Translational Research and Nano – Biotechnology Oral Presentation
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irk-123456789-1565052019-06-19T01:29:35Z Translational Research and Nano – Biotechnology Oral Presentation Abstracts for RECOOP Research Networks' Scientific Review & Multidisciplinary Conference April 8-9 2011 Article Translational Research and Nano – Biotechnology Oral Presentation // Вiopolymers and Cell. — 2011. — Т. 27, № 2, доп. — С. 121-147. — англ. 0233-7657 http://dspace.nbuv.gov.ua/handle/123456789/156505 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Abstracts for RECOOP Research Networks' Scientific Review & Multidisciplinary Conference April 8-9 Abstracts for RECOOP Research Networks' Scientific Review & Multidisciplinary Conference April 8-9 |
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Abstracts for RECOOP Research Networks' Scientific Review & Multidisciplinary Conference April 8-9 Abstracts for RECOOP Research Networks' Scientific Review & Multidisciplinary Conference April 8-9 Translational Research and Nano – Biotechnology Oral Presentation Вiopolymers and Cell |
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Інститут молекулярної біології і генетики НАН України |
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Abstracts for RECOOP Research Networks' Scientific Review & Multidisciplinary Conference April 8-9 |
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Translational Research and Nano –
Biotechnology Oral Presentation // Вiopolymers and Cell. — 2011. — Т. 27, № 2, доп. — С. 121-147. — англ. |
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Вiopolymers and Cell |
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121
Translational Research and Nano –
Biotechnology Oral Presentation
Senior Scientists’ Forum
122
Quantum dot multiplexing technology for cancer diagnosis and
prognosis
1Zhau Haiyen E., 1Hu Peizhen, 2Zhu Guodong, 1Wang Ruoxiang, 3Berel Dror,
3Rogatko Andre, 4Luthringer Daniel, 5Wang Yuzhuo, and 1Chung Leland W. K.
1Uro-Oncology Research Program,
2Department of Urology, School of Medicine, Xi’an Jiaotong University, Xi’an, China;
3Biostatistics & Bioinformatics, Department of Medicine;
4Department of Pathology, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical
Center; Los Angeles, CA, USA;
5BC Cancer Agency, University of British Columbia, Vancouver, B.C., Canada
Haiyen.Zhau@cshs.org
Background: The standard practice in pathology for cancer diagnosis and detection has
been limited to conventional immunohistochemistry (IHC) color imaging coupled with
pathological evaluation of tissue or cell specimens. IHC true color imaging cannot detect
multiple biomarkers at a time on the same specimen due to the inability to un-mix the
signals. A more dynamic detection technology is needed.
Objectives: To develop a multiplexed quantum dot-IHC (mQD-IHC) protocol using QD
light-emitting nanoparticles with composition-dependent tunable emission from visible to
near infrared to expand the pathology from a primarily diagnostic and prognostic based
discipline to the capability of predicting the lethal progression of cancer. Specific aims
are: 1) detect the expression/activation of critical cell signaling proteins at the single cell
level; 2) image the plasticity of human prostate cancer (PCa), such as the ability to
undergo epithelial to mesenchymal transition; and 3) examine the utility of this
technology in clinical PCa specimens to determine its ability to invade and to predict the
metastatic capability of human PCa.
Results: The co-expression or activation of β2-Microlgobulin (β2-M), phosphorylated
cyclic AMP responsive element binding protein (pCREB) and androgen receptor (AR),
assigned as a triple-positive, was examined in 10 PCa tissue specimens from patients
with known metastatic status. The overall median % triple positive for β2-
M+/pCREB+/AR+ cells was 51.5%. When stratified by metastasis, there is a significant
difference in the % triple positive for the samples with metastatic potential (median =
61%) vs. those without metastatic potential (median = 0%); p=0.01 by a Wilcoxon rank
sum test. The results were confirmed in 11 PCa bone metastatic specimens.
Conclusion: Additional tissue specimens with confirmed survival data will be analyzed
and the cell-signaling-network-based mQD-IHC will be automated by Vectra System in a
high throughput manner. By defining the expression/activation of the β2-M/p-CREB/AR
and other relevant cancer progression-associated signaling components using the new
mQD-IHC detection and image analysis protocol, one could expand the pathology from a
primarily diagnostic- to prognostic-based discipline, capable of predicting the lethal
progression of PCa prior to clinical manifestation of distant metastases.
123
Persistence of viral RNA in the brain, hearts and pancreas of
experimentally infected mice with coxsackieviruses
1Bopegamage Shubhada, 1Motusova Jana, 1Stipalova Darina, 1Sojka Martin,
1Badurova Miriama, 1Borsanyiova Maria, 1Marosova Lenka, and 2Sobotova
Zdenka
1Department of Virology, Slovak Medical University,
Limbová 12, 83303 Bratislava, Slovak Republic
2National Reference Centres, Public Health Office of the Slovak Republic,
Trnavska 52, 82645 Bratislava, Slovak Republic
shubhada.bopegamage@szu.sk
The aim of our study was to follow the persistence of viral RNA in selected organs of
experimentally infected with coxsackievirus (CV) B strains from different sources such
as a patient’s sample, an environmental sample and prototype virus strains.
Methods. CD-1 mice were infected with CVB4 diabetogenic strain E2, the prototype
strain of CVB5, isolates from treated sewage waste and isolates from patient´s stool
sample or CSF identified either as CVB4 or CVB5. The viral RNA was detected by RT-
PCR using enterovirus primers specific for the non-coding 5’ region.
Results. We observed presence of RNA in the brain and heart of mice infected with
isolates from patient´s stool or CSF at day 45 post infection (p.i.).
Conclusion. We conclude that CVB4 and CVB5 persist in the brain and heart after oral
infection of CD1 mice. The relevance of viral persistence maybe related viral origin and
the genetics.
Acknowledgements: This work was supported by the Norwegian Financial Mechanism,
Mechanism EEA and Slovak Government and the State Budget of the Slovak Republic (SK
0082)
124
Translational Research and Nano –
Biotechnology Oral Presentation
Young Scientists’ Forum
125
Comparative characteristics action of COX-2 selective inhibition
and COX/LOX dual blockage on ulcerogenic processes in gastric
mucosa of rats
Fomenko I. S., Sklyarov A. Ya., Bondarchuk T. I., Lesyk R. B.
Danylo Halytskyi National Medical University, Lviv, Ukraine
69, Pekarska St., Lviv, Ukraine, 79010
dr_r_lesyk@org.lviv.net
Compounds that combine COX and LOX inhibition are potential new drugs to treat the
inflammation. They act by inhibiting of both PGs and LTs formation, which are involved
in the pathogenesis of gastric ulcer disease. Such combined inhibition avoids some of the
disadvantages of selective COX-inhibitors. The aim of the research was to compare
changes of NO-synthase system and oxidative stress indexes in gastric mucosa of rats
under application of COX-2 inhibitor celecoxib and preparations possessing dual
COX/LOX inhibition darbufelon and 4-{2,5-Dioxo-3-[4-oxo-5-(3-phenyl-allylidene)-2-
thioxothiazo-lidin-3-yl]-pyrrolidin-1-yl}-benzene-sulfonamide on the background of
gastric ulcer induced by the injection of adrenalin. Ulcerative lesions in gastric mucosa of
rats where modeled by intraperitoneal injection of adrenalin (2 mg/kg). Selective COX-2
inhibitor celecoxib, COX-2/5-LOX inhibitor darbufelon and COX/LOX inhibitor 4-{2,5-
Dioxo-3-[4-oxo-5-(3-phenyl-allylidene)-2-thioxothiazo-lidin-3-yl]-pyrrolidin-1-yl}-
benzene-sulfonamide were introduced in a single dose 10 mg/kg (per oz.) on the
background of adrenalin injection. Lipoperoxidation processes were evaluated by MDA
content, activity of enzymes of the antioxidant protection system was evaluated on basis
of determination of SOD and catalase, Griess reagent was used to measure the content of
NO. NOS activity was measured by method of Sumbaev.
Under conditions of modeled gastric ulcer, were observed destructive damage of the
gastric mucosa, activation of NO-synthases, predominantly iNOS (more than 10 times),
accompanied by increase of NO content by 60%, enhancement of lipoperoxidation
processes in the gastric mucosa. Celecoxib owing to its anti-inflammatory and
antioxidant action reduced changes, caused by injection of adrenalin. It decreased MDA
concentration by 78% and activity of iNOS by 57%. Activity of SOD decreased by 34 %
as compared with adrenalin action. Dual acting COX-2/5-LOX inhibitor darbufelon
intensified ulcerogenic action of adrenalin causing increased area of destructive damage
of the gastric mucosa. Concomitantly, indexes of oxidative stress and NO-synthase
system under condition of darbufelon action didn’t change significantly in comparison
with the action of adrenalin alone. Its use as pharmaceuticals needs further investigation
and preclinical testing. Dual COX/LOX inhibitor 4-{2,5-Dioxo-3-[4-oxo-5-(3-phenyl-
allylidene)-2-thioxothiazo-lidin-3-yl]-pyrrolidin-1-yl}-benzene-sulfonamide manifested
pronounced cytoprotective effect under conditions of modeled gastric ulcer. It was
accompanied by sharp decline of the activity of NO-synthases (iNOS activity decreased
more than 2 times) and indexes of oxidative stress.
126
Anticancer activity of novel drugs delivered by newly synthesized
polymeric nanocarriers
1Lehka L. V., 2Panchuk R. R., 3Mitina N., 3Skorokhoda T., 3Moskvin M.,
3Zaichenko О., 4Havrylyuk D. Ya., 4Lesyk R. B., 1,2Stoika R. S.
1 – Ivan Franko Lviv National University, Ukraine
1, Universytetska str., Lviv, Ukraine, 79000
2 – Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine
14/16, Drahomanov St. Lviv, Ukraine, 79005
3 – Lviv National Polytechnic University, Ukraine
12, Bander Str, Lviv Ukraine, 79013
4 – Danylo Halytsky Lviv National Medical University, Ukraine
69, Pekarska St., Lviv, Ukraine, 79010
dr_r_lesyk@org.lviv.net; stoika@cellbiol.lviv.ua
Novel oligoelectrolytes of block, comb-like and branched structure containing
hydrophilic backbone with hydrophobic domains and side branches of controlled length
and functionality were designed and synthesized at Lviv National Polytechnic University.
Their properties allow easy immobilization of various compounds including completely
water insoluble ones. The main goal of this work was to investigate if novel
oligoelectrolyte polymeric complexes can affect cytotoxic action of anticancer drugs
towards tumor cell lines. We compared such action of doxorubicin (Dx) with the action
of novel 4-thiazolidone compound Les-3120 recently synthesized at Danylo Halytsky
Lviv National Medical University. Human Jurkat and CCRF-CEM T-cell leukemia cells
and murine L1210 leukemia cells were used as targets. Antineoplastic activity of Dx-
polymeric complexes was significantly higher than such activity of free Dx. The highest
effect of these complexes was found at low concentrations of Dx (0.1–0.2 ug/ml), while
there were no differences in cytotoxic action of free Dx and Dx-polymer complexes at
high concentrations of Dx (1-2 ug/ml). This conclusion was confirmed by Western blot
analysis of the cleaved forms of caspase-3, caspase-7, as well as their substrates PARP-1
and DFF45 in targeted Jurkat T cells. DAPI staining of human breast adenocarcinoma
MDA-MD-231 cells also showed that Dx-nanocarrier complexes induced more intensive
DNA fragmentation comparing with free Dx.
Although novel 4-thiazolidone compound Les-3120 possesses growth-inhibiting action
towards various mammalian tumor cell lines, its anticancer potential is substantially
restricted by its poor solubility in water media. We demonstrated that application of
olygoelectrolyte carriers resulted in creation of water-soluble forms of this drug with the
same cytotoxic activity, while additional functionalization of Les-3120-nanocarrier
complexes with polyethylene glycol (PEG) increased 5 times their antineoplastic
potential comparing with Les-3120 dissolved in DMSO. This effect can be explained by
high lipophilic activity of PEG-functionalized nanocarriers gaining a capacity of these
drug-nanocarrier complexes to targeted action towards plasma membranes and organelle
(ex. mitochondria) membranes.
This work was partially supported by STCU grant № 4953.
127
Characteristics of colorectal cancer operated at University Hospital
Split, Croatia
Mihanovic Jakov, Tomic Snjezana
Department of Pathology and Department of Surgery, University Hospital Centre Split, Croatia
Mosorska 4, 21251 Zrnovnica, Split, Croatia
mihanovic@gmail.com
Background. Colorectal cancer is the most common malignant disease of digestive tract.
The main risk factors are age over 50, obesity, sedentary lifestyle, low-fiber diet and
high-fat food intake. Clinical presentation includes blood stained stool, changes in bowel
habits and often-obstructive symptoms. Almost 98% of large bowel malignant tumors are
adenocarcinomas. Radical surgery is preferable treatment. The prognosis is most closely
related to the stage of the tumor at the time of diagnosis. Dukes A stage is 100% curable
but survival dramatically drops in stage Dukes C to only 23 %, and in presence of distant
metastasis (Dukes D) to less than 5%. To increase number of cured patients, it is
necessary to improve early detection of colorectal cancer. Implementation of screening
programs such as occult blood test is recommended.
Objectives. The aims of this study were to determine characteristics of colorectal cancer
operated in University Hospital Split during 3-year period.
Methods. Surgically removed colorectal specimens during 3-year period were analyzed
and the following patient data were obtained: sex, age, cancer location and histological
stage. Only adenocarcinomas were analyzed.
Results. A total number of patients operated because of colorectal adenocarcinoma were
576. There were significantly more men (N=345, 60%) than women (N=231, 40 %) (p <
0.001). Most of the patients (89%) were over 50. No one was under 30, and there were 10
patients over 85. Tumor was located on rectum or rectosigmoid junction in 291 patients
(53%), and on the rest of the colon (caecum, ascending, transverse, descending colon and
sigmoid) in 261 patients (47%) without significant difference regarding sex distribution
(p = 0.275). Stage of the tumour in the majority of patients was Dukes B (N=234, 43%),
followed by Dukes C (N=177, 33%) and Dukes D (N=78, 14%). Only 55 (10%) patients
were operated in Dukes A stage. Significant age and sex distribution differences were not
found between patients with different tumor stages.
Conclusion. Only 10% of patients were diagnosed with the earliest stage of the
colorectal cancer. Many patients presented in advanced stage, when surgery is of limited
value and recovery is uncertain. It is necessary to increase percentage of patients
presenting in potentially curable stage. Early detection is a key and it can be
accomplished by screening programs for population over 50, which are at greatest risk
for colorectal cancer. Results of this study put us in east-European transitional countries
average. Further efforts are necessary to improve population education in balanced diet,
regular exercise and early cancer symptoms.
128
Characterization of novel mTOR-kinase splicing isoforms
1Skorokhod Oleksandr , 1Nemazanyy Ivan, 1Panasyuk Ganna, 1Filonenko Valeriy
1,2Zhyvoloup Oleksandr and 1,2Gout Ivan
1
Department of Cellular Signaling, Institute of Molecular Biology and Genetics, Kyiv, Ukraine.
150, Zabolotnogo St., Kyiv, Ukraine, 03680
2
University Collage Londone,
Gower Street London WC1E 6BT, UK.
fiwinner@ukr.net: i.gout@biochemistry.ucl.ac.uk
Background. The mammalian target of rapamycin (mTOR) is a conserved
serine/threonine kinase, which regulates cell growth, survival and metabolism in response
to environmental signals affecting protein synthesis and cytoskeleton dynamics.
Deregulation of mTOR-coordinated signaling has been associated with various human
pathologies, including diabetes, inflammation and cancer. mTOR kinase forms a
multisubunit complex with numerous protein partners. In mammalian cells two distinct
complexes have been identified: mTORC1 in which mTOR is bound to the protein
partner raptor and mTORC2 in which mTOR is bound to another protein partner called
rictor. These protein complexes appear to have distinct biological functions.
Recently, using RT-PCR analysis of various human cell lines our research group has
identified the existence of potential mTOR splice variants. Among them – TOR-β
isoform, have been characterized as a potential oncogene.
Objectives. The aim of our current studies was to prove the existence of additional
mTOR isoforms - TORγ and TORε on RNA and protein levels in mammalian cells.
Results. The bioinformatical analysis of TORγ and TORε primary structure allowed
determining the absence of several functional domains in their structure compared to a
full-length TORα molecule. Both isoforms TOR-γ and TOR-ε were cloned in eukaryotic
vector pcDNA3.1 and stable cell lines were obtained.
Also, a fragment of C-terminal part of mTOR was cloned, overexpressed and purified
from bacteria cells. This recombinant protein was used for rabbit immunization and
TOR-specific polyclonal antibodies generation. The application of generated antibodies
allow to detect the presence in some mammalian cell lines and tissues not only TORα
(290kDa), but also several bands of proteins with lower molecular weight, similar to
TORβ, TORγ and TORε isoforms.
The investigation of phosphorylation status of up-stream and down-stream effectors of
mTOR (Akt, S6K1, S6 protein) in stable cell lines overexpressing TORγ and TORε had
showed that there status was differed compere to wild type cells. Also, we detected that
phosphorylation status of TORγ, like TORα, is rapamycine sensitive, in contrast to TORε
isoform. Finally, analysis of oncogenic properties of TORε and TORγ by soft agar
colonies formation assay had demonstrated that overexpression of TORε, but not TORγ,
lead to 1.5 fold increase in colonies number.
Conclusion. The presented data demonstrate the existence of potential isoforms of
mTOR (TORγ, TORε) with possible oncogenic potential of at least for TORε.
129
Deep proteome characterization as a tool for identification of novel
intraamniotic infection and inflammation biomarkers in preterm
birth patients
1Tambor Vojtech, 1Lenco Juraj, 2Menon Ramkumar, 3Kacerovsky Marian,
1Link Marek and 1Stulik Jiri
1 Institute of Molecular Pathology, University of Defence, Hradec Kralove, Czech Republic
2 Perinatal Research Center, Women´s Hospital, Centennial Medical Center, Nashville, TN, USA
3 Department of Obstetrics and Gynecology, Charles University in Prague, Faculty of Medicine
Hradec Kralove, University Hospital Hradec Kralove, Czech Republic
tambor@pmfhk.cz
Background: Intraamniotic infection and inflammation (IAI) has been demonstrated to
be associated with a significantly increased rate of neonatal adverse outcome, even in the
absence of demonstrable positive cultures. The biochemical and protein composition of
amniotic fluid is altered during pregnancy and reflects both physiological as well as
pathological changes in the fetomaternal compartment. Modern proteomic technologies
are able to detect and characterize even the slightest changes in protein composition of
various biological matrices. Thanks to the ability to both identify and quantify a large
number of proteins, this approach seems to be a very promising one for the detection of
changes in amniotic fluid protein composition and for the identification of possible
biomarkers for the prediction pregnancy related complication.
Objectives: We employed advanced proteomics in identification of novel potential
biomarkers of IAI. The study was performed on amniotic fluid samples from preterm
birth patients with confirmed (n=31) and ruled out (n=26) IAI.
Results: We successfully identified and quantified 847 amniotic fluid proteins (5% FDR)
and selected more than 50 candidates, which showed dysregulated abundance between
the two patient groups. To illustrate, these include neutrophil defensin 3, a range of
histone proteins (H2, H3, H4 etc.), antileukoproteinase and other proteins known to be
involved in host against pathogen response, tissue remodeling and cellular death.
Conclusion: We used multidimensional shotgun proteomics to describe the amniotic
fluid proteome and characterize differences among individual patients group, which
resulted in a rich group of novel biomarker candidates. These are currently being
validated using complementary methods, both antibody-based techniques as well as
targeted proteomics approaches.
130
Translational Research and Nano –
Biotechnology Young Scientist
Poster Presentation
131
Expression of T-type calcium channels during genesis of absence
epilepsy in WAG/Rij rats
Batiuk M.Y., Boldyryev O.I. Shuba Y.M.
International Center of Molecular Physiology, NAS of Ukraine
4, Bogomoletz Str., Kyiv, Ukraine 01024
myhaelis@gmail.com
Background. Childhood absence epilepsy (CAE) is a non-convulsive form of epilepsy
with incidence nearly 2-8 per 100000 children. It is characterized by impaired
consciousness and SWD activity on EEG. Meeren et al. (2002) revealed “cortical focus”
of SWD in somatosensory cortex of the upper lip (S1ULp zone of somatosensory cortex).
Some investigators showed involvement of T-type channels SNPs in disease
susceptibility (Chen, 2003) and increased expression of these channels in thalamus
during disease progression (Broicher, 2008). Furthermore, non-specific blocker of T-
channels ethosuximide inhibits SWD activity.
The aim of this study was to identify links between absence epilepsy and expression of
T-type channels in “cortical focus” and thalamic laterodorsal (LD) nucleus.
We used WAG/Rij rat strain as an animal model of absence epilepsy. We investigated
three T-channels (Cav3.1, Cav3.2, Cav3.3) mRNA expression in laterodorsal (LD) nucleus
and “cortical focus” in WAG/Rij and control Wistar rats using TaqMan Real-Time PCR
system. Three age groups of rats were taken for expression measurements in “cortical
focus” (10 days, 25 days, 6 months) and two groups for measurements in LD nucleus (10
and 25 days).
Results. T-channels mRNAs expression was increased in “cortical focus” of WAG/Rij
rats as compared to control Wistar rats. We observed 1.8-fold increase of Cav3.2 mRNA
level in 10-days old rats, 2.5-fold increase in 25-days old group and insignificant raise in
6-months old rats. We identified almost the same levels of Cav3.1 mRNA in 10-days old
epileptic and control rats, 2-fold increase in 25-days old WAG/Rij rats and 2.5-fold raise
in 6-months old group. There was observed 1.3-fold augmentation of Cav3.3 mRNA
expression in 10-days old group of epileptic rats, 1.4-fold increase in 25-days old group
and the same levels in 6-month old WAG/Rij and Wistar rats. LD nucleus levels of T-
channels mRNAs were decreased in epileptic rats. The levels of Cav3.3 and Cav3.2
mRNAs were the same in 10-days old WAG/Rij and Wistar rats and decreased by 1.6
times in 25 days old epileptic rats. We observed 1.8-fold decrease of Cav3.1 mRNA level
in 10-days old epileptic rats and 3-fold diminution in 25-days old rats.
Conclusion. Elevated expression of Cav3.1 and Cav3.2 in “cortical focus” after puberty
may lead to hyper excitability of neurons and generation of SWD seen in epilepsy.
Expression of T-type channels in LD nucleus was decreased. As neurons of LD nucleus
are over activated during spike-wave discharges, lowering of T-channels expression
might indicate some compensatory mechanism against such over activation. Our data
also show that Cav3.1 and Cav3.3 are the main basis of T-current in LD thalamic neurons
during early ontogenesis.
132
Follow up of viral RNA after coxsackievirus B3 oral and
intraperitoneal routes of experimental infection using recombinant
virus vector
1Baďurová Miriama, 2Precechtelová Jana, 1Sojka Martin, 1Štípalová Darina,
2Gomolčák Pavol, 1Bopegamage Shubhada
1. Slovak Medical University,
12, Limbova 833 03 Bratislava, Slovak Republic
2.Cytopathos s.r.o.,
Limbova 5, 833 01 Bratislava, Slovak Republic
shubhada.bopegamage@szu.sk
Background: Coxsackievirus infections often display a subclinical course or may lead to
only mild disease, but the outcome of the infections may be serious or even fatal.
Coxsackieviruses B3 (CVB3) are more known as aetiological agents of inflammatory
heart diseases and are implicated in the pathogenesis of sub-acute, acute and chronic
human myocarditis or dilated cardiomyopathy
Recombinant CVB3 virus expresses an enhanced green fluorescence protein (eGFP).
eGFP was selected as the optimal marker protein because it is relatively small in size
enabling its incorporation into recombinant CVB and it is relatively resistant to photo
bleaching. Understanding aspects of coxsackieviral diseases such as spread, transmission,
the role of host immune system, the interaction between host and virus and pathogenesis
will help in prevention and control of infection diseases.
Aim: To follow the spread and pathophysiological events after CVB infection using
CVB3 recombinant virus during the 24 hours interval after oral and intraperitoneal (ip.)
infection by studying the presence of viral RNA in the organs of the infected mice and
compare with the eGFP flourescence.
Results: Organs (heart, pancreas, spleen, small intestine and brain) were collected at 2-
hour intervals and studied for the presence of viral RNA by RT-PCR. In ip. infected mice
viral RNA was detected in heart from 2h post infection (p.i.) in 2/3 mice, in pancreas
from 2h p.i. in 1/3 mice, in spleen virus appeared between 4 - 6h p.i.in all 3 mice, small
intestine from 2h p.i.in 3/3 mice and in brain from 2h p.i. in 3/3 mice.
In orally infected mice pancreases were completely negative till 12h p.i., in spleen the
virus was observed from 8h p.i. in 2/3 examinated mice, in small intestine from 2 h p.i. in
1/3 mice and brain and heart showed negativity to 12h p.i mice. Uninfected controls were
negative.
Conclusions: Almost in all organs of ip. infected mice the virus was detected, at 2 h p.i.
Orally infected mice were more protected from virus infection. Virus was presented at
interval 2 h p.i. only in small intestine. All others orally infected organs of mice showed
negativity for presence of virus at this early interval. In ip. infected mice the virus spread
was rapid.
Acknowledgements: We thank Prof. J. M. Galama from Nijmgen, the Netherlands for
scientific discussions, Dr. A. Henke from Jena, Germany for supplying eGFP-CVB3. For
financial support- MZSR code: 2007/03- RUVZBB-01 and the Norwegian financial support
mechanism, Mechanism EEA and Slovak Government - Project SK0082.
133
Peroxisome proliferator-activated receptor-α agonist BAY PP1
attenuates renal fibrosis
1,2,3Boor Peter, 4Celec Peter, 2Martin Ina V., 2,5Villa Luigi, 4Hodosy Július,
1Klenovicsová Kristína, 5Esposito Ciro, 6Schäfer Stefan, 6Albrecht-Küpper
Barbara, 2Ostendorf Tammo, 7Heidland August and 1Šebeková Katarína
1 Department of Clinical and Experimental Pharmacotherapy, Research Base of Slovak Medical
University, Bratislava, Slovakia;
2 Division of Nephrology
3 Institute of Pathology, RWTH University of Aachen, Germany;
4 Institute of Molecular Biomedicine, Comenius University, Bratislava, Slovakia;
5 Unit of Nephrology, Dialysis and Transplantation, University of Pavia, Italy;
6 Bayer Schering Pharma AG, Cardiology Research, Wuppertal, Germany;
7 Department of Internal Medicine, University of Würzburg, Germany.
kata.sebekova@gmail.com
Recent studies have suggested renoprotective effects of peroxisome proliferator-activated
receptor-α (PPAR-α), but its role in kidney fibrosis is unknown. We examined the effect
of a novel PPAR-α agonist, BAY PP1, in two well established rat models of renal
fibrosis: the unilateral ureteral obstruction (UUO) and the remnant kidney model (5/6
nephrectomy). We previously presented the preliminary data in the UUO model. Here we
show further experiments confirming the antifibrotic role of PPAR-α in renal fibrosis.
In healthy animals, PPAR-α was expressed in renal tubular cells but not in interstitial
cells, like e.g. fibroblasts. Upon induction of fibrosis, PPAR-α was significantly down
regulated, and treatment with PPAR-α agonist BAY PP1 significantly restored its
expression. In UUO, treatment with BAY PP1 significantly reduced tubulointerstitial
fibrosis, proliferation of interstitial fibroblasts and TGF-β1 expression, whereas treatment
with a less potent PPAR-α agonist, fenofibrate, had no effects. Treatment with BAY PP1
in the remnant kidney model halted the decline of renal function and significantly
ameliorated renal fibrosis. This is of particular importance, since the treatment was
initiated in already established disease, i.e. a clinically highly relevant situation. In vitro,
BAY PP1 had no direct effect on renal interstitial fibroblasts but reduced collagen,
fibronectin and TGF-β1 expression in tubular cells. Conditioned media of BAY PP1-
treated tubular cells reduced proliferation of fibroblasts in a paracrine fashion.
In conclusion, renal fibrosis is characterized by a reduction of PPAR-α expression, and
the PPAR-α agonist BAY PP1 is capable of restoring it, thereby reducing renal fibrosis.
This is most likely mediated via affecting the cross talk between tubular cells and
fibroblasts. These data suggest that potent PPAR-α agonists could be a novel treatment
option in renal fibrosis.
134
Origin of spontaneous and induced by the chemical mutagens point
mutations and enzymatic control over them
1,2Brovarets O. O., 1,2Hovorun D. M.
1 Institute of High Technologies, Taras Shevchenko Kyiv National University,
2, Hlushkova Ave., Kyiv, 03187, Ukraine;
2 Institute of Molecular Biology and Genetics, NAS of Ukraine,
150, Zabolotnogo Str., Kyiv, Ukraine, 03680
brovarets@list.ru
Background. For half a century, prototropic tautomerism of nucleic acid bases has been
considered an origin of spontaneous point mutations arising in DNA, nevertheless the
elementary physico-chemical mechanisms of its arising still remain uncertain.
Aim. To establish molecular models of the spontaneous and induced by chemical
mutagens transitions and transversions being based on the physico-chemical mechanisms
of the DNA bases tautomerization within Watson-Crick and wobble base pairs using
quantum-chemical calculations.
Results. Physico-chemical mechanisms of the transitions, in particular incorporation and
replication errors in DNA, based on the novel structural ideas in the area of the
spontaneous point mutagenesis were revealed and validated.
Replication errors arise due to tautomerization of Gua·Cyt and Ade·Thy Watson-Crick
base pairs to dynamically stable Gua·Cyt* and Gua*·Cyt or Ade*·Thy and Ade·Thy*
mispairs respectively formed by rare tautomers (marked by an asterisk) and
quasiisomorphic to wobble mispairs, and incorporation errors – due to tautomerization of
Gua·Thy and Ade·Cyt wobble mispairs formed by the bases in their canonical forms to
isosteric Watson-Crick, dynamically stable Gua*·Thy and Ade·Cyt* mispairs involving
rare tautomers accordingly. Transition states of such conversions are planar symmetric
ion pairs stabilized by H-bonds.
It was established that mutations induced by the mutagenic nucleobase analogues –
halogen derivatives of urаcil (5-bromouracil, 5-chlorouracil and 5-fluorouracil), 2-
aminopurine and 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]-oxazin-7-one – arise due to
the decrease of activation barrier of tautomerization and the increase of the population of
mispairs involving rare tautomers.
Physical model of the recognition of Watson-Crick base pairs by DNA-binding proteins
from the major groove side was elaborated. There are strong grounds to suggest that
asparagine and glutamine side chains of these proteins are the best-suited candidates to
participate in the process due to their spatial arrangement about base pairs. This model
allows the inhibition of biosynthesis of mispairs formed by both rare and canonical
tautomers of DNA bases.
We have investigated all possible purine-purine mispairs containing template base only in
anti-configuration. It was established that only those of them, which can easily adopt
Watson-Crick, shape, cause spontaneous transversions arising.
Conclusion. Novel physico-chemical models of spontaneous and induced by chemical
mutagens, namely the analogues of nucleobases, transitions and transversions were
elaborated and analyzed.
135
Pharmacognostic evaluation of Salix spp. honeydew and nectar
honeys and their antioxidant capacity
1Jerković Igor, 2Tuberoso Carlo I. G., 2Bifulco Ersilia and 3Marijanović Zvonimir
1 Dep. of Organic Chemistry, Faculty of Chemistry and Technology,
University of Split, N. Tesle 10/V, 21000 Split, Croatia
2 Dip. Farmaco Chimico Tecnologico, University of Cagliari, via Ospedale 72, 09124 Cagliari, Italy
3 Department of Food Technology, Marko Marulić Polytechnic in Knin,
Petra Krešimira IV 30, 22300 Knin, Croatia
igor@ktf‐split.hr
Rare unifloral nectar and honeydew willow (Salix spp.) honeys from Croatia were
investigated by direct RP-HPLC-DAD method as well as by headspace solid-phase micro
extraction (HS-SPME) and ultrasonic solvent extraction (USE) followed by GC and GC-
MS analyses in order to identify and quantify compounds that can be used as possible
markers of their origin. It is now recognize that many phytochemical compounds can
have health-promoting activities and a honey is known to be rich in both enzymatic and
non-enzymatic antioxidants.
HMF (1), diastase activity and CIE L*a*b*C*h* chromatic coordinates of all the samples
were evaluated. Abscisic acids (ABA) are typical of willow nectar honey, with a
predominance of cis, trans-ABA (2) on trans, trans-ABA (3) [98.2 and 31.7 mg/kg,
respectively]. Kinurenic acid (4) and salicylic acid (5) are useful to mark willow
honeydew honey. The proposed HPLC-DAD method proved to be easy and reliable to
identify the two different Salix spp. honeys, being not affected from any sample
preparation artifact. The use of HS-SPME and USE had advantageous results over the
use of one single technique, as it provided different complementary chromatographic
profiles for a comprehensive screening of the honeydew volatile composition. Salix spp.
nectar and honeydew honeys proved to be two completely different honeys because,
besides color attributes, they show different antioxidant properties and specific
compounds.
O
O OH
OH
N
OH
O CH3
O
O
OH
CH3
OH
CH3 CH3
CH3O OOH
CH3
OH
CH3 CH3
OH
OH
O
1 2 3 4 5
Antioxidant and antiradical activities of willow honeys were evaluated using FRAP and
DPPH tests, respectively. Total antioxidant activity measured with the FRAP assay
ranged from 3.2 to 12.6 mmol Fe2+/kg, and antiradical activity measured with the DPPH
assay ranged from 0.6 to 3.0 mmol TEAC/kg in nectar and honeydew honeys,
respectively.
136
Non – viral nanoscale-based delivery of antisense oligonucleotides
enhances inhibition of the cellular prion expression in vivo.
1Ivanytska L., 2Stadnyk V., 1Kozak M., 3Zaichenko A., 3Mitina N., 1Vlizlo V.
1Institute of Animal Biology NAAS, Lviv, Ukraine,
38, Stusa str., Lviv, Ukraine
2 Eötvös Loránd University ,
Egyetem tér 1-3. Budapest, 1053 Hungary
3Lviv Polytechnik National University, Ukraine
12, Bandery Str, Lviv, Ukraine 79013
ivanytska.lyudmyla@gmail.com
Neurotoxicity from accumulation of misfolded cellular prion (PrPC) is thought to drive
pathogenesis in transmissible spongiform encephalopathies (TSE). There is no cure for
these disorders. It was ascertained that presence of normal PrPC is absolutely necessary
for illness development. Targeting PrPC has the potential to remove substrate for the TSE
pathogenesis so this is a promising strategy for development of means for treatment and
prevention prion infections. In accordance to this the aim of our study was to decrease the
cellular prion expression level in vivo by antisense oligodeoxyribonucleotides (asODNs)
toward prion mRNA.
But instability of asODNs in blood serum and obstacles of biological barriers demand
effective delivery system for in vivo application of the antisense technology. So first
novel telechelic poly dimethylaminoethylmethacrylate (polyDMAEM) oligoelectrolite
(Mw = 5000 g/mol) with end ditertiary peroxide fragment was developed. DNA –
binding ability was checked by UV-spectroscopy and turbidimetry.
For effective inhibition of PrPC gene mRNA translation three asODNs were designed. On
24th h. of incubation L1210 cells with complex asODN and polyDMAEM all three
asODNs showed 95-98% (r<0.01) efficiency in decreasing PrPC expression. Western blot
analysis and immunohistochemistry demonstrate inhibition of PrPC expression in the
spleen of Wistar rats by almost 90% (r<0.05) on 48th h post i. v. injection of asODNs-
polyDMAEM complex. It is observed 80% decrease of PrPC content in the intestine.
Investigated asODN nanoscale delivery system demonstrates most significant results in
rat brain. Thus asODN immobilized on poliDMAEM was capable to overcome the blood-
brain barrier and decrease PrPC expression to 60% from a control level (r<0.01).
Testing of polyDMAEM in vitro on cell line L1210 and primary rat fibroblast cells
showed very low toxicity of polymer. After single i.v. injection of polyDMAEM to rat’s
physical activity and other external manifestations of animal behavior were not changed.
Decrease of feed and water consumption was not observed among all experimental
animals. Results of total protein concentration, activities of marker ferments in serum and
histology investigation of liver, kidneys, spleen, intestine and brain indicate certain
metabolic stress of liver and intracerebral capillary permeability violation on 48 h after
administration of tested compounds.
Our investigations show that polyDMAEM has a high potential for use in CNS delivery
systems. As a result of carried out research there were developed effective asODNs and
their delivery system for inhibition of prion mRNA translation.
137
1H NMR spectroscopy study on the impact of MRPs-rich diet on
urine metabolome in healthy rats
1Kollárová Radana, 2Pronayová Nadežda, 3Boor Peter, 1Šebeková Katarína
1Department of Clinical and Experimental Pharmacotherapy, Slovak Medical University, Bratislava,
2Department of NMR Spectroscopy, Faculty of Chemical and Food Technology, Slovak University of
Technology, Bratislava, 3Institute of Pathology and Department of Nephrology, RWTH University,
Aachen, Germany
kata.sebekova@gmail.com
Non-enzymatic glycation of proteins is a complex series of parallel and sequential
reactions, collectively called the Maillard reaction, occurring during thermal processing
of food. Maillard reaction products (MRPs) are formed via spontaneous reaction of free
amino-groups of proteins and reactive sugars, and render baked and fried food its
characteristic color, odor and taste. Some MRPs are biologically active substances. In
vivo formed analogues of MRPs are called advanced glycation end products (AGEs).
Exaggerate accumulation of AGEs in disease states (e.g. diabetes, chronic renal failure)
alters the structure and function of proteins and elicits negative heath effects via
interaction of AGEs with their specific cell surface receptors. It is accepted that, at least
a part of alimentary MRPs is absorbed into circulation, where they may potentiate the
toxic effects of AGEs. The aim of the study was to investigate the impact of dietary
MRPs on urine metabolome, with regards to presence/absence of MRPs-derived aroma
substances.
MRPs-rich diet was prepared by replacing 25% (wt/wt) of standard rat chow by bread
crusts. Aroma extracted MRPs-rich diet contained 25% of aroma-extracted bread crusts
(obtained via quadruple extraction of bread crust with diethyl ether). 24 male Wistar rats
(180-220g) were randomized into 3 groups (n=8, each) and fed for 3 weeks either by
standard rat chow (controls), MRPs-rich- or aroma extracted MRPs-rich-diet. 24h urine
collection was performed before, and weekly during the experiment. Urine samples were
analyzed using 1H NMR spectroscopy. Obtained data were evaluated using multivariate
statistic analysis.
The composition of urine of the animals on standard rat chow differed from that of the
rats on both MRPs-rich diets. However, at week 1 and 2 metabolomes of aroma-rich and
aroma-extracted diets consuming rats did not differ significantly. The impact of aroma
substances appeared only in samples collected on the last day of the experiment: an
indication of separation between the groups was observed.
It is concluded that consumption of MRPs-rich diet alters urine metabolome. According
to our best knowledge this is the first study dealing with the impact of MRPs-rich diet on
urine metabolome. Identification of single metabolites contributing to between group
differences requires further analyses.
Study was supported by SMU internal grant No.199005 (KK) and CENDO grant
No.194001(KŠ).
138
Evaluation of osteocyte dedifferentiation process
1,4Pejda S., 2Kizivat2 T., 3Fatahi M., 3Igwe J., 4 Horowitz M. C., 3Maye P., 3Rowe
D. W., 3Kalajzic I.
1School of Medicine, University of Split, Croatia,
2School of Medicine University of Osijek, Croatia,
3University of Connecticut Health Center, Connecticut, USA,
4Yale University, New Heaven, Connecticut USA.
spejda@gmail.com
Background: Dedifferentiation is the progression of cells from a more differentiated to a
less differentiated state. Presently, there is no evidence for the ability of mature osteoblast
lineage cells to dedifferentiate. However, if possible, de-differentiation of mature
osteoblast lineage cells could provide an additional source of osteoblast population
capable of proliferation and differentiation. This could become an important anabolic
strategy for older individuals, where a decrease in the osteoprogenitor pool may
contribute to decreased bone formation. Aim: Our aim is to investigate if preosteocytes
and osteocytes under the conditions of high demand can undergo a de-differentiation,
accompanied by dramatic changes in gene expression. This process would generate cells
that could exhibit proliferation potential, followed by differentiation into bone matrix
producing osteoblasts. Methods and Results: To determine whether some of the
outgrowing cells were derived from dedifferentiating osteocytes, we used transgene
lineage markers (DMP1GFP and DMP1Cre). Cell cultures were made from bone chips
using DMP1GFP mice. As the DMP1 is active during an osteocyte stage, we did not
observe GFP expression in cells that grew out of the bone chips. This indicates that these
cells do not express this osteocyte-specific promoter. However, this does not preclude the
possibility that BCOC (bone chip outgrown culture) are derived from osteocytes. To
evaluate whether the BCOC are derived from cells that once were osteocytes, we have
established bone chip cultures from a breeding derived from DMP1Cre x Rosa26R or Ai9
reporter strain in which the GFP-Tomato is expressed only in cells in which Cre is
currently active or was active at some point during differentiation. In contrast to the
DMP1GFP cultures, we observed lacZ or GFP-Tomato in a high proportion of cells that
grew out of the chips just 3-5 days after the initiation of culture. As we have
demonstrated that BCOC’s do not have DMP1 promoter active at that stage in culture
(DMP1GFP negative), these data indicate that the Tomato expression is due to the
historical activation of reporter construct by DMP1Cre. Thus, the cells that are Tomato+
were at some point DMP1Cre+. These indicate that osteocytes can crawl out bone chips
and dedifferentiate in vitro. The BCOC express some of the genes characteristic of
osteoblast/osteocytes and following transplantation their fate is to become embedded into
bone matrix. Conclusion: Utilizing a transgenic model we evaluated the ability of de-
differentiated osteocytes to repopulate the osteoblast niche.
139
Single nucleotide polymorphisms in obesity-related genes
Sliz I, Gogalova V, Smolkova B, Dusinska M, Gasparovic J, Raslova K, Horska A,
Dzupinkova Z, Staruchova M, Volkovova K.
Department of Experimental and Applied Genetics, Slovak Medical University,
Limbova 14, 833 03, Bratislava, Slovakia
sliz.ivan@gmail.com
Background: Numerous connections between chronic metabolic disorders, including
obesity, and cardiovascular diseases (CVD) have been established and are currently well
recognized. Obesity, which represents one of many different cardiovascular risk factors,
exerts effects predominantly on the heart and circulation, on both directly and also by its
association with other known risk factors such as hypertension, dyslipidemia or insulin
resistance. These effects tend to be more prevailing with increased weight gain,
especially in younger subjects even prior becoming obese.
Objective: The aim of this study was to investigate the association between the genotype,
environmental factors and potential health risks resulting from both obesity and CVD in a
group consisting of 2403 randomly selected subjects (1001 men, 1402 women) who were
approximately the same age (40.5 ± 0.7 years) and came from 7 different Slovak regions.
Several following polymorphisms (SNP) primarily contributing to obesity were
genotyped: growth hormone secretagogue receptor (GHSR; rs495225 and rs572169),
beta-1 adrenergic receptor (ADRB1; rs1801252), beta-2 adrenergic receptor (ADRB2;
rs1042713 and rs1042714), 5-hydroxytryptamine receptor 2C (HTR2C; rs6318), leptin
receptor (LEPR; rs1137100) and alpha-ketoglutarate-dependent dioxygenase FTO (FTO;
rs9939609). The frequencies of these variants were analyzed in relation to
anthropometric characteristics like body mass index (BMI) and waist circumference
(WC), a wide range of biochemical parameters such as serum total cholesterol (TC), low
density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C),
non-HDL cholesterol (non HDL-C), total to HDL-C ratio (TC/HDL), triglycerides (Tg)
and glucose (Glu) levels, as well as to volunteers' personal and family anamneses and
other lifestyle data.
Results: Homozygous carriers of variant allele rs1137100 of the LEPR gene had
significantly higher BMI (P = 0.005) and WC (P = 0.001) compared to heterozygotes
combined with reference group. Among the other polymorphisms tested in this study, a
significant association was observed between the homozygous variant genotype of the
FTO gene (rs9939609) and increased blood Glu levels (P = 0.008).
Conclusion: These findings lead us to the suggestion that the rs1137100 variation in the
leptin receptor gene contributes to weight gain and variability in body fat distribution.
The observed presence of the FTO rs9939609 variant associated with increased Glu level,
which belongs to the symptoms of the metabolic syndrome, may support the hypothesis
that variantions in the FTO locus are involved in increasing risk of developing CVD.
140
Differences in pathogenesis of coxsackievirus B4 isolates of different
origin in a mouse model
Sojka Martin, Precechtelova Jana, Badurova Miriam, Stipalova Darina,
Bopegamage Shubhada
Slovak Medical University, Bratislava
Limbova 12, 833 03 Bratislava, Slovak Republic.
shubhada.bopegamage@szu.sk
Background: Coxsackieviruses (CV) are ubiquitous faecal-orally transmitted, small
RNA viruses, belonging to the family Picornaviridae. HEV are now classified into four
species: human enterovirus A-D. These viruses cause a wide variety of diseases and it is
extensively known that non-polio enteroviruses are the most common cause of aseptic
meningitis in adults and in children as well. The presence of mouse coxsackie-adenovirus
(mCAR) receptor, a corresponding receptor to human coxsackie-adenovirus (hCAR), one
of the receptors used by virus in process of target cell entry, has made mouse the most
common experimental model for studying of coxsackieviral infection. Experimental
mouse models are used to study the enterovirus (mainly coxsackievirus) infection.
Objectives: The aim was to study and compare the viral pathogenesis, organ and tissue
tropism of coxsackievirus B4 isolates of different origin.
Results: In the study, CVB 4 isolates of human origin (cerebrospinal fluid and stool) and
isolate from treated wastewater were compared. CD1 mice were infected orally by model
viruses (previously plaque purified) using oral gavage with a defined infectious dose.
Presence of viral RNA in heart, pancreas, brain, small intestine, blood and stool was
observed by means of enterovirus-specific PCR. Histopathological changes of infected
tissues were studied. Significant differences in the presence of virus were found between
viruses of different origin. In CSF isolate infected mice, viraemia occurred at day 10 post
infection (p.i.), while in stool and environmental isolate infected mice viraemia was
present at day 5 p.i. At day 5 and 10 p.i. viruses were present in mice organs, but with
differences depending on the origin of virus. Stool and environmental isolate showed
only cardiotropic and pancreatotropic properties, unlike CSF isolate detected in brains as
well. At day 45 post infection, the virus was detected only in brains of mice infected with
CSF isolate. No significant histopathological changes were observed in infected tissues.
Conclusion: Significant differences in organ tropism of coxsackieviruses of different
origin were observed. The in vivo model infection is of importance for enterovirus
infection study on tissue, organ and host level. A thorough study of model infection and
treatment is the only way how to move forward in our knowledge about enterovirus
pathogenesis, mode of virus adaptation to different tissues and organ tropism.
Acknowledgements: Dr. Z.Sobotova, Bratislava Slovak Republic (for the samples),
Financial support- MZSR code: 2007/03- RUVZBB-01 and the Norwegian financial
support mechanism, Mechanism EEA and Slovak Government - Project SK0082.
141
Persistence of viral RNA in experimental infection of coxsackievirus B5
Stipalova D., Sojka M., Borsanyiova M., Badurova M., Marosova L., and
Bopegamage S.
Department of Virology, Slovak Medical University,
Limbova 12, 833 03 Bratislava, Slovak Republic.
darina.stipalova@szu.sk
Background: Coxsackie B virus (CVB5) is associated often with meningitis sporadic
cases of neurological diseases, and chronic diseases such as cardiomyopathy and
diabetes. Molecular epidemiology of these viruses and its relation to the viral
pathogenesis is important. These viruses exist as circulating heterogeneous populations of
genetic variants as other enteroviruses.
Aim: The aim of our study was to follow the persistence of viral RNA in different organs
of mice infected with CVB5 strains. These were mainly patient’s isolate and an isolate
identified as CVB5 from treated sewage sample (from the area of domicile of the patient)
and the prototype strain in an outbred experimental murine model.
Results: CD-1 mice were infected with CVB5 strain Faulkner, (prototype) CVB5 –
isolate from treated sewage waste and isolate from patient´s stool sample identified as
CVB5. The viral RNA was detected by RT-PCR using enterovirus primers specific for
the non-coding 5’ region. We observed presence of RNA in the brain and heart of mice
infected with the patient’s isolate at day 45-post infection (p.i.).
Conclusion: We concluded that CVB5 persists in the brain and heart after oral infection
of CD1 mice. The relevance of viral persistence maybe related viral origin and the
genetics.
Acknowledgements: Dr. Z.Sobotova, Bratislava Slovak Republic (for the samples),
Norwegian financial support mechnism, Mechanism EEA and Slovak Government -
Project SK0082
142
Determination of excitatory amino acids in brain microdialysates by
capillary electrophoresis-laser induced fluorescence detection
1Tábi T., 1Wagner Zs., 2Zachar G., 2Csillag A., 1Szökő É.
Semmelweis University,
Nagyvárad tér 4., 1089 Budapest, Hungary
1Department of Pharmacodynamics,
2Department of Anatomy, Histology and Embryology,
tamas.tabi@net.sote.hu
Excitatory amino acid neurotransmitters are important in various physiological and
pathological brain processes. Glutamate has mainly gained attention, while involvement
of aspartate in these functions is less well understood. The aim of our studies is to
simultaneously follow the change of aspartate and glutamate concentration in the
extracellular space, analyzing micro dialysis samples from substantia nigra of the
experimental animals. An appropriate analytical method first has been developed.
Difficulties in analyzing biological samples are the limited amount of sample specimens,
low analyte concentration, complex sample matrix, etc. Capillary electrophoresis (CE)
with laser induced fluorescence (LIF) detection is usually used when few microL micro
dialysates are to be analyzed. LIF is regarded to provide selective and sensitive detection.
However, as majority of analytes has no intrinsic fluorescence, derivatization is required,
considerably limiting the expected advantages. Selectivity is rather impaired, since
biofluids contain lots of compounds labeled alongside with the analytes of interest.
Interference also derives from the labeling reagent, and the concentration of the
interfering compounds usually highly exceeds that of the analytes. The impaired
selectivity is accompanied by impaired sensitivity. Furthermore, complex samples require
carefully designed separation conditions to ensure appropriate resolution. Several buffer
additives are commonly used simultaneously to improve selectivity and widen separation
time window. The high sensitivity of LIF is also limited by the unreliable derivatization
reaction at low sample concentration. Labeling at submicromolar concentrations suffers
from the slower reaction rate and the increased competition with hydrolysis reaction of
the derivatizing reagent. The consequence of the incomplete derivatization is the loss of
linear correlation between concentration and peak area or height.
Three labeling reagents have been compared in terms of accuracy and complexity of
separation conditions. In case of all labeling tags, quantitative determination can be
performed down to 100 nM sample concentration. Considering other circumstances,
FITC labeling was used in our further studies. Applying potassium to release
transmitters, about 3 to 4 times increase of the extracellular glutamate concentration and
even higher increase of aspartate has been detected. Even mild stress of animals
stimulated transmitter release, the concentration about doubled in the micro dialysates.
Based on these results, our method is appropriate to follow the change of
neurotransmitter levels in the brain.
143
Gangliosides involvement in apoptotic process
Tomin Andriy, Shkandina Tanya, Bilyy Rostyslav
Institute of Cell Biology, NAS of Ukraine
14/16, Drahomanov St. Lviv, Ukraine, 79005
amtomyn@gmail.com
In our previous research it was shown, that some of human sialidases are activated during
the apoptosis via caspase-involved process. Their main targets are terminal residues of
sialic acid, located on glycotops of plasma membrane glycoproteins. We proved this
process to be important for effective recognition and elimination of apoptotic cells by
phagocytes. Also, one of sialidases, which may be activated during the apoptosis, named
Neu3, is known, as “ganglioside sialidase” for it action towards sialic acid residues of
membrane sialoglycosphingolipids – gangliosides. Gangliosides play significant role in
intercellular communication, and it was presumed that they take part in apoptotic events.
Also, gangliosides are involved in pathology of many diseases, as Guillain-Barré
syndrome and lupus erythematosus, where anti-ganglioside antibodies could be produced,
and autoimmune disorders are caused by failure of apoptotic blebs clearance.
We studied changes, which appear in splingolipid profile of the cell undergoing
apoptosis. Gangliosides were isolated from granulocytes, viable and apoptotic (aged for
24 hours), as described in (Svennerholm, 1964), and analysed by thin-layer
chromatography with different solvent systems (chloroform: methanol: water 65:35:8 and
chloroform: methanol: CaCl2 aqueous for general analysis and propanol: 2M ammonia
7:3 for polysialic gangliosides). Also, sphingolipid samples were digested by ceramide
glycanase (EC 3.2.1.123) from Hirudo officinalis, glycan parts were marked with
anthranilic acid (Sigma), and studied by HPLC analysis.
We revealed some increase of GM2, GM1 and globoside Gb3 in apoptotic cells
simultaneously with decrease of GM3 and Gb4 globoside, which may be caused by
activation of N-acetylgalactosamine-specific glycosidase. Also, the recently described
abzyme (Bilyy, 2010) with sialidase activity, obtained from multiple myeloma patient, is
able to cleave sialic acid from GD3 ganglioside, which may be useful in understanding of
pathology of this disease.
144
Research of europium oxide nanoparticle toxic influence on SPEV
cell culture
Pavlovich E.V., Goncharuk E.I., 1Ganina I.I.
Institute for Problems of Cryobiology and Cryomedicine NAS of Ukraine,
23, Pereyaslavskaya str., Kharkiv, Ukraine, 61015
1Institute of Scintillation Materials of the State Scientific Institution "Institute for Single Crystals" of
NAS of Ukraine,
60, Lenin Avenue, Kharkiv, Ukraine, 61072
lenapavlovich@gmail.com
To understand the influence of nanotechnology on an environment is the study of
nanoparticles interplay at the model of biological objects, such as cell cultures is relevant.
The aim of our investigation was study of the interaction of nano-sized compounds with
cultured cells, by means of morpho-functional state of cells with incorporated NPs
investigation. Effect of NPs at the viability and property to adhesion of SPEV cells were
researched.
Europium oxide nanoparticles (NPs) colloid solution by the «hot solvent» method and
microwave heating were synthesized. For experiments was used NPs without and with
ultrasonic disaggregation.
Assessment of the NPs distribution in cultured porcine kidney epithelium transplantable
line (SPEV) cells was carried out by using a scanning confocal microscope (Carl Zeiss)
at 405 nm excitation. While studying of NPs penetration characteristics on SPEV cells it
was found that investigated NPs luminesced in emission 540-647 nm and were localized
in the cytoplasm and not penetrated in the nucleus.
In the process of estimation of toxic effects in NPs concentrations of 6.8-340 μg/ml on
the viability and adhesive properties of cells it was shown that NPs in concentrations of
6.8 μg/ml did not affect on the viability of cell SPEV. It was found that NPs before and
after disaggregation in 34 and 340 μg/ml concentrations decreased index of cell viability.
After preliminary disaggregation NPs their toxic effects was amplified in the
concentrations NPs 340 mg/ml.
It was shown that washing from NPs are not significantly reduced the toxic effect and
suggested that penetration of NPs occurs in the first minutes of incubation.
Сell suspensions with internalized NPs in the growing medium were incubated, the
adhesive properties of cells was studied during 1-24 hours in the inverted microscope
visually. The ability of cells to adhesion decreases with growing concentration of
internalized NPs. At low concentrations of europium oxide, 17 and 34 mg/ml adhesive
properties of cells are preserved, but these processes proceed more slowly, which
subsequently leads to slow the division. It was shown that NPs concentration 340 μg/ml
irreversibly affect at the processes of adhesion and leading to cell death. Results can be a
basis for prognostic test cultured cells, which has prospects of the assessment of potential
harm nano-sized particles for human health and the environment.
145
Investigation on lanthanides doped β-NaGdF4 nanocrystals -
synthesis and optical characterization
Sojka B., Banski M., Podhorodecki A. , Misiewicz J., 1Afzaal M., 2O'Brien P.
Institute of Physics, Wroclaw University of Technology,
Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland
1Center of Research Excellence in Renewable Energy, King Fahd University of Petroleum and
Minerals, Dhahran, 31261 Saudi Arabia
2The Manchester Materials Science Centre and Department of Chemistry, The University of
Manchester, Oxford Road, Manchester, M13 9PL, UK
artur.p.podhorodecki@pwr.wroc.pl
Multifunctional bio-markers are recently of significant interest due to their huge potential
in bio-medicine applications. Lanthanide doped fluoride nanocrystals (ncs) have already
presented an efficient photoluminescence from variety doped ions giving visible down-
converting (e.g. Eu3+, Tb3+) and infrared up-converting (e.g. Yb3+-Tm3+) emission. Gd
substitution received particular attention due to magnetic properties of this ion. We
propose to extend the functionality of ncs by co-doping of hexagonal NaGdF4 ncs with
Eu3+ and Tb3+ ions simultaneously.
The co-thermolysis of metal trifluoroacetates was used to synthesize Eu3+ and Tb3+ co-
doped β-NaYF4 and β-NaGdF4 nanocrystals. According to TEM and XRD the
nanodimesional samples were highly crystallized and of the desired hexagonal phase with
uniform maximum diameter sizes (~9+1 nm). The photoluminescence (PL), PL
excitation and time-resolved PL were applied to investigate the properties of tunable
emission spectra. Rich sets of emission bands related to f-f transitions of Tb3+ and Eu3+
ions were obtained for both samples. Due to the excitation energy migration and the
cross-relaxation the emission form Tb3+ and Eu3+ can be excited simultaneously or
separately, depends on the excitation energy. The conducted study suggests a strongly
enhancement of the energy migration in β-NaGdF4 matrix resulting in intensive PL from
Eu3+ ions.
The multicolour luminescence of bi-functional β-NaGdF4:Eu3+-Tb3+ nanocrystals
opens a bright future for this material as a bioprobe.
146
Preparation of spr immunosensor chip for fibrinogen and soluble
fibrin determination in human plasma
Storozhylova N. S., Bereznytsky G. K., Makogonenko E. M., Lugovskoi E. V.
O.V.Palladin Institute of Biochemistry NASU,
9, Leontovycha Str., Kyiv, Ukraine, 01601
clearangel@mail.ru
Surface plasmon resonance (SPR) technology is widely used in scientific researches for
investigation of biomolecules interactions in real-time and in medicine for clinical
diagnostics. It provides quantitative information on specificity, concentration and activity
of molecules-analytes, identification of binding targets in biologic fluids, etc. Quickness
and high sensitivity are two important advantages of SPR that allows the quantification of
important proteins in human plasma in 30 minutes [1].
The aim of work was preparation of the biosensor chips with antibodies covalently
immobilized on the chip to quantify of protein markers in plasma of the patients with
cardio-vascular diseases. We used the self-assembled monolayer (SAM), formed on the
gold-plated glass, as a basis for binding of sensor molecules to the chip [1, 2]. SAMs
were prepared from thiols with different length of carbohydrogenic chains – 11-
mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (MH). We investigated
binding properties of chips in dependence on MUA to MH concentration ratio. At first
series of experiments SAMs for immobilization of monoclonal antibodies (monAB) was
prepared at 1:4, 1:2.5, 2:3, and 1:1 MUA to MH concentration ratio. Gold-plated glass
surfaces were treated by 5 mM concentration of thiols in ethanol for 7 days to form SAM.
Thereafter, the COO– groups were activated by 0.2 M carbodiimide. Anti-fibrinogen
monAB II-4d were immobilized in 0.03 M phosphate buffer, pH 7.4, containing 0.3 M
NaCl, at concentration 20 µg/ml. The formed chip was washed with 0.02 M HEPES,
pH 7.4, containing 0.15 M NaCl, 0.005% Tween-20, and 0,005% NaN3. The results of
SPR analysis showed that chips with SAMs of MUA and MH in ratio 2:3 had much
effective binding of fibrinogen (Fig). The biosensor chips for fibrin were prepared in the
same way, but with anti-fibrin monAB 1-ІІІс. Using SPR analysis we tested the obtained
chips for Fig and fibrin binding of model solutions, prepared of purified proteins and
from patients’ plasmas. In the last case protease inhibitors cocktail (539131 Set 1,
Calbiochem) was used for prevention of monAB digestion. Graphs 1, 2 illustrate received
sensograms.
147
In model experiment with human Fig and fibrin as antigen was shown that: a) the
sensitivity of the method was 100 ng/ml and 250 ng/ml, correspondingly and b) the chip
can be regenerated with 50% solution of ethylene glycol in HEPES-buffer. The
immobilized monAB retained the ability to bind Fig and fibrin after 10 regenerations and
storage at +4°C for 30 days.
Thus, using of SAMs with hydrophilic surface, built of thiols with different length of
carbohydrogenic chains, effective and simple method of monAB immobilization, mild
method of regeneration and protease inhibitors cocktail for monAB protection are basis
for preparation of sensitive and stable immunosensore chip for determination of impotent
markers of hemostasis.
References
1. Label-free interaction analysis in realtime using surface plasmon resonance – Technology Note, 23, Biacore
systems, 2007, BR-9004-63, 28-9214-39 AA.
2. A New Approach to Generate Thiol-terminated SAMs on Gold – Jing-Jiang Yu. Agilent Technologies, Inc.2007.
5989-7699EN.
Sensogram 1 shows Fig binding with chip’s surface
C(Feg) = 100 ng/ml
Sonogram 2 shows fibrin binding with chip’s surface
C(fibrin) = 250 ng/ml
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