerosis, cuprizone, demyelination, PARP, MRI
130
Production and testing of anti-ErbB2 F(ab)2-s
Tóth, G., Szöllősi, J., Vereb, G.
Department of Biophysics and Cell Biology, University of Debrecen,
H-4032 Debrecen, Nagyerdei krt. 98.
tothgab@med.unideb.hu
Aim: Trastuzumab, a humanized anti-ErbB2 antibody is a specific targeted therapy
against ErbB2 positive tumors, with a history of both success and a high rate of therapy
resistance. Another humanized antibody, pertuzumab inhibits ErbB2 heterodimerization.
Combined application of the two antibodies may improve treatment outcomes by either
enhancement at the molecular level (e.g. by hypercrosslinking-based enhanced
internalization of ErbB2), or offering sterically complementary docking sites for NK cells
implementing ADCC. Distinction between these routes can be ascertained by also testing
the F(ab)2 fragments of these antibodies. In the first phase, we have generated these
fragments and compaired them in vitro.
Methods: F(ab)2 from trastuzumab and pertuzumab was produced with pepsin-agarose
and separated with gel filtration. Affinity and lack of Fc fragment on F(ab)2 was tested
with immunofluorescence in flow cytometry. In vitro Ki was assessed with an MTT
based assay. ADCC in the presence of the whole antibodies and absence thereof with
F(ab)2 was tested in a real time adherence assay.
Results: Since protein A and protein G still showed binding of cleaved F(ab)2,
chromatography optimized for exclusion size and ionic strength was used to produce
proper F(ab)2 variants with intact antigen binding but no Fc region taggable with
polyclonal anti-Fc. The effect on proliferation of in vitro sensitive and resistant cell lines
BT-474 and JIMT-1 was not affected by removing FC region. The only measurable
difference was a slightly enhanced dimerization inhibition by the smaller pertuzumab
F(ab)2 as opposed to its intact parent antibody. Also, intact antibodies mediated ADCC-
based killing of both tested cell lines, while F(ab)2 fragments did not.
Conclusions: The set of trastuzumab and pertuzumab whole antibodies and their F(ab)2s
are now ready for in vitro and in vivo testing of their possible synergistic effects.
Keywords: ErbB2, trastuzumab, pertuzumab, F(ab)2
131
POLAND
132
Nanobiotechnology and Cancer
Research
133
Functionalization and optical properties of NaYF4 based
nanocrystals doped by lanthanide ions for biomedical applications.
Sojka B., Noculak A., Bański M., Misiewicz J. and Podhorodecki A.
Institute of Physics, Wrocław University of Technology,
Wybrzeże Wyspiańskiego 27,
50-370 Wrocław
artur.p.podhorodecki@pwr.wroc.pl; sojka.bartlomiej@gmail.com
Background: In the field of optical biomarkers for biomedicine applications lanthanide
doped nanocrystals (NCs) are most interesting not only due to their optical properties,
which are significantly better in comparison to organic dyes and even quantum dots, but
also because of the low toxicity and low phonon vibrational host lattices, which promote
strong fluorescence. However, in order to make them biocompatible and water soluble
thier surface must be functionalized.
Aim: The aim of this work was to conduct surface functionalization of NaYF4 based NCs
doped with Europium in order to make them water soluble and ready for bio-conjugation.
Methods: NaYF4 NCs were obtained by the co-thermolysis method with
trioctylphosphine oxide (TOPO) serving both as a solvent and a surface ligand. Among
various approaches (Ligand Oxidation, Surface Silanization, Ligand Attraction) we chose
Ligand Exchange to change the phase of NCs from hydrophobic to hydrophilic. In a
typical experiment we used dimercaptosuccinic acid (DMSA) solved in dimethyl
sulfoxide (DMSO) as the hydrophilic ligand, which substituted TOPO on the surface.
The functionalization process was carried out tens of degrees above the room temperature
under nitrogen atmosphere with vigorous stirring. After transferring to water the optical
properties of NCs were measured by the photoluminescence (PL), PL excitation and PL
decay times experiments.
Results: Luminescent, surface functionalized (and thus water soluble) sodium fluoride
nanocrystals doped with lanthanide ions with nanometer size diameter have been
obtained.
Conclusions: There are numerous factors influencing the success of surface
functionalization in NCs. It is determined that the most important ones are: ligand and
solvent used in synthesis, process temperature, time and atmosphere, the ligand/solvent
ratio and sonication of NCs prior to Ligand Exchange method. Control over them is
crucial in order to prevent aggregation, increase the transfer yield and maintain high
luminescence. Proper optimalization of these factors requires time, but it will result in
unlocking full potential of NCs as optical biomarkers for application in medicine.
Keywords: nanocrystals, lanthanide ions, functionalization, DMSA
134
Synthesis of GDOF:Eu3+ nanocrystalline optical bio-probes by co-
thermolysis method using a new kind of envarionmental friendly
solvent
Noculak A., Bański M., Misiewicz J. and Podhorodecki A.
Institute of Physics, Wrocław University of Technology,
Wybrzeże Wyspiańskiego 27, 50-370 Wrocław
artur.p.podhorodecki@pwr.wroc.pl; 166037@student.pwr.wroc.pl
Background: Considerable interest has been focused on the rare earth doped
nanocrystals (NC’s) due to their interesting optical properties (arising from the 4f
electron configuration), and large possibilities in bio-medical applications as optical
probes in different diagnostic approaches. Because of low phonon vibrational energy and
low toxicity fluorides and oxyfluorides have been recognized as highly efficient
fluorescent host materials for rare earth ions.
Aim: The aim of our work was to obtain nanometer fluoride phosphor with single and
narrow emission band using a new kind of solvent.
Methods: GdOF:Eu3+ nanocrystals with hexagonal or orthorombic structure have been
obtained from trifluoroacetate salts by co-thermolysis method in the presence of cheap
and nontoxic solvent. This method is based on thermal decomposition of precursors
compounds in high temperatures and leads to highly uniform, nanometer-sized particles.
The structural properties of the products were characterized by X-ray diffraction (XRD)
and transmission electron microscopy (TEM). The luminescent properties of the
nanoparticles were investigated by excitation, emission, emission decay and absorption
measurements. The structure and size of GdOF:Eu3+ nanocrystals were controlled by
variable time of synthesis ranging from 15 to 60 minutes and different reaction
temperature. RESULTS: Highly luminescent nanoparticles with controlled size in
nanometer range have been obtained by using low cost and environment friendly method.
Conclusions: It is found that with decreasing size of nanocrystals, surface to volume
ratio increases, increasing the number of ions placed at the NC’s surface. In consequence,
due to different local environments around the rare earth ions (surface or volume), their
relative emission peaks intensity ratio changes. In consequence the color purity of
nanocrystals can be controlled and optimized by nanocrystals size modifications by
changing time and temperature of synthesis. In this way, new and optically efficient
nanocrystalline bio-probes have been obtained.
Keywords: nanocrystals, rare earth ions, GdOF
135
ROMANIA
136
Mother and Child Health
137
Maternal, fetal and neonatal outcomes of pregnancy in women with
type I diabetes
Lazar V., Poalelungi C., Hudita D., Ceausu I.
“Doctor I. Cantacuzino” Clinical Hospital,
Ion Movila Street, no 5-7, Bucharest, Romania
vvirginia.lazar@gmail.com
Aim: the aim of this study was to investigate maternal, fetal and neonatal outcomes of
pregnancy with type I diabetes who delivered in 'Dr. I. Cantacuzino' Clinical Hospital,
Bucharest, Romania. Postal code: 73206
Methods: this was a prospective study. We enrolled 20 women with type I diabetes, who
presented at our clinic before 10 weeks of gestation. We excluded two of them, one due
to first trimester pregnancy loss, and one was lost to follow up. The data was collected
from medical records of pregnant women with type I diabetes, from questionnaires filled
in by the women and from the medical records of the neonates. All participants gave
written informed consent.
Results: the mean age at delivery was 28,1 years. The mean duration of diabetes was 13
years. 22% (4/18) of women presented diabetes chronic complications at the beginning of
pregnancy. 11,1%(2/18) presented also hypothyroidism. 88,88% (16/18) used multiple
insulin injection and 94,4% (17/18) human insulin. The mean concentration of
glycosylated hemoglobin at 10 weeks of gestation was good: 6,3 %, in most women
72,2% (13/18). Preconceptional folic acid supplementation was adequate in 22,2% (4/18)
of the patients. Only 61,1% (11/18) of women performed daily home monitoring of blood
glucose at conception, and only 27,7% (5/18) sought preconceptional guidance. The rate
of preeclampsia was 22,2% (4/18), the rate of preterm delivery:27,7 % (5/18), the rate of
cesarean section was 66,6% (12/18) of which 58,3% (7/12) were delivered by emergency
CS. Maternal mortality rate was 5% (1/18). The rate of macrosomia was 38,88 (7/18),
neonatal morbidity was high, the commonest morbidities were hypoglycemia 55,5%
(10/18) and hyperbilirubinaemia 44,4% (8/18), congenital malformations rate was 11,1%
(2/18),neonatal mortality rate was 5% (1/18).
Conclusions: Women with type 1 diabetes have a poor outcome compared with women
without diabetes, despite the overall good early glycaemic control. They have increased
rates of congenital malformations, preeclampsia, premature delivery, perinatal mortality
and risk of delivering a macrosomic baby. The targets of the St Vincent Declaration of
1989 have not been met. Improvements may be gained by increased prepregnancy care
and in the proportion of pregnancies that are planned.
Keywords: pregnancy, type 1 diabetes, preconceptional care.
138
Risk factors for preterm birth – epidemiologic analysis of 1000
deliveries in 2011 in “Dr.I.Cantacuzino” Ob-Gyn Department –
preliminary results of a prospective study
Poalelungi C., Lazar V., Saulescu O., Abbassi N., Hudita, D., Ceausu I.
”Carol Davila” University of Medicine and Pharmacy, Bucharest,
”Dr. I. Cantacuzino” Hospital, Department of Obstetrics and Gynaecology, Bucharest, Romania
Ion Movila Street, no 5-7, Bucharest, Romania
cristianpoalelungi@yahoo.com
Background: Preterm birth is a major clinical problem associated with perinatal
mortality and morbidity. Several factors may increase the risk of preterm birth and some
of them are purported to predict it.
Objective: The aim of this study was to identify current risk factors associated with
preterm delivery. Analysis of a retrospective study performed in our clinic in 2007-2009
did not show statistically significant data on the factors involved in premature birth. We
found necessary to immediately start a prospective analysis to highlight the hierarchy of
factors involved in premature birth. The data collection was under the guideline of the
RECOOP HST Consortium Mother and Child Health Network Prospective Study.
Results: This is a prospective study which started in January 2011 – part of the larger
multinational prospective study MOCHEA Research Network. We analyzed 1000 births
at Dr. I. Cantacuzino Hospital, Bucharest, Romania. Of these 137 (13.7%) were preterm
births (<37 weeks).
Mean maternal age was 28,2 years. Mean BMI was 25,6.
The factors that remained significantly associated with preterm birth were smoking
(42.3%), anemia (24.8%), vaginal bleeding (16.4%). Prior spontaneous abortion was
found in 29.1%. The medical history revealved urinary tract infections in 14.5% of cases,
diabetes (9.4%) or uterine surgery in antecedents (9%). Twenty mothers (14.5%) have
positive family history of preterm birth.
On the other hand, persistent psychosocial and behavioral factors continue to negatively
influence birth weight. 31% of women who delivered prematurely did not seek or receive
medical care during pregnancy.
Conclusion: Screening tests with prediction model for preterm delivery risk should be
used for all pregnant women.
Prospective analysis allows more accurate identification of risk factors in preterm birth.
This identification is not enough of them; it is necessary to develop predictive models to
help early identification and possibly correction of these risk factors.
Further studies are required to understand the causes of the epidemic of preterm births in
Romania.
Keywords: preterm birth, risk factors, prematurity
139
SLOVAKIA
140
Women’s Health and Cardiovascular
Diseases
141
Vitmin D and systolic blood pressure
1Kramárová P., 2Krivošíková K., 3Krivošíková Z., 3Gajdoš M.
1Slovak Medical University, Faculty of Public Health,
Limbová 12, 833 03 Bratislava, Slovakia
2Comenius University, Faculty of Medicine,
Špitálska 24, 813 72, Bratislava, Slovakia
3Slovak Medical University, Faculty of Medicine,
12, 833 03 Bratislava, Slovakia
riaditel.vvz@szu.sk; martin.gajdos@szu.sk
Aim: There are some evidence on the association of vitamin D (VD) with cardiovascular
risk factors, including hypertension, independently of adiposity. Furthermore, low VD
levels have been reported to predict future hypertension. The aim of our study was to
analyze the interrelations between VD and blood pressure (BP) in healthy subjects.
Methods: The study was carried out during january to december on 429 healthy subjects
(170M/259F). Body mass index (BMI) was calculated. Standard biochemistry, VD and
intact parathormone (PTH) levels were measured. All participants were normotensive.
Results: Subjects were divided into 3 groups according the month of collecting data
(A:January-April, B:May-August, C:September-December). Groups did not differ in
biochemical parameters, PTH and BMI. VD levels variabled in dependence on season.
The highest levels of VD (43.1 ng/ml) and the lowest SBP (117.7 mmHg) were found in
grup B, and on the contrary, the lowest VD levels (29.6 ng/ml) and the highest SBD
(122.9 mmHg) were found in group A. These differences were significant (p<0.002 for
VD and p<0.005 for SBP). VD levels did not significantly correlated with SBP.
Conclusion: The significant relationship between VD and SBP was not confirmed, but
our data suggest some possible role of low VD levels in SBP arise. The study was limited
by number of participants, thus the future extensive study is needed. The confirmation of
the hypothesis, that VD deficit is linked to the hypertension, would offer a cheap and
effective method in reducing blood pressure or preventing of hypertension developmnet
by optimizing vitamin D status. Considering the prevalence of VD deficit worldwide,
even a modest effect on SBP would be of clinical importance.
Key words: vitamin D status, systolic blood pressure
Supported by: Research and development program financed from the European Regional
Development Fund, project ITMS No.24240120033 and by Ministry of Health of SR
under the project No. 2005/27-SZU-05.
142
Mother and Child Health
143
Determinants of mental rotation in gifted children
1, 2Durdiaková J., 2Celec P., 1Ostatníková D.
1Institute of Physiology, Faculty of Medicine, Comenius University,
Sasinkova 2, 81372 Bratislava, Slovakia,
2Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University,
Sasinkova 4, 81108 Bratislava, Slovakia
durdiakova.jaroslava@gmail.com
Aim: Mental rotation is one of the cognitive domains often studied in context of gender
specific testosterone effect.. Low levels of testosterone are associated with higher scores
in mental rotation tests in men but lower scores in women. It is currently unknown
whether mental rotation is also associated with prenatal testosterone or with testosterone-
related genetic polymorphisms. The aim of our study was to analyze associations
between prenatal exposure or acurate testosterone effects and mental rotation in
intellectually gifted boys and girls.
Methods: One hundred forty seven boys and eighty girls aged 10-18 years with IQ>130
were enrolled. Saliva samples were collected and used for ELISA of actual levels of
salivary testosterone. The 2D/4D finger length ratio as an indicator of prenatal
testosterone was measured on both hands and averaged. Amthauer mental rotation test
was used for the assessment of this spatial ability. The CAG repeat polymorphism in
exon 1 of the androgen receptor gene was analyzed using PCR and capillary
electrophoresis.
Results: In boys, mental rotation coefficient was significantly influenced by 2D/4D
finger length ratio (r2=0.029; p<0.05) and the number of CAG repeats in the androgen
receptor gene (r2=0.048; p<0.01). Actual levels of testosterone did not correlate
significantly with mental rotation. However, MANCOVA revealed that after adjustment
of age as a confounding variable, only the effect of the genetic polymorphism was
significant (r2=0.046; p<0.02). In girls, only actual levels of testosterone significantly
correlated with mental rotation (r2=0.093; p<0.01). MANCOVA after adjustment of age
revealed no association.
Conclusion: In intellectually gifted boys mental rotation is determined by genetic
polymorphism of the androgen receptor and not by prenatal or actual testosterone levels.
No significant impact of testosterone exposure or genetic variability in androgen receptor
was proved in intellectually gifted girls.
Keywords: gifted children, mental rotation, testosteron
144
Metabolic changes in mothers during lactation
1Simon Klenovics K, 2Šebeková K
1Institute of Physiology
2Institute of Molecular BioMedicine, Medical Faculty of Comenius University,
Sasinkova 2, 813 72 Bratislava, Slovakia
klenovics.kiki@gmail.com
Aim: Accumulation of fat, changes in lipid profile and decreased insulin sensitivity
represent typical metabolic changes in maternal organism during pregnancy. Lactation
promotes the complete restoration of these physiological changes. The aim of this study
was to compare parameters of insulin sensitivity, lipid metabolism and bone turnover in
three groups of healthy mothers.
Method: In 106 healthy mothers of healthy 5-to-12-month olds infants insulin sensitivity
(HOMA index), lipid profile, concentrations of calcium, inorganic phosphorus and
alkaline phosphatase activity were determined. Mothers were devided into 3 groups
according to their lactation status in time of investigation. Sixty-nine mothers were
breast-feeding (exclusively or partially), the second group of mothers weaned about 3
months before the examination (n=19), and the third group ceased with breast-feeding
about 6 months prior to the study (n=18). Three groups of mothers did not differ
significantly by age, BMI and weight gain during pregnancy.
Results: Breast-feeding mothers had lower plasma fasting glucose (p=0,040) and were
more insulin sensitive (p=0,007) compared to the other 2 groups. Lactating mothers
presented with the most favorable atherogenic profile: they had the lowest plasma
triglyceride concentration (p=0,019), the lowest LDL- (p=0,020) and VLDL-cholesterol
(p=0,019). Groups did not differ significantly in plasma calcium concentration, however
breast-feeding mothers had significantly higher plasma levels of inorganic phosphorus
(p=0,019) and higher activity of alkaline phosphatase (p=0,002).
Conclusions: In the mothers, breast-feeding promotes favorable lipid profile and insulin
sensitivity but it increases bone turnover. These metabolic changes slowly vanish within
a few months after the cessation of lactation.
Key Words: insulin sensitivity, lipid profile, bone metabolism, breast-feeding
145
Attention deficit hyperactivity disorder: Neurometabolite changes
after treatment
1,2Husarova V., 3Bittsansky M., 2Ondrejka I.
1Institute of Physiology, Comenius University Faculty of Medicine, Sasinkova 2, 813 72 Bratislava;
2Clinic of Psychiatry, Martin Faculty Hospital and Comenius University Jessenius Faculty of Medicine,
Kollarova 2, 036 59 Martin;
3Institute of Medical Biochemistry, Comenius University Jessenius Faculty of Medicine, Mala Hora 4,
03601 Martin
nika.husarova@gmail.com
Aim: Attention deficit hyperactivity disorder (ADHD) is the most common
neurobehavioural childhood disorder with the unknown ethiopatogenesis. The cortical-
striatal pathway with the disturbances in catecholaminergic neurotransmission is the most
discussed. Magnetic resonance spectroscopy brought a new light into ADHD
neurobiology by detecting some neurometabolites. The aim of our work was to find out
the 1H MRS neurometabolite changes after two months of atomoxetine or
methylphenidate treatment in children with the ADHD-combined type.
Methods: Twenty-one children (mean age 12.3 years, range 6.1 to 16.8) underwent 1H
MRS examination before and after two months of treatment with methylphenidate (n=10)
or atomoxetine (n=11). The spectra were taken from the dorsolateral prefrontal cortex
(DLPFC, 8 ml) and white matter behind the DLPFC (anterior semioval center, 7.5 ml),
bilaterally using single-voxel 1H spectroscopy (1.5 T Siemens Symphony, PRESS,
TE/TR = 30/3000 ms, 128 averages), and evaluated with the LCModel.
Results: After atomoxetine treatment NAA/Cr (N-acetylaspartate/creatine) decreased in
the left (m0=1.560, SD0=0.121; m1=1.477, SD1=0.158; p=0.021) and Cho/Cr (choline)
increased in the right (m0=0.194, SD0=0.027; m1=0.227, SD1=0.029; p=0.026) DLPFC.
Glx/Cr (glutamate/glutamine/GABA) increased in the left white matter (m0=7.252,
SD0=0.742; m1=7.787, SD1=0.697; p=0.013) after methylphenidate medication.
Conclusion: Decreased NAA/Cr could indicate the reduction of cortical-striatal circuit
hyperactivity sustained by „self-sustaining loop“ after atomoxetine medication.
Methylphenidate could have the effect on tonic dopamine release in mesocortical
pathway via the increased activation of glutamatergic projections with the representation
in the increased Glx/Cr.
Keywords: ADHD, neurometabolite, atomoxetine, methylphenidate
Reasearch was supported by fund of Ministry of Education 2007/57-UK-17 and project
“Centre of excellency for personalized therapy reasearch (CEVYPET)”, code:
26220120053
146
Candidate gene variants in Slovak autism patients
Lakatosova S., Schmidtova E., 1Celec P., 1Ficek A., Kubranska A., Ostatnikova D.
Institute of Physiology, Faculty of Medicine, Comenius University in Bratislava,
Sasinkova 2, 81372 Bratislava, Slovakia
1Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava,
Mlynská dolina, 842 15 Bratislava 4, Slovakia
silvia.lakatosova@gmail.com
Aim: Autism belongs to one of the most genetically influenced neuropsychiatric
disorders. However, the detailed genetic basis is far from being clear. Whole genome
studies pointed out a number of candidate genes. These are mostly involved in
synaptogenesis and neuroendocrine regulations. In this study we have focused on
polymorphisms of oxytocin (OT), oxytocin receptor (OXTR), gamma-aminobutyric acid
receptor (GABRG3), neuroligin (NLGN) and reelin (RELN) in autistic patients from
Slovakia. All these genes were previously shown to be in association with autism.
Methods: After informed consent 90 autistic boys and 85 healthy boys were enrolled into
the study. DNA was extracted from saliva. Four SNP polymorphisms were assessed in
genes OT, OXTR, GABRG3, NLGN4X by RFLP method. In RELN gene (GGC)n STR
polymorphism was genotyped by fragment analysis.
Results: We have found no significant differences in genotype distribution of examined
polymorphisms in OT, OXTR, GABRG3 and NLGN4X genes. Significant difference in
number of GGC repeats in 5´UTR of RELN gene between autistic and control boys
(P=0.001) was found. Slovak autistic boys had higher number of GGC repeats
(9.63±0.082) in comparison with controls (9.18±0.100).
Conclusions: Our finding confirms previous studies showing that higher number of GGC
repeats in the RELN gene was associated with autism. Higher number of these repeats
was found to be associated with decreased RELN expression that may have severe
consequences on brain development and function. This is the first genetic study of autism
in Slovakia. Further genotyping is needed to gain insight about genetic causes of this
neuropsychiatric disorder in Slovakia.
Keywords: autism, candidate genes, reelin
147
Nanobiotechnology and Cancer
Research
148
Nephrotoxic effects of Fe3O4 nanoparticles
1Kollárová R, 1Simon-Klenovics K, 1Vlková B, 1Celec P, 1Šebeková K, 2Boor P,
3Pronayová N
1Faculty of Medicine, Comenius University,
Sasinkova 4, 811 08 Bratislava, Slovakia
2RWTH University Aachen,
Pauwelsstr. 30, D-52074 Aachen, Germany
3Faculty of Chemical and Food Technology, Slovak University of Technology,
Radlinského 9, 812 37 Bratislava, Slovakia
radana.kollarova@gmail.com
Aim: We evaluated the potential nephrotoxic effects of a single dose of i.v.administered
oleic acid coated Fe3O4 nanoparticles (NP) in rats.
Methods: Female Wistar rats were administered either placebo (10% v/v rat serum in
0.9% NaCl), suspension of TiO2 NP (positive control, bimodal 84/213 nm distribution),
or Fe3O4 NP (bimodal 31/122 nm distribution) in doses of 0.1, 1.0 or 10.0 % LD50. Rats
were sacrificed 24h, 7-, 14- and 28-days after NP injection (n=9-10/each group).
Results: Renal function of NP administered rats, as monitored by plasma creatinine and
urea concentrations, creatinine clearance and protein excretion rate, did not differ
significantly in either interval from rats administered placebo. There was no change in
plasma concentrations of uremic toxines (i.e.: asymmetric dimethylarginine, advanced
glycation end-products determined fluorimetrically or as carboxymethyllysine). No
significant changes in the expression of pro-inflammatory (tumor necrosis factor -α) and
pro-fibrotic (transforming growth factor-β1, collagen IV) genes in renal cortex were
revealed. NP administration did not alter significantly the levels of kidney injury
molecule-1, the marker of acute tubular damage. Correspondingly, renal histology did not
reveal significant differences between groups. NMR measurement of T2 relaxation times
of kidney homogenates indicated no NP accumulation.
Conclusions: In young female rats, no obvious nephrotoxic effects were observed after a
single i.v. dose of Fe3O4 NP.
Keywords: Fe3O4 nanoparticles, nephrotoxicity, female Wistar rats
Aknowledgement: Study was supported by 7FP EC EU: NanoTEST (Development of
methodology for alternative testing strategies for the assessment of the toxicological
profile of nanoparticles used in medical diagnostics.), Grant No.: 201335.
149
Translational Research and Drug
Development
150
Role of CCN3 in experimental glomerulonephritis
1,2,3*Boor P., van 1*Roeyen CRC, 4Borkham-Kamphorst E, 1Rong R, 1Kunter U,
1Martin IV, 1Kaitovic A, 1Fleckenstein S, 5Perbal B, 4Weiskirchen R, 1Ostendorf T,
1Floege J
1Department of Nephrology
2Institute of Pathology
3Institute of Molecular Biomedicine, Comenius University, Bratislava, Slovakia,
4Inst. of Clinical Chemistry, RWTH Aachen University, Germany,
5R&D L’Oréal USA, Clark, NJ USA
boor@email.cz
Aim: In contrast to factors promoting mesangial cell proliferation, little is known about
their endogenous inhibitors. During experimental mesangioproliferative nephritis,
glomerular CCN3 (also known as NOV or nephroblastoma overexpressed gene)
expression is reduced prior to the proliferative phase and overexpressed in glomeruli and
serum when mesangial cell proliferation subsides.
Methods: To further elucidate its role in mesangioproliferative glomerulonephritis,
CCN3 was systemically overexpressed by muscle electroporation in healthy or nephritic
rats. This increased CCN3 serum concentrations more than 3-fold for up to 56 days.
Results: At day 5 after disease induction, CCN3-transfected rats exhibited an increase in
glomerular endothelial area and in glomerular mRNA levels of the pro-angiogenic factors
VEGF and PDGF-C. In the mesangioproliferative phase (day 7), CCN3 overexpression
decreased mesangial cell proliferation including expression of α-smooth muscle actin and
matrix accumulation of fibronectin and type IV collagen. In progressive nephritis (day
56), overexpression of CCN3 resulted in decreased albuminuria, glomerulosclerosis and
reduced cortical collagen type I accumulation. In healthy rat kidneys, overexpression of
CCN3 induced no morphological changes but regulated glomerular gene transcripts
(reduced transcription of PDGF-B, PDGF-D, PDGFR- and fibronectin and increased
PDGFR-α and PDGF-C mRNA).
Conclusions: The above data identify a dual role of CCN3 in experimental
glomerulonephritis with pro-angiogenic and anti-mesangioproliferative effects.
Manipulation of CCN3 may represent a novel approach to help repair glomerular
endothelial damage and mesangioproliferative changes.
Keywords: mesangial proliferation, glomerulonephritis, NOV
151
Antiviral activities of natural extracts on coxsackieviruses
Sarmirova S., 1Nossik N. N., Bopegamage S.
Department of Virology, Slovak Medical University,
Limbova 12, 83303 Bratislava, Slovac Republic
1D. I. Ivanovsky Institute of Virology, Russian Academy of Medical Sciences,
Gamaleya street 16, 123098 Moscow, Russia
sona.sarmirova@szu.sk
Aim: The aim of the present work was to systematically study the effect of natural
extracts – Ridostin and Ferrovir for studying their antiviral activity in vitro against
coxsackievirus.
Methods: First the effect of the two preparations on the growth curve of different cell
lines (GMK, Hep-2, L929) was studied, and to determine the optimum concentration for
the antiviral study. The cells were also studied for gross macroscopic changes. The effect
of these antiviral agents on eGFP-CVB3 was studied. Viral titrations were done in cells
pretreated for 24 hrs with the either Ridostin or Ferrovir. Untreated cells infected with
different virus dilutions served as controls.
Results: Different concentrations of Ridostin and Ferrovir did not have an influence on
the attachment efficiency of three cell lines. We observed that the growth of human
epidermoid cell line (Hep-2) was most sensitive to the preparations and mouse cell line
L929 was least sensitive. Ridostin and Ferrovir did not have a negative effect on the cell
lines of human, monkey and mouse origin. Optimal concentrations of these extracts were
evaluated for each cell culture system. Further, antiviral activity of Ridostin and Ferrovir
against coxsackievirus eGFP was studied. Our results demonstrate Ridostin and Ferrovir
exhibited antiviral activity against eGFP-CVB3 in cell line L929, no antiviral effect was
observed in GMK and Hep-2 cell lines. Our results suggest that only mouse fibroblast
cell line L929 was able to induce interferons in the presence of the interferon inducers,
Ridostin and Ferrovir in contrast to GMK and Hep-2 cells.
Conclusion: We conclude that the mechanism of the antiviral effect of the natural
extracts, Ridostin and Ferrovir protecting the cells from coxsackievirus-infection was
mediated via interferon induction.
Keywords: antiviral activity, ridostin, ferrovir, coxsackievirus
152
Cytokines in brain of Coxsackievirus infected mice
Stipalova D., Borsanyiova M., Sarmirova S., 1Sobotova Z, 2Klement
C.,Bopegamage S.
Enterovirus Laboratory Slovak Medical University,
Limbova 12, 83303 Bratislava, Slovak Republic.
1National Reference Centre for Poliomylitis, Authority of Public Health of the Slovak Republic,
Trnavska 52, 82645 Bratislava, Slovak Republic
1Regional Authority of Public Health Banska Bystrica
Cesta k nemocnici 25, 97556 Bratislava, Slovak Republic
darina.stipalova@szu.sk
Aim: To investigate the presence of cytokines IL-4, IL-1β, TNFα in brains of mice
infected with isolates from different origins and coxsackievirus B5 (CVB5) prototype.
Methods: CD-1 mice were orally infected with patient’s isolate (aseptic meningitis) and
an S1B5 isolate from treated sewage sample (from the area of domicile of the patient)
both identified as CVB5 and the CVB5 (Faulkner) prototype strain. Brains were collected
on days 5, 10 and 45 post infection (p.i.), one part was snap frozen and other part was
fixed in formalin and embedded in paraffin. Presence of cytokines was analyzed in tissue
sections by immunohistochemical staining using polyclonal/monoclonal cytokine specific
antibodies. Viral RNA was detected by reverse transcriptase PCR and nested PCR.
Results: IL-1 β was present in the brains of all the infected mice at day 5, 10, 49 p.i. All
3 cytokines IL-4, IL-1β, and TNFα were detected at day 5 and 10 p.i. in S2 B5 infected
mice and day 45 p.i. of S1B5 infected mice. CVB5 showed mild induction of cytokines
IL-1 and TNFα. Viral RNA was observed in 1/15 mice in CVB5 infected mice whereas
3/15 in the S1B5 infected mice and 7/15 in S2B5 infected mice.
Conclusion: Cytokine induction depended on the virus origin. Mice infected with
environmental isolate S1B5 and patient’s isolate S2B5 induced proinflammatory
cytokines as well as the IL-4, which can be related to the presence of RNA in the brain of
these mice.
Key Words: brain, coxsackievirus, isolates, cytokines.
153
UKRAINE –AMOSOV
154
Women’s Health and Cardiovascular
Diseases
155
Plethysmography analysis according to age and gender
Zaporozhko I. A., Zubchuk V. I., 1Nastenko E. A.
National Technical University of Ukraine "KPI",
16/2, Yangelya str., Kyiv, 03056, Ukraine,
1National M.Amosov Institute of Cardiovascular Surgery NAMSU Intercollegiate Medical Engineering
Faculty, Kyiv, 03056, Ukraine
Inna.Zaporozhko@gmail.com
Pulse waves (PW), recorded on the small blood vessels of extremities, give an integral
information about the CVS and other organs associated with the CVS. In particular, important
task is to determine parameters values of the CVS and PW for both men and women in the norm
and pathology for different ages. Measured parameters are - blood pressure (systolic and
diastolic), heart rate (HR), peripheral blood oxygen saturation (SpО2) and the pulse wave
dynamics. The purpose is to build normal parameters of a cardiovascular system CVS for people
with different age and sex for further creation computer express diagnostic system. The aim was
to develope method for PW forms analysis and to build quantitative measures for pulse wave
shape. The research of the need to consider gender as important information in cardiovascular
parameters classification was made. Pulse waves registration is provided by using the pulse
oximeter UtasOxi 200. Sensors are placed on index fingers vessels of both hands of the patient.
Pulse waves averaged over selected ensemble and transformed to normalized amplitude and
duration for further classification of forms of PW. Methods: The research involved 188 people,
including 87 women and 101 men. Cluster analysis and regression analysis were used to study
the dependence of age and cardiovascular parameters in healthy people. These procedures were
provided for further solving classification task with use of artificial neural networks, which
makes it possible to estimate the biological age according to indicators of cardiovascular and
other anthropometric data. NN are also used for evaluating normal parameters for further
pathologies detection. As a quantitative measure of pulse wave shape changes proposed an index
of pulse wave shape, which is calculated as the ratio of the harmonic components of Fourier
expansion for normalized PW. Results: Proposed index of pulse wave shape gives a numerical
estimate of changing the shape of PW of men and women with age, and pulse wave deviation
from the norm. When using statistical methods to determine the optimal number of parameters
for building a linear model for assessment of biological age, gender was not important criterion,
and only had a high correlation with anthropometric indices and the level of SpO2. But further
cluster analysis and attempts to build an assessment of biological age by training neural networks
identified the need to consider gender as required parameter to construct an accurate model of
age changes based on CVS classification. Error of biological age estimation on test data was near
3 years and significant parameters were index of PW shape, SpO2, diastolic arterial pressure and
body mass index. Conclusion: Ability to analyze changes of PW form index with other
parameters gives background information to assess the functional state of subjects and formation
of diagnostic conclusions. Gender should be considered as required parameter to construct an
accurate model of changes in PW forms with age. Taking in account this difference of norm for
CVS parameters for man and woman diagnosis classification should consider this to provide
reliable result. To increase the accuracy of classification there is a need to build bigger training
set.
156
Gender based post CABG circulatory aspects
Shapovalova V. Rudenko M
M. Amosov National Institute of Cardiovascular Surgery National Academy of Medical Science of
Ukraine. 6 M. Amosov St. 03180 Kyiv,Ukraine
shapovalya@mail.ru; n‐rudenko@yandex.ru
Aim: To study the influence of morpho-functional circulatory gender aspects in the usage
of compensatory resources of heart and vessel regulation in critical conditions in early
period after CABG.
Methods: We used the database of patients in early period after CABG, 95 were men and
20 were women. We used ECG, ECHO, and blood pressure test and biochemical blood
readings. We also used a control group of 96 patients: 48 men and 38 women. We
compared 24 main values of systemic and coronary circulation and oxygen delivery
value.
Results: The average age of investigated women was slightly higher (nearly by 20%) and
was 57,4 an 52,8 years. Average height, weight, and body surface area in men were
higher by 6, 19 and 12%.The main difference was in absolute geometrical values of the
LV: end systolic and end diastolic values, and in the thickness of the posterior wall,
interventricular septum and stroke volume. The average reading of LV beating in women
was approximately 22% lower than in men. Women had a 7% decrease in DAP. This is
explained by 39%, decrease in general peripheral resistance in women. Due to the
differences in heart rate the heart index and the LV index minute work did not differ
greatly. But the LV beating work index was higher in men by 24%.Pressure growth speed
in the LV in the isometric phase was higher in women by 33%.Peripheral resistance
index, was higher in men by 10%. In both groups it exceeded physiologically normal
value. The oxygen extraction was higher in women. The myocardium energy usage
coefficient was by 27% lower in women and the compensatory conditions were by 20%
lower.
Conclusions: The study proves that the myocardium mass index of the LV in women is
smaller by 10-15 g/m2. In conditions of critical compensatory mechanisms the
combination of decreased general peripheral resistance index and increased heart rate
allows to create same integral readings in both genders due to 33% smaller LV beating
rate in women.
Keywords: CABG, peripheral resistance index, heart rate.
157
Gender based hemodynamics regulatory reserves
Shapovalova V.V., Rudenko M.L
M. Amosov National Institute of Cardiovascular Surgery National Academy of Medical Science of
Ukraine. 6 M. Amosov St. 03180 Kyiv,Ukraine
shapovalya@mail.ru; n‐rudenko@yandex.ru
Aim: The aim of this work study the gender differences in correlation of time intervals of
mechanical systole and diastole of the left ventricle depending on the heart rate, which
show the regulatory reserves of coronary circulation.
Methods: The materials were based on screening of patients in early postoperative
periods after CABG and heart valve replacement, including 517 men and 193 women.
We used ECG, ECHO. We also used a control group with practically healthy men and
women who didn’t have any circulatory disorders. The control group consisted of 96
patients: 48 men and 38 women.
Results: We used the EVR-endocardial valuation ratio as a measure of adequate
extracardial coronary circulation,
EVR=
SPTI
DPTI
,
where DPTI – diastolic “pressure time” index; SPTI – systolic “pressure time” index,
яwhich is the energy measure that the myocardium uses during one contraction cycle.
Compensatory condition of myocardium energy usage:
DPTI = D·(APср. – EDPLV).
Work coefficient during one contraction cycle
SPTI = Sm·ASP,
Conclusion: We created regression equations for men and women Sm/D(HR) THE
correlation coefficient R in all cases was statically significant (p < 0,01).
Resulted in
Men : Sm/D = 0,0143·HR – 0,487; R2 = 0,61;N = 517
Women: Sm/D = 0,012·HR – 0,5708; R2 = 0,71; N = 193
Coronary circulation in women is more endured and tolerant to increase in heart rate.
Prior to surgical treatment of valve disorders and IHD gender differences in circulation
control mechanisms are not seen due to heart disorders, but adequate surgical treatment
restores gender differences in control. The main circulatory control mechanism in women
in early postoperative stages is to control integral traits of circulation, specifically heart
productivity, minute blood flow, left ventricle productivity. Women are more sensitive to
diastole/systole circulatory disorders
Key Words: circulation, systole, diastole, heart rate
158
Gender based circulatory aspects in cardiosurgery
Shapovalova V. Rudenko M.
M. Amosov National Institute of Cardiovascular Surgery National Academy of Medical Science of
Ukraine.
6, M. Amosov St. Kyiv,Ukraine, 03680
shapovalya@mail.ru; n‐rudenko@yandex.ru
Aim: To study the gender differences in hemodynamic in early postoperative period
(PoP) in patients with valve disorders with increase of left ventricle(LV) size.
Methods: In early PoP we studied the hemodynamics in men and women with increased
or normal LV. We evaluated following groups 93 women, of which 27 – normal (group
1), 20 – moderate increase (group 2) and 46 – severe increase (group 3) preoperative end
diastolic index(EDI) of LV; 207 men, of which 47 – normal (group 1), 35 –moderate
increase (group 2) and 125 –severe increase (group 3) preoperative EDI of LV. We used
ECG and ECHO.
Results: In the analyzed groups 60% men and 49% of women had increased EDI of LV.
Heart failure in group 1 was 34% men and 59.3% women, group 2 17.1% and 30%,
group 3 28.8% and 34.8% In female groups 2 and 3 compared with group 1 the beating
index increased by 10-14%, in combination with high heart rate it lead towards high heart
index in all groups. Contraction activity in groups 2 and 3 was 32% higher than group1.
The frequency of heart failure in men was 28 and 40% in women, thus, in women it was
almost one third. In all three groups EDI and heart rate was by 20-30% lower in men.
Conclusions: The main reason of formation of gender-related hemodynamic specifics,
without regards of preoperative changes is considered to be the mass of contracting
myocardium. Its smaller size is compensated by the growth of heart rate and contracting
activity of the heart. But, in case of dilatation of LV women tend to have higher
frequency of heart failure, which shows smaller compensatory reserves. Most
characteristics in men and women tend to be the same, mainly due to compensatory
mechanisms.
Keywords: end diastolic index, heart rate, left ventricle
159
Nanobiotechnology and Cancer
Research
160
Influence of nanocomplex and electromagnetic field on nonlinear
dynamics of carcinosarcoma walker-256
2,4Rykhalskiy A. Y., 2Dzyatkovskya I. I., 2Romanov A. V., 2,4Nikolov N. A.,
2, 4Orel V. E., 3Shevchenko A. D., 5Bogatyreva G. P., 1MaximenkoV. B.,
2Dzyatkovskya N. N.
1National M.Amosov Institute of Cardiovascular Surgery, NAMS Ukraine, 03680, Kyiv, Amosov Str, 6,
Ukraine
2National Cancer Institute, MH Ukraine, 33/43 Lomonosov Street, 03022, Kyiv, Ukraine
3G.V. Kurdyumov Institute for Metal Physic, NASU, 36, Acad. Vernadsky Blvd. 03680, Kyiv, Ukraine
4National Technical University of Ukraine 'Kyiv Polytechnic Institute’, MESYS Ukraine, 37 Prospect
Peremogy, 03056, Kyiv, Ukraine
5V.Bakul Institute for Superhard Materials, NAS Ukraine, 2Avtozavodskaya Street, 04074, Kyiv,
Ukraine
alex@kpi.ua
Aim: The study examines the effects of anticancer treatment by nanocomplex of
nanodiamonds with anthracycline antitumor antibiotic doxorubicin and alternating
electromagnetic field on nonlinear dynamics of the growth of carcinosarcoma Walker
256.
Methods: Nanodiamonds with mean diameter < 10 nm was obtained by a detonation
wave. Mehano-magneto-chemical synthesis of nanocomplex from nanodiamonds with
doxorubicin (Pfizer, Italy) was processed in mechanomagneto reactor «MMR» (NCI,
Ukraine). The magnetic properties were studied by magnetometer using a «Vibrating
Magnetometer 7404 VSM» (Lake Shore Cryotronics , Inc., USA) .The animal tumors
irradiated locally by 40 MHz with an initial power of 100 W by «Magnetotherm»
(Radmir, Ukraine).Nonlinear kinetics of tumor volume was evaluated by growth factor
according to autocatalytic equation. Statistical processing of numerical results was
carried out using Statistic 6.0 (© Stat Soft, Inc. 1984–2001) computer program with
Student`s test.
Results: Detonation nanodiamonds were weak soft ferromagnetic with saturation
magnetic moment ms=0.15842 emu/g. Anthracycline antitumor antibiotic doxorubicin
after mechano-magneto-chemical treatment was paramagnetic with ms=+0. 06836emu/g
in contrast to conventional drug which is diamagnetic with ms= -0.01200emu/g. Compleх
from nanodiamonds with doxorubicin after mechano-magneto-chemical synthesis was
weak soft ferromagnetic with ms=0.058182 emu/g. In the research of animals with
Walker 256 carcinosarcoma was shown, that nanocomplex on the basis of detonation
nanodiamonds with doxorubicin after mechano-magneto-chemical synthesis had a greater
antitumor effect on 10% (p <0.05) than conventional doxorubicin during combined
treatment by local electromagnetic irradiation of tumor.
Conclusions: Nanocomplex from nanodiamonds with doxorubicin after mechano-
magneto-chemical synthesis was weak soft ferromagnetic with saturation magnetic
moment ms=0.058182 emu/g and initiated greater antitumor effect on 10% than
conventional doxorubicin during combined treatment by local electromagnetic irradiation
of tumor.
161
Translational Research and Drug
Development
162
Gender influence on professional orientation and progress of
bachelors education in technical university
1Ovcharenko A., 2Maksymenko V.
1 National Technical University of Ukraine "KPI", 16/2, Yangelya str., Kyiv, 03056, Ukraine,
ilikanet@ukr.net
2 National M. Amosov Institute of Cardiovascular Surgery NAMSU Intercollegiate Medical Engineering
Faculty, Kyiv, 03056, Ukraine
maksymenko.vitaliy@gmail.com; v.maksimenko@kpi.ua
Some women have psychological barriers in chose of professions, inclines toward no
technical fields. Frequent asked questions are: does gender influence on choose of
profession? Does gender important for progress in career and education? What are
competitive characteristics of male and female groups?
Aim: To approve females competitive capability in all professional fields in compare
with males and to destroy female’s psychological barriers in choose of any professions.
Methods: Evaluation of engendered influence on chooses of profession and on the
progress in education was performed on the group of 1919 students of seventh faculties at
technical university. Criteria for study with an application of correlation analysis
included engendering structure of departments, excellent and send down results in
education, stipendiary and non stipendiary students in 14 male and female groups at 7
faculties (covered medical technical oriented departments) included: Medical-
Engineering Faculty (IMEF); Faculty of Electronics (FE); Information and Calculating
Technologies Faculty (ICTF); Radio Engineering Faculty (REF); Device – Building
Faculty (DBF); Engineering and Physical Faculty (EPF); Faculty of Heat and Power
Engineering (FHPE).
Results: Significantly more male students choose technical professions, twice
predominate females. More female in technical professions choose medical oriented
specializations, if they have this opportunity. Despite of their medical prefers, female
results significantly much better by all investigated parameters of education at technical
faculties (even in heavy industry), then in mail group of study. Medical engineering
faculty contained highest per centage of women (>50%) and twice more scholarship
(grant) holder in female group in comparison with mails, what is higher then at all other
medical and technical oriented faculties. In mean time, male easier reach “excellent”
results at this faculty then at others, and have almost the same “excellent” level with
female;
Conclusions: In spite of women have psychological barriers in choose of engineering
profession and more often go in to medical fields, they have more capabilities then male
groups of students as for medical and as for technical disciplines during period of
education. Female’s competitive capability in education superior to male in all
professional fields.
Keywords: Engendering, psychology, capability, profession, education
163
UKRAINE – DHLNMU
164
Mother and Child Health
165
Glycodelin and prognosis of pregnancy
Shurpyak Serhiy
Department of Obstetrics, Gynecology, Perinatology, Danylo Halytskyy Lviv National Medical
University
69 Pekarska St. 79010 Lviv, Ukraine
shurpyak_serhiy@yahoo.com
Aim: stable high frequency of miscarriage (10-25%) requires new approaches to the
prediction of this pathology.
Methods: A definition of the content α-2-microglobulin of fertility (Glycodelin), which
is produced by endometrial glands and is considered as a possible marker of
complications of pregnancy, the serum in 6 - 10 weeks of pregnancy and the threat of
premature delivery in 22 - 25 weeks of gestation. Quantifying the glycodelin in serum
was conducted by ELISA.
Study involved 69 patients, 10 applied for abortion at will, 20 with miscarriage that
began, 19 with pregnancy that does not develop, verified by ultrasound, 20 pregnant
with threat of preterm delivery in 22 - 25 weeks of gestation.
Results: serum level of glycodelin in women with physiological pregnancy in 6 - 10
weeks amounted to 120,4 ± 6,85 ng / ml, with the threat of pregnancy loss was reduced to
98,6 ± 3,45 ng / ml (p <0 , 05 compared with control and pregnancy, that does not
develop - 136,3 ± 7,35 ng / ml). With the threat of early preterm delivery was observed to
increase glycodelin 139,5 ± 6,45 ng / ml (p <0.05 compared with control and risk of
miscarriage in the first trimester).
Сonclusions: Changes in production of glycodelin in pathological pregnancy in
trimesters I. and II. are going in different directions: in the I. trimester miscarriage occurs
in background reduction, and in II. trimester - against the backdrop of increasing
production of glycodelin. Women with pregnancy not developed in the I trimester was
characterized by normal levels glycodelin in serum. Determination of glycodelin serum
for I and II trimesters is an important non-invasive method of monitoring the course of
gestational process.
Key words: preterm delivery, glycodelin, α-2-microglobulin of fertility, miscarriage
166
The current status and perspectives of umbilical cord blood banking
in Ukraine
Nasadyuk K. M.
Department of Biochemistry, Danylo Halytsky Lviv National Medical University
69, Pekarska Street, 79000 Lviv, Ukraine
nasadyukch@gmail.com
Due to the high rates of hematologic malignancies the development of cord blood banking is of crucial
medical and social importance in Ukraine.
Aim of the study was to evaluate the current status of cord blood banking and clinical
application in Ukraine.
Methods: literary review and sociological (opinion poll).
Results: The first cord blood transfusions for managing blood lost in Ukraine are dated
1939 and the first cord blood bank in our country was created in 1984 in Kharcov, being
perhaps the first European experience of cord blood banking. But it existed 5 years and
again the cord blood banking in Ukraine launched in 2004. At the moment 4 cord blood
banks offer their services to expecting parents in Ukraine but all are autologous. Hence,
in Ukraine cord blood is being collected only in about 0.3% births. The only report on
umbilical cord blood hematopoietic stem cell transplantation in Ukraine with its 47
million population is dated 2009 and concerns a 2 year old patient with hypoxic brain
injury, whereas literary data gives evidence that autologous umbilical red blood cells and
plasma are currently used for cardiopulmonary bypass in neonates undergoing surgery
due to the congenital heart defects.
The opinion poll on the subject “cell therapy”, “cord blood banking” (2010), which
included 1000 respondents (aged 18-60; males and females 1:1) as well as 1000 pregnant
women and 200 obstetricians from different regions of Ukraine showed that both
population and specialized medical staff realize the biologic value of cord blood and
support the umbilical cord blood banking.
Conclusions: The Ukrainian population and medical community are ready to support the
creation of the public cord blood bank. The necessity of its integration with the world
registries is emphasized by the national peculiarities of HLA-phenotype and high rates of
migration of the Ukrainian population to Europe and USA.
Keywords: umbilical cord blood, hematopoietic stem cells, hematopoietic stem cell
transplantation, cord blood bank
167
Experimental hypothyroidism increased exposure of LFuc and
DGlcNAc sugar determinants in rat adrenal gland
Lutsyk S. A., Shchur M. V.
Department of Histology and Embryology, Faculty of General Medicine, Danylo Halytsky Lviv National
Medical University, 69 Pekarska st., Lviv, 79010, Ukraine
lutsyk@meduniv.lviv.ua
Aim: To investigate influence of experimental hypothyroidism on tissue glycoconjugates
of rat adrenal gland.
Methods: Hypothyroidism was induced in 150-180 g male Wistar rats by
supplementation of their daily food allowance with 5 mg/kg of antithyroid drug
mercazolil (1-methyl-2-mercapto-imidazole) during 30 days. Control rats were
maintained in the same conditions on standard diet. Animals were sacrificed by diethyl
narcosis overdose. Adrenal glands were removed immediately, fixed in 4% neutral
formalin and embedded in paraffin. Sections 5-7 µm thick was subjected to routine
lectin-peroxidase-diaminobenzidine staining protocol. Lectin set included Laburnum
anagyroides bark agglutinin (LABA, specific to LFuc), Sambucus nigra agglutinin (SNA,
specific to NeuNAc(α2-6)DGal), and wheat germ agglutinin (WGA, specific to
DGlcNAc > NeuNAc).
Results: In control rats it was detected selective LABA affinity to vascular endothelium
of adrenal medulla. Under experimental hypothyroidism endothelium of adrenal cortex,
non reactive in control rats, was strongly labelled with LABA. Moreover,
semiquantitative evaluation of medullary vessels endothelial reactivity revealed increased
LABA binding. Hypothyroidism induced reduction of SNA, and enhancement of WGA
reactivity apparently due to impairments of sialilation in association with unmasking of
DGlcNAc residues. Hovewer, remodelling of SNA and WGA receptor sites was less
significant in comparison with that for LABA.
Conclusions: Hypothyroidism has significant influence on glycoconjugate processing in
rat adrenal gland, inducing enhanced exposure of LFuc and DGlcNAc determinants in
association with reduced terminal NeuNAc residues. Vascular endothelium of adrenal
cortex and medulla demonstrated heterogeneity in lectin binding.
Keywords: hypothyroidism, adrenal gland, lectin histochemistry, fucosoglycans.
168
Impairments of rat ovaria and endometrium in experimental hyper-
and hypothyroidism as detected by lectin probes
Sogomonian E. A ; Turkevych M.
Department of Histology and Embryology, Faculty of General Medicine, Danylo Halytsky Lviv National
Medical University, 69 Pekarska st., Lviv, 79010, Ukraine
lutsyk@meduniv.lviv.ua
Aim: To investigate influence of experimental hyper- and hypothyroidism on
carbohydrate determinants of rat ovaries and endometrium by means of lectin
histochemistry.
Methods: 20 lectins of different affinities, including 5 new lectin preparations, purified
from fungi, were used for the histochemical examination of carbohydrate determinants in
rat ovary and endometrium under experimental hyper- and hypothyroidism. Lectin
histochemistry was supplemented with general morphology and estrous cycle studies.
Results: Hyperthyroidism induced ovarian luteinisation, increased content of decidual-
like cells and of collagen fibers in endometrium with no reliable influence on estrous
cycle. Hypothyroidism was accompanied with retardation of estrous cycle, leucocyte
infiltration of ovarian and endometrial stroma. Lectin binding in the ovaries of control
rats was restricted predominantly to zona pellucida and corona radiata of growing
follicles, luteocytes and leucocytes. Within endometrium lectins strongly labeled its
luminal surface, epitheliocytes and secretion of uterine glands, decidual-like cells,
leucocytes and collagen fibers. Both hyper- and hypothyroidism induced significant and
specific redistribution of DMan, LFuc, NeuNAc, DGlcNAc, DGalNAc and DGal sugar
determinants of ovaria and endometrium; hyperthyroidism induced more severe
alterations of tissue glycoconjugates in comparison with hypothyroidism; endometrium
was more susceptible to thyroxin-modulated impairments compared to ovaria.
Conclusions: The data give new insight into pathogenic mechanisms of thyroid disorder
influences on female reproductive organs demonstrate suitability of lectin histochemistry
methods for monitoring the efficacy of hormonal disballance correction therapy, as well
as applicability of new lectin preparations for the selective labeling of ovarian and
endometrial constituents.
Keywords: hyperthyroidism, hypothyroidism, rat, ovary, endometrium, lectins.
169
Nanobiotechnology and Cancer
Research
170
Novel thiazolopyridines synthesis as anticancer agents
Chaban T. I., Ogurtsov V. V., Klenina O. V.
Danylo Halytsky Lviv National Medical University,
Pekarska Str., 69, Lviv, 79010 Ukraine
chabantaras@ukr.net
The synthesis innovations that are enabling the development of the condensed
heterocycles with the antitumor efficiency are essential challenge of nowadays.
Thiazolo[4,5-b]pyridines structural variants syntheses with an eye toward the discovery
of compounds having improved or novel properties as drugs is a perspective and relevant
strategy.
Aim:3H-thiazolo[4,5-b]pyridines synthesis by 5,7-dimethyl-3H-thiazolo[4,5-b]pyridine-
2-оne structural modification in 3rd and 6th core heterocycle positions and their antitumor
action evaluation.
Methods: organic synthesis, 1H-NMR spectroscopy, pharmacological screening.
Results: 5,7-dimethyl-3H-thiazolo[4,5-b]pyridine-2-оne (1) was selected as the starting
compound its synthetic route development by [3+3]cyclocondensation of acetylacetone
with 4-iminotiazolidon-2 has been previously reported. The acidic character of its 3rd
proton position led us to design 3-(5,7-dimethyl-2-oxo-thiazolo[4,5-b]pyridine-3-yl)-
propionitrile (2) under cyanoethylation reaction which has been further utilized by
hydrolysis for 3-(5,7-dimethyl-2-oxo-thiazolo[4,5-b] pyridine-3-yl)-propionic acid (3)
preparation:
N
S
N
O
CH3
CH3 OH
O
N
H
S
N
O
CH3
CH3
N
CH2
N
S
N
O
CH3
CH3 N H+
pyridine
1 2 3
The next stage includes the core heterocycle structural modification in its 6th position.
5,7-dimethyl-6-arylazo-3Н-thiazolo[4,5-b]pyridine-2-ones were yielded by appropriate
α-arylazoacetones treatment with 4-iminothiazolidone-2:
S
N
HNH
O
CH3 OH
N
CH3
ON
RH4C6
S
N
H
O
N
CH3
CH3
N
NRH4C6
S
O
O
N
H
S
N
S
O
O
N
H
S N
N
Et
R=H(4), 2CH3(5), 4-CH3(6), NO2(7), COOH(8),
(9) (10)
The compounds structures were established by 1H-NMR spectroscopy. Their antitumor
activity evaluation was carried out in National Cancer Institute, Bethesda, MD USA on
60 human cancer cells lines. Compound 4 was shown to posses the highest cytostatic
effect on some cell lines of non-small cell lung cancer, CNS and breast cancer, and
melanoma the growth inhibition being 7.76% (cell line UACC-62), 18.2% (NS 578T) and
23.89% (EKVX).
Conclusions: The core heterocyclic system can be used as a promising framework for the
anticancer candidate drug development.
171
Generation of novel nogalamycins through targeted gene disruption
1Klymyshin D.O., 2Nimets O.J.,2Honchar M.A., 2Fedorenko V.O.
1Danylo Halytsky Lviv National Medical University, Pekarska str. 69, Lviv, Ukraine, 79010;
2Ivan Franko National University of Lviv, Hrushevskuy str. 4, Lviv, Ukraine, 79005;
dedima@rambler.ru
Aim: Among anthracycline derivatives, nogalamycins showed superior cytotoxicity and
antitumor activity and also proved to be effective against breast cancer clinically. We
aimed in generation of novel nogalamycins by targeted gene disruption of snoaE, snoaM,
snoaL genes from Streptomyces nogalater Lv65.
Methods: Enzymatic manipulation of DNA and Southern hybridizations were carried out
according to manufacturer’s directions (Stratagene, NEB, MBI Fermentas, Boehringer
Mannheim). Oligonucleotide primers were purchased from Sigma-Genosys. PCR was
performed in a thermocycler MiniCycler (MJ Research). Intergeneric Escherichia coli –
Streptomyces conjugation was used to introduce plasmid DNA into S. nogalater strains.
Results: We were interested in applying gene replacement techniques to sno-genes
controlling cyclisation steps of the initial polyketide backbone of nogalamycin, thus
leading to novel bioactive compounds. To disrupt snoaE, the spectinomycin resistance
cassette aadA was used. The S. nogalater mutant with a replaced snoaE gene was
obtained. The recombinant strain was shown to accumulate novel compounds.
Complementation results suggest that snoaE disruption had a polar effect on downstream
sno-genes. SnoaM and SnoaL disruption was achieved after a second crossover
introducing the deleted snoaM, snoaL genes into the chromosome. In this case
nogalamycin was not detected in extracts of the recombinant strains. Additional copies of
the snoaE, snoaM, snoaL genes, cloned in pKC1218E plasmid, increased nogalamycin
production in the wild-type strain S. nogalater Lv65.
Conclusions: Our work has showed that the cyclisation in the nogalamycin biosynthesis
occurs prior to the three aglycon cyclases, and that their targeted disruption leads to
dramatic effects on antibiotic production and leads to generation of novel antitumor
compounds.
Keywords: breast cancer, antitumor antibiotics, anthracyclines, nogalamycin.
172
Translational Research and Drug
Development
173
Biological activity of α-alkylacroleines derivatives
Marshalok O. I., 1Karpiak N. M.
Danylo Halytskyy Lviv National Medical University, 69 Pekarska Str., Lviv 79010, Ukraine
1National University “Lviv Polytechnic”, 12 Stepan Bandera Str., Lviv 79013, Ukraine
olga.marshalok@i.ua
Aim: There are many important biologically active substances among heterocyclic
compounds. Thus, acrolein dimer is used as starting material in antibiotics preparation.
Our research is concerned with α-Alkylakrolein dimers and their derivatives
investigation.
Methods: α-Alkylakrolein dimers were obtained by corresponding aldehyde dimerization
at 170-190 °C:
where R = –СН3, –C2H5.
2,5-dimethyl-3,4-dihydro-2H-pyran-2-methanol (1) and 2,5-diethyl-3,4-dihydro-2H-
pyran-2-methanol (2) were obtained via Cannizzaro mechanism from corresponding α-
alkylakrolein dimer reaction with NaOH 40% aqueous solution at 50 °C:
Structure of synthesized compounds was confirmed by IR– and NMR–spectroscopies.
The bacteriostatic and bactericidal activities were determined towards Gram-negative
(Escherichia coli, Salmonella enteritidis) and Gram-positive (Staphylococcus aureus 209
and Streptococcus) cultures by the diffusion method in agar. As the standards amikacin
and ciprofloxacin were used. Fungicidal activity was determined towards Candida
albicans, Aspergillus fumigatus and Penicillium chrysogenum using a Saburaud medium.
A standard disc with pimafucin served as the control.
Results and Conclusions: The data obtained showed that α-alkylacrolein dimers and
their derivatives have acceptable biological activity in comparison with standards that
allow using them as antiseptic agents. Using PASS program it was determined that
investigated substances have high probability of broad spectrum anticancer activity (85%
prediction accuracy).
Table. Results of predictable screening of biological activity
Probable activity
Substance
Apoptosis
agonist, %
Anticarcinogenic,
%
Antioxidant,
%
Antineoplastic,
%
α-Alkylakrolein dimers 71 52 53 –
1, 2 – – 82 78
Na salts of 2,5-dialkyl-3,4-dihydro-
2H-pyran-2-carbonic acid
68 69 78 –
174
Alterations of rat liver carbohydrates in streptozotocin-induced
diabetes mellitus as detected by lectin histochemistry
Pankevych L. V., Fetsych M.
Department of Histology and Embryology, Faculty of General Medicine
Danylo Halytsky Lviv National Medical University, 69 Pekarska st., Lviv 79010, Ukraine
lutsyk@meduniv.lviv.ua
Aim: To use a set of lectins with different carbohydrate affinities for the investigation of
rat liver glycoconjugate impairments under the influence of streptozotocin-induced
diabetes mellitus.
Methods: The lectin panel included 7 conventional lectins – Con A, SNA, RCA, WGA,
PNA, SBA, and HPA, supplemented with the original fucose-specific Laburnum
anagyroides bark agglutinin (LABA). Tissue samples were fixed in 4% neutral formalin,
embedded in paraffin and subjected to routine lectin-peroxidase-diaminobenzidine
staining protocol.
Results: In control rats it was detected strong reactivity of Con A, LABA, SBA and SNA
with cytoplasmic granularity of hepatocytes, of RCA, WGA and HPA - with vascular
endothelium, of WGA and HPA - with bile capillaries. Experimental diabetes was
associated with the redistribution of Con A and LABA receptor sites from centrolobular
hepatocytes to hepatocytes with peripheral localization. Diabetes mellitus induced
exposure of lectin reactivity within the hepatocyte and endothelial cell nuclei. Endothelial
lining of sinusoidal hemocapillaries, of central veins and portal tract vessels also
demonstrated significant and differential rearrangement of carbohydrate determinants.
Diabetes-induced activation of Kupffer cells was associated with exposure of SNA, PNA
and SBA receptor sites within these cells cytoplasm, completely non-reactive in control
specimens.
Conclusions: These results give new insights into the pathogenic mechanisms of diabetes
induced impairments of hepatic carbohydrates, and demonstrate applicability of original
fucose-specific lectin preparation for experimental histopathology research.
Keywords: Streptozotocin, Diabetes mellitus, Rat liver, Lectins
175
Prooxidant activity of amaranth oil provides its membrane
protective effects under normal and adrenalin induced stress
conditions
Semen K. O., Yelisyeyeva O. P., Kaminsky D. V., 1Ostrovska H. V.
1st Department of Internal Medicine, Danylo Halytsky National Medical University, Lviv, Ukraine
1 Taras Shevchenko National University, Kyiv, Ukraine
khrystyna_semen@yahoo.com
Aim: to study the physical chemical properties of plasmatic membranes (PM) of rat
hepatocytes and pro-/antioxidant balance in blood of rats supplemented for one month
with Amaranth oil (AmO) under normal conditions and after adrenalin induced stress.
Methods: white male rates were divided into two groups: 1, control; 2, supplemented
with AmO 60 µl/kg per day for one month. Half of the animals from each group
underwent adrenalin (350 µg/kg) induced stress 30 min before decapitation. Monolayers
of membranes were prepared by I. Langmuir. Time of stable membrane functioning (tst),
surface pressure () and boundary potential jump (Δφ) were evaluated during
interaction with neurotensin under normal and stress condition. Pro-/antioxidant balance
was assessed spectrophotometrically in rat blood samples by the levels of TBARS,
conjugated dienes, oxidative modification of proteins, catalase and SOD activities.
Results: Increase in tst of PM of rat hepatocytes was noted after AmO use under normal
conditions and after adrenalin induced stress. Intensity of neurotensin molecules
incorporation into PM from “Amaranth” rats increased and remained at the control level
even after adrenalin stress. More distinct biphasic distribution of peptide-membrane
interaction process was clearly observed with AmO supplementation. That effect was
abolished by adrenalin, more prominently in control animals. The study of pro-
/antioxidant balance in blood showed modulation of antioxidant enzymes activity aimed
at mild prooxidant effect with AmO use that was further maintained after adrenalin stress
exposure.
Conclusions: AmO was shown to have membrane protective action both under normal
and adrenalin-induced stress conditions. Modification of membrane fatty acid
composition during activation of free radical reactions can be an essential mechanism
involved in these effects, as it was underpinned by slight prooxidant activity triggered by
AmO. Demonstrated AmO actions can be important for improvement of adaptive
potential and heart rate variability in clinical settings.
Keywords: Amaranth oil, plasmatic membrane stability, neurotensin, adrenalin induced
stress, mild prooxidant activity
176
Mouse lacking pituitary tumor transforming gene show elevated
exposuure of DGalNAc sugar determinants
Varyvoda O. Ye.
Department of Histology and Embryology, Faculty of General Medicine , Danylo Halytsky Lviv
National Medical University, 69 Pekarska st., Lviv, 79010, Ukraine
lutsyk@meduniv.lviv.ua
Aim: To investigate influence of pituitary tumor transforming gene (pttg-1) knockout on
glycome of murine parenchimal organs by means of lectin histochemistry.
Methods: Kidney, liver, lungs and testes of murine strain BL6/C57 with pttg-1 knockout
(pttg-KO) were compared to the wild type animals (pttg-WT). Animals for both groups
were kindly provided by Dr. S.Melmed (Cedars Sinai Medical Center). Tissue samples
were fixed in 4% neutral formalin and embedded in paraffin. General morphology was
studied after haematoxylin and eosin staining. Carbohydrate determinants were labelled
with soybean agglutinin (SBA, specific to DGalNAc), wheat germ agglutinin (WGA,
specific to DGlcNAc > NeuNAc), both lectins conjugated to peroxidase, with subsequent
diaminobenzidine visualization.
Results: General morphology studies revealed no significant changes in liver and lungs
of pttg-KO mice in comparison to control. Kidneys of experimental animals were
characteristic with glomerular compactization and enhanced urinary space of Bowmans
capsules. In testes it was detected dislocation of Sertoli cells, decreased population of
spermatogenic cells, impairments of syncytial complexes in between them. Kidneys and
testes thereafter were subjected to lectin histochemistry investigation. It was identified
enhanced exposure of DGalNAc sugar residues (SBA receptor sites) within Golgi
complex of secondary spermatocytes, in brush border of renal tubules and on the lumenal
surface of collecting ducts. Redistribution of WGA receptors included reactivity shifting
from basal spermatogonia to adlumenal spermatogenic cells, apparently due to the
enhanced exposure of DGlcNAc residues on the final stages of spermatogenesis.
Conclusions: This study suggests that pttg-1 gene plays pivotal role in carbohydrate
processing in mammalian organism.
Keywords: pttg-1 knockout, glycoconjugate processing, lectin histochemistry.
177
UKRAINE – IMBG
178
Women’s Health and Cardiovascular
Diseases
179
Polymorphisms of ESR1 gene involvement in stroke
1,2Kucherenko A., 2Kravchenko S., 2Livshits L.
1. Taras Shevchenko Kiev National University, Educational and Scientific Centre "Institute of
Biology", 03022, Kyiv, 2 Ac. Glushkov av.
2. Institute of Molecular Biology and Genetics of the NASU, Department of Human Genomics,
03680, Kyiv, 150 Zabolotnogo str.
livshits@imbg.org.ua
Estrogen is known to play role in vascular function and lipid metabolism regulation and
to be a neuroprotector. Two single nucleotide polymorphisms in ESR1 gene – c.454-
397C>T (PvuII) and c.454-351 A>G (XbaI) - are thought to influence gene transcription
and thus change cell sensitivity to estrogen action.
Aim: To establish possible involvement of c.454-397 C>T and c.454-351 A>G
polymorphisms into stroke development we investigated this SNPs in: case group -
patients with ischemic stroke (n=183), control group I - individuals from the general
population (n=100), control group II - healthy individuals elder then 65 years (n=88),
from different regions of Ukraine.
Methods: Blood samples for DNA analysis were obtained after informed consent.
Genotyping was performed by PCR followed by RFLP analysis.
Results: Homozygous -397C genotype frequency was significantly higher (P<0.05) in
women from case group (34.1%) compared to women from control I (20.8%) and control
II (20.8%) groups.
There was a significant difference (P<0.05) in -351G/G genotype frequency between
women (17.6%) and men (10.6%) from case group. Frequency of -351G/G genotype was
significantly higher (P<0.05) in women from case group (17.6%) comparing to women
from control II (5,7%). Linkage disequilibrium between -397 C>T and -351 A>G was
shown (P<0.0001); CG and TA alleles are in phase (r2=0.557). Pairwise comparisons
using Fisher exact test revealed significant difference (P<0.05) of CG/CG genotype
frequency in women from case group (17.7%) comparing to women from control II
(5.7%). Individuals with this genotype have 3.5-fold higher risk of stroke development
(OR = 3.54; CI-95%: 1.043 – 16.03).
Conclusions: Our findings suggest a possible role of studied ESR1 gene polymorphisms
in stroke development in female.
Keywords: ESR1 gene, polymorphism, stroke
180
Discovering inhibitors of protein kinase ASK1
Volynets G. P., Bdzhola V. G., Yarmoluk S. M.
Institute of Molecular Biology and Genetics, NAS of Ukraine,
150 Zabolotnogo Str., 03143, Kyiv, Ukraine
galina.volinetc@gmail.com
Aim: Apoptosis signal-regulating kinase 1 (ASK1) has recently emerged as an attractive
therapeutic target for treatment of several cardiovascular diseases. Earlier in vivo
experiments demonstrated that cardiac hypertrophy induced by AngII is associated with
enhanced ASK1/proapoptotic signaling. ASK1 deficiency attenuated cardiac
inflammation and fibrosis under diabetic conditions. The main aim of our research is to
identify the small molecule inhibitors of ASK1.
Methods: To discover the protein kinase ASK1 inhibitors we have performed screening
program, using both in silico and in vitro approaches. AutoDock and DOCK software
were used to conduct receptor-ligand flexible docking. The best-scored compounds of
different chemical classes were taken for the kinase assay analysis.
Results: In vitro observations revealed that derivatives of 2-Thioxo-thiazolidin-4-one
exhibited inhibitory activity towards ASK1. The most active compound inhibited ASK1
with IC50 = 2 μM. Then, in-depth study of this chemical class was performed using the
pre-selected library of 2-thioxo-thiazolidin-4-one derivatives. Ten best-scored
compounds were selected for the kinase assay analysis. Compound 2-{5-[5-(3,4-
Dichloro-phenyl)-furan-2-ylmethylene]-4-oxo-2-thioxo-thiazolidin-3-yl}-propanoic acid
inhibited ASK1 with a Ki of 340 nM. Our preliminary selectivity studies demonstrated
that this compound seems to be selective inhibitor of ASK1. In silico analysis of the
complexes of ASK1 with compounds indicated, that the peculiarity of the active
compound in the comparison to other nine derivatives is its ability to bind simultaneously
to the part of kinase domain known as “hinge region” and the phosphate-binding region
of the ATP-binding cleft.
Conclusion: Obtained results suggest that the core structure of identified active
compound can be used for further optimization and developing more potent and selective
inhibitors of ASK1.
Keywords: Apoptosis signal-regulating kinase 1, inhibitor, virtual screening.
181
Mother and Child Health
182
The premises for taurine biosynthesis in human placenta
1,2Romanets K. L., 1Martsenyuk O. P., 1Obolenskaya M. Yu.
1Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, Kyiv, Ukraine
2Taras Shevchenko National University, Kyiv, Ukraine
kate_romanets@yahoo.com
Aim: Taurine (Tau) is a sulfonic acid that is synthesized from cysteine (Cys) by
consequent action of cysteine dioxygenase (CDO) and cysteine sulfinic acid
decarboxylase (CSAD). Tau is a multifunctional moiety with its particular role in
development of fetal central nervous system, endocrine glands and maintenance of
oxidative status. Up-regulation of Cys production in human placenta under
hyperhomocysteinemia raises a question concerning the further Cys metabolism. So the
main goal of our research was to fill the gap in the study of taurine biosynthesis in human
placenta.
Methods: Expression of CDO, CSAD at the mRNA level was determined by reverse
transcriptase and quantitative polymerase chain reaction in real time. Total amount of
taurine in placental samples was determined by using amino acid analyzer. The objects of
the study were placentas of the first and the third trimesters of gestation.
Results: Total amount of taurine in term placenta was 0.64mg/g tissue, which consists
29% of the total amino acid content. For the first time the expression of CDO and CSAD
genes was ascertained in human placenta. The amount of CDO-specific mRNA (product
length of 380 nucleotides) was 6,65*106 number of copies per mg of total RNA in term
placenta and 2,95*105 number of copies per mg of total RNA in the first trimester one.
The level of CSAD mRNA (amplicon of 268 nucleotides) was 1,18*103 number of
copies per mg of total RNA in the samples from the third and 1,66*102 number of copies
per mg of total RNA in the samples from the first trimester of gestation. It is suggested
that taurine is synthesized more intensively in term placenta than in the first trimester
placenta.
Conclusions: On the basis of CDO and CSAD genes expression in human placenta we
suggest that the definite amount of Tau may be synthesized in this organ. It is likely that
activity of the synthesis substantially increases during the pregnancy.
Keywords: Taurine biosynthesis, human placenta, CDO, CSAD.
183
Model of one-carbon metabolism in human placenta
Rodriguez R., Obolenskaya M.
Institute of Molecular Biology and Genetics NASU, Kyiv, Ukraine, 03680
northernwizard@ya.ru
Background: Folate-related one-carbon unit metabolism (FOCM) is a complex
metabolic network with the tetrahydrofolate (THF) and methionine (Met) cycles in its
core connected with the synthesis of purines and pyrimidines, methylation reactions and
synthesis of cysteine (Cys). C677T polymorphism of MTHFR gene and the elevated
content of homocysteine (Hcy) in human placenta are associated with obstetrical
pathologies, particularly preeclampsia while ex vivo Hcy increases apoptosis and
decreases proliferation.
Objective of our study was to analyze the response of mathematical FOCM model for
C677T MTHFR heterozygosity, duplicate Hcy content and joint action of both factors.
Methods: Cross-database and literature search for placental-specific expression of
FOCM enzymes; Metatool software and COBRAToolbox for construction a
stoichiometric model of FOCM on the basis of placental-specific reactions (folate cycle -
9 reactions, methionine cycle - 4, -transsulfuration - 2, glutathione metabolism – 4,
taurine synthesis -3, reactions of in/output of metabolites).
Results: The stoichiometric model of FOCM features 7 key metabolites (THF,
methylene-THF, formyl-THF, Met, Hcy, Serine and Cys) and 15 elementary flux modes
consisting from 3 to 17 reactions with corresponding flux values for each. The simulation
of MTHFR C677T genotype (65% enzyme activity) induces the least changes in the
network, while its combination with double Hcy content induces decrease of metabolic
fluxes for purine synthesis and increase for Cys synthesis in parallel with the decrease of
flux values for Cys consumption and glutathione (GSH) synthesis. The latter is crucial
for cell's red-ox status.
Conclusion: The adverse effect of Hcy on proliferation and apoptosis may be partially
explained by the failure of purine synthesis and by oxidative stress due to accumulated
Cys autooxidation and decrease of GSH – main antioxidant moiety in the cells.
Keywords: placenta, FOCM, model, Hcy, MTHFR
184
Nanobiotechnology and Cancer
Research
185
Structural-functional characterization of human CHI3L1
Iershov Anton V., Kavsan Vadym M..
Institute of Molecular Biology and Genetics NAS of Ukraine,
150 Zabolotnogo St., 03680 Kyiv, Ukraine
a.yershov@yahoo.com
Aim: Chitinase 3-like 1 (CHI3L1, HC gp-39, YKL-40) belongs to glycosylhydrolase
protein family 18. Expression of CHI3L1 was found increased significantly in various
tumors in comparison with corresponding normal tissues. CHI3L1 can decrease the
doubling time of 293 cells, it allows the anchorage independent growth in soft agar, and
stable CHI3L1 expression makes 293 cells tumorigenic, stimulating the initiation of
tumors after xenograft transplantation into the Wistar rat brains. The aim of this work
was to perform structural-functional characterization of CHI3L1 and find key motifs
responsible for CHI3L1 activity.
Methods: Protein coding region of CHI3L1 cDNA sequence was amplified from plasmid
containing CHI3L1 insertion and cloned in pcDNA3.1 expression vector at EcoRI and
XhoI restriction sites. Site-directed mutagenesis of CHI3L1 heparin-binding region and
chitin-binding site was performed by QuikChange or overlap extension strategy. Tyrosine
residues were replaced by serine, lysine or arginine were replaced by glutamate. 293 cells
were transfected with obtained plasmid constructs, and colony formation assay was used
to assess oncogenicity in vitro.
Results: CHI3L1 mutants were generated and confirmed by sequencing. Mutations in
heparin-binding site of CHI3L1 reduced colony formation efficiency of transfected 293
cells, while mutations in chitin-binding site did not have significant effect.
Conclusions: Our results suggest that heparin-binding site of CHI3L1 is involved in
CHI3L1-driven enhancement of oncogenicity of 293 cells. Further characterization of the
mechanisms of CHI3L1 activity is necessary for elucidation of the role of CHI3L1 in
tumor formation and progression.
Keywords: CHI3L1, heparin-binding site, site-directed mutagenesis
186
Optimization of enzyme biosensor for fructose determination
Pyeshkova V. N., Dudchenko O. Y., Dzyadevych S. V.
Institute of Molecular Biology and Genetics of Ukrainian National Academy of Sciences,
150 Zabolotnogo Str., 03680 Kyiv, Ukraine
victoriya.p@gmail.com
Fructose concentration in human fluids can indicate different diseases connected with
fructose metabolism disorders in human organism. For example, high fructose
concentration in blood of patients indicates hereditary fructosemia; in urine indicates
essential fructosuria; in intestine - fructose malabsorption.
Today various methods are available for fructose determination, but most of them suffer
from diverse disadvantages: expensive equipment, time-consuming process, insufficient
selectivity etc. Nowadays, much attention is devoted to development of biosensors,
modern analytical devices based on the combination of biological component and
suitable transducers. The combination of enzymes with electrodes creates enzyme
electrochemical biosensors which can provide simple, accurate, high specific, sensitive,
fast, convenient and rather cheap determination of substrate. Moreover biosensors can be
easy miniaturized what makes them applicable for analysis in situ and in vivo.
Aim: The main aim of our work was study and optimization of analytical characteristics
of conductometric enzyme biosensor for fructose determination.
Methods: Enzyme electrochemical methods of fructose determination
Results: The new enzyme conductometric biosensor for fructose determination in liquid
samples has been developed by immobilizing fructose dehydrogenase using
glutaraldehyde on the surface of gold electrodes. Effect of pH of phosphate-acetate buffer
solution and different concentrations of mediator in buffer solution (0.5 – 10 mM
ferricyanide) on responses of fructose biosensors were investigated. The linear range of
fructose determination was from 0.005 to 2 mM. Time of fructose concentration
measuring was 1-2 min.
Conclusions: The new fructose biosensor showed good selectivity and high signal
reproducibility. The developed fructose biosensors can be applied in medical diagnostics
in future.
Keywords: fructose intolerance, fructose dehydrogenase, biosensor.
187
Structural models of the complete HIV-1 poly(A) region
Potyahaylo A. L., Zarudnaya M. I., Kolomiets I. N., Hovorun D. М.
Institute of Molecular Biology and Genetics, NAS of Ukraine,
150, Zabolotnoho str., Kyiv, Ukraineб 03680
apotyahaylo@gmail.com
Aim: In HIV-1 retrovirus, identical core poly(A) sites are present at both the 5 and 3
end of HIV-1 pre-mRNA. The usage of a real 3 poly(A) site is promoted by upstream
sequence elements (USEs) which are uniquely present at this end of HIV-1 transcript.
Polyadenylation signal (PAS) is partly occluded by base pairing in the upper part of
polyA hairpin which can form at both ends of HIV-1 transcript. To our knowledge there
is no structural model of HIV-1 complete 3 poly(A) region including the core poly(A)
site and the USEs. Based on our observation that the minor USEs are located in a G-
quadruplex-forming segment, we studied here the secondary structure of the 3 terminal
region of HIV-1 pre-mRNA stretching from the G-rich segment to the end of core
downstream element (DSE).
Methods: We have performed secondary structure prediction and phylogenetic analysis
of poly(A) region for about 1500 HIV-1 isolates of different subtypes and recombinant
forms. Predictions of the secondary structure were performed by mfold program (Zuker
2003).
Results: Folding results are stored in our database CESSHIV-1 (abbreviated from
Control Elements Secondary Structures of HIV-1 genome) which is available online at
http://www.cesshiv1.org. Based on our phylogenetic findings, we have proposed two
common structural models of complete 3 poly(A) region of HIV-1 pre-mRNA: a domain
and an in-line structures.
Conclusions: In proposed structural models of complete 3 poly(A) region of HIV-1 pre-
mRNA both the core downstream element and the upstream sequence element in HIV-1
3′ poly(A) region are exposed in single stranded segments of hairpins or domains and are
available for protein factors of polyadenylation machinery. Information on HIV-1
poly(A) region structure may facilitate development of therapeutic strategies targeting
polyadenylation process of viral transcript.
Keywords: HIV-1 genome; RNA folding; poly(A) site
188
Serum cell-free DNA as diagnostic marker in patients with for renal
cell carcinoma (RCC)
Skrypkina I., Tsyba L., Morderer D., Nikolaienko O., 1Vozianov S.,
1Romanenko A., Rynditch A.
Institute of Molecular Biology and Genetics NASU
150 Zabolotnogo str., Kyiv, Ukraine, 03680
1Institute of Urology AMSU,
9-A Yuriy Kotsubinsky str., Kyiv, Ukraine, 04053
i.skrypkina@imbg.org.ua
Aim: The presence of tumor DNA in cell-free DNA (cfDNA) circulating in the plasma or
serum of cancer patients was first demonstrated 30 years ago. Since then, overall plasma
DNA concentration in cancer patients and genetic or epigenetic alterations specific to
tumor DNA have been investigated in patients diagnosed with different types of cancer.
The objective of this study was to evaluate the putative significance of serum cell-free
DNA for renal cell carcinoma (RCC).
Methods: In this study blood plasma samples were collected from 28 patients with
histologically confirmed RCC and 15 healthy control individuals. Determination of
plasma concentration of cfDNA was carried out using quantitative real-time PCR and
measuring fluorescence intercalation of SYBR Green I.
Results: We isolated plasma DNA from 2 mL plasma after phased
centrifugation/fractionation of blood cells to obtain serum, which does not contain DNA
fractions of leukocytes. cfDNA concentration was measured by quantitative PCR assay.
Average DNA concentrations for the healthy individuals and RCC patients were 25,4 and
58,6 ng/ml, respectively. Elevated plasma DNA levels were also detected in patients with
either stage I or II disease. All plasma DNA samples also were quantitatively assessed by
direct SYBR Green I staining in tablet analysis. DNA concentration value in RСС plasma
was a median of 254.2 ng/ml, for the control - 57.2 ng/ml (P < 0.001). The observed
concentrations of circulating DNA differ depending on the analysis methods, but the
concentration of DNA in patients is higher than in healthy individuals.
Conclusions: The current results indicated that cell-free DNA represents a serum-based
diagnostic and prognostic biomarker for RCC.
Keywords: renal cell carcinoma, cell-free DNA, diagnosis, prognosis.
This work was funded partially by „Scientific and technical (innovation) projects the
NAS of Ukraine” № 44/11-I, 2011
189
Translational Research and Drug
Development
190
PH domain of Bcr-Abl interacts with PLCε in the nucleus
Tyutyunnykova A. P., Telegeev G. D.
Institute of Molecular Biology and Genetics of NAS of Ukraine,
150, Zabolotnogo Str.,Kyiv, Ukraine, 03680
anna.tyutyunnykova@gmail.com
Bcr-Abl, the product of a chromosomal translocation t(9;22), has been demonstrated to
be a key protein responsible for the pathogenesis of Ph-positive leukemia. Three forms of
Bcr-Abl proteins have been observed: p190 Bcr-Abl, p210 Bcr-Abl, and p230 Bcr-Abl.
The only structural difference among various Bcr-Abl chimeras is the presence of Dbl
homology (DH) and pleckstrin homology (PH) domains in p210 Bcr-Abl and p230 Bcr-
Abl, and their absence in p190 Bcr-Abl. p210 Bcr-Abl and p230 Bcr-Abl are responsible for
chronic myelogenous leukemia while p190 Bcr-Abl is associated with acute lymphoid leukemia
suggesting the important role of the DH and PH domains in leukemogenesis. Our resent data
demonstrated that Bcr-Abl PH domain binds a number of proteins including
phospholipase Cε (PLCε). Also it has been shown that p210 Bcr-Abl is localized in
perinuclear area. But the fact of interaction between PH domain of Bcr-Abl and PLCε in
the living cell should be confirmed as well as the role of this interaction should be
explained.
Aim: The aim of this study is to discover the interaction between PH domain of Bcr-Abl
and full-length PLCε in the living cell.
Methods: Two expression constructs (mRFP-PLCε and GFP-PH) were used for
transfection into Cos-7 cells. The fluorescence of both proteins in living cells was
detected by confocal microscopy. Results obtained were confirmed with further Western-
blot analysis.
Results: We have found that both proteins are expressed in Cos-7 cells and are co-
localized in nucleus, while PH domain alone localizes in perinuclear area.
Conclusion: Our results suggest that PH domain of Bcr-Abl and full-length PLCε co-
localize in nucleus. This may indicate the important role of PH domain of Bcr-Abl in
intracellular localization of PLCε. Using these data we can predict other proteins
localized in perinuclear area that can be important partners in signaling pathway
involving p210 Bcr-Abl and PLCε.
Keywords: Leukemia, Bcr-Abl, phospholipase Cε.
191
UKRAINE – ICB
192
Mother and Child Health
193
Autophagy and viability of ovarian tumor cells
Shuvayeva G., Igumentseva N., 1Isidoro C., Stasyk O.
Department of Cell Signaling, Institute of Cell biology, NAS of Ukraine,
14/16 Drahomanova Str., Lviv, Ukraine, 79005
1Laboratory of Molecular Pathology and Nanobioimaging, Department of Medical Science,
Amedeo Avogadro University, Novara, Italy.
shuvayeva@cellbiol.lviv.ua
Aim: to study autophagy inhibition to enhance therapeutic effect of enzymotherapy based
on arginine (ARG) deprivation for ovarian cancer treatment.
Methods: cell viability test, MDC staining, immunofluorescence, fluorescence
microscopy, RNA interference, western blot analysis.
Results: we demonstrated that autophagic response in human ovarian carcinoma SKOV3
cells upon ARG deprivation can be inhibited by chloroquine (CQ), a non-toxic
antimalarial drug, proposed as inhibitor of autophagy in vivo. CQ decreased SKOV3 cells
viability under ARG withdrawal suggesting a prosurvival role of autophagy. We
observed that resistant to taxol SKOV3 cells are sensitive to this drug upon ARG
deprivation. Analysis of cell viability demonstrated that CQ and taxol negatively affected
SKOV3 cell viability even after ARG resupplementation. We also showed that
transcriptional silencing of autophagic protein Beclin 1 led to a decrease in cell survival
under ARG deprivation and to the decrease in their proliferative potential upon ARG
resupplementation.
Conclusions: we demonstrated that autophagy has an important prosurvival role in the
response of cultured SKOV3 cells to ARG deprivation. We propose that ARG
deprivation-based combinational treatment with CQ and taxol may produce a stronger
anticancer effect as a second line therapy for difficult to cure ovarian cancers.
Keywords: arginine deprivation, autophagy, ovarian cancer, chloroquine, taxol.
194
Nanobiotechnology and Cancer
Research
195
Effect of arginine deprivation on leukemic cells
1,2Chen O., 1Lyniv L., 1Barska M., 2Sybirna N., 1Stasyk O.
1 Institute of Cell Biology of NASU,
14/16 Drahomanova Str., Lviv, Ukraine, 79005
2 Ivan Franko Lviv National University,
4, Hrushevskyi Str., Lviv 79005, Ukraine
oleh.chen@gmail.com
Aim: to elucidate the effect of combinational arginine deprivation-based enzymotherapy
(ADBE) with exogenous nitric oxide donor, sodium nitroprusside (SNP), on proliferation
and viability of human acute lymphoblastic leukemia (ALL) cells and normal peripheral
blood lymphocytes (PBL) in vitro.
Methods: Trypan Blue Test, MTT Assay, Annexin V Staining Assay, Western Blot
Analysis, Separation and Cultivation of Blood Cells.
Results: Several human ALL cell lines and PBL were treated with recombinant human
arginase (rhARG) for 24, 48 and 72 hours. ADBE effectively decreased viability of all
tested leukemia cell lines but had not significantly effect of PBL. SNP (0.05 mM)
statistically enhanced cytotoxicity of ADBE for leukemic cells. PBL were more resistant
to this dose of SNP and SNP IC50 concentration for PBL in full and arginine-deficient
medium was not significantly different. ADBE effectively induced apoptosis in ALL
cells but not in PBL cells. NO donor sped up the apoptotic process. Arginine anabolic
precursor, citrulline (0.4 mM), supported proliferation of two leukemia cell lines (CEM-
T4 and Namalva) but not Jurkat cells and significantly decreased cytotoxicity of SNP.
Simultaneously, ADBE with or without citrulline supplementation induced expression of
arginine biosynthetic enzymes argininosuccinate synthase (ASS) in ALL cells.
Conclusions: The obtained results suggest that ADBE may have clinical potential as an
inhibitor of leukemic cells proliferation. We demonstrated for the first time that
exogenous NO-donor can be potentially used as a secondary agent under ADBE in vivo
to compensate for NO limitation as an arginine catabolite.
Keywords: arginine deprivation-based enzymotherapy (ADBE), sodium nitroprusside
(SNP), leukemic cells, normal lymphocytes (PBL).
196
Nanocomposites carrying Dx and NSE as Doxil alternative
1,2Lehka L. V., 1Panchuk R. R., 2Chumak V. V., 1Skorokhyd N. R., 3Ryabtseva A.,
3Zaichenko O. S., 1,2Stoika R. S.
1 Institute of Cell Biology NASU,
79005, Lviv, Drahomanov Str. 14/16
2 Ivan Franko Lviv National University, Biology Faculty,
79005, Lviv, Hrushevskogo Str. 4
3 Lviv National Polytechnic University,
79013, Lviv, St Yuri Square 2
lilya_lehka@mail.ru
Aim: Doxil is a clinically used PEGylated liposome with encapsulated doxorubicin (Dx).
It possesses much lower cardiotoxicity comparing to free Dx, however, it is 100 times
more expensive. A novel nanoscaled PEGylated polymeric drug carrier was synthesized
and functionalized by the biologically active lipid N-stearoylethanolamine (NSE). Here
we evaluated the efficiency of such carrier (Dx-NSE-NC) in Dx delivery to tumor cells.
The protective role of NSE at the action of that carrier with encapsulated Dx in vivo was
also predicted.
Methods: Fluorescent microscopy, flow cytometry, Western-blot analysis of
proapoptotic proteins, in vivo toxicity studies of Dx-NSE-NC, in vivo studies of treatment
efficiency of Dx-NSE-NC towards L1210 murine leukemia.
Results: Functionalizing by NSE of Dx-NC increased its antineoplastic potential by 40%
compared to non-functionalized Dx-NC. NSE functionalizing was even more effective
than PEGylation of Dx-NC. Such functionalizing of Dx-NC led to both acceleration of
Dx penetration to the nucleus of human tumor HeLa cells and more rapid accumulation
of Dx in these cells. Dx-NC-NSE also accelerated apoptosis. It caused early activation of
caspase-3 and -7, and cleavage of their substrates PARP-1, DFF45 in 6 h, while non-
functionalized Dx-NC and Dx caused such changes only in 9 h. 90% of mice receiving
lethal dose of Dx (20 mg/kg) as Dx-NSE-NC, remained alive, while all mice receiving
the same dose of Dx and Dx-NC, died. Dx-NC-NSE efficiently inhibited growth of
murine L1210 leukemia in vivo in cumulative dose 1 mg/kg that is 10 times less than
dose of free Dx (10 mg/kg) needed to achieve the same treatment effect.
Conclusions: N-stearoylethanolamine functionalizing of novel PEGylated polymeric
nanocarrier with encapsulated doxorubicin significantly enhanced the anticancer action of
this drug, and provided this carrier with protective effect towards normal cells in vivo.
Potentials of novel nanocarrier of doxorubicin as a cheaper alternative of Doxil are
considered.
Keywords: polymeric nanocarriers, doxorubicin, N-stearoylethanolamine, treatment,
apoptosis, murine L1210 leukemia.
197
Amino acid depletion: differences in 2D and 3D cultures
Vynnytska-Myronovska B., Kurlishchuk Y., Bobak Y., 1Kunz-Schughart L.,
Stasyk O.
Department of Cell Signaling, Institute of Cell Biology, NAS of Ukraine
14/16,Drahomanov Str., Lviv, Ukraine, 79005
OncoRay, National Center for Radiation Research in Oncology,
TU Dresden, Fetscherstr., 74, PO Box 41, Dresden, Germany, 01307
bozhena@litech.net
Aim: To compare the effects of arginine, leucine, lysine or methionine starvation on
human epithelial cancer cells growth, viability, proliferative potential and apoptosis
induction when grown in monolayer versus spheroid culture.
Methods: Monolayer cells were maintained under regular culture conditions. Spheroids
were obtained using a standard liquid overlay technique. Amino acid deprivation was
achieved by incubating cells in the formulated media lacking arginine, leucine, lysine or
methionine. Cell growth, viability and proliferative potential were assessed by Trypan
blue exclusion test and MTT analysis. Apoptosis induction was analyzed by Western-
blot-mediated detection of fragmented PARP and cleaved caspases.
Results & Conclusions: We revealed that in monolayer culture the effects of starvation
for individual amino acids differed, with methionine and arginine having the most severe
impact on cancer cell viability and proliferative potential. At the same time, we are first
to show that cancer cells spheroids preserve growth potential even after prolonged amino
acid starvation, despite the signs of mitochondrial and receptor-mediated caspase-
dependent apoptosis induction. This suggests that in 3D culture human cancer cells are
much more resistant to individual amino acid starvation relative to monolayer cells. The
differences in the cell capacity to preserve growth potential upon individual amino acid
starvation between monolayers and spheroids of one cell line might depend on the
disparity in hypoxia-mediated signaling pathways or in regulatory networks that control
protein translation during amino acid limitation between these types of cell cultures.
Keywords: human epithelial cancer cells, monolayer culture, spheroid culture, amino
acid starvation, apoptosis.
198
Overcoming drug resistance by new delivery platform
1,2Senkiv Yu., 2Heffeter P., 3Zaichenko O., 2Berger W., 1Stoika R.
1 Institute of Cell Biology, NAS of Ukraine,
Drahomanov Str. 14/16, 79005, Lviv, Ukraine
2 Institute for Cancer Research, Medical University of Vienna,
Borschkegasse 8A, 1090, Vienna, Austria
3 Lviv National Polytechnic University,
Bandera Str. 12, 79013, Lviv, Ukraine
yu.senkiv@gmail.com
Aim: Drug resistance is widely spread in pathogenic microorganisms and tumor cells
under chemotherapy. Development of effective remedies for drug transportation to
various target cells is of great significance. New polymeric platform was tested for
Doxorubicin (Dx) delivery to drug-resistant human tumor cells.
Methods: Intracellular accumulation of Dx, FACS analysis, cell cycle, Western-blot
analysis, DNA comet assay.
Results: Application of the developed nanocomposite (NC) significantly enhanced the
intracellular accumulation of Dx. Such treatment resulted in about 10-fold higher Dx
levels in human lung carcinoma SW1573 cells, comparing with the action of free Dx. 6-
fold increase in the intracellular accumulation of the NC-bound Dx was observed in the
Pgp-overexpressing drug-resistant 2R160 subline of these cells. We found that 3 h
treatment of tumor cells with the NC-Dx much more effectively enhanced DNA comet
tail moment of treated cells comparing with the action of free Dx. The application of such
drug delivery platform led to more pronounced stimulation of cell stress pathways such
as up-regulation of p53 and phosphorylation of mitogen-activated protein kinase p38.
These changes were accompanied by a distinct G2/M arrest of cells surviving after 24 h
NC-Dx treatment.
Conclusions: Dx delivery by means of new polymeric platform decreases up to 10 times
drug concentration necessary to achieve similar antineoplastic action of this drug in the
inhibition of cell proliferation and G2/M arrest. Such way of Dx delivery permitted
overcoming resistance of various human tumor cells to the action of this anticancer drug.
Perspectives of application of new drug delivery system in cancer chemotherapy are
considered.
Keywords: chemotherapy, doxorubicin, cancer, nanocarrier, drug accumulation.
199
Tumor cells phagocytosis under arginine starvation
Lyniv L, Tomin A, Bilyy R, Barska M, Chen O, Stasyk O.
Institute of cell biology of NASU,
Drahomanov str. 14/16, Lviv, 79005, Ukraine
liliana‐lyniv@mail.com
Arginine (ARG) deprivation-based enzymotherapy is a novel anticancer strategy for
treatment of haematological tumors. It is also known that ARG (as a component of Arg-
Gly-Asp sequences) is essential for proper function of surface proteins involved in cell
recognition/clearance by macrophages. We showed that ARG withdrawal inhibited
proliferation/viability and induced apoptosis of human leukemic cells.
Aim: to evaluate possible effect of ARG starvation on recognition/clearance of dying
leukemic cells by immune-competent cells.
Methods: MMT, in situ apoptosis detection and phagocytosis assays, lectin
cytochemistry. Jurkat cells were used as experimental model.
Results: We tested 2 distinct phagocytosis assays involving human monocytes-derived
macrophages (MoMa) and THP-1-differentiated macrophages with Arg-deprived, Lys-
deprived, intact and apoptotic Jurkat cells serving as “prey”. It was shown that 24h Arg-
deprivated cells were more efficiently phagocyted by macrophages than the intact ones,
resembling the phagocytosis of apoptotic (UV-irradiated) Jurkat cells. Lys-deprived cells
did not exhibit increased phagocytosis. Combined administration of Arg and nitrite oxide
donor sodium nitroprusside (SNP) allows maintaining phagocytic activity of
macrophages. Moreover, SNP did not promote survival of tumor cells. Interestingly,
Phagocytis Index for Arg-starved cells was higher than Apoptotic Index that was detected
by AnnexinV-FITC/PI staining. Apoptosis mediated by Arg-starvation was accompanied
by changes in cell surface carbohydrates. It was demonstrated significant reduction of
exposition of DGlcNAc-, NeuNAc-and βDGal, NeuNAc-containing glycoconjugates.
Conclusions: We report for the first time that ARG-deprivated tumor cells are
efficientlly cleared by macrophages. We propose that combination of ARG starvation
with NO donor should support phagocytic activity of macrophages under ARG starvation
in vivo.
Keywords: arginine deprivation, macrophages, phagocytosis.
200
Novel nanocarrier enhances doxorubicin action
1Shlyakhtina Ye.A., 1Boiko N.M., 1Senkiv Yu.V., 2Zaichenko O.S., 1Stoika R.S.
1 Institute of Cell Biology, NAS of Ukraine,
Drahomanov Str. 14/16, 79005, Lviv, Ukraine
2 Lviv National Polytechnic University
Bandera Str. 12, 79013, Lviv, Ukraine
lisaschliakhtina@gmail.com
Aim: Most anticancer agents cannot effectively discriminate between healthy and
malignant cells. Doxorubicin (Dox) is one of the most employed anticancer agents whose
clinical application is limited by the acute and chronic cardiotoxicity. Application of
novel drug delivery systems is an actual goal in pharmaceutics. Here we investigated the
effectiveness of action of Dox transported by novel developed nanosized polymeric
carriers whose PEGylated shell should inhibit the access of degrading proteases and
nucleases, thus, minimizing drug recognition and elimination by blood cells.
Methods: cell toxicity assay, DNA laddering assay, Western-blot analysis, fluorescence
microscopy and experimental tumor models in vivo.
Results: The results of in vitro study showed that the application of carriers with
immobilized doxorubicin (OC-Dox) reduced proliferation of L1210, Jurkat, MCF-7 and
L929 cell lines approximately 10 times more intensively than free Dox. Fragmentation of
DNA in cancer cells under the influence of OC-Dox was expressed much better than in
cells treated with free Dox used in same concentration. OC-Dox also caused activation of
the effector caspase-3 and cleavage of its substrate PARP-1 much faster than free Dox
did. Due to such properties, OC-Dox induced apoptotic cell death in much lower
concentrations comparing with free Dox. High efficiency of the nanoscale drug delivery
system has been confirmed in vivo. OC-Dox possessed a capability of inhibiting growth
of NK/Ly lymphoma without causing general toxicity towards the organism.
Conclusions: Application of new polymeric nanocarriers for drug delivery allows a
significant reduction of current dose while maintaining antitumor effect, which can
provide significant reduction of adverse effects of tumor chemotherapy.
Keywords: nanocarriers, drug delivery, cancer chemotherapy, doxorubicin, apoptosis.
201
Translational Research and Drug
Development
202
Canavanine: a new look at an old drug
Kurlishchuk Y., Vynnytska-Myronovska B., Bobak Y., Stasyk O.
Institute of Cell Biology, NASU;
14/16, Drahomanov Str., Lviv, Ukraine, 79005
kurlishchukyuliya@gmail.com
Arginine analogue of plant origin canavanine (Cav) exhibits antitumor activity in vitro
and in vivo, but has high overall toxicity. Our recent data suggest that malignant cells
become selectively more sensitive to low dose Cav treatment upon arginine deprivation
in comparison with pseudonormal cells. However, the effect of arginine biosynthetic
precursor citrulline (Cit) on Cav toxicity for cancer cells remained to be evaluated.
Aim: to investigate the effect of Cit supplementation on cancer cell sensitivity to Cav
treatment under arginine-deprived conditions.
Methods: MTT assay, reverse transcription PCR and Western-blot analyses. Human
epithelial colorectal cancer cell lines (HCT-116, HT-29) were utilized as models.
Results: Cit addition to arginine-free medium partly restored growth of tested cell lines.
Remarkably, 0.1 mM Cav inhibited proliferation of malignant cells under such
circumstances and promoted cell death as efficiently as upon arginine withdrawal without
Cit. We revealed that inability of colorectal cancer cells to utilize Cit for arginine
resynthesis under Cav treatment resulted from to the decrease in the expression of
argininosuccinate synthetase (ASS), the key enzyme of arginine synthesis. Stepwise
application of tested impacts (incubation in arginine-free medium for 24 h before Cav
supplementation) increased cancer cell sensitivity to Cav relative to simultaneous
arginine starvation and Cav treatment. However, supplementation of arginine free
medium with Cit 24 h before Cav treatment was sufficient to induce ASS expression and
partly protect the cells from Cav toxicity.
Conclusion: Canavanine application in arginine deprivation-based anticancer therapy
overcomes one of the significant drawbacks of such treatment in vivo, namely, the
induction of ASS expression resulting in decreased tumor sensitivity to starvation. The
mode of Cav application as a component of combinational therapy has to be further
elucidated.
Kywords: arginine deprivation, canavanine, colorectal cancer.
203
Anticancer effects of novel 4-thiazolidone derivatives
1Krupak V.I., 1Hrydzuk O.S., 1Filyak Ye.Z., 2Kaminskyy D.V., 2Lesyk R.B. and
1Stoika R.S.
1Institute of Cell Biology, NAS of Ukraine,
14/16, Drahomanov St., Lviv, Ukraine, 79005
2Danylo Halytskyy Lviv National Medical University,
69, Pekarska St., Lviv, Ukraine, 79010
krupak.icb@gmail.com
Aim: Synthetic compounds based on 4-thiazolidone ring are very perspective in cancer
chemotherapy. We used three structurally related compounds (Les-28, Les-236, Les-
3183) that were synthesized at Danylo Halytskyy Lviv National Medical University.
Under Drug Discovery and Development Program at National Cancer Institute (USA) it
was shown by that they possess potential antineoplastic activity with highest effect
towards human breast cancer cell lines. In our study, we have examined the anticancer
action of these compounds more precisely.
Methods: MTS-cell viability assay, soft agar assay, wound healing assay, fluorescent
microscopy, targeting experimental tumor in vivo and study of histological sections after
hematoxylin and eosin staining.
Results: We found that 4-thiazolidone derivatives Les-28, Les-236 and Les-3183
suppressed proliferation of human breast carcinoma cells in vitro: MCF-7 cell line
(IC50~0.1 uM) and MDA-MB-231 and 4T1 cell lines (IC50~20 and 25 uM,
respectively). Moreover, these compounds reduced substrate-independent cell growth and
cell migration in vitro, and also induced apoptosis.
In spite of very low toxicity in vivo (8 injections to each of 6 Balb/c mice causing a
cumulative LD50>800 mg/kg), all tested compounds in dose 20 mg/kg caused tumor
growth inhibiting effect in model tumor (4T1 mice breast adenocarcinoma). Les-3183
was the most active towards this tumor. Analysis of histological sections revealed
necrotic lesions in control tumor, as well as in tumor under treatment with Doxorubicin
(1 mg/kg) or Les-3183 (20 mg/kg). However, Les-3183 induced strong immune response
and inhibition of tumor neovascularisation.
Conclusions: 4-thiazolidone based compounds under study caused various anticancer
effects (inhibiting of cell proliferation and migration, inducing of apoptosis, inhibiting of
tumor growth) observed at low general toxicity in vivo. Moreover, one of them
stimulated immune response and inhibited neovascularization.
Keywords: 4-thiazolidone, anticancer treatment, experimental tumors.
204
Overexpression of (His)6-tagged human arginase I in Saccharomyces
cerevisiae and purification of the enzyme using nickel affinity
chromatography
1Romanova M., 1Zakalskiy A., 1Zakalska O., 1,2Gonchar M.
1Institute of Cell Biology, NAS of Ukraine,
14/16, Drahomanov St., Lviv, Ukraine, 79005
2 University of Rzeszow, Rzeszow, Poland
rom‐ma@ukr.net
Aim: Some of actively proliferating cancer cells (e.g. many hepatocarcinomas,
melanomas, carcinomas, including well-known cervical carcinoma HeLa, as well as some
types of leukemia) are strongly dependent on exogenous arginine supply. Arginine
deficiency causes antiproliferative effect in vitro and in vivo and often leads to the death
of such cancer cells whereas normal cells move into a quiescent phase. For the treatment of
some cancers, it is promising the use of specific enzymes that catalyze degradation of arginine.
Arginase I (EC 3.5.3.1; L-arginine amidinohydrolase) is the main enzyme of the urea
cycle that catalyses the hydrolysis of arginine to urea and ornithine.
Methods: The recombinant strain Saccharomyces cerevisiae capable of overproducing
human liver arginase was constructed. The corresponding gene HsARG1 was cloned
using efficient copper inducible expression system. The active enzyme was accumulated
in S. cerevisiae cells to the level of approx. 15-30 µmol· min−1·mg−1 protein. The highest
expression was obtained after 6-8 h of induction.
Results: The (His)6-tagged enzyme was purified from the cell-free extract of the
recombinant strain by metal-affinity chromatography on Ni-NTA-agarose or
monodisperse Ni-IDA-modified poly(glycidylmethacrylate-co-ethylene dimethylacrylate)
microspheres. The specific activity of the enzyme was increased more than 70-fold up to
2000 µmol·min−1·mg−1 protein. The yield of the enzyme after Ni-affinity chromatography
was approximately 80%. The obtained enzyme preparations are almost homogeneous.
Conclusions: For the first time, (His)6-tagged form of human arginase I was
overexpressed in S. cerevisiae cells under the control of CUP1 promotor. It was shown
that (His)6-tag bound to the N-terminus does not disturb the arginase enzymatic activity,
but allows easy purification procedure by Nickel-chelating chromatography on Ni-NTA-
agarose or newly synthesized monodisperse Ni-IDA-modified
poly(glycidylmethacrylate-co-ethylene dimethylacrylate) (PGMA) microspheres.
Keywords: Arginase, cancer, chromatography.
205
UKRAINE – PALLADIN
206
Women’s Health and Cardiovascular
Diseases
207
Synthetic peptides 69-78 and A195-205 are specific inhibitors of
fibrin polymerization and potential antithrombotic agents
1Pozniak T. A., 1Pydiura M. O., 1Urvant L. P., 2Andreev S. M., 1Lugovskoy E. V.,
1Komisarenko S. V.
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine,
2Institute of immunology of FMBA of Russian Federation (Moscow),
Tanya_kow@list.ru
Aim: localization of a new fibrin functional site using synthesized peptides as inhibitors
fibrin polymerization
Methods: Turbidity analysis and transmission electron microscopy were used to study
the effect of synthesized peptides on fibrin polymerization, fibrinogen was obtain from
human blood plasma by salting out with Na2SO4, human fibrin was obtained by
dissolving in 20 mM acetic acid.
Results: The peptides imitating the amino acide sequences of the fibrin molecule:
NPDESSKPN(69-78), BQPDSSVKPY(228-236), BRPFFPQ(455-460),
ALPSRDRQHLPL(195-205) were synthesized and their effects on the process of fibrin
polymerization in fibrinogen+thrombin, fibrin desA, fibrin desAB solution were
investigated. Turbidity data showed that synthesized peptides A195-205 and 69-78, but
not B228-236 B455-460 retarded fibrin polymerization. IC50 value for A195-205 and
69-78 were 2.44·10-4 M and 1.5·10-5 M, respectively. Electron microscopy data show
that peptide A195-205 inhibits the stage of fibrin protofibril formation and peptide 69-
78 – the step of protofibril lateral association.
Conclusions: The new active sites were localized in the coiled-coil fragment of the fibrin
molecule: site 69-78, and site A195-205 which are suggested to be important for the
preservation of native structure of fibrin molecule to realize early stages of fibrin
polymerization. The fibrin fragment 69-78 takes part in fibrin protofibril lateral
association. The spatial localization of the fragment A195-205 in the fibrin(ogen)
molecule suggest that this site may be considered as a base for the binding of C-
connector portion of C-region to the bulk of the fibrin molecule namely: with the coil-
coiled region.
Therefore, the synthesized peptides A195-205 and 69-78 may be used for the
development of antithrombotic drugs.
Keywords: fibrinogen, fibrin, polymerization, inhibition, synthesized peptides
208
Mother and Child Health
209
Vitamin D3 and mineral metabolism in pregnansy associated with
pathology and during neonatal period
Labudzynskyi D. O., Shymanskyy I. O., Riasniy V. M., Apukhovska L. I.,
Veliky M. M.
Laboratory of Medical Biochemistry, Palladin Institute of Biochemistry of NAS,
9 Leontovich St., 01601 Kyiv, Ukraine
konsument3@gmail.com
Aim: To establish the character of changes in the vitamin D3 and mineral metabolism in
newborn infants related to the maternal D3 levels under different pathologies that
accompany pregnancy.
Methods: 37 pregnant women with preexisting Type 1 diabetes (12.4±7.5 yrs), 30
women with the late toxemia of pregnancy, 35 pregnant women with hypofunction of
parathyroid glands and 15 controls with no pathology during pregnancy, well matched for
age, were enrolled in the study. Blood serum 25(OH)D3 levels (ELISA) and markers of
mineral metabolism were examined between 18th and 24th gestational weeks in all women
and in their newborn infants at 14th and 21st days after delivery.
Results: The blood serum content of 25 (OH) D3 decreased to 28.7 ± 1.2 nM/L in
pregnant women with diabetes vs. 65.0 ± 6.4 nM/L in controls. Diabetes caused
impairments of mineral metabolism as it is evident from the reduction of serum calcium
(Ca2+) and phosphorus (Pi) to 1.76 ± 0.10 and 1.40 ± 0.10 respectively vs. 2.21 ± 0.02
and 1.85 ± 0.09 mM/L in control. These changes in pregnant women suffering from
diabetes led to vitamin D3 deficits and altered mineral metabolism in newborns. They
exhibited reduction of 25(OH)D3 by 25 % compared with infants from healthy mothers.
The levels of Ca2+ and Pi were shown to be decreased by 34 and 33% respectively. At the
same time, the activity of alkaline phosphatase was upregulated in serum by 79 %.
Vitamin D3 availability and the indices of mineral metabolism were partially normalized
in newborns from diabetic mothers that were given D3 at a dose of 2000 IU/day. Similar
changes of 25(OH)D3, Ca2+
, Pi and alkaline phosphatase activity were observed in
newborns from women with toxemia and hypoparathyroidism.
Conclusions: Thus, deficiency of vitaminD3 in pathologies that accompany pregnancy
results in the reduction of D3 content and in impairment of mineral metabolism in
newborns. The alterations observed can be eliminated by treatment with adequate doses
of vitamin D3.
Keywords: vitamin D3, pregnancy, diabetes, toxemia, hypoparathyroidism
210
Effect of parental smoking conditions on the Tlr2 and Tlr4 gene
expression in children’s nasal and oral cavity epithelial cells
1,2Minchenko D. O., 2Vankhanova T.O., 1Danilovskyi S.V., 1Minchenko O.H.
1Palladin Institute of Biochemistry,
9 Leontovycha St., Kyiv 01601, Ukraine;
2Bogomolets National Medical University,
13 Taras Shevchenko Bulv., Kyiv, 01601 Ukraine
xanrok@yahoo.com
Aim: The aim of this study was to investigate the expression level of human Toll-like
receptors (TLR), structurally related to Drosophila Toll which play a fundamental role in
pathogen recognition, activation of innate immunity and mediate the production of
cytokines necessary for the development of effective immunity, in nasal and oral cavity
epithelial cells from children of smoking and non smoking parents.
Methods: The nasal and oral cavity epithelial cells from children of smoking and non-
smoking parents were used for RNA extraction. Complementary DNA was synthesized
from these RNAs and used for analysis of Toll-like receptors (TLR2 and TLR4) mRNA
expression by quantitative polymerase chain reaction. Beta-actin mRNA expression was
used as control of RNA quantity.
Results: We showed that expression level of TLR2 and TLR4 transcripts was much
higher in nasal cavity epithelial cells as compared to oral cavity cells. Moreover, the
expression level of TLR2 mRNA is significantly reduced in both nasal and oral cavity
epithelial cells from children of smoking parents as compared to children of non-smoking
parents. However, the TLR4 transcript level strongly reduces preferentially in nasal
cavity epithelial cells from children of smoking parents.
Conclusions: Thus, our results showed that children of smoking parents showed
dramatically reduced Toll-like receptor-2 transcript levels both in nasal and oral cavity
epithelial cells and reduced TLR4 transcript predominantly in the nasal cavity cells as
compared to children of non-smoking parents, which could be responsible for the
suppression of pathogen recognition ability, activation of innate immunity, and
production of cytokines necessary for the development of an effective immune response.
Keywords: Tlr2, Tlr4, nasal cavity, epithelial cells, children, perinatal smoking
211
Nanobiotechnology and Cancer
Research
212
ERN1 signalling system as a new target for anticancer therapy
Danilovskyi S.V., Minchenko D.O., Karbovskyi L.L., Kharkova A.P.,
Minchenko O.H.
Palladin Institute of Biochemistry,
9 Leontovycha St., Kyiv 01601, Ukraine
sergius03@gmail.com
Aim: The aim of this study is to investigate the expression level of retinoblastoma related
genes contribution to endoplasmic reticulum–nuclei-1 (ERN1)-mediated glioma
proliferation for identification possible target sites needed for new anti-cancer therapeutic
strategies.
Methods: Expression of retinoblastoma (RB1), RB-like-1 (RBL1) and different
retinoblastoma related genes (RBAP48, RBAP46, RNF40, CTIP, KDM5A, JARID1B,
EID1, E2F1, and E2F3) was studied in glioma cells with ERN1 loss of function by qPCR
under hypoxia and glutamine or glucose deprivation (16 hrs) using qPCR.
Results: The blockade of the ERN1 enzyme function increases the expression levels of
retinoblastoma, retinoblastoma-like 1 and most retinoblastoma related genes: EID1,
JARID1B, E2F1, E2F3, RBAP48, and CTIP, does not change RNF40 and RBAP46 and
decreases KDM5A. Moreover, hypoxia reduces the expression levels of RB1, EID1, and
E2F1 in glioma cells with ERN1loss of function only. At the same time, the expression
levels of RBL1, E2F3, RBAP46, RBAP48, and CTIP decrease in hypoxia, while
JARID1B and RBBP2 increase in both types of cells, but much stronger in cells with
suppressed function of ERN1. The expression level of JARID1B and KDM5A mRNA is
also enhanced in glutamine deprivation condition, but this effect is amplified by the
blockade of the ERN1 enzyme function. The expression levels of RB1, EID1, RBAP48,
and E2F3 are decreased in glutamine deprivation condition only in ERN1-deficient
glioma cells, but RBL1, CTIP, RBAP46, and E2F1 decrease in both tested cell types with
more significant effect in ERN1-deficient cells.
Conclusions: Thus, blockade the ERN1 signalling enzyme contributes significantly to
the suppression of glioma growth via overexpression of RBL1 and retinoblastoma related
genes EID1, JARID1B, E2F1, E2F3, RBAP48, and CTIP that could be used as new the
targets for anticancer therapy.
Keywords: RB1, RBL1, EID1, JARID1B, E2F1, E2F3, RBAP48, CTIP, ERN1, glioma
cells
213
Magnetic nanoparticles for cardiovascular – and
neuronanotechnology
Каsatkina L., Krisanova N., 1Dudchenko N., 1Brik O., Sivko R., Borisov A.,
Chunihin O., Borisova Т.
Department of Neurochemistry, Palladin Institute of Biochemistry, NAS of Ukraine;
9 Leontovicha Street, Kiev, 01601, Ukraine
Semenenko Institute of geochemistry, mineralogy and ore formation NAS of Ukraine
ludmilka.kasatkina@gmail.com
Aim: Synthesized superparamagnetic nanoparticles covered with certain types of
polymers and native protein complex with magnetic nanoparticles are a very perspective
for usage in nanoneurotechnology. The aim of this work was to study interaction of
polymer-covered nanoparticles of magnetite with brain nerve terminals and blood
platelets as a potent selective tool for drug delivery, contrast agent in magnetic resonance
imaging, selective/local hyperthermia enhancement therapy of brain cancer and
manipulation of platelet aggregates in circulatory system.
Methods: Spectrofluorimetry with potential-sensitive and pH-sensitive fluorescent dyes,
flow cytometry, radiolabeled assay (L-[14C]glutamate), photon correlation spectroscopy.
Results: Using photon correlation spectroscopy, binding of synthesized nanoparticles of
magnetite covered with dextran, hydroxyethyl starch, oxidized hydroxyethyl starch, and
chitosan with nerve terminals and platelets was demonstrated. We showed that polymer-
covered nanoparticles did not influence the potential of the plasma membrane of nerve
terminals and platelets. Acidification of synaptic vesicles of nerve terminals and
secretory granules of platelets did not change in the presence of nanoparticles. Also,
synthesized nanoparticles did not influence glutamate transport in nerve terminals and
platelets. In contrast, native protein complex with magnetic nanoparticles, ferritin, in not
indifferent for functional state of nerve terminals and platelets significantly altering
glutamate transport.
Conclusions: On the base of the experimental data on binding of synthesized
nanoparticles with nerve terminals and platelets, certain types of covering polymers were
selected for manipulation with externally applied magnetic field. It was concluded that
synthetic nanoparticles of magnetite covered with definite polymer and native protein
complex with magnetic nanoparticles are a perspective instrument in
nanoneurotechnology for selective drug delivery and for hyperthermia therapy of brain
cancer by externally applied magnetic field.
Keywords: polymer-covered magnetic nanoparticles, ferritin, synaptosomes, platelets
214
Translational Research and Drug
Development
215
Anticoagulant effects of Echis multisquamatis venom fibrinogenase
1,2Chernyshenko V. O., 1Rebriev A. V., 3Mikhalovska L. I., 2Maksymovych I. S.
1. Protein Structure and Functions Dept., Palladin Int. of Biochemistry,
Leontovych Str., 9, Kyiv-01601, Ukraine
2. Educational and Scientific Centre «Institute of Biology», National Taras Shevchenko University,
64, Volodymyrska Str, Kyiv, Ukraine
3. The School of Pharmacy and Biomolecular Sciences, Brighton University, UK
bio.cherv@gmail.com
The aim of our work are purification and characterization of the fibrinogenase from
crude venom, study of its effects on human blood plasma and purified fibrinogen
coagulability, influence on fibrinolysis and on platelets activation and aggregation.
Methods: Q-sepharose and Heparine-agarose chromatography and Laemmli protein
electrophoresis were used. MALDI-TOF spectrometry was done on Voyager DE PRO.
Inhibitory assay using chromogenic substrate S2238 was conducted on Multiscan EX. N-
terminal analysis was carried out using ABI Procise 494HT Protein Sequencer (for
cleavage site determination). Aggregation of washed platelets was studied on
aggregometer Solar-AP2110, shape and granulation of platelets on flow cytometer
COULTER EPICS XL, fibrin polymerization – using turbidimetry on spectrophotometer
SF-2000.
Results: A new serine protease with fibrinogenolytic activity from the venom of E.
multisquamatis was purified. The target of fibrinogenolysis was ВβR42-A43 peptide
bond. 50% of truncated desBβ(1-42)2 form in fibrinogen solution decrease
polymerization speed 1.8-fold. Fibrinolysis of clot formed with truncated fibrinogen was
2.1-fold faster then the control one. Fibrinogenase and truncated fibrinogen had no
influence on platelets shape, granulation or the rate and level of platelets aggregation.
However, interaction of platelets in the presence of desBβ(1-42)2 fibrinogen is weaker
and is followed with disaggregation. The enzyme has no obvious hemorrhagic effects and
doesn’t affect plasminogen, prothrombin, protein C and factor X.
Conclusions: By its characteristics, E. multisquamatis venom fibrinogenase is a
prospective fibrinogen-depletive agent.
Keywords: Fibrinogen depletion, fibrinogenase, fibrinogen
216
Protective effect of precursors of ubiquinone biosynthesis on
mitochondria under effect of doxorubicin
Kuchmenko O., 1Burlaka A., Petukhov D., Donchenko G.
Palladin Institute of Biochemistry of NAS of Ukraine,
9 Leontovicha str, 01601, Kyiv, Ukraine
1Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NAS of Ukraine,
45 Vasyl’kivs’ka str., 03022, Kyiv, Ukraine
kuchmeb@yahoo.com
Doxorubicin (D) plays a major role in cancer chemotherapy. Its use has been hampered
by adverse effects. Ubiquinone (CoQ) is an important electron and proton carrier and
antioxidant. Its biosynthesis may be inhibited under number of pathologies.
Aim: The aim was to study the state of mitochondrial electron transport chain (ETC)
components, CoQ content and redox state, superoxide anion radicals and NO production
rates, and matrix metalloproteinase-2 and -9 activities in rat liver and heart tissues under
treatment with D, CoQ10 medical, and complex preparation of modulator and precursors
of CoQ biosynthesis.
Methods: Doxorubicin was administered intraperitoneally in dose of 2.2 mg/kg daily for
8 days. Experimental rats in addition to Doxorubicin received per os α-tocopherol
acetate, 4-hydroxybenzoic acid, and methionine (EPM complex) and CoQ10.
Results: Treatment with EPM complex and CoQ10 in addition to Doxorubicin
administration exerts a protective effect on liver and heart cells’ mitochondria, evidenced
by restoration of electron transport in ETC, which is expressed as decreased nitrile
complexes formation with Fe-S-proteins and increased ubisemiquinone content. It should
be stressed that the protective effects of EPM complex on mitochondrial ETC under
Doxorubicin administration is on par with those of CoQ10. Concurrently, matrix
metalloproteinase-2 and -9 activities are decreased, which gives evidence of lessened
extracellular matrix destruction. CoQ10 appeared to be more efficient at this if compared
to EPM complex.
Coclusions: The expiremental data obtained may become the basis of development of
approaches to correction of adverse effects of Doxorubicin. These data may be used to
substantiate the application of these biologically active substances within frameworks of
complex treatment of oncological pathologies.
Key words: coenzyme Q, doxorubicin, mitochondria
217
On inhibition of fibrin polymerization by Вβ121-138 peptide
Storozhylova N.S., Urvant L.P., Bereznytsky G.K., 1Ushenin Yu.V.,
Kolesnikova I.M., Makogonenko E.M., Lugovskoi E.V.
O.V. Palladin Institute of Biochemistry NASU, 9 Leontovich Str., Kyiv 01601, Ukraine;
1V.E. Lashkaryov Institute of Semiconductor Physics, 41 Nauki Av., Kyiv 03028, Ukraine;
nstorozhylova@gmail.com
Aim: Previously site of lateral association of protofibril (SLA) in Вβ118-138 fragment of
fibrin has been identified. Synthetic Вβ121-138 peptide inhibited the fibrin
polymerization. For clarifying the inhibition mechanism we investigated the location of
peptide binding sites onto fibrin molecule.
Methods: The interaction between Bβ121-138 peptide and mAb I-3с, fibrinogen (Fg),
fibrin desA (fb) and desAB (f0) was investigated by SPR method using Plasmon-6
(produced by Institute of Semiconductor Physics, NASU).
Results: It was found that Bβ121-138 bound to mAbs I-3с, immobilized to the chip, with
KD = 1,6 mM. The control peptid AαP195-L205 did not react with mAb I-3с. The
binding of Bβ121-138 peptide with Fg, fb and f0 was investigated using Fg-specific
mAb II-4d immobilized to the chip. Fg immobilized to mAb II-4d was step-by-step
transformed to fb, then into f0 using batroxobin and thrombin, respectively, and binding of
the peptide to each of these units was measured. Bβ121-138 peptide bound with the same
level to Fg and both forms of fibrin. Comparison of peptide binding to fb and desA Х
fragment of Fg, deprived of αC-regions, showed that value of peptide binding with X
fragment was 5.8 times grater than with fb.
Time, s
0 200 400 600 800 1000 1200 1400
R
es
po
n
se
, a
rc
s
0
50
100
150
200
250
Buffer
1 mM
0,5 mM
A195-205
Fig. Binding of Bβ121-138 peptide with mAb I-3с immobilized to the chip
Conclusions: 1) The conformation of Bβ121-138 peptide in solution is identical to that in
Bβ-chain of fibrin molecule. 2) The binding site(s) of Вβ121-138 peptide, probably, are
located within a coiled-coil region of molecule. 3) The ability of the peptide inhibits the
fibrin polymerization and binds to the coiled-coil region of molecule indicate its
important role in the process of polymerization at the stage of lateral association of
protofibrils.
Keywords: fibrinogen, lateral association, Вβ121-138 peptide, SPR
218
RECOOP Visegrad Scholarship Program
General Information:
Visegrad Scholarship http://visegradfund.org/scholarships
Visegrad Scholarship Program (VSP)
The International Visegrad Fund offers Master’s and Post‐Master’s scholarships awarded to
selected scholars for periods of 1 or 2 semesters (with the exception of Master’s scholarships
within the In‐Coming scheme where 1– to 4‐semester scholarships can be awarded). Given the
annual budget of €1,448,000, the Fund will award about 400 semesters in total in the academic
year 2011/2012 (more information).
Applicants whose current (i.e. at the time of applying) university or employer is further than 1,500 km
from the selected host university/institute are eligible for a one‐time travel grant (see the Instructions).
Citizens of the following countries can apply:
Albania (AL), Armenia (AM), Azerbaijan (AZ), Belarus (BY), Bosnia and Herzegovina (BA),
Croatia (HR), the Czech Republic (CZ), Georgia (GE), Hungary (HU), Macedonia (MK), Moldova
(MD), Montenegro (ME), Poland (PL), the Russian Federation (RU), Serbia (RS), Slovakia (SK)
and Ukraine (UA). The same rules are applicable to Kosovar scholars.
The scholarship program is not applicable to, nor does it cover full studies within joint programs (i.e.
double– or multiple‐degree). The scholarship does not cover studies/research at other institutions or in
other countries than those specified in the application form. The scholars are expected to work on their
study/research projects at the host university/institution for the entire scholarship period. All applicants
must have finished at least 4 semesters of university at the time of applying.
The following scholarship schemes are available:
Intra‐Visegrad Scholarships
In‐Coming Scholarships
Out‐Going Scholarships
Scholarship Program for Belarusian Students
Scholarship Program for Ukrainian Students
Visegrad Scholarships at OSA Archivum (separate program)
If selected each scholar receives the scholarship funding at the beginning of each five‐month period
(semester) upon a written confirmation from the host university/institution
Deadline for all scholarship applications is 31 January. Results are announced by mid‐May.
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