Abstracts
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Інститут молекулярної біології і генетики НАН України
2015
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irk-123456789-1527752019-06-17T17:36:28Z Abstracts GDRI meeting "From Molecular to Cellular Events in Human Pathologies" 2015 Article Abstracts // Вiopolymers and Cell. — 2015. — Т. 31, № 5, доп. — С. 1-38. — англ. 0233-7657 http://dspace.nbuv.gov.ua/handle/123456789/152775 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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GDRI meeting "From Molecular to Cellular Events in Human Pathologies" GDRI meeting "From Molecular to Cellular Events in Human Pathologies" |
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Інститут молекулярної біології і генетики НАН України |
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Abstracts // Вiopolymers and Cell. — 2015. — Т. 31, № 5, доп. — С. 1-38. — англ. |
| series |
Вiopolymers and Cell |
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2025-07-14T04:16:14Z |
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2025-07-14T04:16:14Z |
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1837594391218225152 |
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1
New cytotoxic agents and their combinations for the treatment of chemoresistant
glioblastoma and mantle cell lymphoma
S. Avdieiev1, L. Gera2, D. Havrylyuk3, R. Hodges2, R. Lesyk3, V. Ribrag4, Y. Vassetzky4
1Institute of Molecular Biology and Genetics, NASU, Kyiv Ukraine; 2University of
Colorado Denver, USA; Danylo Halytsky Lviv National Medical University, Lviv
Ukraine; 4Gustave Roussy Institute, Villejuif, France
Background. Glial tumors are driven by multiple molecular aberrations, chromosomal
instability and tumor heterogeneity; therefore combined multitarget anti-cancer therapy
aimed simultaneously at different elements of tumor formation mechanisms might be
more effective and will promote the extension of patients’ life. Results. Several
bradykinin antagonists (BA) and 4-thiazolidinones (TD) were analyzed for their cytotoxic
effect using different glioblastoma (GB) and mantle cell lymphoma (MCL) cells. BKM-
570 appeared to be the most effective agent with IC50 4 μM and 3,3 μM in rat glioma C6
and human glioblastoma U251 cell lines, correspondingly. ERK1/2 and AKT1
phosphorylation was suppressed in U251 cells after treatment by this compound, thus,
growth-repression effect of BKM-570 could be mediated by the modulation of MAPK-
and PI3K-signaling cascades. Temozolomide (TMZ), a first-line anti-gliomic drug used in
clinics, has only a temporary positive effect and severe side effects in GB patients. We
have shown that combination of 1 µM BKM-570 with only 10 µM temozolomide (TMZ),
led to about 80% growth reduction of C6 and U251 cells, compared to temozolomide used
alone. Thus, BKM-570 significantly potentiates TMZ cytotoxicity. Screening of 4-
thiazolidinones revealed ID28 to be the potent suppressor of C6 and U251 cells growth
(IC50 4 μM and 15 µM, correspondingly). ID4523 demonstrated the highest activity in C6
cells with IC50 0.13 μM. Treatment of MCL cells by this compound and its chemical
produced IC50 values of 0.27 µM for ID4526 and 0.16 µM for ID4527 as compared to
0.37 µM for doxorubicin. Recombinant proteins with cytotoxic properties are promising
therapeutic when used with chemotherapy and immunotherapy. Glioma-associated protein
CHI3L2 down-regulates proliferation of U251, as well as 293 cells. Furthermore, CHI3L2
protein inhibits cell viability of U251 cells more effectively than TMZ in therapeutic
concentrations. Combination of CHI3L2 and BKM-570 resulted in additive cytotoxic
effect in U251 cells. CHI3L2-mediated decrease of cell viability is associated with G1/S
transition arrest. We also analyzed the impact of CHI3L2 on key components of cell cycle
machinery, namely pRb, cyclin D, p53, and p21. CHI3L2 provoked a dramatic reduction
of pRB phosphorylation and a significant decrease of cyclin D1 expression. Moreover,
p53 expression level was substantially increased. We also demonstrated the upregulation
of cyclin-dependent kinase inhibitor p21, thus G1/S cell cycle arrest in CHI3L2 treated
cells could be due to activation of pRB and p53 and downregulation of cyclin D.
Conclusions. BKM-570 has a potent cytotoxicity against glioma cells and significantly
potentiates anti-gliomic drug TMZ. TD treatment suppresses of human and rat gliomas
and MCL cells growth. CHI3L2 protein, which is overexpressed in human gliomas,
inhibits glioma cells viability and enhances the cytotoxic properties of chemotherapeutic
agents, thus, could be considered as a potential component for complex chemotherapy.
Reduced cell viability after CHI3L2 treatment could be due to activation of pRB and p53,
an downregulation of cyclin D1. Acknowledgments. This work was supported in part by
a travel grant of the Boehringer Ingelheim Fonds ‘Multitargeted complex therapy of
human gliomas and lymphomas’ and FP7 Project 294932 ‘COMBIOM’.
2
DNA polymerase Iota participates in clustered damage repair
E. A. Belousova and O. I. Lavrik
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the
Russian Academy of Sciences, 630090 Novosibirsk, Russia; fax: +7 (383) 333-3677
sheffield@ngs.ru
Multiple DNA lesions occurring within one or two turns of the DNA helix known as
clustered damage are a source of double-stranded DNA breaks, which represent a serious
threat to the cells. Repair of clustered lesions is accomplished in several steps. If a
clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base
excision repair mechanism (BER). One of the BER stages involves specialized DNA
polymerases after excising of DNA damage. Here, we investigated DNA synthesis
catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA
substrates were used as model DNAs: partial DNA duplexes containing breaks of different
length, and DNA duplexes 5-formyluracil (5-foU) and uracil as a precursor of
apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we shown
that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes
having breaks of different length as substrates. In addition, we found that DNA
polymerase iota could catalyze DNA synthesis during repair of clustered damage via the
BER system by using both undamaged and 5-foU-containing templates. We found that
hPCNA (human proliferating cell nuclear antigen) acting here as a replication protein
increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.
This study was supported by the Russian Scientific Foundation (Grant No. 14-24-00038)
and the Russian Foundation for Basic Research (Grant No. 14-04-00268).
3
Modulation of GluN2B subunit-containing NMDA receptors expression and Spatial
long-term memory in medial septal immunolesioned rats
G. Beselia1, R. Solomonia1,3, M. Dashniani1, M. Burjanadze1, M. Mepharishvili1,3,
N. Chkhikvishvili1, S. Mataradze1,2, L Kruashvili1, T. Naneishvili1,2
Beritashvili Center of Experimental Biomedicine, 14 Gotua Street, 0160 Tbilisi,
St.Andrew the Firs-Called Georgian University of Georgian Patriarchate, 53a
Chavchvadze av. 0162 Tbilisi, Georgia;Institute of Chemical Biology Ilia State
University, 3/5 K. Cholokashvili Avenue, 0162 Tbilisi, Georgia
The hippocampus is important in the formation of spatial memory in both humans and
animals. The N-methyl-D-aspartate (NMDA) type of glutamate receptors in the
hippocampus has been reported to be essential for spatial learning and memory as well as
for the induction of synaptic plasticity. Evidence accumulated from recent studies
suggests that GluN2A and GluN2B subunit-containing NMDA-Rs preferentially
contribute to the induction of hippocampal LTP and LTD. Using a Morris water maze
(MWM) task, the LTP-blocking GluN2A antagonist had no significant effect on any
aspect of performance, whereas the LTD-blocking GluN2B antagonist impaired spatial
memory consolidation.
The present study was designed to investigate the effect of selective immunolesions of
cholinergic and GABA-ergic SH projection neurons [using 192 IgG-saporin (SAP) or
GAT-1 saporin (GAT), respectively] on spatial memory assessed in MWM and NMDA
receptor GluN2B subunit expression in the rat hippocampus. We used MWM training
protocol with eight training trials. One day after training, probe test with the platform
removed was performed to examine long-term spatial memory retrieval. We found that
immunolesion of medial septal cholinergic or GABAergic neurons did not affect spatial
learning as exhibited by a decreased latency to find the hidden platform across the eight
training trials. Trained control and SAP treated rats spent significantly longer than chance
(15 s) performances such as swimming time in test sector (where the hidden platform was
located). Moreover, they spent significantly longer in test sector than in the opposite
sector, confirming the establishment of long-term memory. In contrast, the preference for
test sector was abolished in medial septal GAT treated rats. Because GAT treated rats
learned the location of the hidden platform during training, the results suggest that GAT
treated rats could not remember the training a day later. We found that the expression
level of NR2B subunit of NMDA receptor in the hippocampus was decreased significantly
in the GAT treated group compared with the control and SAP treated groups.
In conclusion, our findings suggest that immunolesion of medial septal GABAergic
neurons can interrupt hippocampus�dependent spatial memory, possibly through
modulation of NMDA receptor subunit expression in the hippocampus. Moreover, our
finding that selective lesions of medial septal GABAergic neurons affects probe-test
performance but not spatial learning, suggests that septohippocampal GABAergic
projections are involved specifically in the consolidation or retrieval, but not in the
acquisition of long-term spatial memory.
4
Radiobiological effects modeling by using plant test-systems
M. E. Gogebashvili, N. I. Ivanishvili
Laboratory of Radiation Safety Problems
I.Beritashvili Center of Experimental Biomedicine
One of main feature of radiobiological effects at various structural-functional level of the
organization is their cascading formation. In the formation of radiobiological reactions
each level of the organization of living organism is characterized by specific mechanisms
of post–radiation recovery.
While with respect to cells, tissues and organs we face the realization of reparative,
regenerative and other compensative mechanisms, with respect to population, population
recovery rates are the leading criteria.
Various types living organisms differ from each other by radiosensitivity. The mentioned
feature varies even within one species (individual radiosensitivity). Hence, in order to
estimate the results of irradiation correctly, radiosensitivity study with respect to
organization level of living organisms will be reasonable. Although there is modern
equipment determining the damaging influence of various physical-chemical factors,
adequate estimation of injury level is practically impossible. At the same time the
structures that are widely used in identification of quantitative parameters of radiation
hazards are found in living organisms. The inhibition of cell proliferation activity or entire
inactivation is one of the most visible effects among the processes that are detected in the
result of damaging impact of radiation exposure. It is only natural that exploration of
specific bio-subjects targeted for modeling and the study of their individual
radiosensitivity enables to form general radiobiological concept. The diversity of plant
organisms and unique structures existence in their organization enable to use them as bio-
models. From this point of view it is essential to find such subjects that reveal
radiobiological effects even when their reaction is not adequate to damage level. In the
present work we have discussed the effectiveness of the use of plant test-systems in order
to study such radiobiological processes as: transmission of radiation damage in to cellular
generations, regulations of detection of somatic embryogenesis, and radio-resistance of
separate parts of secondary metabolism.
Key words: plant test-system, ionization radiation, radiobiological effects
5
Evaluation of the spatial prevalence of cancer in conditions of an inadequate data of
population
H. Grebenchuk
Beritashvili Center of Experimental Biomedicine, Tbilisi
H.Grebenchuk@lifescience.org.ge
We have introduced the concept of the spectral coefficient of the incidence rate, allowing
to assess the incidence rate under the circumstances of uncertain population data. The
quantitative relationship of this indicator with a standard (population) incidence rate has
been displayed and the main qualities of this connection have been studied.
The independence of the ratio between the standard and spectral coefficients of the
incidence from the tumor site induces has been determined. It has been shown that this
ratio is equal to ratio between signed numbers of cancer cases in area to be study and
region in whole.
It has been determined that the value of the standard (population) and the spectral
coefficients for the large populated areas are approximately equal, so their ratio is
approximately equal to one.
It’s estimated that the time series of standard, as well as spectral coefficients of the
incidence for specific populated areas can’t be distributed by Poisson random variables.
The algorithm for calculation of the Cochran-Student confidence interval from the
extension of the initial array has been built. The confidence intervals have been calculated
for the mean values of the spectral coefficients of the incidence of all sites of tumors in all
districts of Georgia.
The clearly pronounced spatial heterogeneity of the spectral coefficients of the incidence
of some localization of tumor has been shown, reaching the value of 5 – 6, allowing us to
suggest the existence of cancer-causing agents in a number of areas.
Key words: cancer spatial prevalence, evaluation of incidence spectral rate, estimation of
confidence intervals.
6
Antimicrobial peptides as anticancer agents
M. Grigolava, B. Vishnepolsky, M. Pirtskhalava
Laboratory of Bioinformatics of I. Beritashvili Center of Experimental Biomedicine
There is a considerable interest in developing anticancer agents with new mode of action.
Many natural or synthetic cationic peptides have been reported to show anticancer
activity. They are united in a separate class of anticancer peptides (ACP). Compared with
the traditional cancer treatments such as chemotherapy or radioactive treatment, peptides
with high specificity against cancer cells may present the way of killing cancer cells while
protecting normal cells and helping patients to recover rapidly. Since ACPs are not
directed to a specific extracellular or intracellular receptor, some mechanisms of
resistance can be impaired. The number of approved peptide-based drugs has been
increasing from the last few decades, which reflects the potential of peptides as
therapeutics. In order to assist the scientific community engaged in developing anticancer
drugs, information on ACPs and anticancer proteins scattered in the literature have been
collected and stored in the “CancerPPD” database
(http://crdd.osdd.net/raghava/cancerppd/).
Antimicrobial Peptides (AMP), produced by the innate immune system in response to
infectious agents, are currently considered for drug development. Many AMPs show
broad-spectrum toxicity against both bacteria and cancer cells. The electrostatic
interaction of ACPs with negatively charged components of plasma membrane of cancer
cells is believed to play a crucial role in the cancer-selective toxicity of ACPs. AMPs and
most ACPs have similar characteristics: small length, positive charge, amphyphaticity.
The mechanism of their action is not fully understood so far. Most ACPs, like AMPs,
exhibit membrane-lytic mode of action. However, some ACPs and AMPs induce
apoptosis via disruption of mitochondrial membrane; consequently, AMP and ACP can be
united in the same class of membrane-active peptides.
Anticancer /Antimicrobial drug design has focused on peptides with desired properties
through the set of known or predicted peptide sequences. Effective predictive methods
will allow investigators to conduct task-oriented design of new peptides, and decrease
costs of new drug production. This requires data regarding the peptide’s chemical
structure and antimicrobial/anticancer activities. We have developed a Database of
structure and Antimicrobial/Cytotoxic Activities of peptides (DBAASP) (http//:
dbaasp.org). DBAASP is the depository of information for structure/activity study
providing the information and analytical resources to the scientific community in order to
develop new peptide drugs. The "Prediction" utility allows to reveal a potential
antimicrobial activity for the queried peptides based on amino acid sequence. This utility
is based on the machine learning algorithm and peptide parameters such as hydrophobic
moment, charge density, depth-dependent potential, etc. The predictive model was
optimized by the positive and negative training set of peptides. Positive set was formed by
AMPs from DBAASP (Training-AMP). The negative one consisted of randomly selected
fragments of non-secretory proteins from UniProt (training-UniProt). Two different
positive sets were used for testing : test set from DBAASP (test-AMP) and test set from
CancerPPD database (test-ACP). The accuracy of predictions for both testing was 92% for
test-AMP and 90% for test-ACP. ). The predictive model developed on the basis of AMP
is successfully working in the case of anticancer peptides. These results give additional
proof that the data on AMP can be used for the design of anticancer drug.
7
PARP1 and PARP2 catalyze poly(ADP-ribosyl)ation of DNA strand break termini.
Ibtissam Talhaoui1,*, Natalia A. Lebedeva2,*, Mikhail M. Kutuzov2, Maria V. Sukhanova2,
Bakhyt T. Matkarimov3, Murat K. Saparbaev1, Olga I. Lavrik2,4
and Alexander A. Ishchenko1
1Groupe «Réparation de l’ADN», Université Paris Sud, Laboratoire «Stabilité
Génétique et Oncogenèse» CNRS, UMR 8200, Gustave Roussy, F-94805 Villejuif
Cedex, France.
2SB RAS Institute of Chemical Biology and Fundamental Medicine, Lavrentiev Av.
8, 630090 Novosibirsk, Russia.
3Nazarbayev University Research and Innovation System, Astana 010000,
Kazakhstan.
4Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St.,
Novosibirsk 630090, Russia.
Poly(ADP-ribose) polymerases (PARPs) use nicotinamide adenine dinucleotide (NAD+)
to catalyze the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to
the acceptor amino acid residues of nuclear proteins. PARP1 is activated up to 500-fold
when bound to DNA strand breaks. In vivo, when DNA is in the chromatin form, PARPs
work on single- and double-strand DNA breaks (SSB and DSB) by recruiting and
assembling DNA repair factors. Here, we investigated interactions of PARP enzymes with
the DNA repair intermediates of base excision repair (BER) and nucleotide incision repair
(NIR) pathways. Our results revealed that both mammalian PARP1 and PARP2 can
covalently modify DNA oligonucleotide duplexes by addition of multiple poly(ADP-
ribose) units to 3' extremities of DNA. Therefore, DNA can be regarded as a substrate for
PARP1 and PARP2 which catalyze a post-replication modification of DNA. The covalent
DNA PARylation is a reversible process since PARG removes the PAR polymer from
DNA with high efficiency and restores native DNA structure. Finally, this newly
discovered type of post-replication modification of DNA mediated by PARPs provides an
heuristic insight into molecular mechanisms involved in DNA repair, transcription and
chromatin dynamics in eukaryotic cells.
8
Mechanisms of heat stress-induced cell senescence.
N. Petrova1,2, A. Velichko1, O. Kantidze1
1Laboratory of Structural and Functional Organization of Chromosomes, Institute of
Gene Biology RAS, 119334 Moscow, Russia. e-mail:
2Department of Molecular Biology, Moscow State University, 119991 Moscow,
Russia.
kantidze@gmail.com
The aim of this study was to investigate delayed effects of heat stress on mammalian
cells.
Methods: Comet assay and modified PFGE were used to measure DNA damage. Indirect
immunofluorescence was applied to track DNA damage response events. FISH was used
to quantify the levels of hyper-replication of certain genomic loci. Gene expression
analysis was performed using qRT-PCR and Western blot hybridization.
Results: In the present study we demonstrated that acute heat stress (HS) induces cell
senescence in human cells – HS results in a robust G2/M cell cycle arrest within
approximately one cell cycle. Heat-treated human cells acquire most of the cell
senescence marks (proliferation arrest, senescence-associated (SA) beta-gal activity,
changes in cell size/morphology, etc.) in few days after the treatment. Interestingly, HS
does not lead to formation of SA heterochromatin foci (SAHF) – only ring-like
heterochromatin domains are formed. We demonstrated that HS-induced cell senescence
state is maintained by p21. We found that persistent DNA damage response, which
depends on DNA hyper-replication, is the trigger of HS-induced cell senescence.
Conclusion: Heat stress induces p21-dependent cell senescence.
Funding: The work was supported by RFBR (grants 13-04-00604, 14-04-
32169).
9
Mechanism of suppression of limbic motor seizures by activation of the thalamic
reticular nucleus
N. A. Khizanishvili, I. G. Bilanishvili, M. G. Barbakadze, Z. I. Nanobashvili
Department of Neurophysiology,
Ivane Beritashvili Experimental BioMedicine Centre, Tbilisi, Georgia.
At present, a number of forms of epileptic attacks are intractable. The search for
alternative possibilities for therapy of such disorders motivated ones to study such
“antiseizure” approaches as electrical stimulation of profound structures of the brain.
Several studies have indicated that thalamic nuclei are involved in seizure development
and expression. For example, it is well-established that thalamocortical systems
participate in the generation of pathological rhythms with 3-4 Hz frequency. Neurons in
the thalamic reticular nucleus (TRN) are involved in the mechanisms of synchronization
and generation of this type of seizure activity. The TRN forms a shell around the dorsal
thalamus and consists of GABA-ergic neurons, which provide a strong inhibitory input on
thalamic relay cells.
Because our recent data [Exp. Neurol. 2003.] provide the first evidence that stimulation
of TRN can act to suppress limbic motor seizures, objective was to explore a possible
mechanism of blocking the limbic motor seizure reactions induced by activation of the
TRN.
Experiments were carried out on adult cats (n = 4). Simultaneous recordings were made
expra- and intracellularly in the thalamic ventrolateral nucleus and TRN.
Our experiments support the hypothesis that the activity of the TRN neurons is
potentiated in the course of activation of this structure and inhibition of generalization of
the seizures can be based on this phenomenon. Thus, stimulation of the TRN appears to be
a rather valuable methodical tool that can open up prospects in the development of new
anticonvulsive strategies in the treatment of temporal lobe epilepsy.
10
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with
apurinic/apyrimidinic sites in DNA
Anastasiya A. Kosova, Svetlana N. Khodyreva, Olga I. Lavrik
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the
Russian Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk, 630090, Russia
kosova.anastasiya@gmail.com
Aim. Apurinic/apyrimidinic (AP) sites are some of the most frequent DNA damages and
the key intermediates of base excision repair. Certain proteins can interact with the
deoxyribose of the AP site to form a Schiff base, which can be stabilized by NaBH4
treatment. The aim of this study was to identify the human cell extract protein specifically
interacting with AP DNA containing single-stranded regions. Methods. The Schiff base-
dependent crosslinking of proteins with AP DNA (borohydride trapping) in combination
with gel electrophoresis and MALDI-TOF MS was used to identify the protein.
Chromatography was used to enrich the cell extract in the target protein and to purify the
protein. Results. We performed borohydride trapping of human cell extract proteins with
several types of AP DNA. In the case of single-stranded AP DNA and AP DNA duplex
with both 5� and 3� dangling ends, the major crosslinking product had apparent
molecular mass of 45 kDa. Using peptide mass mapping based on mass spectrometry data,
we identified the protein forming this adduct as an isoform of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). GAPDH is a glycolytic enzyme with many additional putative
functions, such as interaction with nucleic acids, including damaged DNA, and DNA
repair enzymes. We investigated interaction of GAPDH purified from HeLa cells and
rabbit muscles with different AP DNAs. In spite of the ability to form a Schiff-base
intermediate with the deoxyribose of the AP site, GAPDH does not display the AP lyase
activity. In addition, along with borohydride-dependent adducts with AP DNA containing
single-stranded regions, GAPDH was shown to form also stable borohydride-independent
crosslinks with these DNA. NAD+ inhibits GAPDH–AP DNA adduct formation. GAPDH
was proven to crosslink preferentially to AP DNA cleaved via β-elimination mechanism
(spontaneously or by AP lyases) as compared to DNA containing the intact AP site. The
level of GAPDH–AP DNA adduct formation depends on oxidation of the protein SH-
groups; disulfide bond reduction in GAPDH leads to loss of its ability to form adducts
with AP DNA. Conclusion. Formation of stable adducts with AP sites by GAPDH may
be involved in regulation of DNA metabolism. One can assume that under the oxidative
stress GAPDH undergoes disulfide bond formation that results in enhancement of its
DNA binding capacity and translocates to the nucleus. When the pool of NAD+ is
exhausted due to the PARP-1 activity, GAPDH can bind with DNA and, if the unrepaired
AP site is present, GAPDH can be trapped in the stable covalent complex that would
hamper DNA repair.
The work was supported by the Russian Scientific Foundation projects no. 14-24-00038,
14-14-00501, “Molecular and Cellular Biology” program of the Russian Academy of
Sciences, and Russian Foundation of Basic Research grant no. 13-04-01426.
11
mRNA and DNA selection via protein multimerization:
YB-1 as a case study
Dmitry A. Kretov1,2, Patrick A. Curmi1, Loic Hamon1, Sanae Abrakhi1,
Bénédicte Desforges1, Lev P. Ovchinnikov2 and David Pastré*,1
1 - Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques,
INSERM
U829 and Université Evry-Val d’Essonne, EA3637, Evry, 91025 France.
2 - Institute of Protein Research, Russian Academy of Sciences,
Pushchino, Moscow Region
142290, Russia.
Translation is tightly regulated in cells for keeping adequate protein levels, this task being
notably accomplished by dedicated mRNA-binding proteins recognizing a specific set of
mRNAs to repress or facilitate their translation. However the binding of isolated mRNA-
binding proteins to short mRNA sequences/structures requires a high protein affinity to
these specific sites, which is not a common feature among mRNA-binding proteins.
Indeed many of them rather display a weak specificity to short and redundant sequences.
Here we examined an alternative mechanism by which mRNA-binding proteins inhibits
the translation of specific mRNAs, using YB-1, a major translation regulator, as a case
study. We found using a single molecule approach that YB-1 triggers a homo-
multimerization process based on a cooperative binding to mRNA. By doing so, YB-1
may inhibit translation of the mRNAs on which it has formed multimers. This novel
mechanistic view on mRNA selection may be shared by other proteins considering the
elevated occurrence of multimerization among mRNA-binding proteins. Interestingly, we
also demonstrate how, by using the same mechanism, YB-1 can form multimers on
specific DNA structures, which could provide novel insights into YB-1 nuclear functions
in DNA repair and multi-drug resistance.
12
The Study of Structure and Function of Muscle Giant Proteins
R. Kupatadze, N. Gachechiladze J. Gogorishvili, K. Kuridze, T. Shonia,
L. Lobzhanidze, M. Zaalishvili.
Department of Biophisics, I.Beritashvili Center of ExperimentalBiomedicine, Tbilisi
Besides the main contractile proteins the muscle contains the giant proteins with high
molecular mass - Titin (skeletal muscle) and Smitin (smooth muscle )with molecular
weights 3000 kD and 2000 kD accordingly.
Smitin, likewise Titin, has similar molecular morphology and location within the
contraction apparatus, but its role in the smooth muscle tonic contraction is unexplained
(there, in contrast to the striated muscle, the sarcomere is not distinctly formed and
contraction character is different).
By means of Reverse sieve super pure preparations of Titin and Smitin have been
received.
By the method of sedimentation analysis it was shown that speed of sedimentation of
Smitin (C-titin) is less than that of Titin (Smitin S20,ω =9.3S;Ttitin S20,ω =13.8S) which is
also due to the higher molecular mass of Titin.
The analysis of viscosimetrical data shows that the characteristic viscosity for Titin and
Smitin is not high (Smitin ρcharac=0.21; Titin ρcharac=1.32). The apparent low viscosity
obtained during the experiment, is typical for globular proteins but we believe that this
fact is explained by the elastic properties of the investigated proteins.
Calorimetric studies show that thermal denaturation of Titin starts at 400C and ends at
800C. Transition temperature is T=59.40C and for Smitin is starts at 500C and ends at
800C. Transition temperature is T=66.10C.
The influence of Smitin (C-titin) on Mg2+-activated ATPase activity of chicken smooth
muscle (stomach) Actomyosin in different area conditions (ionic strength, pH, different
concentrations of Smitin) was studied. It was shown that Smitin, likewise Titin, causes the
increasing of Mg2+-activated ATPase activity of Actomyosin. Obtained results confirm
that in smooth muscle Smitin has the same effect on Actomyosin ATPase activity as Titin
has on skeletal muscle ATPase activity. Smitin stipulates muscle elastic properties, on the
one hand, and on the other hand it is the “scaffold” for the proteins participating in muscle
contraction, forming the supermolecular complex with these proteins.
Our work is the first step for explanation of a role of those proteins in cell cytoskeleton
formation and their participation in the process of muscle contraction.
13
Human tyrosyl-DNA phosphodiesterase 1: new activities and development of enzyme
inhibitors as anticancer drugs
O. Lavrik1,2
1Institute of Chemical Biology and Fundamental Medicine, 630090, Russia,
Novosibirsk, Lavrentiev Av., 8
2Department of Natural Sciences, Novosibirsk State University, 630090, Russia,
Novosibirsk, Pirogov St., 2
lavrik@niboch.nsc.ru
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is responsible to process topoisomerase 1
(TOP1) – DNA adducts as well as to hydrolyze a variety of other DNA 3′-substituents.
We have shown recently that TDP1 can initiate repair of apurinic/apyrimidinic (AP) sites
located in the internal positions of DNA generating breaks with the 3΄- and 5΄-phosphate
termini [1-3]. This activity was not observed for TDP1 SCAN mutant responsible for
neurodegeneration. Polynucleotide kinase phosphatase, Polβ and DNA ligase coordinated
with XRCC1 are completing repair of the AP sites. The AP site cleaving activity of TDP1
is shown to be stimulated by the key modulator of base excision repair (BER) –
poly(ADP-ribose)polymerase 1 (PARP1). The data suggest a role of TDP1 in the new
APE1-independent BER pathway in mammals. This activity can contribute to repair of
AP sites particularly in ssDNA structures or in the context of cluster-type lesions. The
specific real-time fluorescent detection of the AP-site cleaving activity of TDP1 was
developed, which is not sensitive to AP site cleavage by APE1 and useful to evaluate a
biological significance of new TDP1 function to initiate cleavage of AP sites. TDP1 is a
promising target for antitumor therapy based on TOP1 poison-mediated DNA damage.
The row of novel benzopentathiepins and other compounds were synthesized and tested as
TDP1 inhibitors using an original oligonucleotide-based fluorescence assay [4]. The
benzopentathiepines showed IC50 values in the range of 0.2–6.0 µM. The specificity of
these inhibitors was investigated by using other target DNA repair proteins such as
PARP1, PARP2, AP-endonuclease 1 and DNA polymerase beta. The study of cytotoxicity
of these compounds revealed that their action leads to the apoptotic cell death of cancer
cells. Therefore the new class of very effective inhibitors of TDP1 was elaborated, which
are very prominent to improve cancer therapy based on TOP1 poison–mediated DNA
damage.
Financial Support: Ministry of Education and Science of Russian Federation, State
Agreement RFMEFI60414X0018, CDRI program “From Molecular Events to
Human Pathology”
References
1. Lebedeva N.A., Rechkunova N.I., Lavrik O.I. FEBS Lett., 2011, 585, 683-686.
2. Lebedeva N.A., Rechkunova N.I., El-Khamisy S.F., Lavrik O.I. Biochimie, 2012, 94,
1749-1753.
3. Lebedeva N.A., Rechkunova N.I., Ishchenko A.A, Saparbaev M., Lavrik O.I. DNA
Repair, 2013, 12, 1037– 1042.
4. Zakharenko A., Khomenko T., Zhukova S., Koval O., Zakharova O., Anarbaev R.,
Lebedeva N., Korchagina D., Komarova N., Vasiliev V., Reynisson J., Volcho K.,
Salakhutdinov N., Lavrik O. Bioorg. Med. Chem,. 2015, 23, 2044-2052.
14
Postsynaptic reactions of cat somatosensory cortex neurons in response to painful
stimualation and analgesia
T. Labakhua, T. Janashia, G. Gedevanishvili
Ivane Beritashvili Center of Experimental Biomedicine, Tbilisi, Georgia
We studied effects of electrical stimulation of the substantia nigra (SN), locus coeruleus
(LC), raphe nuclei (RN), substantia innominata (SIn), nucleus caudatus (NC) and central
grey (CG) on postsynaptic processes evoked in neurons of the cat somatosensory cortex
by excitation of nociceptive and non-nociceptive afferent inputs (intense stimulation of
the dental pulp and moderate stimulation of the thalamic ventroposteromedial nucleus,
VPMN, respectively). We analyzed intracellularly recorded activity of cortical cells
activated exclusively by stimulation of nociceptors and cells activated by both nociceptive
and non-nociceptive influences (“nociceptive” and “convergent” neurons). In neurons of
both groups, stimulation of both nociceptive afferents and thalamic VPMN resulted in the
development of successions of EPSP – action potential (AP) or their series – IPSP (IPSP
duration 200–300 msec). Conditioning electrical stimulation of the above-mentioned
nuclei induced suppression of synaptic reactions that occur in cortical neurons in response
to stimulation of nociceptive inputs. The maximum decrease in the amplitude of the IPSP
was observed at test intervals of 600 to 800 msec when stimulated nuclei containing
biogenic amines and 100-150 msec at conditioning electrical stimulation of cholinergic
structures. We observed certain parallelism between conditioning influent ions of CG
activation and effects of systemic injections of morphine. Discusses is the physiological
significance of presumebly dendritic action potentials observed in our experiments.
Decrease in the amplitude or complete postsynaptic inhibition of IPSP in cortical neurons
under different treatments associate with the occurrence of convulsive epileptic activity,
and at the painful action with analgesic effect. Discussed are the mechanisms for
modulatory influences exerted by conditioning stimulation of SN, LC, RN, NC, SIn and
CG on the somatosensory neurons, activated upon excitation of high-threshold
(nociceptive) afferent inputs. Such modulation is probably based on changes developing
in both pre and post-synaptic intracortical mechanisms.
15
Broken proto-oncogenes AML1 and MLL leave the inherent chromosome territories
in human lymphoid cells treated with DNA topoisomerase II poison etoposide
N. A. Lomov1,2, M. A. Rubtsov1,2, D. A. Alexeyevsky3,4, Y. S. Vassetzky2,5, S. V. Razin1,2,6
and O. V. Iarovaia2,6
1Department of Molecular Biology, Faculty of Biology, M.V.Lomonosov Moscow
State University, Moscow, Russia; 2LIA 1066 French-Russian Joint Cancer Research
Laboratory, Villejuif, France–Moscow, Russia; 3National Research University Higher
School of Economics (HSE), Faculty of Humanities, School of Linguistics, Moscow,
Russia; 4A.N. Belozersky Institute Of Physico-Chemical Biology, Lomonosov
Moscow State University, Moscow, Russia; 5UMR8126, Université Paris-Sud, CNRS,
Institut de cancérologie Gustave Roussy, 94805 Villejuif, France ; 6Institute of Gene
Biology RAS, Moscow, Russia
ma_rubtsov@mail.ru
Aim: Today it is known that treatment of cells with DNA topoisomerase II poisons (such
as etoposide) leads to the formation of double stranded DNA breaks in breakpoint cluster
regions (BCRs) of known proto-oncogenes such as MLL or AML1. And that incorrect
repair of these breaks may cause chromosomal rearrangements which in turn may induce
the so-called “treatment-related” leukaemias. But the exact mechanisms of these
rearrangements remain unclear. Our experimental data support the “breakage-first” theory
supposing that DNA breaks formed at distant locations might be brought together and
produce inter-chromosomal translocations. Methods: We have treated cultured human
lymphoid cells (Jurkat) with etoposide or camptothecin and then visualized the
chromosome territories and proto-oncogene parts upstream and downstream of
corresponding BCR by using 3D-FISH technique. In order to increase statistical
significance of results, we have analyzed hundreds of cells in each experiment. To
facilitate the task we have wrote a computer software in order to analyze three-
dimensional confocal images automatically. The first step of analysis is the detection of
cells by positioning of uniform cylinders overlapping the cells in optimal places within
image. Next step is the detection of signals as groups of the brightest pixels within each
cell. Visual control of random subsample of images confirmed the correctness of the
automatic detection of both cells and signals. Final step is the measuring the distances
between determined signals. Results: New software allowed us to obtain number of data
and reliable statistics. We have found that exposure of Jurkat cells to etoposide resulted in
frequent cleavage of MLL and AML1 genes, with the flanks of the break located distant
from each other and are often found outside the inherent chromosome territories.
Therewith it was no differences between 5'- and 3'- flank of the breaks. It was also
observed that spatially separated flanks of proto-oncogenes are more frequently located
outside chromosome territory than non-separated ones. Treatment of cells with
camptothecin does not affect the integrity of MLL and AML1 genes. Conclusion: Our
findings demonstrate that flanks of the breaks introduced in BCR of MLL or AML1 gene
under inhibition of DNA topoisomerase II acquire additional mobility inside the nucleus
and tends to be on the edge or even outside the inherent chromosome territory, which
might allow the meeting and incorrect joining with its translocation partners.
The work was partially supported by the Russian Foundation of Basic Research (grant
14-04-93105_CNRS_а)
16
Squalenoylated-siRNA for the treatment of thyroid and prostate carcinomas
Giorgia Urbinati1, Ali Hafiz1, Didier Desmaële2, Patrick Couvreur2
and Liliane Massaad-Massade1
1 UMR 8203 CNRS, laboratory of Vectorology and anti-cancerous therapeutics,
Gustave Roussy, 94805 Villejuif, France.
2 UMR 8612 CNRS, Institut Galien, 5, rue Jean-Baptiste Clément 92296 Chatenay-
Malabry France
We aim at introducing a personalized treatment for patients affected by cancers
harbouring fusion oncogenes (FO). FO are the products of chromosomal rearrangements
that are responsible for the development of more than 20% of cancers. In our laboratory,
we focused on two pathologies harbouring FO: papillary thyroid carcinomas (PTC) where
RET/PTC is found in 70% of cases and prostate cancers (PCa) harbouring TMPRSS2-
ERG present in 50% of biopsies. Because FO are only present in cancer cells, they
represent a promising target for personalized therapies based on small-interfering RNA
(siRNA). siRNA, short oligonucleotides able to knockdown genes at mRNA level, are
highly specific and present low toxicity; however their administration is a major challenge
because the biological efficacy is hampered by their poor stability in biological
environments. It is therefore necessary to implement methods for their protection, such as
vectorization. In vitro, siRNA designed across the RET/PTC and TMPRSS2-ERG
sequences, inhibited specifically the expression of the corresponding oncogenes, and
impaired cell viability of both PTC and PCa cells, respectively. Induction of apoptosis
was also observed. To deliver and protect siRNA, we covalently linked them to squalene
(SQ) and the resulting bioconjugate was able to self-assemble into stable nanoparticles.
The siRNA-SQ nanoparticles injected intravenously in mice were able to reduce
drastically tumour growth in PTC and PCa xenografts following inhibition of RET/PTC
and TMPRSS2-ERG oncogenes and oncoproteins, respectively. This strategy opens new
therapeutic perspectives for the use of siRNAs-squalene nanoparticles as an innovative
nanomedicine for patients affected by cancer pathologies harbouring FO.
17
The primer RNA packaging complex of HIV-1: understanding its assembly and
disassembly
Fawzi Khoder Agha, Toufique Islam Khandekar, Martine Comisso, and Marc Mirande
Institute for Integrative Biology of the Cell (I2BC), CNRS, Université Paris-Saclay,
CEA, CNRS, Université Paris-Sud, 1 avenue de la Terrasse, 91190 Gif-sur-Yvette,
France
The replication phase of HIV-1 viral RNA involves reverse transcription, which converts
the viral RNA into corresponding double stranded DNA. This step is performed by
reverse transcriptase using the tRNA3
Lys of the host cell as a primer. We previously
showed that tRNA3 Lys packaging into HIV-1 particles involves the assembly of the
packaging complex GagPol:mLysRS: tRNA3
Lys. The mLysRS, or mitochondrial form of
lysyl- tRNA synthetase, is hijacked from the host cell by GagPol polyprotein of HIV-1
and is selectively incorporated into the budding virion particle. It serves as a carrier for
tRNA3
Lys. The catalytic domain of mLysRS interacts with the TF (Transframe) and IN
(Integrase) domain proteins of Pol domain of the GagPol polyprotein. A better
understanding of the packaging complex may lead to the design of inhibitors at this stage
of the HIV-1 replication. The precursor of Pol protein is very difficult to express and
purify because of its tendency to aggregate and undergo proteolysis. This study was
designed to reconstruct the packaging complex in vitro with a surrogate of Pol protein
containing only the TF and IN domains separated by an artificial linker. The purpose is to
analyze the binding affinity of Pol surrogate to mLysRS in the presence or absence of
tRNA3 Lys and to further analyze if the binding of tRNA3
Lys to mLysRS is affected in the
presence of the Pol surrogate. The interaction between the Pol surrogates containing
spacers of different lengths was quantified using a FRET-HTRF assay. This should help in
determining a minimum distance between the TF and IN domains which should be
sufficient for a proper binding with the mLysRS. The interaction of tRNA3 Lys with
mLysRS was then quantified in the presence of a suitable Pol surrogate, using a
fluorescence polarization assay. We determined that the binding of tRNA3
Lys to mLysRS
is strengthened in the presence of Pol surrogate. This analysis allowed us to express and
purify a derivative of Pol that can form a binding complex with mLysRS and tRNA3
Lys,
and that can substitute to GagPol in further biochemical and structural studies of the
GagPol:mLysRS: tRNA3
Lys packaging complex. This surrogate of Pol will be a powerful
tool in the understanding of this essential process in HIV-1 replication.
18
Sigma-receptor-1 and mGluR5 may participate in Rac-dependent oncogenesis
through modulation of macrophage activity
N. Natsvlishvili., L. Shanshiashvili, D. Mikeladze
I.Beritashvili Center of Experimental Biomedicine, Tbilisi, Georgia, Ilia State University,
Tbilisi, Georgia
A defining feature of tumor inflammation is the polarization of M1 into M2 macrophages
which promotes tumor growth, angiogenesis, invasion, and metastasis. The mechanism(s)
that control the M1-M2 transition remain to be determined. During inflammatory states,
activated innate cells, as well as tumor cells, release amino acid glutamate that induce
innate cell migration by activating class I/5 metabotropic glutamate receptors
(mGluR1/5). Mutation of mGluR1/5 detected in several tumors triggers a downstream
signaling cascade leading to activation of Rho family GTPase, Rac1, and Rac2. Rac
overexpression in tumors may play a role in cancer progression. Targeting the Rac
pathway is an approach to cancer treatment.The sigma1R (Sig1R) is an endoplasmic
reticulum (ER)-resident molecular chaperone expressed at the mitochondria-associated
ER membranes (MAM). Cell treatment with sigma antagonists causes rounding,
detachment and growth inhibition of several cancer cells, however whether Sig1R protects
cancer cells from death remains to be elucidated.To investigate the interplay between
SigR1, Rac, and mGluR5, immunoprecipitation experiments were performed. Sig1R
interacted with the Rac1 and Rac2. This interaction was increased in the presence of
sigma-agonists, decreased by sigma-antagonists. Sig1R-Rac interaction was enhanced by
GppNHp, suggesting that active Rac interacted with Sig1R. Specific
association/dissociation of Sig1R with an inositol-3-phospate receptor (IP3R) and BiP was
also found. p21-activated kinases (PAK) downstream effectors of Rac. PAK can
phosphorylate various regulatory proteins, including pro-apoptotic Bad. Sigma-agonists
increase PAK-dependent phosphorylation, causing dissociation of Bad from mitochondria,
thus sigma-ligands could operate through SigR1/Rac complex by changing the PAK
activity. Rac1 is be involved in the assembly and activation of ROS-generating enzyme -
NADPH oxidase (NOX). ROS are increased in the presence of sigma-agonist
(pentazocine), and sigma-antagonist (haloperidol) eliminated this effect, suggesting that
that sigma-ligands could operate through SigR1/Rac complex by changing the NOX
activity. Sigma 1-receptor induces immunosuppression through modulation of IP3R and
Rac in macrophages. In activated macrophages, the binding of SigR1 to IP3R sigma
ligands was shown to be decreased, and binding of SigR1 to Rac2 was increased. SigR1
was immunoprecipitated with mGluR5 suggesting that intracellular glutamate receptor
may be part of a macromolecular complex in MAM. We next transfected mGluR5 cDNAs
into macrophages. This decreased the binding of SigR1 to IP3R. Immunostimulants, like
LPS, induced the association of SigR1 with Rac only in non-transformed cells., whereas
sigma-agonists increased the binding of SigR1 to Rac in mGluR5-overexpressing
macrophages that had a decreased sensitivity to the immunostimulants. Our data suggest
that Sig1R dissociates from IP3R, and then associates with Rac1 and Rac2, and through
this association/dissociation, sigma ligands can regulate Rac activity. The intracellular
glutamate receptor may participate in induction of SigR1-Rac association. This
association could lead to activation of downstream effectors (Pak and NOX) of Rac, to
initiation of actin rearrangement and production of ROS. This may induce the M1→M2
transition and immunosuppression contributing to progression of tumorigenesis.
19
Translation elongation factor eEF1A1 and its oncogenic variants
Boris Negrutskii and Anna El’skaya
Laboratory of Protein Biosynthesis,
Institute of Molecular Biology and Genetics,
150 Acad. Zabolotnogo Str., Kyiv, Ukraine
negrutskii@imbg.org.ua
Aim. To find specific features of oncogenic proteins eEF1A2 and PTI-1 which make them
different from very similar non-oncogenic translation factor eEF1A1 and, thus, to predict
possible ways of their involvement in cancer development.
Methods. Complexes of translation elongation factor eEF1A1 and its very similar proto-
oncogenic or oncogenic analogs eEF1A2 and PTI-1 correspondingly, with both
translational and non-translational partners were studied by crystallography, atomic force
microscopy, confocal microscopy, low-speed sedimentation and pull-down assays.
Results. Different shape of actin bundles produced by eEF1A1 or eEF1A2 is shown. PTI-
1 protein is not capable of forming actin bundles due to its inability to dimerize, so it
cannot be sequestered by actin compartments in cell.
eEF1A1 rather than oncogenic eEF1A2 is able to interact with Ca2+calmodulin and other
regulatory proteins. Ca2+-calmodulin interferes with eEF1A1 binding to translational
(tRNA) and non-translational (actin) partners.
PTI-1 protein retains similar to eEF1A1 translation elongation activity as well as ability to
bind nucleotide exchange factor eEF1B. Surprisingly, eEF1B cannot stimulate nucleotide
exchange in PTI-1. Based on our recent crystallographic data we built a molecular model
of nucleotide exchange in eEF1A explaining inability of PTI-1 to be regulated by eEF1B.
Conclusion.
eEF1A1 and its oncogenic analogs can fulfill similar functions but the oncoproteins are
suggested to perform these functions in non-controlled way, that may be one of specific
features of cancer cells.
20
Transient receptor potential channel inactivation by non-steroidal anti-
inflammatory drugs
I. Nozadze, N. Tsiklauri, E. Abzianidze,G. Gurtskaia, M. G. Tsagareli
Laboratory of Pain and Analgesia, Beritashvili Center for Experimental Biomedicine,
Tbilisi, Georgia
Transient receptor potential (TRP) cation channels have been extensively investigated as
targets for analgesic drug discovery. These channels are the largest group of sensory
detector proteins expressed in nerve terminals and pain receptors, and are activated by
temperature and chemicals that elicit hot or cold sensations. Stimuli include menthol,
cinnamaldehyde (CA), gingerol, capsaicin, allyl isothiocyanate (AITC) (a natural
compound of mustard oil), camphor, and eugenol. The thermal thresholds of many TRP
channels are known to be modulated by extracellular mediators, and released by tissue
damage or inflammation, such as bradykinin, prostaglandins, and growth factors.
Antagonists of these channels are likely promising targets for new analgesic drugs at the
peripheral and central levels. Because some non-steroidal anti-inflammatory drugs
(NSAIDs) are structural analogs of prostaglandins, we examined three widely used
NSAIDs (clodifen, ketorolac, and xefocam) on the activation of TRPA1 and TRPV1
channels using thermal paw withdrawal (Hargreaves) test and mechanical paw withdrawal
(von Frey) test in male rats. Thermal withdrawal latencies and mechanical thresholds for
both hind paws were obtained with 5, 15, 30, 45, 60, and 120 min intraplantar post-
injection of CA and AITC or vehicle. Twenty minutes prior to the start of the experiment,
clodifen, ketorolac or xefocam were pre-injected in the same hindpaw and animals were
examined by these two tests. After pretreatment of all three NSAIDs in the ipsilateral
(injected) hindpaw that produced strong antinociceptive effects, CA, AITC, and capsaicin
caused significant decreases in latency of the thermal withdrawal reflex compared with
vehicle or the contralateral hindpaw. The same findings were observed for the paw
withdrawal threshold. In approximately 30 min the effects of CA, AITC, and capsaicin
returned to baseline. The findings in this study are different from our previous results,
where TRPA1 agonists CA and AITC and TRPV1 agonist capsaicin produced
hyperalgesia for nearly 2 h and resulted in facilitation of these withdrawal reflexes. Thus,
we show for the first time an inactivation of TRPA1 and TRPV1 channels by NSAIDs to
channel agonists in two behavioral assays.
21
Topokaryotyping – a novel approach to study nuclear organization
Jurisic, C. Robin, V. Ogryzko
CNRS UMR 8126, Universit Paris-Sud 11, Institut Gustave Roussy
114, rue Edouard Vaillant, Villejuif, France, 94805.
vogryzko@gmail.com
The aim of this study was to develop a new approach to study nuclear organization at the
single cell level.
Methods: Proximity Utilizing Biotinylation was used to label chromatin proximal to
nuclear envelope, by coexpressing BirA-emerin (NE protein emerin fused to Biotin ligase
BirA) and BAP-H2A histone (H2A fused to Biotin acceptor domain BAP, targeted
specifically by BirA for biotinylation). After biotin was removed from the medium and
cells were allowed to enter mitosis, mitotic spreads were generated and the location of
biotinylated chromatin was visualized via streptavidin-Cy3 staining. The quality of the
spreads was sufficient for partial identification of the chromosome in the spread by
karyotyping; moreover, the streptavidin labelling can be combined with FISH for more
conclusive chromosome identification.
Results: In the present study we demonstrated that the chromatin domains that are
proximal to nuclear envelope appear as discrete bands on mitotic chromosomes.
Importantly, the band patterns for individual chromosomes differ from one mitotic spread
to another, pointing to variability of chromosome domain localization in the interphase
nuclei and thus to importance of the study of nuclear organization at the single-cell level.
Treatment of cells with various stressors affects the pattern distribution in the cell
population.
Conclusion: We have developed a new approach to study nuclear organization at the
single cell level.
Funding: The work was supported by the ARC grant n° SFI20121205936.
22
Functional state of red blood system as a possible predictor of individual
radiosensitivity
G. Ormotsadze, D Nadareishvili
I.Beritashvili Center of Experimental Biomedicine. Departement of problem of
radiation safety.
Tbilisi, Georgia
The results of already completed and present large scale radio-epidemiological studies
ranked the late - stochastic and tissue effects prognosis, prevent and mitigate issue among
priority problems of fundamental and applied radiobiology.
As it is known, that if early radiobiological tissue effects are mainly dependent on
absorbed dose, individual features of adaptive response of organism have an important
role in the formation of late and very late effects of radiological impact.
Hence, the study of mechanisms of interindividual variability in the regulation of
functional systems of the organism and determination of their possible casual relationship
with the final outcome of radiation impact is a prospective way of the detection of
individual radiation risk predictors
There were studied red blood system functional state in physiological norm and early
postradiation period .
The functional state of RBS was determined by using the specially developed method
based on analysis of the dynamics of peripheral blood erythrocytes spectra (PBE PS) -
distribution of PBE according to their spherulation degree (Q) and volume (V)).
Spherulation degree is considered as biological age and along with the volume determines
the probability of their elimination from circulating bed. In connection with these
positions distribution P=P(V,Q) express mechanism of production-aging-elimination of
PBE and according to their dynamics we can discuss about the character of these
processes.
In animals exposed with lethal and sublethal doses of ionizing radiation, at the initial stage
of post -radiation period (6-10 days) PBE co-operative (massive) elimination and the
involvement of young red blood cells with short life span are observed. Right short life
span factor is considered as one of the main mechanisms in the development of radiation
anemia.
On the other hand, it was detected that massive involvement in circulation of immature
red blood cells that have diminished living status, elimination of the old fraction of PBE
and erythropoiesis tension are observed during the further period of stress influence on
animals.
From the position of the obtained results, the role of PBE PS regulation mechanisms in the
development of radiation anemia is of great interest, while elucidation of inter- individual
mechanisms of functional state regulation of RBS lays out new ways for prognosis of late
effects of radiation impact.
Keywords: radiation impact, red blood system, erythrocytes of peripheral blood .
23
Roles of RNA and RNA-binding proteins in TDP-43 and FUS aggregation
S. Abrakhi1, D. Kretov1,2, B. Desforges1, D. Pastré1 and L. Hamon1
1- INSERM U1204, Université d’Evry Val d’Essonne, 91025 Evry, France
2- Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow
Region,142290, Russia.
The formation of insoluble TDP-43 and FUS cytoplasmic inclusions are critical for the
onset and progression of devastating neurodegenerative disorders like amyotrophic lateral
sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). These proteins are
predominantly nuclear but, as many other proteins involved in mRNA metabolism and
transport, they actively shuttle in and out of the nucleus under physiological conditions.
However, in affected neurons, they are found enriched in the cytoplasm and form
cytoplasmic inclusion.
If the mechanisms driving the onset of ALS and FTLD remain unknown, one can assume
that the loss of normal TDP-43 and FUS functions is probably involved in neuron
degeneration. First, these proteins bind to a huge number of mRNAs and, by doing so,
regulate the translation efficiency. Second, TDP-43 and FUS are components of RNA
transport granules which have important functions in neurons. For example, ALS linked
mutations of TDP-43 impair the transport of RNA granules to distal neuronal
compartments. Finally, due to their RNA recognition motif and their prion-like domain,
both proteins are recruited in cytoplasmic stress granules upon stress conditions in a
reversible manner. However, in pathological conditions, stress granule dynamics is
deregulated, with the appearance of RNA granules of different morphologies and which,
in contrast with stress granules, can no longer be dissociated after stress.
The role of TDP-43 and FUS in devastating neurodegenerative disorders explains the
numerous studies on this subject both at the animal and cellular level but strikingly,
structural analysis of the protein-RNA complexes has received little attention except for
the potential amyloid characteristic of the filaments observed in inclusions of ALS or
FTLD patients. To that end, we propose here a structural analysis at the single molecule
level using Atomic Force Microscopy (AFM) of TDP-43-RNA and FUS-RNA complexes.
We will take advantage of the high resolution imaging possibilities of AFM to detect
potential differences in the binding mode of these proteins to RNA and the influence of
RNA-binding proteins present in RNA granules on the stability of the TDP-43- and FUS-
RNA complexes.
24
Reconfiguration of extended DNA fragment harboring alpha-globin gene domain in
chicken erythroid cells.
S. V. Ulianov1,2, A. A. Galitzina 2, I. M. Flyamer1,2, E. E. Khrameeva 3, S. V. Razin 1,2
1Institute of Gene Biology RAS
2Lomonosov Moscow State University
3Research and Training Center on Bioinformatics, Institute for Information
Transmission Problems, RAS
sergey.v.razin@usa.net
The chicken domain of alpha-globin genes represents a useful model to study regulation
of globin genes expression. Current evidence suggests that spatial organization of
genomic domains plays and important role in regulation of gene expression. We have
previously found that activation of globin gene transcription correlates with a spatial
reconfiguration of the alpha-globin gene domain resulting in an assembly of the active
chromatin hub. Here, in order to get more information on a possible relation between
transcription and large-scale chromatin folding, we have studied the spatial configuration
of an extended (2.5 Mb) segment of chicken chromosome 14 harboring the domain of
alpha-globin genes in cultured lymphoid cells, cultured erythroid cells, and cultured
erythroid cells stimulated to a terminal differentiation resulting in activation of globin
gene transcription. The results obtained demonstrate that active transcription of globin
genes correlates with a limited decompaction of a relatively large area harboring the
alpha-globin gene domain, although the domain itself became more compact apparently
due to the formation of an active chromatin hub, ACH. Importantly, organization of the
area under study into topologically-associated domains and spatial separation of active
and repressed chromatin compartments remain virtually the same in all three cell lines
studied.
The research was supported by the Russian Science Foundation (project #14-24-00022)
25
Macromolecular complexes in invadopodia formation.
Sergii Kropyvko1, Tetyana Gryaznova1, Olga Gubar1, Valentyna Kryklyva1, Mariia
Burdyniuk1, Mykola Dergai1, Liudmyla Tsyba1, Yegor Vassetzky2, Alla Rynditch1
1 Institute of Molecular Biology and Genetics, 150 Zabolotnogo Street, Kyiv 03680,
Ukraine,
2 Institut Gustave Roussy 39, rue Camille-Desmoulins, 94805 Villejuif, France
rynditch@imbg.org.ua
Aim. Invasive cancer cells form membrane protrusions, invadopodia, that degrade
extracellular matrix and facilitate cell invasion and metastasis. Invadopodia contain an
actin-rich core surrounding by adhesion and scaffolding proteins which presence
correlates with invasive behavior of human cancer cells. Key players include the adaptor
proteins Tks4 and Tks5, the actin regulators cortactin and N-WASP, the tyrosine kinase
Src and the transmembrane metalloprotease MT1-MMP. In spite that in the last two
decades significant advances in our knowledge of the structure and development of
invadopodia have been made, detailed mechanisms by which they facilitate cell migration
is not yet available. Methods. We used pull-down assay and Wester blot analysis,
immunofluorescence and confocal microscopy, invadopodia gelatin degradation assay.
Results. We have identified a series of new Tks4 binding partners including adaptor
proteins of the ITSN family (ITSN1 and ITSN2) and small adaptor molecules Crk and
Grb2, the Amphiphysin protein family (Amph1 та Amph2), kinase Src and phospholipase
C gamma 1 (PLCg1) and also another member of the Tks family - Tks5. It may indicate
the possible role of Tks4 in transport and sorting of cell vesicles. Current data is supported
by interaction with the proteins of Amphiphysin family (Amph1 та Amph2), as their main
functions are membrane trafficking and remodeling. Adaptor proteins Crk, Grb2 and
ITSNs are important for the actin cytoskeleton rearrangements and signal transduction.
Tks4 also may participate in these processes as we found that Tks4 formes complexes
with above mentioned proteins. Moreover, we have identified and characterized new Tks4
isoform - Tks4-beta. We suggested that an active state of Tks4 is regulated via
intramolecular interactions between its proline-rich motifs and own SH3-domains. We
have shown the interaction between ITSNs and other prominent component of
invadopodia WIP. Data from immunofluorescent analysis revealed co-localization of
ITSN1 and WIP at the sites of invadopodia formation and in clathrin-coated pits. We have
also demonstrated that the key endocytic protein ITSN1 and WIP and N-WASP can form
a complex in cells. Together, these findings provide insights into the molecular
mechanisms of invadopodia formation and identify ITSNs as scaffold proteins involved in
this process.
26
Disassembly of interphase prenucleolar bodies are driven by the maturation of pre-
rRNA
Y. R. Musinova1, O. M. Lisitsyna1, D. V. Sorokin2, E. A. Arifulin1, S. A. Golyshev1,
E. V. Sheval1
1A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow
State University, 119992 Moscow, Russia. e-mail:
2Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University,
Botanická 68a, 602 00 Brno, Czech Republic
evsheval@gmail.com
Aim: Cell nucleus is a complex structure, harboring a variety of discrete subnuclear
organelles, collectively referred to as nuclear bodies (NBs). Recent studies are beginning
to elucidate the molecular mechanisms responsible for the assembly and maintenance of
several NBs, but little is known regarding the mechanisms of NB disassembly.
Methods: Here, we have used the model of interphase prenucleolar bodies (iPNBs),
nucleoplasmic bodies which form from the material of destroyed by hypotonic treatment
nucleoli after return of cells to isotonic culture medium. The iPNB disasembly occurs
during several hours after the formation and synchronously in all cells, which makes the
system convenient for studying the mechanisms of NB disassembly.
Results: The nucleoplasmib B23-containing bodies – iPNBs, were easily seen in cells
during 5 hours after return from hypotonic buffer to isotonic medium. During this time,
iPNB number and size were gradually reduced. iPNBs demonstrated preferentially
constrained diffusion, and there was no directed motions of iPNBs to the nucleoli. The
major nucleolar protein B23 was transferred from iPNBs to nucleoli by diffusion. The
maturarion of pre-rRNA occured inside iPNBs, and inhibition of pre-rRNA maturation led
to the stabilization of iPNB structure and inhibition of B23 transfer to the nucleoli.
Conclusion: It seems that molecules of pre-rRNA played the 'seeding' role inside iPNBs,
and their maturation and subsequent delivery from iPNBs led to the disassembly of these
structures.
Funding: The work was supported by Russian Science Foundation (grant 14-15-00199)
and Russian Foundation for Basic Research (grant 14-04-01650).
27
The peculiarities of anti-HSP60 antibodies at thyrod cancer progression
L. Sidorik1, L. Yakovenko1, I. Kroupskaya1, M. Roiuk1, S. Chornyy1, A. Tsisarenko1,
I. Tykhonkova1, P. Pogribnyy2, V. Gurtovyy3, V. Usenko3
1Institute of Molecular Biology and Genetics NAS of Ukraine, Kyiv, Ukraine
2 Kavetskiy Institute of Experimental Pathology, Oncology and Radiobiology NAS of
Ukraine, Kyiv, Ukraine
3 Morphological Laboratory BIONTEK, Dnipropetrovsk, Ukraine
sidorik@imbg.org.ua
Background. The study of anti-Hsp60 antibodies levels in several tumors is a topic of
growing interest. It was demonstrated that levels of circulating anti-Hsp60 antibodies and
Hsp60 could be used as biomarkers for carcinogenesis, and prognosis factor for survival
and treatment susceptibility. The nature of the anti-Hsp60 immune response in malignant
transformation and role of antibodies in that process need to be elucidated.
Our aim was to investigate the possible biological activity of anti-Hsp60 antibodies (both
polyclonal and autoantibodies, purified from thyroid patients sera on Protein G Sepharose)
on different continuous cell lines.
Methods. 25 patients sera (20-57 years old, 18 – follicular adenoma, 6 – papillary and 1
follicular carcinoma) and 12 healthy donors (control) were used for determination of anti-
Hsp60 antibodies level by ELISA.
The anti-Hsp60 antibodies from highly reactive sera were purified by affinity
chromatography method. Polyclonal anti-Hsp60 developed in our Laboratory were used in
the study of biological activity of such antibodies in MTT-test.
Results. The increased anti-Hsp60 autoantibodies level was detected by ELISA in sera
more than 50% of patients with follicular adenoma and autoimmune thyroiditis, and in
86% of thyroid cancer patients sera, respectively. IgG antibodies purified from highly
reactive sera recognized target antigen (human Hsp60) in Western-blot analysis. Increased
anti-Hsp60 antibodies level and Hsp60 expression in thyroid cancer tissue were associated
with lymphoid infiltration and sclerotic changes of thyroid tissue.
First we investigated the influence of polyclonal anti-Hsp60 antibodies on different
continuous cell lines (WRO, KTC-2, TPC-1, A431 and 3LL) vitality. We established
dose-dependent and time-dependent anti-Hsp60 antibody effect on dehydrogenises cell
activity. We also determined decreased level of THF-α production in MAEC supernatant
and IL-8 production in total lyzates of continuous cell lines (Namalva, A549, A431W)
after 24 hours incubation with polyclonal anti-Hsp60 antibody in vitro. However,
polyclonal Hsp60 antibodies didn’t affected IL-10 production in MAEC.
Conclusion. Significant increase of anti-Hsp60 antibodies level was determined in sera
of patients with thyroid pathology. The highest titers of anti-Hsp60 antibodies in sera of
patients with thyroid cancer has been revealed. The biological (cytokine-induced and
vitality-suppressed) activity of anti-Hsp60 antibodies was observed firstly on different
continuous cell lines. The results obtained could serve as a base for new diagnostic tool
development at thyroid tumors and cancer diagnosis verification.
28
Regulation of DNA-repair, nitrosative stress-related and proteasomal gene
expression by 1,4-dihydropyridines in diabetic animals
K. Ošiņa1, E. Rostoka2, N. Sjakste2, T. Sjakste1,2
1 Institute of Biology, University of Latvia, Miera Street 3, Salaspils LV2169, Latvia
2Latvian Institute of Organic Synthesis, Aizkraukles Street 21, Riga LV1006, Latvia;
DNA damage by endogenous free radicals and ineffective repair of these lesions is one of
the etiological factors of diabetes mellitus (DM). Oxidative stress and abnormal
production of nitric oxide lead to increased DNA breakage in DM. In DM production of
NO by inducible NO synthase (iNOS) is dramatically increased, but endothelial NO
synthase (eNOS) is uncoupled and produces superoxide radical. One of treatment
strategies of DM can be aimed on decrease of production of reactive oxygen species with
consequent DNA damage prevention, normalization of NO production and DNA repair
enhancement. 1,4-dihydropyridine (1,4-DHP) derivatives appear to be prospective as
novel drugs with above spectrum of effects. The aim of our work was to evaluate the
impact of a group of novel 1,4-DHP on expression of genes involved in NO production,
DNA repair and proteolysis. Induction of DM model by injection of streptozotocin to rats
increased expression of iNOS gene in kidneys and blood cells, decreased expression of
eNOS in kidneys cells; DNA-repair related genes PARP1 and γH2AX were up-regulated in
blood cells, DNA breakage level was increased in nucleated blood cells. NO synthases.
Three of the tested compounds - AV-153, metcarbatone and etcarbatone increased eNOS
gene expression in kidneys of diabetic animals, the effect seems to be favourable in DM
conditions. Some of the tested compounds (etcarabatone, glutapyrone and J-9-125)
enhanced iNOS gene expression in kidneys of intact animals, and further enhanced the
increased iNOS expression in kidneys of diabetic animals, except etcarbatone.
Etcarbatone, metcarbatone and glutapyrone normalized iNOS expression in blood cells of
diabetic animals. NOS-independent NO production. The enzyme xanthine deoxygenase
(XD) is involved in NOS-independent production of NO from nitrite in diabetic animals,
several 1,4-DHP inhibit this process. Most of the studied compounds except etcarbatone
increased the level of XD gene expression both in intact and STZ-treated animals. This
apparent contradiction might be due to feedback arising when XD enzyme is inhibited by
a compound. DNA repair. We have observed increased DNA breakage in animals treated
with AV-153; it also enhanced effects of DM on DNA integrity and expression of DNA
repair-coupled genes (PARP1 and γH2AX). Etcarbatone decreased expression of these
genes in blood of diabetic animals, although it increased the DNA breakage induced by
diabetes. Glutapyrone per se increased expression of PARP1 and γH2AX in kidneys, but
decreased in blood. J-9-125 and metcarbatone increased expression of DNA repair genes
in kidneys of both intact and diabetic animals, however metacarbatone was the only
compound able to reduce DNA breakage in diabetic animals. Proteasomes. Ubiquitin-
proteasome system dysfunction is an important factor in DM pathogenesis. In our model
we did not detect modification of the proteasomal PSMA6 gene expression by DM
conditions. Some of 1,4 DHP (etcarbatone, glutapyrone) enhanced expression of the gene
in intact animals, however almost all compounds produced increase of its expression in
diabetic animals. Conclusions. 1,4-DHP can interfere with expression of genes involved
in DNA-repair, nitrosative stress-related and ubiquitin-proteasome system. Effects of the
compounds are dependent on structure and organ-specific. Some of the 1,4-DHP appear to
be prospective for normalization of DM-induced metabolic abnormalities.
29
Extracellular ubiquitin regulates regeneration of leucopoiesis
R. Sujashvili1, I. Ioramashvili1, K. Aptsiauri2, N Gvinadze2
Department of Biophysics1, Department of Neurotoxicology2
Iv.Beritashvili Centre of Experimental Biomedicine
14 Gotua, Tbilisi, Georgia, 0160
Deregulation of protein catabolism mediated by ubiquitin-proteasome system is involved
in pathogenesis of different malignancies. Dividing cells are more sensitive to defects in
protein degradation. Therefore direct control of ubiquitin-proteasome system components
and its targets opens new potential ways for cancer therapy. Behaviour of extracellular
ubiquitin in mammal cells is poorly presented in literature. Our earlier experiments
revealed that in vivo injected extracellular ubiquitin inhibits mitotic activity of hepatocytes
in healthy rats, but significantly stimulates it in alcoholic liver after partial hepatectomy.
Moreover, we showed that extracellular ubiquitin caused significant changes in dynamics
of regeneration of leucopoiesis. Mitotic activity in bone marrow of test group animals
injected by ubiquitin was decreased by about 53% as compared with the intact animals’ of
control group. In the presented work we exposed the influence of in vivo injected
extracellular ubiquitin on spontaneous regeneration of leucopoiesis in modelled
leucopenia in Wistar female rats with weight about 120–150gr. Calculation of nuclear cell
ratio in bone marrow smears stained with azure-eosin was performed. Animals were
divided into two groups. In the first, control group of animals inhibition of haematopoiesis
was achieved by means of 100mg/kg cyclophosphamide LD50 50-200mg/kg injection.
Bone marrow samples from the first group of rats had been taken at 0h (intact), 24h, 48h,
72h, 96h and 168h time points after injection of cytostatic. The second, test group was
injected by 200 μg/ml Ubiquitin 72 hours later after cytostatic induction. Bone marrow
samples from the second test group of rats had been taken at 6h, 24h and 96h points after
injection of ubiquitin. 5000 cells per sample were counted. Calculation of number of
nucleic figures per 1000 cells (‰) was carried out. Animals were anesthetized by ether
before decapitation. Treatment of animals performed in accordance with regulations
established by the Centre animal’s ethic committee (Protocol N06/13.10.2014). The most
noticeable cytological effect induced by ubiquitin in bone marrow is a significant increase
of per mille at 6 and 24 hour points after ubiquitin injection. Ratio of nuclear cell to total
cell count is increased 2 times in both cases as compared with the same (72h and 96h)
hour points of control group. Finely, per mille reaches the value of intact animals cell
count at 7Th day after cyclophosphamide injection. So, after cytostatic injection clearly
marked cytopenia in peripheral blood and depletion of nuclear cell index was achieved
through the next 24 hours that was followed by elevation of nuclear cell per mille at 48h
point. The second depletion of nuclear cells at 72h point has been removed by means of
injected ubiquitin. Supposedly, extracellular ubiquitin enhances proliferative activity of
stem cells and acts as a cell cycle regulator in bone marrow after chemically induced
cytopenia, retains bone marrow cells in compartments of differentiation and maturation
and prevents their early passage to peripheral blood. Farther investigation of extracellular
ubiquitin effect on regeneration of leucopoiesis may elucidate new possible pathways of
ubiquitilation in hematopoietic cells, mechanisms of regulation of leucogenesis in norm
and pathology and provides excellent perspectives to create new methods of targeted
therapy for hematological disorders.
30
Detection the specificity of poly(ADP-ribose) polymerase 1 and poly(ADP-ribose)
polymerase-2 interaction with DNA strand breaks at single molecule level
Maria V. Sukhanova1,2, Sanae Abrakhi2, Vandana Joshi2, Rashid O. Anabaev,1
Mikhail M. Kutuzov1, David Pastré2, Patrick Curmi2, Loic Hamon2 and Olga I. Lavrik 1
1 LBCE, Institute of Chemical Biology and Fundamental Medicine SD RAS, 630090,
Novosibirsk, Russia,
2 INSERM U829, Université Evry-Val d’Essonne, F-91025, Evry, France
sukhanovamv@mail.ru
The aim of this study was to investigate the interaction of PARP1 and PARP2 with single
DNA damage site in the context of long DNA substrates.
Methods: The interaction PARP1 and PARP2 with long DNA fragments containing DNA
breaks was studied using atomic force microscopy (AFM) and fluorescent titration.
Results: PARP1 and PARP2 are eukaryotic nuclear proteins which are implicated in
synthesis of poly(ADP-ribose) (PAR) after DNA damage detection. We directly
visualized and compared the binding of PARP1 and PARP2 to long DNA fragments
(1200-bp) containing only double-strand breaks (DSBs) or DSB along with a unique nick
(nicked 1200-bp). AFM data show that PARP1 specifically binds to both nick and DSBs.
At the same time, this protein binds to undamaged DNA, but to a lower level than to
breaks. In contrast to PARP1, PARP2 binds weakly to undamaged DNA and to DSBs, and
localizes mainly to nick site. These results correlate with fluorescence measurement data
revealing that the binding affinity of PARP2 to 1200 dsDNA is 5-fold weaker in
comparison to the same DNA fragment containing a nick. Interestingly, AFM assay clear
demonstrate that PARylated PARP1 and PARP2 are still able to interact with DSB and
nick.
Conclusion: Our AFM observations at single-molecule level implicate PARP2 in the
recognition of nick and suggest its role in single-strand break repair. It is appears that the
length of the PAR polymer formed by PARP1 and PARP2 proteins is influenced by the
initial binding affinity of the proteins for the damaged DNA site.
Funding: The work was supported by INSERM, the Genopole Evry, program GDRI of
CNRS and Russian Foundation for Basic Research (project no. 15-54-16003).
31
Nature of hyperpolarizing afterpotentials accompaning epileptic discharges
V. Tsomaia, N. Nachkebia, T. Labakhua
Lab. Neurobiology of Sleep-Wakefulness Cycle,
I. Beritashvili Centre of Experimental Biomedicine
The aim of present investigation was to explain the inhibitory mechanisms in neocortical
neurons leading to the arrest of epileptic ictal activity.
Experiments were performed on adult cats. All surgical procedures were conducted under
pentobarbital (36 mg/kg) anaesthesia. Ictal activity was induced by repetitive electrical
stimulation of the sensory-motor region of cerebral cortex, or local application of
penicillin on the surface of sensory-motor region of cerebral cortex. Glass microelectrodes
filled with 2M potassium citrate were used for intracellular recording of neuronal
electrical activity.
Hyperpolarizing after-potentials of penicillin – induced paroxysmal depolarizing shifts in
neocortical neurons of the cat were investigated. On the bases of studying the membrane
conductance changes at different points of hyper-polarization and the role of various ionic
currents in its generation the conclusion was drawn that paroxysmal depolarizing shifts of
hyperpolarizing after-potentials is a composite nature. The membrane resistance value
was measured by recording the increase in membrane potential induced by injecting
hyper-polarizing current pulses. At the beginning of the post-ictal hyper-polarization the
resistance was markedly reduced to 45±10 % (n=30) of its ictal value, suggesting that this
phase of post-ictal inhibition was caused by an IPSP with increased conductance. The
initial component conditioned by increased membrane permeability to chloride,
presumably a synaptic GABA-A response, the next component conditioned predominantly
by potassium current, representing presumably a GABA-B response and the final
component comprising mainly a calcium-activated potassium current. However, the
reduced membrane resistance gradually returned to the initial level (90±10%, n=30),
while the hyper-polarization persisted for a long time. It is possible that the activation of
an electrogenic pump might be responsible for this continuing hyper-polarization.
32
Aminoacyl-tRNA synthetases as biomarkers for cancer diagnostics
M. Tukalo, A. Kondratov, A. Yaremchuk, O. Gudzera
Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine,
150, Zabolotny St., 03143 Kyiv, Ukraine,
mtukalo@imbg.org.ua
Recent evidence suggests that aminoacyl-tRNA synthetases (ARSs) exhibit remarkable
functional versatility and their noncanonical functions have been pathologically linked to
cancers. ARSs seem to have critical mechanistic roles in a variety of cellular processes
that are relevant for disease development and pathology, and these roles may be used as
one possible avenue for improvement of diagnostics.
To uncover the biomarkers related with tumorigenesis and behavior of cancer we have
studied of differently expressed genes of four or six aaRSs in tissues of colon and kidney
cancers by the quantitative polymerase chain reaction (Q-PCR) method.
In the case of kidney cancer from 18 samples of tumor tissue (ccRCC), increased
expression (more than 2 fold) of seryl-tRNA synthetase (SARS) was observed in 10, and
only two were noted its slight decrease. The tendency for co-expression of SARS, leucyl-
tRNA synthetase (LARS) and histidyl-tRNA synthetase (HARS) in ccRCC has also been
shown.
In the case of human colon cancer we have observed the expression profile for LARS,
HARS, SARS and lysyl-tRNA synthetase (KARS) in 16 primary cancer samples. We
have found that genes of LARS, SARS and KARS underwent most changes and tendency
for expression with mutual exclusivity of KARS and SARS and LARS in colorectal
cancer has been shown The LARS and SARS expression decreased significantly (from 2
to 60 fold) in 38 % of samples (6 of 16) in comparison with surrounding normal tissue
according to Q-PCR data. The KARS expression was increased significantly (from 2 to
3000 fold) in 50 % (8 from 16) and was decreased in 25% of cancer samples. These genes
might be used for diagnosis of colon tumors.
33
Nuclear organization, oncoviruses and cancer
Tatiana Tsfasman1,2, Diego Germini1,2, Rawan El-Amine1,2, Olga Iarovaia1,2,
Stephanie Bury-Moné3, Marc Lipinski1 and Yegor S. Vassetzky1
1UMR8126, Université Paris-Sud, CNRS, Institut de cancérologie Gustave Roussy,
Villejuif, France
2 CNRS LIA1066 “Laboratoire Franco-Russe de Recherche en Oncologie »,
Villejuif- Moscow
3Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
4ENS, Cachan, France
vassetzky@igr.fr
Background: Environmental factors play an important role in most human cancer.
Burkitt’s lymphoma (BL), a rare B-cell lymphoma caused by specific chromosomal
translocations resulting in the juxtaposition of the CMYC oncogene with an
immunoglobulin gene locus, is a typical example of cancer strongly affected by
environmental factors. BL is associated with the Epstein Barr virus, human
immunodeficiency virus (HIV), malaria and exposure to a Euphorbiaceae plant. The
molecular mechanisms of these environmental factors remain largely unknown.
Results: One of the major enigmas to solve is why all these factors specifically induce
Burkitt’s lymphoma, and no other malignancies? Our data indicate that this these factors
perturb the nuclear organization of B-cells inducing the prolonged colocalization of
potential translocation partners, the IGH and CMYC loci.
Conclusions: HIV- and EBV induce changes in the nuclear architecture of B cells. This
may specifically provoke BL-specific translocations.
How to become famous and popular in science
(All scientometric tricks in 5 minutes for everyone)
Iryna O. Tykhonkova
Institute of Molecular Biology and Genetics, NAS of Ukraine,
150, Zabolotnogo St., Kyiv, Ukraine, 03680
i.o.tykhonkova@imbg.org.ua
Impact-factor and h-index are more often used on the pre expertise phase of scientist’
activity estimation or evaluation of the represented projects and grants. The formula for
the indexes calculation is so simple and it is used by different scientometric databases.
For the correct presentation the achievements, a scientist must have only one profile in
Scopus, ResearcherID, Researchgate, Google scholar and Orcid. Check you visibility at
http://www.scopus.com/ for profiles merging go to http://www.scopusfeedback.com/
http://www.researcherid.com
https://www.researchgate.net
https://scholar.google.com.ua/
http://orcid.org/
Calculation of Impact Factor and h-index at http://biopolymers.org.ua/I_b.pdf
34
The study of nuclear localization of amh and sox9a genes in male and female gonadal
tissues of zebrafish
M. Karapetian,1 E. Zaldastanishvili, 1 T. Zaalishvili,2 G. Zaalishvili 1
1 Animal Molecular Biology Laboratory, Agricultural University of Georgia
2 Laboratory of Genome Structure and Function,
I. Beritashvili Center of Experimental Biomedicine
Zebrafish (Danio rerio) is widely used model organism in numerous research fields.
Different RNA FISH techniques have been optimized for zebrafish, however DNA FISH
on tissue cryosections was never adapted for this organism. The main goals of present
research were to optimize DNA FISH on tissue cryosections of zebrafish testis and
ovaries for the study of nuclear positioning of amh and sox9a genes in gonadal tissues.
DNA FISH was successfully optimized for cryosections of gonadal tissues using BAC
clones. Positioning analysis of amh and sox9a genes reveals that both loci are found
predominantly on the nuclear periphery in male germ cell nuclei. Along with this we
evaluated the interallelic distances for both genes and showed that mostly homological
loci are localized together at the nuclear periphery. Similar distribution however was not
observed in ovarian tissues. However it should be mentioned that only oocytes on early
stages of development were suitable for FISH analysis in our experimental conditions.
35
Some cellular and molecular mechanisms of radiation damage in animal and cellular
model systems
Sanikidze T, Kiparoidze S.
Departement of problem of radiation safety
I.Beritashvili Center of Experimental Biomedicine, Tbilisi, Georgia
Far tissue effects of ionizing radiation on living organisms represents primary radiation
damages and the body's whole hierarchy responses adaptive-regulatory mechanisms
interference, which outcome is importantly determined by the the individual features of
the metabolism. We have studied the metabolism of post-irradiation changes in the
irradiated rats (disposable irradiation 6 Grey) and Jurkat cells culture (2 Grey during 1
minute).
After 24 hours incubation of radiated Jurkat cells population amount of gaploid
(apoptotic G 0 stage) cells was increased by 400 %, diploid cells (G 0/G1 stage) –
increased by 17 %, tetraploid cells (G2/M) decreased by 73 %, amount cells in S phase
decreased by 35 % in comparison to the control group. It was revealed intensive
production of oxygen and nitrogen free radicals and inactivation of antioxidant enzymes
(superoxidismutasa, catalasa and glutathione peroxidase). Statistically significant
dependence between intensification of cellular apoptosis level, amount of tetraploid cells
and intensity of oxidative stress was revealed.
In irradiated rats blood alteration in the antioxidant enzimes activity, parameters of lipid
metabolism, NO-s content and its modulatory activity, also increase proapoptotic factor p-
53 in rats hepatocytes were revealed on the different stages after radiation.
We concluded that free radical oxidation plays important role in the radiation-induced cell
and tissue damage.
36
Mitochondria-targeted antioxidants prevent TNFα induced endothelial cell damage
Vlada Zakharova, Ivan Galkin, Valerya Romashchenko Olga Pletjushkina,
Boris Chernyak, Roman Zinovkin, Ekaterina Popova, Vladimir Skulachev
Lomonosov Moscow State University, Russia
t-vlada@mail.ru
Using mitochondria-targeted antioxidants made of plastoquinone and penetrating cations
(SkQ family), we investigated the role of mitochondrial reactive oxygen species (ROS) in
the TNF-induced cytoskeleton reorganization and apoptosis in endothelial cells.
SkQ, as well as the classic antioxidant N-acetylcysteine and Trolox significantly
suppressed apoptosis induced by TNF [1]. Their action was directed at the suppressing the
release of cytochrome c. We showed that SkQ treatment led to an increased level of Bcl-2,
and reduced levels of Bax and p53 [1]. Nevertheless, SkQ had no on Bcl-2 mRNA
expression, but it enhances Bcl-2 phosphorylation thus contributing to the inhibition of
protein ubiquitination. We have also shown that SkQ prevents Bcl- 2 proteolysis induced
by TNF. We assume that SkQ may affect activation of the redox sensitive stress kinases
that are involved in the phosphorylation of the Bcl-2 family proteins.
In our system we plan to evaluate the influence of mitochondria-targeted antioxidants on
DNA damage induced by TNF. The results will be presented at the conference.
We showed that SkQ as well as N-acetylcysteine and Trolox inhibited TNF-induced
monolayer endothelial permeability for dextran with a molecular weight of 65–85 kDa.
Endothelial monolayer permeability induced by TNF is accompanied by cytoskeleton
reorganization. We observed that SkQ prevented TNF -induced release of VE- cadherin
and β- catenin from the contact area, as well as disassembly of the annular peripheral
beam of actin microfilaments. TNF-activated matrix metalloproteinases (MMP) cleaved
extracellular fragment of VE- cadherins. We observed MMP-dependent decrease in the
overall VE- cadherin level in the cells and the appearance of its cleavage product in cell
medium induced by TNF. These effects were markedly suppressed by SkQ.
Thus, it was shown that TNF-dependent endothelial cell damage was largely dependent on
ROS production in mitochondria, thus indicating promising angioprotective action of
SkQ.
Reference
[1] I.I. Galkin, O.Yu. Pletyushkina, R.A. Zinovkin, V.V. Zakharova, I.S. Birjukov,
B.V.Chernyak, and E.N.Popova, Mitochondria-targeted antioxidants prevent TNFα-
induced endothelial cell damage. Biochemistry (Moscow), 79(2), (2014),169–177.
37
Detection of genetic disorders during the radiation impact on human organism
A. Zedginidze1, T. Nikuradze1 E. Namchevadze2
1 I.Beritashvili Center of Experimental Biomedicine.
Departement of problem of radiation safety.
Tbilisi, Georgia
2 E.Andronikashvili Institute of Physics,
Tbilisi, Georgia
Being a strong mutagen, Ionizing Radiation first of all causes changes in genetic apparatus
of the living organisms . That’s why genetic indices are excellent biomarkers for detection
of the effects of Ionizing Radiation. The actual problem is to find the optimal combination
of different biomarkers. We used the genetic methods ( chromosomal
aberrations, comet assay and level or micronuclei in lymphocytes and
buccal exfoliative cells ) for assess the mutagenic effect on the organism
individuals in different conditions exposed to ionizing radiation. Irradiation
of individuals is possible not only in nuclear disaster, or environmental contamination, but
also during routine diagnostic procedure (e.g. computed tomography scanning, etc). In
recent years the use of ionizing radiation for treatment and diagnostic purposes has
significantly increased during recent years, Although , the general radiobiologic
principles underlying external beam and radionuclide therapy are the same , there are
significant differences in the biophysical and radiobiological effects.. The existence of
genetic disorders is important to sort out the risk groups, because radiation as the mutagen
has cancerogenic features. We revealed the heterogeneity of different organism response
to irradiation.
Determination of absorbed dose, identification of various genetic disorders in individuals
exposed to identical doses of radiation, offers the opportunity to judge the individual
biological effect and is very important for individual preventive activities.
The work was supported by the Georgian natiional programme, ISTC and IAEA projects
Key words irradiation. ,biomarker, radiotherapy
38
Neuronal porosome complex in norm and pathology. Electron microscopic study
Mzia G. Zhvania1,2, Nadezhda J. Japaridze1,3, Mariam Kssovreli1
1Ilia State University, Tbilisi, Georgia, 2I. Beritashvili Center of Experimental
Biomedicine,Tbilisi, Georgia, 3New Vision University, Tbilisi, Georgia
In all cells, cellular cargo destinated for secretion is packaged in membranous vesicles that
transiently dock and establish continuity at the base of cup-shaped membranous structures
called “porosomes” and neurons are not exception. Via porosomes vesicles release
intravesicular contents to the outside of the cell during secretion. It is suggested that in
each type of secretory cell special content of secretory vesicles, different speed of release
and different volume of content release dictates specific size of porosomes. In earlier
atomic force microscopic and electron microscopic studies, it was shown that in the
neurons, reprensenting fast secretory cells, 40–50 nm synaptic vesicles are docked at
roughly 10 nm in diameter neuronal porosomes. Recent EM 3D tomography in rat brain
also reveals the presence of 12–17 nm permanent presynaptc densities in which 35-–50
nm synaptic vesciles are found docked. Moreover, the inside-out ultrahig-resolution
atomic force microscopic study of presynaptic membrane preparations of isolated
synaptosomes displays the presence of the inverted cup-shaped 10–17 nm neuronal
porosomes. Neuronal porosomes possess a central plug, that is absent in porosomes in
other kinds of secretory cells. This plug interacts with proteins at the periphery of the
structure, conforming to an eightfold symmetry; each of them is connected with spoke-
like elements to the central plug that is involved in the rapid opening and closing of the
neuronal porosome to the outside. The central plug has been further examined in various
conformations: fully pushed outward, halfway retracted, and completely retracted into
porosome cup – has been demonstrated. Recently we described the morphology of
porosome in different brain structure of various mammals (rat, cat, dog). In this electron
microscopis study we evaluate if rat porosome structure/size is altered as a result of
pathological and other conditions, specifically as a result of chronic restraint stress.
Finally, to further understand the structure of the neuronal porosome complex, and the
bare protein backbone of the complex for future single-particle cryo-EM studies, we
evaluated the size of this complex from high-detegent solubilized synaptosome membrane
preparations. For to assess the effect of hypokinetic stress on porosome structure,
conventional electron microscopic methods were used. For to evaluate the isolated
neuronal porosome complex, it was immunoisolated from synaptosomes, using SNAP-25
specific antibody conjugated to protein A-sepharose. For all cases the morphometric
analysis of porosome diameter and depth was made. The one-way ANOVA was
performed on the diameter and depth. The results revealed that the parameters of
porosome, especially diameter, are very heterogenous. However despite the dynamic
nature of neuronal porosome, the ranges of dimension (diameter 12–16 nm, depth – 5–20
nm) remain the same in normal conditions and after influence of chronic restraint stress or
white noise. Results of studies of isolated complex demonstrate, that althouight the eigth –
fold symmetry of the immunoisolated porosome is maintained, and the central plug is
present, there is a loss in the average size of the porosome, possibly due to a loss of lipids,
proteins or both from the complex. In view of this, proteomics and lipidomics on the
isolated neuronal porosome using our current procedure using elevated detergent for
synaptosome solubilization, will be carried out to determine whether there is loss of lipids,
proteins, or both from the structure
39
Scientific Program. VIII International meeting “From Molecular to Cellular Events
in Human Pathologies”, 18–20 October 2015, Tbilisi, Georgia
MONDAY 19 OCTOBER
9:00–9:30 Opening of the meeting.
I. Beritashvili Center of Experimental Biomedicine,
David Nadareishvili, Olga Lavrik, Patrick Curmi
9:30–9:50 Keynote lecture David Nadareishvili (Georgia) Functional state of red blood
system as a possible predictor of individual radiosensitivity
9:50–11:30 Session 1. Chromatin and nuclear organization.
9:50–10:10 Sergey Razin (Russia) Reconfiguration of extended DNA fragment harboring
alpha-globin gene domain in chicken erythroid cells
10:10–10:30 Yegor Vassetzky (France) Nuclear organization, oncoviruses and cancer
10:30–10:50 Evgeny Sheval (Russia) Disassembly of interphase prenucleolar bodies are
driven by the maturation of pre-rRNA.
10:50–11:10 M. Karapetian (Georgia) The study of nuclear localization of amh and
sox9a genes in male and female gonadal tissues of zebrafish
11:10–11:30 Vasily Ogryzko (France) Topokaryotyping –a novel approach to study
nuclear organization
11:30–11:35 Iryna Tykhonkova (Ukraine) How to become famous and popular in
science (All scientometric tricks in 5 minutes for everyone)
11:30–11:50 Coffee Break
11:50–13:00 Session 2 Cellular mechanisms of physiological processes and
disorders
11:50–12:10 N. Gachechiladze (Georgia) The study of structure and function of muscle
giant proteins
12:10–12:25 Vlada Zakharova (Russia) Mitochondria-targeted antioxidants prevent
TNFα induced endothelial cell damage
12:30–12:50 N. Khizanishvili (Georgia) Mechanism of suppression of limbic motor
seizures by activation of the thalamic reticular nucleus
12:45–13:00 Dmitry Kretov (France) mRNA and DNA selection via protein
multimerization: YB-1 as a case study
13:00–13:50 Lunch
13:50–16:00 Session 3. Radiobiology, DNA Damage and Repair
13:50–14:10 Olga Lavrik (Russia) Human tyrosyl-DNA phosphodiesterase 1: new
activities and development of enzyme inhibitors as anticancer drugs
14:10–14:30 M. Gogebashvili (Georgia) Radiobiological effects modeling by using plant
test-systems
14:30–14:45 Ekaterina Belousova (Russia) DNA polymerase Iota participates in
clustered damage repair
14:45–15:05 A. Zedginidze (Georgia) Detection of genetic disorders during the radiation
impact on human organism
15:05–15:25 Alexander Ishchenko (France) PARP1 and PARP2 catalyze poly(ADP-
ribosyl)ation of DNA strand break termini
15:25–15:40 Anastasiya Kosova (Russia) Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) interacts with apurinic/apyrimidinic sites in DNA
40
15:40–16:00 Nikolajs Sjakste (Latvia) Regulation of DNA-repair, nitrosative stress-
related and proteasomal gene expression by 1,4-dihydropyridines in diabetic animals
TUESDAY 20 OCTOBER
9:00–12:00 Session 4 Cellular mechanisms of physiological processes and
disorders II
9:00–9:20 R. Sujashvili (Georgia) Extracellular ubiquitin regulates regeneration of
leucopoiesis
9:20–9:40 Loic Hamon (France) Roles of RNA and RNA-binding proteins in TDP-43
and FUS aggregation
9:40–10:00 G. Beselia (Georgia) Modulation of GluN2B subunit-containing NMDA
receptors expression and Spatial long-term memory in medial septal immunolesioned rats
10:00–10:20 T. Labakhua (Georgia) Postsynaptic reactions of cat somatosensory cortex
neurons in response to painful stimualation and analgesia
10:20–10:40 Alla Rynditch (Ukraine) Macromolecular complexes in invadopodia
formation
10:40–11:00 M. Tsagareli (Georgia) Transient receptor potential channel inactivation by
non-steroidal anti-inflammatory drugs
11:00–11:20 Mzia Zhvania (Georgia) Neuronal porosome complex in norm and
pathology. Electron microscopic study
11:20–11:40 V. Tsomaia (Georgia) Nature of hyperpolarizing afterpotentials
accompaning epileptic discharges
11:40–12:00 Marc Mirande (France) The primer RNA packaging complex of HIV-1:
understanding its assembly and disassembly
12:00–12:20 Coffee Break
12:20–13:15 Session 5 Cancer and Pathologies I
12:20–12:40 H. Grebenchuk (Georgia) Evaluation of the spatial prevalence of cancer in
conditions of an inadequate data of population
12:40–13:00 Boris Negrutskii (Ukraine) Translation elongation factor eEF1A1 and its
oncogenic variants
13:00–13:15 Nikolai Lomov (Russia) Broken proto-oncogenes AML1 and MLL leave the
inherent chromosome territories in human lymphoid cells treated with DNA
topoisomerase II poison etoposide
13:15–14:00 Lunch
14:00–15:35 Session 5 Cancer and Pathologies II
14:00–14:20 M. Grigolava (Georgia) Antimicrobial peptides as anticancer agents
14:20–14:40 Liliane Massade (France) Squalenoylated-siRNA for the treatment of
thyroid and prostate carcinomas
14:40–14:55 Stanislav Avdieiev (Ukraine) New cytotoxic agents and their combinations
for the treatment of chemoresistant glioblastoma and mantle cell lymphoma
14:55–15:15 N. Natsvlishvili (Georgia) Sigma-receptor-1 and mGluR5 may participate in
Rac-dependent oncogenesis through modulation of macrophage activity
15:15–15:35 Mikhail Tukalo (Ukraine) Aminoacyl-tRNA synthetases as biomarkers for
cancer diagnostics
15:35–16:30 Coffee Break + Posters
16:30–19:00 Round Table and Closing ceremony
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