Conference Program
October 11 (SUNDAY)
11.00–16.00
Registration and Coffee (Institute of cell biology, Drahomanov str. 14/16),
16.00–18.00
City tour
18.00
Lviv Opera House
October 12 (MONDAY)
8.30–10.00
Registration (The House of Scientists, Lystopadovogo Chynu str.6)
9.00–9.10
Opening Ceremony (The House of Scientists, Lystopadovogo Chynu str.7)
9.10-9.30
Andriy
Sibirny
DEVELOPMENT
OF
CELL
BIOLOGY
AND
BIOTECHNOLOGY RESEARCH IN INSTITUTE OF CELL BIOLOGY, NAS OF UKRAINE
(LVIV): CURRENT STATE AND PERSPECTIVES
9.30–11.30
Session 1 (Cell Biology, Chairs Andriy Sibirny, Bartosz Grzegorz)
9.30–9.45
Volodymir Antoniuk PURIFICATION AND PROPERTIES L-FUCOSE-
SPECIFIC
LECTINS
FROM
DIFFERENT
SOURSES
AND
THEIR
VALUE
FOR
HISTOCHEMISTRY
9.45–10.00
Yuliya Kurlishchuk MOLECULAR MECHANISMS INVOLVED IN HUMAN
CANCER CELLS’ RESPONSE TO ARGININE DEPRIVATION
10.00–10.15
Olexandr
Korchynskyi
PROINFLAMMATORY
CONTROL
OF
SKELETOGENIC SIGNALING PATHWAYS
10.15 –10.35
Maria Jolanta Redowicz MYOSIN VI IN THE NUCLEUS: A SEARCH FOR
ITS ROLE IN THE NUCLEOLUS
10.35 –10.55
Rostyslav
Stoika
NANOBIOTECH
RESEARCH
ACTIVITY
AT
DEPARTMENT OF REGULATION OF CELL PROLIFERATION AND APOPTOSIS
(INSTITUTE OF CELL BIOLOGY, NAS OF UKRAINE)
10.55 –11.10
Alexander
Petrenko
PROLIFERATIVE
AND
DIFFERENTIATION
POTENTIALS OF HUMAN ADULT MESENCHYMAL STROMAL CELLS IN THREEDIMENSIONAL CULTURE. THE EFFECT OF STORAGE AT CRYOGENIC AND AMBIENT
TEMPERATURES
11.10–11.30
Izabela
Sadowska-Bartosz
PROTEIN MODIFICATIONS
1
NITROXIDES
PREVENT
UNDESIRED
11.30–12.00
12.00–13.30
Coffee Break
Session 1 (Cell Biology, continued, Chairs Rostyslav Stoika, Maria Jolanta
Redowicz)
12.00–12.15
Lyudmila Sidorik HSP60 AND ANTI-HSP60 ANTIBODIES AT THYROD
CANCER PROGRESSION
12.15–12.30
Yuriy Kit MYOSIN 1C-LIKE PROTEIN REVEALED IN BLOOD SERUM IS
A NOVEL POTENTIAL MARKER OF AUTOIMMUNE DISORDERS
12.30–12.45
Maxim Lootsik HEMOSTATIC CHITOSAN: PREPARATION, PROPERTIES,
VERIFICATION OF HEMOSTATIC ACTIVITY
12.45–13.00
Rostyslav Panchuk NOVEL MECHANISMS OF MODULATION OF
ANTICANCER
ACTIVITY
OF
LANDOMYCIN
ANTIBIOTICS
BY
SPECIFIC
ANTIOXIDANTS
13.00–13.15
Volodymyr
Shablii
ONTOGENY
MULTIPOTENT CELLS BASED ON THEIR
OF
PLACENTA-DERIVED
IMMUNOPHENOTYPE AND GENE
EXPRESSION PROFILE
13.15–13.30
Yuriy Shkorbatov MICROWAVE IRRADIATION AND MAGNETIC FIELD
EFFECTS ON PUFFING PATTERN IN DROSOPHILA MELANOGASTER
13.30–14.30
Lunch
14.30–14.50
Dariya Fedorovych IDEAS OF PROFESSOR GEORGIY SHAVLOVSKY
AND THEIR DEVELOPMENT AT THE INSTITUTE OF CELL BIOLOGY, NAS OF
UKRAINE
14.50–16.30
Session 2 (Biotechnology Chairs Volkmar Passoth, Patrick Fickers)
14.50–15.10
Volkmar Passoth NON-CONVENTIONAL YEASTS FOR BIOFUEL AND
ANIMAL FEED PRODUCTION
15.10–15.25
Kostyantyn Dmytruk YEAST ENDOMYCES MAGNUSII AS A PROMISING
PRODUCER OF ISOBUTANOL
15.25–15.45
Patrick Fickers IMPROVEMENT OF ERYTHRITOL SYNTHESIS IN
YARROWIA LIPOLYTICA BY METABOLIC ENGINEERING
2
15.45–16.00
Olena Kurylenko THE ROLE OF CYTOSOLIC AND PEROXISOMAL
TRANSALDOLASES AND TRANSKETOLASES IN HANSENULA POLYMORPHA XYLOSE
METABOLISM AND FERMENTATION
16.00–16.15
METABOLISM
Marta Semkiv GENETIC ENGINEERING OF CYTOSOLIC CARBON
FOR
IMPROVED
GLYCEROL
PRODUCTION
IN
YEAST
SACCHAROMYCES CEREVISIAE
16.15–16.30
Liudmyla Kozeko WHOLE-MOUNT GUS STAINING ANALYSIS OF HEAT
SHOCK PROTEINS HSP90 AND HSP70 EXPRESSION IN ARABIDOPSIS THALIANA
SEEDLINGS
16.30–17.00
Coffee Break
17.00–18.00
Poster Session
18.00-18.10
Closing Ceremony (Best Poster Presentations Award)
18.10–21.30
Get-Together Party, Concert
October 13 (TUESDAY)
8.30–20.00
Excursion to Pochayiv Lavra (monastery) and Kremenets (with lunch)
3
ORAL PRESENTATIONS
4
DEVELOPMENT OF CELL BIOLOGY AND BIOTECHNOLOGY RESEARCH
IN INSTITUTE OF CELL BIOLOGY, NAS OF UKRAINE (LVIV): CURRENT
STATE AND PERSPECTIVES
Andriy Sibirny1,2
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005 Ukraine;
2
Department of Biotechnology and Microbiology, Rzeszow University, Zelwerowicza 4, Rzeszow 35-601
Poland.
Email address: sibirny@cellbiol.lviv.ua
1
Institute of Cell Biology was founded as the independent institution of NAS of Ukraine 15 years ago,
in 2000. Predecessor of the Institute, Lviv Division of Kiev Institute of Biochemistry, NAS of Ukraine, was
established in 1969. During last 15 years of autonomous existence, Institute of Cell Biology passed
complicated way which included continuation of already renowned investigations as well as establishing
the directions of research. All studies in the Institute could be divided into two categories of basic research:
molecular microbiology and molecular oncology. Both mentioned directions of basic research also resulted
in potential biotechnological applications. Institute currently consists of four Departments (Molecular
Genetics and Biotechnology, Regulation of Cell Proliferation and Apoptosis, Analytical Biochemistry, Cell
Signaling Mechanisms) and three laboratories (Metabolic Engineering, Molecular Genetics of Prokaryotes,
Intercellular Interactions). The Ukrainian Society for Cell Biology, the all-Ukrainian scientific
organization, is registered at the Institute. Institute published numerous articles in the leading international
journals, including Nature Medicine, Developmental Cell, Autophagy, Molecular Biology of the Cell,
Journal of Biological Chemistry, Metabolic Engineering, Journal of Biomedical Nanotechnology,
Biotechnology and Bioengineering, Microbial Cell Factories, Applied Microbiology and Biotechnology,
International Journal of Cancer, Anticancer Drugs, Amino Acids, PLoS One, FEMS Yeast Research and
many others. Institute participates or participated in fulfillment of many domestic and international grants,
including INTAS, NATO, CRDF, FIRCA, STCU and others.In addition, Institute has contacts with
Ukrainian and foreign biotechnological companies as Lviv yeast factory, Ladyzhyn enzyme plant, Archer
Daniels Midland Co. (USA) and others. Institute collaborates also with many Ukrainian and foreign
research institutions, especially with Rzeszow University, Poland, which is the partner in cross-border
research project. Other foreign partners are Warsaw University of Life Sciences; Wroclaw University;
Nencki Institute of Experimental Biology of Polish Academy of Sciences; Uppsala University; Lund
University; Dresden Technical University; Marburg University, Institute of Plant Genetics, Gatersleben,
Germany; Keck Graduate Institute, Beaverton, USA; Groningen University, The Netherlands, Lille
University, France, Korean Institute of Bioscience and Biotechnology, Daejon, Nara Institute of Science
and Technology, Japan; King Mongkut Bangkok Technical University, Thailand, and others. Institute is
known as the organizer of international scientific meetings as 12th International Congress on Yeasts, two
specialized international symposia on yeasts, bilateral Ukrainian-Polish J. Parnas and R. Weigl
conferences. Institute of Cell Biology is one of the first Ukrainian research institutions which implemented
system for evaluation of achievements of individual scientists and research groups based on internationally
recognized criteria, such as h-index, citation index, summary impact factors of publications. Candidates for
defense of PhD theses are required to possess at least two international publications of which in at least one
the candidate is the first author.
Thus, in spite of complicated times in which we live, the Institute continues to be a leading
Ukrainian institution with recognized international reputation.
5
Session 1
(Cell Biology, Chairs Andriy Sibirny, Bartosz Grzegorz)
6
PURIFICATION AND PROPERTIES L-FUCOSE-SPECIFIC LECTINS FROM DIFFERENT SOURSES
AND THEIR VALUE FOR HISTOCHEMISTRY
Volodymir Antonyuk
Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine.
Email address: antonyukvo@gmail.com
α-L-Fucopyranosyl residues are widely distributed in the sugar chains of glycoconjugates:
glycoproteins, glycolipids, and proteokeratan sulfate. In many cases, the fucosyl residues constitute parts of
important antigenic determinants such as the blood group antigens H, Lea, and Leb and the stage-specific
embryonic antigens. Fucosylated sugars often play pivotal roles in many cellular processes. At the same
time, the lectins in this group are rare in nature. As result long-term investigation we have found a number
of new L-fucose-specific lectins in plants, mushrooms and fish roe. This review summing long-term
authors and collaborations search, purification and investigation fucose-specific lectins.
Search for new lectin was made among higher plants, true fungi and fish roe. Lectins purification
were realized by affinity chromatography on synthezised sorbents followed by gel filtration or ion
exchange chromatography. For determined molecular mass polypeptide chains SDS-polyacrylamide gel
electrophoresis under reducing conditions was carried. Hemagglutination inhibition tests were carried out
for determination carbohydrate specificity.
New L-fucose-specific lectins were detected in Laburnum anagyroides bark, Perca fluviatilis and
Lucioperca lucioperca roe, Peziza badia and Peziza vesiculosa fruiting bodies. The main physicochemical
characteristics, interaction with human and animal erythrocyres, carbohydrates and glycoproteins were
established. SDS-polyacrylamide gel electrophoresis Laburnum anagyroides bark lectin revealed one
protein band with molecular weight of 26 kDa, the MW of native lectin were about 110 kDa. The obtained
lectin contains 13.1 % of carbohydrates. Lectin agglutinates native human erythrocytes with a preference
for the O(H) blood group. The lectins Perca fluviatilis and Lucioperca lucioperca roe are heterotetramers
with molecular weight of 50 kDa. The Perca fluviatilis lectin have lower sensitivity for human erythrocytes
as Laburnum anagyroides bark lectin, but better differentiate human blood group antigens. In spite, Peziza
badia and P. vesiculosa lectins not differentiate human blood group antigens. Unlike previous lectins, P.
badia and P. vesiculosa lectins binds with human thyroglobulin and mannofucogalactans of aphyllophoric
fungi; this suggests that the lectins interacts with L-fucose located inside the polysaccharide chain of the
glycoconjugates.
Interestingly, seed and bark Laburnum anagyroides, Sarothamnus scoparius and Ulex europaeus,
Perca fluviatilis roe containing else one lectin with different carbohydrate specifity. Are discussed causes
these phenomenons.
Purified lectins were used in the histochemical and cytochemical studies. For example, lectin
Laburnum anagyroides bark was used in сomparative lectin histochemical analysis of the duodenal glands
in various mammals, at lectin histochemical study human placenta in health and the weakness of labor
activity, as a marker of thin gut inflammation and other.
The lectins Perca fluviatilis and Lucioperca lucioperca roe were used for investigated distribution of
glycoconjugates of the rat kidney in the dynamics of postnatal ontogenesis of the development diabetic
nephropathy.
Although all the purified lectins have L-fucose specificity, they substantially differ the thin
carbohydrate specificity and ability to interact with carbohydrate-containinng glycoconjugates. Therefore
they all are important for scientific investigation.
7
MOLECULAR MECHANISMS INVOLVED IN HUMAN CANCER CELLS’
RESPONSE TO ARGININE DEPRIVATION
Yuliya Kurlishchuk1,2, Yaroslav Bobak1, Bozhena Vynnytska-Myronovska1, Leoni A. KunzSchughart2,3, Maria J. Redowicz4, Oleh Stasyk1
1Institute of Cell Biology, National Academy of Sciences of Ukraine, 14/16 Drahomanov Str,
79005 Lviv, Ukraine
2OncoRay – National Center for Radiation Research in Oncology, TU Dresden, Fetscherstr. 74,
01307 Dresden, Germany
Helmholtz Zentrum Dresden Rossendorf, Institute of Radiooncology, Dresden, Germany
4Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Str, 02-093
Warsaw, Poland
Email address: kurlishchukyuliya@gmail.com
Arginine deprivation abrogates proliferation of cancer cells both in vitro and in vivo. For
several types of cancer, the efficacy of enzymatic depletion of arginine as metabolic anticancer
therapy was verified in a number of Phase I/II clinical trials. However, a common challenge is the
ability of individual tumors, initially defined as sensitive to the lack of arginine, to acquire
resistance to arginine deprivation. One of the options to overcome such resistance is to target the
molecular pathways involved in the cancer cells’ response to nutrient limitation in combination
with arginine deprivation. We were first to show that the lack of arginine induces endoplasmic
reticulum (ER) stress in several established human cancer cell lines derived from solid tumors.
Despite a prolonged ER stress (up to 72 h), arginine deprivation did not result in profound
apoptosis when compared with the classic ER stress inducer, tunicamycin. Instead, Akt- and
MAPK-dependent pathways were activated which may oppose pro-apoptotic signaling under
arginine deprivation. Moreover, ER stress and apoptosis in arginine-starved cancer cells could be
critically augmented by supplementation of the natural arginine analogue canavanine. In
conclusion, we suggest that the ER stress pathway is a novel target for the development of more
efficient combinational anti-cancer approaches based on arginine deprivation.
8
PROINFLAMMATORY CONTROL OF SKELETOGENIC SIGNALING PATHWAYS
Olexandr Korchynskyi1,2, Khrystyna Malysheva1,3, Volodymyr Krupak1, Karien de Rooij2, Clemens
Löwik2, Rostyslav Stoika1
1
Institute of Cell Biology of NAS, 14/16, Drahomanov St., Lviv 79005, Ukraine;
2
Leiden University Medical Center, 2, Albinusdreef, 2333 ZA Leiden, The Netherlands;
3
Insitute of Animal Biology of NAAS, 38, V. Stusa St., Lviv 79034, Ukraine.
Email address: olexkor@hotmail.com
Rheumatoid arthritis (RA) is a severe autoimmune disease that affects up to 1.8% of adult population
of the world. Chronic inflammation leads to a persistent pain in joints frequently leading to patient’s
disability. During RA development, synovial fibroblast-like cells (FLS) start overgrowing and invade into
surrounding tissues in joint, thus, forming a pannus and leading to a severe destruction of the articular
cartilage and frequently to bone erosions. The BMP and Wnt/β-catenin pathway are key signaling pathways
that induce and support cartilage and bone formation and maintenance. Recently, we have discovered and
characterized two novel transcriptional repressors of BMP-Smad pathway that contribute to inflammationinduced bone destruction. We hypothesize that pituitary tumors transforming gene 1 (PTTG1) and its
partner PTTG1 binding protein 1 (PTTG-BP1) represent a novel key system in regulation of homeostasis of
joint tissues at RA pathogenesis. According to our preliminary data, PTTG1 overexpression leads to drastic
inhibition of Wnt/β-catenin signaling in target cells. Overexpression of PTTG-BP1 inhibits early
osteogenesis and shRNA-mediated knockdown of PTTG1 and PTTG-BP1 potentiates osteoblast
differentiation. We also found that inhibitory effect of PTTG1 converges on direct modulation of
functioning of β-catenin/TCF4 transcriptional complex. Such result suggests that PTTG/PTTG-BP1 axis
can serve as a new negative regulator of bone and potentially cartilage homeostasis. In parallel, we found
that PTTG1 deficiency in mice leads to alterations in kidney morphology and function. In addition, we
have studied possible contribution of PTTG1 interaction with Wnt pathway into kidney morphogenesis and
development of autoimmune nephropathologies. To validate our hypothesis and understand functional
impact of PTTG/PTTG-BP1 system in regulation of joint homeostasis and RA pathogenesis, the following
studies are in progress: 1) detection of PTTG and PTTG-BP1 and monitor activation of BMP, Wnt and NFκB pathways in synovial tissues of early and progressed disease RA patients versus healthy individuals
and/or other disease patients; 2) in vitro investigation of the molecular mechanism(s) mediating inhibition
of Wnt signaling by PTTG/PTTG-BP1 axis and their modulation by the pro-inflammatory stimuli; 3) in
vivo validation of functional impact of PTTG1/PTTG-BP1 in triggering and/or progression of experimental
autoimmune diseases in joints and kidneys of experimental animals.
Our work will shed light on the molecular mechanisms and functional impact of PTTG/PTTG-BP
axis in regulation of BMP, Wnt and NF-κB signaling pathways during osteoporosis and autoimmune
diseases in joints (rheumatoid arthritis) and kidney (nephropathies) progression. The results are of potential
significance for development of novel strategies in diagnostics and treatment of these diseases.
9
MYOSIN VI IN THE NUCLEUS: A SEARCH FOR ITS ROLE IN THE NUCLEOLUS
Jolanta Nowak1, Łukasz Majewski1, Magdalena Sobczak1, Justyna Karolczak1, Krzysztof Skowronek1,
Marta Lenartowska2, Robert Lenartowski2, Maria Jolanta Rędowicz1
1
Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw,
Poland;
2
Faculty of Biology and Environment Protection, Nicolaus Copernicus University, 1 Lwowska St., 87100 Toruń, Poland.
Email address: j.redowicz@nencki.gov.pl
Myosin VI (MVI), the only known myosin walking towards the minus end of actin filaments, is
involved in numerous cellular processes associated with the actin cytoskeleton. Remarkably, MVI was also
found within the nuclei of several cell types, including neuronal PC12 cells, and the C2C12 myotubes and
rat skeletal muscle as well as in neonatal rat cardiomyocytes. Its nuclear role still remains poorly
understood. Since MVI acts through interactions of N-terminal motor domain with actin filaments and of
C-terminal globular tail (GT) domain with cell/tissue specific binding proteins, we performed a search for
MVI partners by means of a pull down assay on cell lysates with GST-tagged MVI-GT as a bait. It was
followed by mass spectrometry that allowed for identification of numerous proteins involved in interaction
with nucleic acid and the related processes that are potential novel MVI binding partners. In PC12 cells,
among the identified putative partners was nucleolin, a major nucleolar protein which is implicated in the
ribosomal DNA (rDNA) transcription, rRNA maturation and ribosome assembly. MVI-nucleolin
interaction was further confirmed by immunocytochemistry and proximity ligation assay. Additionally, we
showed that other nucleolar proteins involved in ribosome biogenesis, namely fibrillarin and RNA
polymerase I engaged in ribosome biogenesis also interacted with MVI. As revealed by the immunogold
technique, MVI is present within all nucleolar compartments, also at the nucleoplasm-nucleoloplasm
border, indicating its involvement in the shuttling between nucleus and nucleolus, and in nucleolar
processes. Indeed, immunocytochemistry and electron microscopy revealed profound changes in
organization of the endoplasmic reticulum in MVI-depleted PC12 cells. Similar observations we made for
MVI-depleted C2C12 myoblasts. Also, profound changes in the nucleolus organization were found in
MVI-depleted PC12 cells. In light of these data, we speculate that MVI could be engaged in maintenance of
nucleolar structure as well as in regulation of ribosome biogenesis, and thus in the ER network
organization.
10
NANOBIOTECH RESEARCH ACTIVITY AT DEPARTMENT OF REGULATION OF CELL
PROLIFERATION AND APOPTOSIS (INSTITUTE OF CELL BIOLOGY, NAS OF UKRAINE)
Rostyslav Stoika
Institute of Cell Biology (ICB), NAS of Ukraine, Drahomanov Street 14/16, 79005 Lviv, Ukraine.
Email address: stoika@cellbiol.lviv.ua
Several NanoBioTech projects are presently realized in the Department at the ICB.
Project 1: Imaging of formalin-fixed, paraffin-embedded labeled tissues animals.
Thermodestruction of mouse melanoma tissue was achieved after applying specific nanocrystals (NC)
developed at Wroclaw Technical University (Poland) and then functionalized with specific lectins at the
ICB. That approach can be effectively used before conducting technically complicated and costly
experiments on whole animal imaging. Studies are in progress for detection of functionalized NC using Xray excitation for X-ray compatible detection systems. Collaboration in that field has been started with the
NanoBioTech-interested laboratories at Cedars-Sinai Medical Center (Los Angeles, USA) and the Institute
for Brain Research, University of Zagreb School of Medicine (Croatia).
Project 2: Biomedical applications of novel polymeric nanoparticles with a reactive shell
suitable for bio-functionalization. Enhanced anticancer activity and circumvention of drug resistance
mechanisms was achieved by applying novel polymeric/phospholipidic nanocarriers synthesized at Lviv
National Polytechnic University (Ukraine). Novel oligoelectrolyte-based non-viral gene delivery systems
for genetic transformation of the eukaryotic (mammalian, plant, yeast) cells were also created. Active
collaboration in estimating the bio-compatibility of the developed nanomaterials, namely their negative side
effects (cardio-, hepato-, and nephrotoxicities) is also carried out with scientists of Lviv National Medical
University.
Project 3: Biomedical and biotechnological application of novel super-paramagnetic nano- and
microparticles developed at the Institute of Macromolecular Chemistry (Prague, Czech Republic).
Improvement of the bio-compatibility and enhancement of the engulfment of the created particles were
demonstrated after functionalization of their shell. Novel affinity sorbents were also created on the basis of
the developed particles.
Key words: nanomaterials, treatment, diagnostics, anticancer drugs, tumor cells, apoptosis.
Acknowledgements: The collaborators of the ICB, as well as scientists of other mentioned research
institutions are acknowledged for their work on the NanoBioTech projects listed above. Many thanks to
Cedars-Sinai Medical Center’s International Research and Innovation Management Program, the
RECOOP HST Association, and its President Dr. Sandor Vari for their support of the research activity of
collaborators of the Department.
11
PROLIFERATIVE AND DIFFERENTIATION POTENTIALS OF HUMAN ADULT
MESENCHYMAL STROMAL CELLS IN THREE-DIMENSIONAL CULTURE. THE EFFECT
OF STORAGE AT CRYOGENIC AND AMBIENT TEMPERATURES.
Alexander Petrenko1, Vitaly Muzenko1, Elena Rogulska1, Vedeney Zaikov1, Svetlana Mazur1, Dmitry
Tarusin1, Hermann Ehrlich2, Yuri Petrenko1
1
Institute for Problems of Cryobiology and Cryomedicine NAS, 61023, Kharkov, Ukraine;
2
Institute of Experimental Physics, TU Bergakademie Freiberg, 09599 Freiberg, Germany.
Email address: alexander_petrenko@cryo.org.ua
Mesenchymal stromal cells (MSCs) can differentiate into various cell types, which make them
attractive for regenerative medicine. Preservation of MSCs seeded and cultivated in scaffolds at cryogenic
or ambient temperatures can serve as ready-to-use transplantation units for tissue repair.
The aim of the study was investigation of ability to proliferation and multilineage differentiation of
MSCs in three-dimensional culture within alginate microspheres (AMS) and porous scaffolds. As porous
scaffolds for MSCs alginate-gelatin macroporous cryogel sponges and demineralized chitinous skeletons of
marine sponge Ianthella basta were used.
MSCs isolated from adult human bone marrow, dermal and adipose tissues after expansion in
monolayer culture had similar phenotype and growth patterns as well as ability to induced multilineage
differentiation. After encapsulation in AMS MSCs had spherical shape and during 3-D culture stopped
proliferate, but possessed viability and capability to differentiation toward adipogenic, osteogenic and
chondrogenic lineages. MSCs encapsulated in AMS could be successfully cryopreserved by both
conventional cryopreservations with 10% of dimethyl sulfoxide and vitrification protocols. Besides,
encapsulation in AMS delayed cell death during storage at ambient temperatures.
After seeding into porous scaffods MSCs attached to the pore walls spread and proliferated.
Response of MSCs cultivated into porous scaffolds to conventional cryopreservation protocol depended on
cell adhesion and spreading as well as structure of scaffolds per se. Between observed three types of
scaffold the lowest cryoresistance had MSCs growing as sheets on chitinous marine demosponge Ianthella
basta. After cryopreservation in various scaffolds survived MSCs retained abilities to proliferation and
differentiation into osteogenic and adipogenic lineages. The data obtained indicate that cryo-banking of
MSCs cultivated into tissue engineered scaffolds is feasible for the future regenerative medicine projects.
12
NITROXIDES PREVENT UNDESIRED PROTEIN MODIFICATIONS
Izabela Sadowska-Bartosz1, Grzegorz Bartosz2
1
Department of Biotechnology and Microbiology University of Rzeszów,
Zelwerowicza 4, 35-01 Rzeszów, Poland;
2
Department of Biochemistry and Cell Biology, University of Rzeszów,
Zelwerowicza 4, 35-01 Rzeszów, Poland.
Email address: grzegorz.bartosz@gmail.com
Nitroxides are promising compounds for prevention of undesired protein modifications. Seven
nitroxides of different structures were studied for the ability to prevent glycoxidation of bovine serum
albumin incubated with three monosaccharides (glucose, fructose, and ribose). Glycoxidation was
estimated by fluorimetric parameters of protein modifications (formation of advanced glycation end
products [AGEs], dityrosine, N'-formylkynurenine, and kynurenine) and enzyme-linked immunosorbent
assay for AGEs. From among the nitroxides tested, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), 4carboxy-TEMPO, and 4-hydroxy-TEMPO offered significant protection against glycoxidation induced by
glucose and fructose, while 3-carbamoyl-PROXYL was not protective, enhancing glycoxidation. Lower
protection was observed for glycoxidation induced by ribose where only 3-carbamoyl-PROXYL and 4amino-TEMPO showed some protection. Loss of electron spin resonance signal of the nitroxides was
observed during glycoxidation indicating occurrence of free radical reactions in this process. We compared
also the efficiency of 11 nitroxides, derivatives of 2,2,6,6-tetramethylpiperidine-1-oxide (TEMPO) and
2,2,5,5-tetramethylpirrolidine-1-oxyl (PROXYL) in prevention of nitration and oxidation of model
compounds and human serum albumin (HSA). Most nitroxides were very efficient in preventing loss of
fluorescein fluorescence induced by peroxynitrite (PN) (IC50 in the nanomolar range) and preventing HSA
nitration. The loss of fluorescein fluorescence was demonstrated to be due to nitration. Nitroxides were
more effective in prevention nitration than oxidation reactions. They showed a concentration window for
preventing dihydrorhodamine (DHR) 123 oxidation but exerted a prooxidant effect at both high and low
concentrations. No prooxidant effect of nitroxides was seen in prevention of DHR123 oxidation induced by
SIN-1. In all essays hydrophobic nitroxides (especially 4-nonylamido-TEMPO and 3-carbamolyldehydroPROXYL) showed the lowest efficiency. An exception was the prevention of thiol group oxidation
by PN and SIN-1 where hydrophobic nitroxides were the most effective, apparently due to binding to the
protein. Nitroxides showed low toxicity to MCF-7 cells. Most nitroxides, except for the most hydrophobic
ones, protected cells from the cytotoxic action of SIN-1 and SIN-1-induced protein nitration. These results
point to potential usefulness of nitroxides for prevention of PN-induced oxidation, glycation and,
especially, nitration.
13
Session 1
(Cell Biology, continued, Chairs Rostyslav Stoika,
Maria Jolanta Redowicz)
14
HSP60 AND ANTI-HSP60 ANTIBODIES AT THYROD CANCER PROGRESSION
Sidorik L.1, Yakovenko L.1, Kroupskaya I.1, Markelova O.1, Tykhonkova I.1, Pogribnyy P.2,
Gurtovyy V.3, Usenko V.3
1
Institute of Molecular Biology and Genetics NAS of Ukraine, Kyiv, Ukraine;
2
Kavetskiy Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine;
3
Morphological Laboratory BIONTEK, Dnipropetrovsk, Ukraine.
Email address: sidorik@imbg.org.ua
BACKGROUND. The molecular chaperones (HSPs) are involved in numerous diseases, including
cancer, revealing changes of their expression and cellular localization. The expression of Hsp60 in
neoplasia has been implicated in the regulation of apoptosis, as a modulator of p53 function, in the immune
response against tumors, and in multidrug resistance. A wide range of tumor cells or tissues have been
shown to express atypical level or localization of HSPs. For example, Hsp60 expression in breast or gastric
cancer is associated with poor prognosis and resistance to chemotherapy or radiation therapy. Such
observations have led to suggestions that HSPs could be used as biomarkers.
Molecular chaperon (chaperonin) Hsp60 is a special interest due to recently observed proinflammation potential of Hsp60 and anti-Hsp60 autoantibodies which have been detected in circulation of
diseased and healthy persons as well.
Hsp60 expression in thyroid cancer has not been studied extensively. Understanding the specific
roles of such chaperone in thyroid carcinogenesis has important implications regarding tumor behavior and
potential prognostic implications. The aim of our study was to evaluate the possible changes in Hsp60
cellular content and localization in thyroid gland’ cells in normal state and upon pathology by quantitative
immunohistochemical analysis and to exam the correlation between anti-Hsp60 autoantibodies level and
the degree of thyroid gland lesions in this group of patients.
METHODS. Sera from 49 patients (46 females and 3 males, 20-57 years old, 12 – nodular
hyperplasia of thyroid gland, 12 – Hashimoto’s thyroiditis, 18 – follicular adenoma, 6 –papillary and 1
follicular carcinoma) and 12 healthy donors (as a control) were used for determination of anti-Hsp60
antibodies level by ELISA. To evaluate the content and localization of Hsp60 in thyroid tissue of such
patients and upon pathology we used immunohystochemical analysis. As a control, autopsy material of 12
thyroid tissue without morphological signs of thyroid pathology was used. Hsp60 expression was evaluated
according to the percentage of positively stained cells.
RESULTS. The increased anti-Hsp60 autoantibodies level has been detected in sera more than 50%
of patients with non-malignant thyroid gland pathology (Hashimoto’s thyroiditis, nodular hyperplasia and
follicular adenoma) and in 86% of TC patients sera by ELISA method. This finding correlates with the
degree of thyroid gland lesions in this group of patients using immunohistochemical studies.The increased
Hsp60 expression in thyroid cancer tissue in comparison with normal ones was heterogenic and associated
with lymphoid infiltration and sclerotic changes of thyroid tissue.
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 have been revealed. Changes of Hsp60 expression and cellular localization in thyroid cancer tissue
have been detected in comparison with normal ones. The increase of Hsp60 expression and elevated level
of anti-Hsp60 autoantibodies was associated with morphological signs of pathology - lymphoid infiltration
and sclerotic changes of tissue.
The data obtained could serve as a scientific basis for new diagnostic and therapeutic tools
development.
15
MYOSIN 1C –LIKE PROTEIN REVEALED IN BLOOD SERUM IS A NOVEL POTENTIAL
MARKER OF AUTOIMMUNE DISORDERS
Myronovkij S.1, Negrych N.2, Nehrych T.2, Stoika R.1, Souchelnytskyi S.3,4,5, Kit Y.1
1
Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov St., 14/16, 79005,
Lviv, Ukraine;
2
Danylo Halytsky Lviv National Medical University, Pekarska St., 69, 79010, Lviv, Ukraine;
3
Center for Translational Molecular Medicine, College of Medicine, Qatar University, Doha Qatar;
4
Oranta CancerDiagnostics AB, Uppsala, Sweden;
5
Odesa State Medical University Karolinska, Odesa, Ukraine.
Email address: kit@cellbiol.lviv.ua
Serum, derived from plasma with clotting factors removed, contains 60–80 mg of protein/ml in
addition to various small molecules including salts, lipids, amino acids, and sugars. The major protein
constituents of serum include albumin, immunoglobulins, transferrin, haptoglobin, and lipoproteins. In
addition to these major constituents, serum also contains many other proteins that are synthesized and
secreted, shed, or lost from cells and tissues throughout the body. It is estimated that up to 10,000 proteins
may be commonly present in serum, most of which would be present at very low relative abundances.
Blood plasma is known to react on al changes in the human body. Therefore, serum obtained from plasma
has been extensively explored for detection of markers of disease, with strong success. Proteome analysis
of clinical samples aims at characterizing disease-specific changes in the protein expression profile of an
affected tissue. We aimed to explore serum proteins for detection of markers of autoimmune disorders.An
appropriate sample preparation is essential for obtaining reliable results in a proteomic analysis. 2,2,2trichloroacetic acid (TCA)-induced protein precipitationis the most popular and preferred method for
sample preparation for proteome analysis. For the preparation of proteins for proteome analysis, is
frequently used to concentrate proteins and to remove interfering compounds. Extraction of the entire
protein from the complex biological samples like serum requires a multi-step robust protocol, and TCAinduced precipitation is widely use.
Using two-step purification procedure including treatment of blood serum with 10% trichloroacetic
acid followed precipitation of soluble substances with acetone in ratio 1:9, we isolated two proteins with
molecular masses 66 and 48 kDa. MALDI/TOF tandem MS mass spectrometry analysis of a 66 kDa
polypeptide showed its high structural similarity to a blood serum albumin (AAX63425). The second 48
kDa polypeptide was identified as a unconventional myosin IC isoform b (NM 001080950.1). Myosin 1c
(Myo1c) is a member of the unconventional class I myosins of vertebrates, which directly link the plasma
membrane with the microfilament cortical web. This myosine has not been described in blood serum
earlier. This protein is implicated in such cell functions as cytoskeleton organization, cell motility and
nuclear transcription. Myo1c is abundantly expressed in murine B lymphocytes and is preferentially located
at the plasma membrane, especially in peripheral processes such as microvilli. The data suggest that Myo1c
is involved in the cytoskeleton dynamics and membrane protein anchoring or sorting in B lymphocytes.
Screening of blood serum sets toward the presence there Myo1c revealed it high level in blood serum of
multiple sclerosis and rheumatoid arthritis patients versus low concentration in blood serum of SLE
patients and healthy donors. This protein was not detected in blood serum of 12 patients with diabetes type
1.
Conclution: We isolated from human blood serum a 48 kDa protein which was identified as a
fragment of cellular unconventional myosin 1C (isoform_b). Preliminary data demonstrated positive
correlation of the level of Myo 1c in blood serum with appearance of some autoimmune disorders.
16
HEMOSTATIC CHITOSAN: PREPARATION, PROPERTIES,
VERIFICATION OF HEMOSTATIC ACTIVITY
Lootsik M.D.1, Bilyy R.A.1,2, Lutsyk M.M.2, Stoika R.S.1
1
Institute of Cell Biology, NAS of Ukraine, Drahomanov st., 14/16, Lviv, Ukraine;
2
D.Halytskij National Medical University, Pekarska st. 69, Lviv, Ukraine.
Email address: lootsik@cellbiol.lviv.ua
Among numerous hemostatic agents used in practical medicine, chitosan preparations known under
the trademark “Celox” are superior in arresting of external bleeding, especially severe one. Recently, we
developed new laboratory tests for detection of hemocoagulating/ hemostatic activity of chitosan
preparations. They include test on in vitro coagulation of blood sample stabilized with anticoagulants
(heparin, EDTA, citrate, oxalate etc.) by powdered chitosan and in vivo determination of bleeding arrest
after cutting off distal part of mouse or rat tail and application of chitosan bandage on the wound. These
tests revealed that commercial chitosans supplied as chemical reagents do not exhibit
hemocoagulating/hemostatic effect. They can be transformed to active hemostatic material by specific
processing which includes dissolving of chitosan sample in 3% acetic acid, its precipitation with ammonia,
collection of sediment, and its dissolution by adjustment to pH5.0 with diluted acetic acid. Then
precipitation of chitosan at neutral pH with alcohol at 55-60% concentration, dessication of the sediment on
flat surface, collection of dried material and grinding it to powder with particles less then 0.2 mm. Samples
of hemostatic chitosan were prepared in our laboratory from chitin of crab shells or chitosan of SigmaAldrich company (low mol.weight grade) and their activity was compared with original “Celox” (Medtrade
Products Ltd, UK). Our chitosan samples coagulated heparinized blood in 45 sec when it was taken in
proportion 30 mg per 1 ml of blood. The initial reagent grade chitosan showed no coagulating effect, while
“Celox” coagulated blood in 2 min due to coarse form of powder, and after its grinding to fine powder
coagulation rate was 45 sec. Bleeding after cut off of mouse tail was stopped in 10 min of bandage
application (in control – 30 min).
The following properties are useful for evaluation of chitosans as the hemostatic agents:
1) poured volume, the values were 2.5 -6.0 ml/g for our preparations, 1,9 ml/g for “Celox”, 1,7 ml/g
for chitosan reagent; 2) swelling in water (as x-fold increase in volume of distinct mass of material), for
“Celox” and active samples - 100 fold, samples with law activity - 40 fold, inactive chitosan -6 fold; 3) pH
of water suspension: in our samples – 5.2-5.5, “Celox” – 4.7, chitosan reagent –5,5. Poured volume and
swelling index are closely related and reflect porosity of chitosan particles. It is advisable to obtain material
with higher porosity which provide more effective blood clot formation due to rapid absorption of blood
plasma, rapid increase of viscosity and attachment of blood cells to surface of chitosan particles. We also
revealed that hemostatic chitosan provides clotting of defibrinated blood. This indicates that dehydration of
blood due to swelling of particles and absorption of cells on their surface are principal factors in the
mechanism of chitosan-induced blood clotting, activation of blood platelets (viscose metamorphosis) and
fibrinogen are less important in that case.
The application of chitosan prepared by proposed process for control of bleeding, as well as for
treatment of wounds and burns needs their approval in clinical conditions. The research aspects of its
implication are also of great importance.
17
SIGNALLING FUNCTION OF β-CATENIN DURING CARDIOGENESIS AND NOVEL
MECHANISMS OF MODULATION OF ANTICANCER ACTIVITY OF LANDOMYCIN
ANTIBIOTICS BY SPECIFIC ANTIOXIDANTS
Rostyslav Panchuk1, Liliya Lehka1, Jurgen Rohr2, Petra Heffeter3, Walter Berger3,
Rostyslav Stoika1
1
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street 14/16, 79005 Lviv, Ukraine;
2
University of Kentucky, College of Pharmacy, 789 S. Limestone St. Lexington;
3
Institute of Cancer Research, Vienna Medical University, Borschkegasse, 8a, Vienna 1090, Austria.
Email address: rpanchuk@ukr.net
In the last decade antioxidants have gained an enormous popularity in cancer treatment, as they were
considered to be crucial for lowering of side effects of anticancer drugs towards cancer patients and thus
increasing their quality of life. However, a number of cases indicate that use of antioxidants can worsen
health conditions of cancer patients due to different reasons, which are still poorly understood.
The main aim of current study was to investigate modulatory effect of various antioxidants – catalase
(H2O2 scavenger), mannitol (hydroxyl radicals’ scavenger), superoxide dismutase (O2- scavenger) and
thiol-type ROS scavenger N-acetylcysteine on molecular mechanisms of anticancer activity of angucycline
antibiotics of landomycin family towards human Jurkat T-leukemia cells.
Previously it was reported that landomycins (Ls) cause massive ROS burst (5-fold increase of
DCFDA fluorescence) already at 1h after addition to cell culture, which is almost completely eliminated
after cell pre-treatment with nonspecific ROS scavenger N-acetylcysteine. For better understanding, what
exactly ROS are involved in Ls-induced apoptosis, specific scavengers for H2O2, OH* and O2- were
applied. It was found that catalase was the strongest scavenger of Ls-driven ROS, leading to 2-fold
decrease in level of DCFDA fluorescence, thus indicating on crucial role of hydrogen peroxide in Lsinduced oxidative burst. Mannitol (OH- scavenger) possessed much weaker activity (1.3-fold decrease of
DCFDA fluorescence), while SOD (superoxide anions scavenger) had almost no effect here, pointing out
on a secondary role of hydroxyl radicals and absence of any impact of superoxide radicals in Ls-induced
ROS burst. Simultaneous addition of catalase, mannitol and SOD has demonstrated the highest ROSscavenging effect at all observed timepoints. However, Annexin V/PI double staining assay has revealed
that all tested ROS scavengers, except NAC, provide very weak cytoprotective activity towards LE-treated
cells, despite strong decrease of ROS production. Thus, more attention was given to NAC, as it was the
only compound studied, able to completely inhibit both ROS production and apoptosis under LE treatment.
It was revealed that NAC was able to completely inhibit Ls entry into target cells, as revealed by
drug autofluorescence studies, while other tested ROS scavengers failed to do it. NMR studies of
landomycin E (LE)-NAC solution have revealed appearance of extra peak with Mw=865 Da, which was
identified as 1:1 LE-NAC adduct, where NAC was covalently bound by SH-group to 4th carbon atom in
quinone group of LE aglycon. Thus, all previously revealed NAC cytoprotective effects towards LE are
explained not by ROS scavenging by NAC, but by formation of extracellular covalent LE-NAC adduct,
which physically can’t enter the cells. Summarizing, ability of NAC to form covalent adducts with
quinone-containing anticancer drugs should be of crucial importance while developing the chemotherapy
courses for cancer patients in clinical oncology, as NAC can inhibit therapeutic activity of these drugs and
thus enhance tumor progression instead of stopping it.
18
ONTOGENY OF PLACENTA-DERIVED MULTIPOTENT CELLS BASED ON THEIR
IMMUNOPHENOTYPE AND GENE EXPRESSION PROFILE
V.A. Shablii1, M.D. Kuchma1,2, G.M. Svitina1,3, І.Ya.Skrypkina2, T.V. Bukreieva1, V.M. Kyryk4, P.O.
Areshkov2, Yu.М. Shablii1, G.S. Lobintseva1
1
Institute of Cell Therapy, Kyiv, Ukraine;
2
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine, Kyiv, Ukraine;
3
Educational and Scientific Centre “Institute of Biology”, Taras Shevchenko National University of
Kyiv, Kyiv, Ukraine;
4
State Institute of Genetics and Regenerative Medicine of Academy of Medicine of Ukraine, Kyiv,
Ukraine.
Email address: v_shabliy@ukr.net
Primary cultures have distinct immunophenotype from their in vivo progenitors. Culture conditions
induce expression of markers specific for ontologically different cell types that complicate the
establishment of origin of obtained cells. The aim of this study was to investigate placenta-derived
multipotent cells (PDMCs) that possess several trophoblast related features. PDMCs were obtained by
explants primary culture methods. PDMCs simultaneously expressed both mesenchymal (vimentin (Vim),
alpha-smooth muscle actin (a-SMA), ERG, CD90, CD73, CD105) and trophoblast markers (pancytokeratin (pCK), cytokeratin 7 (CK7), chorionic gonadotropin (CG), CDX-2, EOMES). Moreover, it was
observed the expression of both trophoblastic and nontrophoblastic types of CGβ genes in obtained
cultures; some of PDMC-lines expressed fusogenic gene ERVW-1 and trophoblast related transcription
factor GSM1.
The colonies acquired from single cells culturing were either positive or negative for CK7. The
expression of CK7 decreased during subculturing and disappeared at 3nd passage. The CK7low clones had
high proliferation potential and were maintained in culture up 8 passages. We obtained the four CK7 low
clones (Ep4C1, Ep18C2, Ep18C4, Ep18C9) which possessed similar immunophenotype profile except
several markers. Expression profile of CK7low-derived clones that included CDX2, EOMES, POU5F1,
VASA, SPP1, and GSM1 was heterogeneous, only Ep4C1 expressed all of those genes. In contrast to VASA,
another primordial germ cell marker IFITM3 was common for Ep18C2, Ep18C4, and Ep18C9.
Interestingly, ERVW-1 that involved in syncyalization was expressed in all propagated single-cell derived
clones although we have not detected any evidence of cell fusion and multinuclearity of cells. Interestingly,
all of CK7low-derived clones had great potential to osteogenic and adipogenic differentiation in contrast to
primary culture of PDMC.
To investigate the origin of PDMCs the immunohystochemistry of full term placenta, first trimester
placental tissue and cultured full term placental explants was made. CDX-2 and EOMES were not detected
in full-term placental tissue but in first trimester placental tissue they were expressed in cytotrophoblast and
stromal cells. ERG expression mainly was observed in endothelial cells of vessels in full term and first
trimester placental tissue. Although alone ERG positive cells were located distinct of vessels and coexpressed a-SMA. Interestingly, ERG –positive cells co-expressed proliferative marker PCNA in placental
explants. Proliferated cells were appeared after culturing of placental tissue fragments and were either
CK7-positive or negative. Furthermore the expression of epithelial and trophoblast markers (CG, CK7) in
stromal cells of full term placental tissue was induced under culture condition We conclude that PDMCs
belong to mesodermal cells possibly of perivascular origin with some trophoblast features.
19
MICROWAVE IRRADIATION AND MAGNETIC FIELD EFFECTS ON PUFFING
PATTERN IN DROSOPHILA MELANOGASTER
L.A. Shakina1, N.N. Kolchigin2, Y.G. Shckorbatov1
1
V.N. Karazin Kharkiv National University, Institute of Biology, pl. Svobody, 4, Kharkov, 61022,
Ukraine;
2
V.N. Karazin Kharkiv National University, School of Radiophysics, Department of Theoretic
Radiophysics, pl. Svobody, 4, Kharkov, 61022, Ukraine.
Email address: yuriy.shckorbatov@gmail.com
In our previous work we have demonstrated the effect of microwave radiation of low intensity on the
puffing pattern in Drosophila melanogaster polytene chromosomes (Shakina, et al., 2008). In this work we
investigated the effects of successive exposure to microwave radiation and static magnetic field on puffing
pattern in polytene chromosomes of Drosophila melanogaser. Drosophila eggs were exposed to
microwaves of 36.64 GHz frequency, 100 µW/sm2 intensity, exposure time 30 sec.; and also to static
magnetic field of 25 mT intensity, exposure time 5 min. The dimensions of puffs in giant chromosomes in
Drosophila larvae salivary gland at 0 h prepupal stage was measured after orcein staining. Puffs 63F, 71CE,
72CD (chromosome 3L); and 82EF, 83E, 93D (chromosome 3R) were investigated. The microwaveinduced decrease of puffing activity was demonstrated in puffs 63F, 71CE, 82EF. The magnetic fieldinduced decrease of puffing activity was shown in puffs 83E, 93D. The puffs 50CD and 72CD/73A not
react on magnetic nor microwave field. After successive influence of microwaves and magnetic field the
puffing activity increased relatively to “microwave only” variant in puff 71CE/73A. Thus, the Drosophila
puffing activity answers to low-level microwave irradiation and medium intensity magnetic field in a
specific way.
Shakina L.A. et al., Central European Journal of Biology, 2011, 6:524-530.
20
IDEAS OF PROFESSOR GEORGIY SHAVLOVSKY AND THEIR DEVELOPMENT AT THE
INSTITUTE OF CELL BIOLOGY, NAS OF UKRAINE
Fedorovych D.V.1, Sibirny A.A.1,2
1
Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Street 14/16, 79005
Lviv, Ukraine;
2
Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, 35-601
Rzeszow, Poland.
Email address: fedorovych@cellbiol.lviv.ua
Professor Georgiy Shavlovsky was well known scientist in the field of microbial physiology,
biochemistry, genetics and molecular biology with emphasis of non-conventional yeasts. The main
directions of his research activities were studies of biosynthesis of riboflavin and flavin nucleotides,
regulation of these processes and connected processes such as biosynthesis of purine nucleotides, classical
and molecular genetics of flavinogenic yeasts. The basic researches of riboflavin biosynthesis in yeasts and
influence of various factors on this process was started at the Department of Microbiology of the Lviv State
University in 1960th. Subsequently, this line of research was successfully developed at the Institute of Cell
Biology, NAS of Ukraine (that time, Lviv Division of the Institute of Biochemistry). Investigation of
riboflavin biosynthesis at the institute progressed from studying physiology of flavinogenic
microorganisms up to starting to decipher the molecular mechanisms of riboflavin biosynthesis regulation
resulted in construction of overproducers of riboflavin and its derivatives. Using developed methods of
classic genetics and generated riboflavin auxotrophs, new reactions of riboflavin biosynthetic pathway were
identified. The tentative pathway was confirmed, in collaboration and competition with foreign groups,
especially group of Dr. A. Bacher from Munich Technical University, after isolation of the corresponding
enzymes and intermediates of riboflavin synthesis, followed by cloning of the structural genes involved in
the pathway. Most important achievements of Prof. G.M. Shavlovsky in this field were the proof that GTP
cyclohydrolase catalyzes the first reaction of riboflavin synthesis and identification of ribose-5-phosphate
or its derivatives as precursor in riboflavin synthesis.
It was shown that regulation of riboflavin biosynthesis by iron in the flavinogenic yeast Pichia
guilliermondii occurs mainly at the transcriptional level. Several groups of the mutants affected in the
regulation of riboflavin biosynthesis by iron ions were isolated in yeast P. guilliermondii using original
approaches and were found to be defective in regulation of iron uptake too. Many types of mutants
involved in regulation of riboflavin synthesis were isolated, however, their molecular nature remained
unknown. Studying of riboflavin extrusion and transport leaded to identification of two transport systems
and several P. guilliermondii genes potentially encoding their main components.
During last decades, molecular biology methods were developed for yeast Candida famata resulting
in cloning and identification of genes of flavin biosynthesis. Exploring of this yeast species leaded to
discovering of a transcriptional activator Sef1p that regulates both riboflavin biosynthesis and iron
acquisition. A riboflavin overproducing C. famata strain that over-expressed genes SEF1 as well as RIB1
and RIB7 (the first and last steps of riboflavin synthesis) was constructed. Its fed-batch fermentation
allowed to obtain more than 16 g of riboflavin/L, suggesting a strong industrial potential of the constructed
producer and overall process designed on base of its use. C. famata recombinant strains with 960-fold
increased FMN production and more then 1000-fold increased FAD production, as compared to the
parental strain of C. famata were obtained. Exploring of molecular mechanisms of regulation of flavins
biosynthesis and their transport are of fundamental and industrial interests.
21
Session 2
(Biotechnology Chairs Volkmar Passoth, Patrick Fickers)
22
NON-CONVENTIONAL YEASTS FOR BIOFUEL AND ANIMAL FEED PRODUCTION
Volkmar Passoth
Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, S-75007 Uppsala,
Sweden.
Email address: volkmar.passoth@slu.se
Yeasts are a highly diverse group of organisms, with an amazing biotechnological potential.
Bioethanol, mainly produced by yeasts, is the worldwide major biofuel. However, yeasts can also be
utilised to generate other biofuels, animal feed or chemicals. Biodiesel is currently generated from first
generation resources – oil plants, including soya, oil palms or rape, which can also be used as food.
Moreover, their energy yield per covered area is low and cutting of rain forest area for oil plant production
has been reported. In contrast, oleaginous yeasts can accumulate lipids to more than 50% of their biomass,
and their fermentation does not compete with the utilisation of arable land.
We identified a number of ascomycetous and basidiomycetous yeasts as promising to convert
lignocellulose hydrolysate to lipids. In fed-batch fermentations 7 g lipids per L and more were reached.
Ascomycetous yeasts e.g. Lipomyces starkeyi showed a better capacity to produce lipids from xylose than
basidiomycetes e.g. Rhodotorula glutinis. On the other hand, R. glutinis generated more unsaturated fatty
acids (10% and more of linolenic acid- C18:3).
Animal feed production to a large scale consumes food-grade resources and requires often more
energy than transportation. We have tested to utilise oil of L. starkeyi grown on lignocellulose hydrolysate
as fish feed. Utilisation of lignocellulose for the production of feed and valuable chemicals is a key to a
sustainable society and will be discussed.
23
YEAST ENDOMYCES MAGNUSII AS A PROMISING PRODUCER OF ISOBUTANOL
Kostyantyn Dmytruk1, Olena Kurylenko1, Barbara Kshanovska1, Andriy Sibirny1, 2
1
Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Street 14/16, 79005
Lviv, Ukraine;
2
Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, 35-601
Rzeszow, Poland.
Email address: dmytruk@cellbiol.lviv.ua
Compared to the traditional biofuel, ethanol, higher alcohols (C4 and C5) offer advantages as
gasoline substitutes because of their higher energy density and lower hygroscopicity. A synthetic approach
to produce the longer chain alcohols as next-generation biofuels was implemented in Escherichia coli and
Saccharomyces cerevisiae. These host organisms have fast growth rates and are facultative anaerobes,
allowing for a flexible and economical process design for large-scale production. However, importing and
the expression of heterologous pathways may lead to metabolic imbalance, whereas the accumulation of the
heterologous metabolites may cause cytotoxicity (Atsumi et al., 2008).
Therefore, screening for native microorganisms like non-conventional yeasts with excellent growth
behavior and capacity for producing higher alcohols by fermentation is of great interest. Around 50
laboratory yeast strains belonging to 19 genera and 40 species were subjected to the cultivation under semiaerobic conditions in shake flasks to test their ability to produce higher alcohols. It was shown that yeast
Endomyces magnusii is able to produce high amounts of isobutanol reaching 409 mg/L. Yeast
Saccharomycopsis fibuligera also produced isobutanol though in less extend, up to 76 mg/L. The last yeast
species also produces trace amounts of amyl alcohol (37 mg/L). Further increase of isobutanol production
could be achieved by molecular approaches. However, molecular tools for yeast E. magnusii are poorly
developed and genome sequence is not known. In this work, the transformation procedure applying
dominant selective marker gene natNT2 conferring resistance to nourseothricin was optimized for E.
magnusii. Strong constitutive homologous promotor of TEF1 gene encoding translation elongation factor 1
was cloned by chromosome walking.
Isobutanol is synthesized in yeasts by the valine Ehrlich degradation pathway. The first enzyme of
isobutanol synthesis de novo is acetolactate synthase, encoded by ILV2 gene. Recently, enhanced
isobutanol production in S. cerevisiae has first been demonstrated by overexpression of the endogenous
genes involved in valine biosynthesis, namely ILV2, ILV3, ILV5. The isobutanol production was further
increased by simultaneous overexpression of ARO10 together with ADH2 gene (Brat et al., 2012). Aro10 is
known as a broad-substrate-specificity decarboxylase involved in Ehrlich pathway and is a promising
candidate enzyme for activation of ketoisovaleriate decarboxylation to increase isobutanol production. In
our research both ILV2 and ARO10 genes were overexpressed under control of TEF1 promotor in E.
magnusii. Resulted strains were characterized by increase in isobutanol production.
Atsumi S. et al., Nature. 2008, 451:86–89.
Brat D. et al., Biotechnology for Biofuels. 2012, 5:65.
24
IMPROVEMENT OF ERYTHRITOL SYNTHESIS IN YARROWIA LIPOLYTICA BY
METABOLIC ENGINEERING
Frédéric Carly1, Jean-Marc Nicaud2, Patrick Fickers1,3
1
Biotechnology and bioprocesses, Univeristé libre de Bruxelles, Av Roosevelt 50, 1050 Brussels,
Belgium;
2
INRA, UMR1319 Micalis, F-78350 Jouy-en-Josas, France;
3
Microbial Processes and Interaction, University of Liège – Gembloux AgroBio Tech, Passage des
déportés 2, 5030 Gembloux, Belgium.
Email address: pfickers@ulg.ac.be
Erythritol is a 4-carbon polyol with sweetening properties that is found in a number of natural
products such as seaweeds, fungi and fruits. It is frequently used in the food industry as a sugar substitute
due to its interesting properties i.e. it is 70% as sweet as sucrose, non-caloric and it does not affect
glycemia. Currently, erythritol is mainly produced by fermentation using osmophilic microorganisms,
cultivated at low pH and high osmotic pressure. One of these organisms is Yarrowia lipolytica, a nonconventional yeast naturally occurring in different types of food but also in lipid-rich or hypersaline
environments. Its main characteristics are the ability to degrade hydrophobic substrates, a great tolerance to
salinity and pH and the ability to produce high amounts of proteins and metabolites of interest. These
properties, along with a GRAS status, helped Y. lipolytica to emerge as an important microorganism for a
number of biotechnological and food-related applications. The aim of the present work was to optimize
erythritol production from glycerol by the non-conventional yeast Y. lipolytica by using a genetic
engineering approach and to develop an efficient process of production in bioreactor.
25
THE ROLE OF CYTOSOLIC AND PEROXISOMAL TRANSALDOLASES AND
TRANSKETOLASES IN HANSENULA POLYMORPHA XYLOSE METABOLISM AND
FERMENTATION
Olena Kurylenko1, Olena Krasovska1, Justyna Ruchala2, Kostyantyn Dmytruk1, Andriy Sybirny1, 2
Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine,
Drahomanov Street, 14/16, Lviv 79005 Ukraine;
2
Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow 35601 Poland.
Email address: olenkakurylenko@gmail.com
1
Lignocellulosic biomass is a cheap and abundant feedstock for production of liquid biofuels. Xylose
is the second major fermentable sugar of lignocellulosic biomass, however, only several microorganisms
are able to metabolize xylose and ferment this monosaccharide to ethanol. The methylotrophic yeast
Hansenula polymorpha is one of the most thermotolerant yeast species known with ability to ferment
xylose at elevated temperatures (up to 50oC). However, the efficiency of xylose conversion to ethanol by
natural H. polymorpha strains is quite low due to complexity of xylose utilization pathway, which includes
reactions of xylose entering pentose phosphate pathway (PPP), PPP per se and glycolysis. In contrast to
other microorganisms, the role of two key enzymes involved in PPP, namely transketolase and
transaldolase, in xylose metabolism and alcoholic fermentation in H. polymorpha still remain unknown.
This methylotrophic yeast contains, in addition to cytosolic transaldolase (gene TAL1) and transketolase
(gene TKL1), also peroxisomal transketolase (known as dihydroxyacetone synthase, gene DAS1) and
putative peroxisomal transaldolase with unknown functions (gene designated by us as TAL2).
In the wild type strain of H. polymorpha overexpression of DAS1 and TAL2 genes turned out to be
beneficial for xylose alcoholic fermentation. Both knockout strains did not show growth retardation on
xylose as carbon source but were impaired in xylose fermentation as compared to the wild-type strain. The
impact of TAL1 gene on xylose utilization in H. polymorpha seems to be more pronounced as tal1Δ
mutants were unable to grow in xylose-containing medium. It is interesting to note that overexpression of
TAL2 gene restored growth of tal1Δ mutants on xylose, confirming the role of both Tal1 and Tal2
transaldolases in utilization of this pentose in H. polymorpha.
26
GENETIC ENGINEERING OF CYTOSOLIC CARBON METABOLISM FOR IMPROVED
GLYCEROL PRODUCTION IN YEAST SACCHAROMYCES CEREVISIAE
Marta Semkiv1, Kostyantyn Dmytruk1, and Andriy Sibirny1,2
1
Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine,
Drahomanov Street, 14/16, Lviv 79005 Ukraine;
2
Department of Biotechnology and Microbiology, University of Rzeszow, Cwiklinskiej 2, Rzeszow 35-601
Poland.
Email address: sibirny@cellbiol.lviv.ua
Glycerol is used in cosmetic, paint, automotive, food, pharmaceutical industries. Despite increase of
glycerol accumulation as by-product of biodiesel production, it is unprofitable to purify this polyol for
subsequent application in food and cosmetic industry. There are known Candida yeast strains effectively
converting glucose to glycerol, however, they need aeration which elevates process costs. Therefore there
is an interest in development of microbial or yeast strains effectively converting cheap feedstocks to
glycerol under anaerobic conditions. Facultative anaerobic yeast Saccharomyces cerevisiae can be a good
platform for this goal.
In S. cerevisiae glycerol synthesis occurs from dihydroxyacetone phosphate by subsequent action of
glycerol-3-phosphate dehydrogenase (Gpd1) and glycerol-3-phosphate phosphatase (Gpp2).
Dihydroxyacetone phosphate is predominantly isomerized to glyceraldehyde-3-phosphate by triose
phosphate isomerase (Tpi1) and subsequently converted to pyruvate, which is further converted to ethanol
by pyruvate decarboxylase and alcohol dehydrogenase (most active isoform is Adh1). To redirect
consumed carbon toward glycerol instead of ethanol we aimed to construct recombinant strains with
simultaneous decrease of Tpi1and Adh1 and increase of Gpd1 and Gpp2 activities.
To decrease TPI1 or ADH1 gene expression recombinant S. cerevisiae strains with shortened to 25
bp versions of corresponding gene promoter were constructed. Constructed strains revealed decreases in
Tpi1 or Adh1 activities and 2-2.5 fold increase in glycerol production. Combination of these modifications
further increased glycerol/ethanol ratio but it also severely impaired recombinant strain growth and glucose
consumption level.
In order to enhance the activities of enzymes involved in glycerol synthesis, we transformed S.
cerevisiae with vector containing hybrid GPD1-GPP2 ORF (encoding artificial fusion of Gpd1 and Gpp2
enzymes) under the control of strong constitutive promoter of the alcohol dehydrogenase gene (ADH1).
Glycerol production reached 4 folds increase in the best of obtained recombinant strains. Combination of
GPD1-GPP2 fusion overexpression and decrease in Tpi1 activity resulted to 5-fold increase of glycerol
production as compared to the WT strain. In future, combination of all mentioned modifications is planned.
27
WHOLE-MOUNT GUS STAINING ANALYSIS OF HEAT SHOCK PROTEINS HSP90 AND
HSP70 EXPRESSION IN ARABIDOPSIS THALIANA SEEDLINGS
Liudmyla Kozeko, Galyna Ivanenko, Elizabeth Kordyum
Department of cell biology and anatomy, M.G.Kholodny Institute of Botany, National Academy of
Sciences of Ukraine, Tereshchenkivska str. 2, 01601, Kyiv, Ukraine.
Email address: liudmyla.kozeko@gmail.com
Heat shock proteins (HSP) 70 and 90 kDa are involved as chaperones in a wide range of cellular
processes in different cellular compartments, including, cytosol, plastids, mitochondria, and endoplasmic
reticulum (ER) under both normal and stress conditions. HSP70s are essential for protein folding and
translocation, as well as preventing aggregation and refolding of non-native proteins. HSP90 family differs
from the other chaperones by its substrates which are mainly regulatory proteins. However, expression of
the individual genes might have specific pattern. The purpose of this study was to investigate the general
patterns of different HSP90 and HSP70 gene expression in Arabidopsis thaliana seedlings under the
normal conditions. We used seeds of A. thaliana transgenic lines with the GUS reporter gene for HSP90.2,
HSP90.5, HSP90.7, HSP70.3 and HSP70.5 from NASC. 11-12-day old seedlings grown on the 0,5 MS
medium at 22°C and 16 h light cycle were studied. After staining for GUS activity, seedlings were
examined as whole-mount preparation.
It was shown that HSP90.2 was intensively expressed in cotyledon and true leaves (especially in the
vascular system and apical region), stipules, hypocotyls and weakly expressed in roots.
HSP90.5 expression was revealed in guard cells and in the root quiescent center, meristem and
cortex of the elongation and mature zones.
HSP90.7 was intensively expressed in guard cells and in the root meristem and cortex of the
elongation and mature zones and weakly expressed in stipules.
Intensive expression of HSP70.3 was detected in guard cells and in the root meristem and cortex of
the elongation and mature zones, weak one – in cotyledons and true leaves.
HSP70.5 was intensively expressed in leaf hydatodes, stipules as well as cortex of the root mature
zone, and weakly expressed in the root meristem.
Thus, the patterns of GUS staining showed the organ- and tissues-specific character of gene
expression for HSPs localized in different cellular compartments, namely: HSP90.2 and HSP70.3 in the
cytoplasm/nucleus, HSP70.5 in mitochondria, HSP90.5 in plastids, HSP90.7 in ER. It is of interest and
important that these chaperons function mainly in intensively dividing cells and in cells being in the active
physiological state.
28
POSTER SESSION
29
APOCYNIN DECREASES ROS PRODUCTION AND INDUCES TRANSITION FROM
SUSTAINED TO TRANSIENT EGF-DEPENDENT Akt SIGNALING IN Ruk/CIN85
OVEREXPRESSING HUMAN BREAST ADENOCARCINOMA MCF-7 CELLS
Bazalii A.V., Drobot L.B.
Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine (NASU) 9
Leontovicha Street, Kyiv, 01601, Ukraine.
Email address: bazalii@biochem.kiev.ua
It is well recognized up to date that physiological concentrations of ROS function as signaling
molecules to mediate various responses including tumor cell proliferation, migration, differentiation, and
gene expression, usually through the sustained activation of signaling pathways. NADPH oxidases are the
major source of ROS. They have specific subcellular localizations required for localized ROS production.
Currently, apocynin is considered to be one of the most specific inhibitor of NADPH oxidases, which
blocks the assembly of the active enzyme complex. This inhibitor is widely used to study the involvement
of NADPH oxidases in the control of cellular biological responses as well as the mechanisms involved in
enzymes regulation.
Our previous data demonstrated that stable expression of adaptor protein Ruk/CIN85 in weakly
invasive human breast adenocarcinoma MCF-7cells deficient of Ruk/CIN85 led to a more rapid and
prolonged EGF-dependent activation of Akt kinase and was followed by increased motility in both
transwell migration and wound healing assays. Treatment of Ruk/CIN85 overexpressing cells with PI3K
inhibitor LY294002 abolished Ruk/CIN85-dependent changes in cell motility.
Using chemiluminescence analysis, it was found that human breast adenocarcinoma MCF-7 cells
overexpressing adaptor protein Ruk/CIN85 (subclone G10) are characterized by increased production of
reactive oxygen species (ROS) in comparison with wild-type cells and G10 cells treated with Ruk/CIN85specific shRNA lentivirus. Treatment of G10 cells with apocynin decreased ROS production to the level of
control cells. These data suggest that ROS produced by MCF-7 cells with Ruk/CIN85 overexpression are
derived through NADPH oxidases. It was also shown that apocynin induces transition from sustained to
transient EGF-dependent Akt activation in G10 cells that is characteristic for control cells, and substantially
suppressed G10 cells migration. Interestingly, these effects of apocynin were associated with potent
increase of EGFR autophosphorylation at Y845 and Y1068. At the same time, apocynin did not influenc
EGFR autophosphorylation in the control cells.
The data obtained can suggest that ROS produced by NADPH oxidases are signaling components
upstream to Akt kinase that mediates the increased migratory potential of Ruk/CIN85 overexpressing
MCF-7 cells.
30
INSERTIONAL TAGGING OF THE SCHEFFERSOMYCES STIPITIS GENE INVOLVED IN
REGULATION OF GLUCOSE AND XYLOSE ALCOHOLIC FERMENTATION
Krzysztof Berezka1, Orest Hryniv2, Johanna Blomqvist3, Tomas Linder4, Mattias Carlsson4, Justyna
Ruchała1, Kostyantyn Dmytruk2, Volkmar Passoth4, Andriy Sibirny1,2
1
Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4,
Rzeszow 35-601, Poland;
2
Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine,
Lviv 79005, Ukraine;
3
Department of Chemistry and Biotechnology, Uppsala BioCentrum,
Swedish University of Agricultural Sciences, Uppsala, Sweden;
4
Department of Microbiology, Uppsala BioCentrum,
Swedish University of Agricultural Sciences, Uppsala, Sweden.
Email address: kberezka@gmail.com
Amid several known and profoundly studied microbial bioethanol producers the yeast
Scheffersomyces (Pichia) stipitis seems to be particularly promising in terms of alcoholic fermentation of
both glucose and xylose, the main constituents of lignocellulosic biomass hydrolyzates. However, the
ethanol yield and productivity, especially from xylose, are still insufficient to meet the requirements of a
feasible industrial technology; therefore, construction of more efficient S. stipitis ethanol producers is of
great significance.
The aim of this study was to isolate insertional mutants of S. stipitis with altered ethanol production
from glucose and xylose and to determine gene(s) affecting their fermentative capabilities. Mutants
obtained by random insertional mutagenesis were screened for their growth abilities on solid media with
different sugars and for resistance to 3-bromopyruvate (3-BP). Shaking cultures were carried out in order to
measure sugar consumption and ethanol formation rates. Subsequently, the most interesting strains were
analyzed to determine the genetic background of the observed alterations.
Of more than 1 300 screened mutants, 17 were identified to have significantly changed ethanol
yields during the fermentation. In one of the best fermenting strains, a single insertion event resulted in the
enhancement of ethanol formation in media with either glucose or xylose. Insertion was found within the
ORF of a gene homologous to Saccharomyces cerevisiae gene YDL119C, encoding a not yet described
mitochondrial transporter. We propose to designate this gene in S. stipitis as TMI1 (from Transport into
MItochondria). Wild-type phenotype was restored via complementation of the insertional mutation by the
wild allele of TMI1 gene. Thus, gene TMI1 is apparently involved in the control of hexose and pentose
alcoholic fermentation in S. stipites.
31
THE CATABOLITE INACTIVATION AND DEGRADATION OF THE CYTOSOLIC ENZYME
FRUCTOSE-1,6-BISPHOSPHATASE IN THE METHYLOTROPHIC YEAST PICHIA PASTORIS
Nina Bulbotka1, Kateryna Levkiv1, Olena Dmytruk1, Andriy Sibirny1,2
1
Institute of Cell Biology, National Academy of Sciences of Ukraine,
Drahomanov Street 14/16, 79005, Lviv, Ukraine;
2
Department of Biotechnology and Microbiology, University of Rzeszow,
Zelwerowicza 4, 35-601 Rzeszow, Poland.
Email address: sibirny@cellbiol.lviv.ua
Many enzymes of methanol metabolism of methylotrophic yeasts are located in peroxisomes
whereas some of them are of a cytosolic localization. Shift of methanol-grown cells into a glucosecontaining medium induces fast inactivation of peroxisomal and cytosolic enzymes of methanol
metabolism. Inactivation of peroxisomal enzymes occurs due to the autophagic degradation (pexophagy)
whereas mechanisms of the inactivation of cytosolic enzymes like fructose-1,6-bisphosphatase (FBPase),
formaldehyde and formate dehydrogenases remain unknown. In baker’s yeast, the catabolite degradation of
FBPase occurs after shift of glucose-starved cells into a glucose-containing medium. It was shown that
FBPase is degraded by the proteasome-dependent pathway after glucose starvation of the yeasts for 1 day
and by the vacuole-dependent pathway (autophagy) after glucose starvation of the cells for 3 days. We
studied mechanisms of FBPase degradation in the methylotrophic yeast Pichia pastoris.
The wild type strain of P. pastoris GS200, the protease-deficient strain SMD1163 (pep4, prb1) and
the strain with deletion of the gene coding for glucose sensor Gss1p were used in this research. We
analyzed the activity and the degradation of FBPase with adding of proteasome inhibitor MG132 and
without it. The study of changes of the activity of FBPase showed decrease in the FBPase activity in
GS200 and SMD1163 strains and the minor change of the activity of FBPase in the Δgss1 strain with and
without the inhibitor. We also compared the FBPase activity of the mutants defected in autophagy pathway
(∆mon1, ∆ypt7, ∆ccz1) with that in the wild-type strain of P. pastoris.
The GS200, SMD1163 and Δgss1 strains were also analyzed by the Western blot analysis for the
study of degradation of the FBPase. The results showed decrease in FBPase quantity in the GS200 strain
and the minor decrease this protein in the SMD1163 strain after transfer of cells from methanol medium in
a glucose containing medium not depending on duration of a glucose starvation. The amount of this
enzyme did not show significant changes in the Δgss1 strain indicating the need of glucose sensing for
FBPase degradation.
From the received results we make a preliminary conclusion that cytosolic enzyme of methanol
catabolism, FBPase, is degraded both using proteasomal and vacuole-dependent pathway (autophagy) what
doesn’t depend on duration of a glucose starvation.
32
MECHANISMS OF ANTINOPLASTIC ACTION OF NEW HEYEROCYCLIC COMPOUNDS ON
BASE OF 4-THIAZOLIDIDNONES
V.V. Chumak1, M.R. Fil1, R.R. Panchuk1, N.O. Manko2, D.Ya. Havrylyuk3, R.B. Lesyk3,
R.S. Stoika1
1
Institute of Cell Biology, NAS of Ukraine, Dragomanova Str., 14/16, Lviv, 79005, Lviv, Ukraine;
2
Ivan Franko Lviv National University, Hrushevskogo Str., 4, Lviv, 79005, Lviv, Ukraine;
3
Danylo Halytskyi Lviv National Medical University, Pekarska Str., 69, Lviv, 79010, Lviv, Ukraine.
Email address: virachumak@gmail.com
This work is devoted to the determination of mechanisms of antineoplastic action of novel synthetic
4-thiazolidinone derivatives which were synthesized in the Danylo Halytsky Lviv National Medical
University (head prof. Lesyk R. B). The main aim of the work was to find a relationship between the
structure of 4-thiazolidinones molecules and the mechanisms of apoptosis induction in tumor cells in vitro.
3 groups of 4-thiazolidinone derivatives were used. They differ in the type of chemical groups: 1) isatin
containing; 2) cyminal containing; 3) "hybrid" substances containing cyminal and 3,5-dypirazolin. All
studied 4-thiazolidinones induce apoptosis in tumor cells in vitro. The results of the study of the 1st group
of substances showed that halogen presence in the 5th position of isatin leads to 20-30-fold enhancement of
cytotoxic activity of compounds compared with the tiazolidynones containing nonhalogenated isatin.
Besides, it was shown that the most effective compound of this group – ID-3833 – demonstrates timedependent and dose-dependent effect on the apoptosis induction in tumor cells. In compounds of 2nd group
a replacement of benzylidene group from ID-3166 on the cyminal fragment in the compound ID-3506 was
performed. This chemical modification leads to 2-fold increase in the cytotoxic activity of substance ID3506, rapid and intense (1 hour) ROS generation by tumor cells, more earlier and intensive apoptosis
induction. The effect of 4-tiazolidinones of the 3rd group with 3,5-dypirazolin in the 4th position of the
thiazolidinone was accompanied by an increase in cytotoxic effect of "hybrid" substance ID-3661. This
compound is 3-5 times more active than the “parental” substances ID-3120 and ID-3372, and 14 times
more active compared with 2-substituted isomer ID-3713. ID-3661 induces in vitro apoptosis of mixed type
in contrast to the “parental” compounds involving mitochondrial (ID-3120) and ER dependent (ID-3372)
apoptosis. For the first time it is shown that 4-substituted 4-thiazolidinone derivative possess higher
cytotoxic potential than its 2-substitutes isomer. Colloidal form of the drug ID-3661 effectively blocks the
growth of ascite Nemet-Kellner lymphoma in mice and prolonged the lifetime of experimental animals on
3.4 – 4.1 times compared with control. Side effect of ID-3661 treatment such as cardiotoxicity in mice, was
eliminated by using antioxidant selenomethionine without a decrease in efficiency of the therapeutic action
of the drug. Thus, the type and position of chemical groups in the 4-thiazolidynone molecules play a crucial
role in the mechanisms of apoptosis induction in tumor cells and determine the antineoplastic properties of
these novel drugs.
Acknowledgement to XV-XVIII WUBMRC competitions.
33
MOLECULAR MECHANISMS OF NOVEL HETEROCYCLIC 4-THIAZOLIDONES ACTION
Nataliya Finiuk1, Julia Senkiv1, Dmytro Kaminskyy2, Roman Lesyk2, Rostyslav Stoika1
1
Institute of Cell Biology of National Academy of Sciences of Ukraine, Drahomanov St., 14/16, 79005
Lviv, Ukraine;
2
Danylo Halytsky Lviv National Medical University, Pekarska 69, 79010 Lviv, Ukraine.
Email address: nataliyafiniuk@gmail.com
Introduction. Conventional anticancer drug discovery and development have focused on the
cytotoxic agents. 4-thiazolidones and related heterocycles have been demonstrated to be a perspective
source for innovative anticancer agents.
Methods. In ours study we have used cytotoxicity assays (MTT and thrypan blue), Fluorescent
microscopy, Western blot analysis and FACS (for cell cycle investigations).
Results. The anticancer activity and effective inhibiting concentration of the compound with ID4368
was tested towards 8 cell lines of different origin. It was no observed viability inhibition of pseudo-normal
cells (HEK 293T, KS 483 and L929) after effect of compound ID 4368. Anticancer effect of synthesized
compound was studied towards HL-60, HL-60/ADR, Jurkat, L1210 and MCF-7 cell lines. Compound
possessed the cytotoxic effect in nanomolar concentrations. Leucemia cells appeared to be more sensitive
towards compound ID 4368 (IC50 = 120 nM). Studying the anticancer effect of compound ID 4368 on the
adriamycin-resistant cell line with overexpression of P-glycoprotein (HL-60/ADR) growth it has been
observed that this cell line is also sensitive to compound (IC50 = 1000 nМ).
However, HL-60/ADR cells were more resistant to Doxorubicin treatment (IC50 = 8.8 μМ).
Supravital double staining of the HL-60 cells, treated with compound ID 4368 (0.1 and 1 μM), with
FITC conjugated annexin V and propidium iodide revealed that tested compound induced apoptosis in
leukemia cells
The pathway of compound ID 4368 action is connect with the accumulation of ROS, both of oxygen
and nitrogen species, in HL-60 and HL-60/ADR cells. Compound ID 4368 leads to mitochondria-mediated
apoptosis (activation of ERK1/2, EndoG and Bcl-2) and effected to the G0/G1 arrest of the cells.
Conclusions. Compound ID 4368 can inhibit cancer cells growth even being applied in nanomolar
concentrations, in addition, it possess low toxicity towards pseudo-normal cells. Comparison of different
cancer cell lines sensitivity to the tested compounds showed that leukemia panel is the most sensitive to the
compound ID 4368 effect. Summarizing, mitochondria could be of high importance in the apoptosis
inducing after compound ID 4368 effect on tumor cells. Besides, compound ID 4368 involved in G0/G1
arrest of the cells, inhibiting cell division.
34
THE NONINVASIVE TECHNOLOGY FOR SCREENING OF INDIVIDUALS WITH RISK OF
ORAL CANCER
Natalia Yurchenko1, Switlana Kirieieva1, Michael Sidorenko1, Natalia Pershko1, Volodymyr Protsyk2,
Anna Boychuk3, Olena Korolchuk3
1
Department of Biotechnical Problems of Diagnostics, Institute of Cryobiology and Cryomedicine of
NAS of Ukraine, Prospect Nauki 42/1, 03028, Kyiv, Ukraine;
2
National Institute of Cancer of the Ministry of Health, Vasylkivska street 45, 03022,
Kyiv, Ukraine;
3
Clinic of St. Nicholas Street. Teshebayeva 18, 03029, Kyiv, Ukraine.
Email address: natalipershko@gmail.com
The analytical epidemiology associates oral squamous cell carcinoma (OSCC) with long-term
smoking tobacco, being alcohol, exposure to various carcinogens (Hayat et al,2007). The aim of this work
was investigation of the state of tumor-distant oral mucosa and buccal epithelium in patients with oral
squamous cell carcinoma (OSCC) and patients with epidemiological risk of oral cancer. Biopsy specimens
of tumor-distant mucosa and buccal scrapes (brush biopsy) were taken from 18 patients with OSCC (male,
age from 43 to 77 years; smoking-drinking status 15/18, > 20 years, > 20 cigarettes per day; long-term
professional contact with mutagen). Buccal scrapes of patients with epidemiological risk of oral cancer
were studied in 30 volunteers (male, age from 21 to 70 years; smoking-drinking status, > 20 years, > 20
cigarettes per day; moderate drinkers). Buccal scrapes from 10 healthy males (age from 19 to 25 years,
nonsmokers, nondrinkers, volunteers) were used as controls. The alterations in tumor-distant oral mucosa
were classified according to the UICC criteria. Tissue sections of tumor-distant mucosa were
immunohistochemically stained using monoclonal antibodies: for p53 (clone DO-7) and En Vision “Daco
Cytomation”. Buccal scrapes were stained by Papanicolaou (Papanicolaou Stains, Sigma). The state buccal
epitheliocytes were estimated by technology, that to include the estimate of cytological stage differentiation
of cells, index of differentiation (ID) and presence of atypical cells (Patent of Ukraine №21957, 2007
10.04.). In tumor-distant oral mucosa revealed the progression of histopathological phenotype: hyperplasia
(2 case), mild dysplasia (2case), high dysplasia (3case), carcinoma in situ (4 case), invasive carcinoma
(7case). p53 was detected in 16/18 cases of tumor-distant mucosa. Negative expression of p53 was found in
tumor- distant mucosa in cases of hyperplasia. In buccal epithelium from both patients with OSCC and
patients with epidemiological risk of oral cancer detected similar disturbance in differentiation and
maturation of epitheliocytes of oral mucosa, significant decrease ID of cells (control group, 392±10.4; rick
group, 288.3±30.7; patients group, 254.0±16.7; p< 0,05) and detected presence of cells areas with
predominant cells on 3rd and 2nd cell differentiation stage and appearance of basal cells1(1 stage, range,
control group, 1.0%-5.0%, rick group, 3.0%-9.0%, patients group 15.0%-17.0%) and frequent atypical cells
in areas of orange coloring. The patients with group of rick with revealed cellular deregulation in
differentiation of epitheliocytes in buccal mucosa (predominance of 2nd and 3rd stage of differentiation),
appearance of basal cells, atypical cells, fields of orange coloring and decrease of ID up to 300 were take to
individuals with high rick of oral cancer. They may be defined more correctly by molecular diagnostics.
This noninvasive technique may be used for mass screening of individuals with risk of oral cancer and
monitoring of oral mucosa of the patients with OSCC after treatment.
Hayat M.J. et al., Oncologis. 2007, 12(1): 20-27.
35
ASSOCIATION OF CELLS EXPRESSING OF MARKER PROTEINS OF THE EPITHELIAL
STEM/PROGENITOR CELLS p63, Cк5/14, CD44 IN THE FORMATION OF THE
TRANSFORMED PHENOTYPE IN ORAL MUCOSA FROM PATIENS WITH PRECANCEROUS
LESIONS AND RISK OF ORAL CANCER
Switlana Kirieieva, Natalia Yurchenko, Michael Sidorenko, Natalia Pershko
Department of Biotechnical Problems of Diagnostics, Institute of Cryobiology and Cryomedicine of NAS
of Ukraine, Prospect Nauki 42/1, 03028, Kyiv, Ukraine.
Email address: natalipershko@gmail.com
Recent studies suggest that the proteins p63, Ck5/14 and CD44 are markers of the epithelial
stem/progenitor cells and typically expressed by cells in the basal layer of normal squamous epithelium of
oral mucosa, that also niche of the epithelial stem/progenitor cells.
The aim of this study was estimated the expression markers of epithelial stem cells p63, Cк5/14,
CD44 and cell biology them in epithelium of oral mucosa from patients with epidemiological risk of oral
cancer and with precancerous lesions.
The study include biopsy specimens of oral mucosa from men that nonsmoking (control group, 10
cases, material forensic medical examination), with smoking habits (group of epidemiological risk;
13cases, material forensic medical examination, of precancerous lesions (37 cases). The alterations of oral
mucosa were classified according to the UICC criteria and expression of p53. The histopathological
alterations and precancerous lesions in oral mucosa were: hyperplasia (5 cases), leucoplakia (5 cases),
dysplasia low (5 cases), dysplasia mild (9 cases), dysplasia height (5 cases), Ca in citu (8 cases). In the oral
mucosa from individuals with risk found of the fields with histopathological progression. There were:
hyperplasia (2 cases), dysplasia low (2 cases) dysplasia mild (2 cases), dysplasia height (4 cases).
Immunohistochemicaly were study the expression of proteins p63, Cк5/14, CD44 in the epithelial layers of
oral mucosa. The tissue sections were immunohistochemically stained using monoclonal antibodies: for
p63 (clone 4А4, antibody to all isotype), for Ck5/14 (clone LL002), for CD44 (clone 156-3C11) and p53
(clone DO-7, antibody to wild/mutant type); visualization Mouse/Rabbit PolyVue (TM) HRP/DAB
Detection System, (Diagnostic BioSystems, USA) and counted according to the epithelial layers as labeling
index (LI, %). Basal cells of normal epithelium of the oral mucosa strongly expressed marker proteins of
epithelial stem/progenitor cells p63, Cк5/14, CD44 as the predominant (control group basal layer, p63,
range LI(%) 34.6%-63.0%; Cк5/14, range LI(%) 26.0%-39.7%; CD44 range LI(%) 35.0%-61.0%; in
suprabasal layers, range LI (%), 3.0%-10.0%;1.0%-5.0%; 0.0%-2.0%,respectively). In oral mucosa from
individuals with smoking habits and in precancerous lesions showed anomaly distribution of cells that
expressed markers of the epithelial stem/progenitor cells p63, Cк5/14, CD44 above basal cell layer, in
suprabasal layers and fields of cells with atypia. The anomal suprabasal immunoexpression and Labeling
Index (LI %) p63, Ck5/14, CD44 is strongly associated with high grade dysplasia and Ca in situ
(suprabasal layers, height dysplasia, range LI(%) p63, 30.6%-51.5%; Ck5/14, 20.5%-46.6%; CD44, 36.2%44.2%; Ca in situ, p63, 48.8%-56.3%; Ck5/14, 36.0%-44.2%; CD44, 38.2%-42.2% These results indicate
an involvement of cells that expressed markers of stem/progenitor cells p63, Ck5/14, CD44 in neoplastic
transformation.
It is to confirm the participation in formation of transformed phenotype and biology of oral cancer.
The architectural alteration of cells that expressed markers of stem/progenitor cells p63, Ck5/14, CD44 and
distribution in the suprabasal epithelial layers of oral mucosa represent high risk of development of oral
cancer and can be used for differential diagnostic dysplasia.
36
THE EFFECT OF INOCULUM CONCENTRATION ON RIBOFLAVIN
ACCUMULATION BY THE RIBOFLAVIN OVERPRODUCING STRAIN #91 OF THE YEAST
CANDIDA FAMATA
Rafał Kordiaka1, Maciej Kluz1, Andriy Sibirny1,2
University of Rzeszow, Department of Biotechnology and Microbiology,
Zelwerowicza 4, 35-601 Rzeszow, Poland,
2
Institute of Cell Biology, National Academy of Sciences of Ukraine,
Drahomanov Street 14/16, 79005 Lviv, Ukraine,
Email address: rafal.kordiaka@wp.pl
1
Some microorganisms are able to overproduce riboflavin (vitamin B2), the precursor of the essential
coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Ascomycete fungus
Ashbya gossypii, yeast Candida famata and bacterium Bacillus subtilis are mainly used for the industrial
production of this vitamin. Riboflavin is manufactured for use as a vitamin for human and animal nutrition
and as a food colorant, e.g. for soft drinks and yogurts. Recently, applying a combination of random
mutagenesis and metabolic engineering, a riboflavin overproducing strain of C. famata was constructed.
The aim of our work was optimization of cultivation conditions, namely, the inoculum concentration
optimal for maximal accumulation of riboflavin by the isolated recombinant strain C. famata #91.
First step of experiments was conducted in in 300 ml shake flasks cultivated in Innova Lab Shaker
(New Brunswick Scientific Co.) at 28°C with 250 rpm for 5 days. Yeast cells were cultivated in OP-5m
medium with the addition of 1, 5, 10, 15, 20 and 25% cell inoculum. The highest riboflavin accumulation
(1.24 g/L in 96 h) was obtained during cultivation of strain #91 in OP-5m medium after adding 20%
inoculum. The next step was directed to optimize riboflavin production in a 1.3 L laboratory glass
fermentor (BioFlo®/CelliGen® 115, New Brunswick Scientific Co.). The batch operation mode was
adopted to obtain a high cell density and high concentration of riboflavin. The inoculum in a concentration
of 5, 10, 15, 20% was added to 0.5 L of optimized medium OP-5m. The dissolved oxygen (DO)
concentration was monitored using the fermentor control unit based on polarographic DO probe. Air was
used for culture aeration. Foam was suppressed by sterile antifoam Structol 673. The bioreactor was
operated at 28°C and 600 – 1000 rpm. The initial pH of the medium was 5.5. Samples were withdrawn
twice per day, biomass and riboflavin concentrations in the medium were determined. The following results
on riboflavin accumulation were obtained after 116 h cultivation after adding different inoculum
concentrations: 5%, 5.67 g/L; 10%, 6.18 g/L; 15%, 5.9 g/L; 20% , 6.18 g/L. Thus the 20% inoculum
addition appeared to be the most appropriate for maximal riboflavin accumulation. During accumulation of
6.16 g of riboflavin per L after adding 20% inoculum, biomass was achieved 52.6 mg/mL. Thus,
optimization of cultivation conditions led to a noticeable improvement in riboflavin production by the
recombinant C. famata strain.
37
SUPEROXIDE DISMUTASE ACTIVITY IN NICOTIANA TABACUM EXPRESSING HYBRID
CYANOBACTERIAL Δ12-ACYL-LIPID DESATURASE GENE AFTER HYPERTHERMAL
STRESS
Kyrpa-Nesmiian T.М., Sheludko Y.V
Institute of Cell Biology and Genetic Engineering NAS of Ukraine,
Zabolotnogo str. 148, Kyiv 03143, Ukraine.
Email address: t-kirpa@ukr.net
Reaction of plant cell membrane ensemble to temperature fluctuations is an important factor
providing constancy of intracellular environment and metabolic pathways. Desaturases are enzymes
responsible for formation of double bonds in fatty acids of membrane lipids that increase membrane
fluidity and raise phase transition temperature from gel to liquid crystal state.
Increased desaturation of membrane lipids correlates to low temperature resistance. However, this
can affect various physiological aspects of plant cells and, among them, cause enhanced sensitivity to
hyperthermal stresses. The aim of our research was studying on several physiological reactions of tobacco
plants expressing hybrid gene of Synechocystis sp.PCC 6803 Δ12 (desA) acyl–lipid desaturase fused with
thermostable lichenase reporter gene from Clostridium thermocellum under hyperthermal conditions.
Transgenic N. tabacum (cv. Wisconsin) line expressing the gene described above under control of
constitutive 35S CaMV promoter was obtained and characterized recently (Gerasymenko I.M. et al., 2015).
Analysis of fatty acids exhibited increase of linoleic acid portion (C18:2; Δ9,12) accompanied with
decrease of α-linolenic acid (C18:3; Δ9,12,15) relative amount. Transgenic plants expressing desaturase
gene exhibited enhanced levels of superoxide dismutase (SOD) activity after cold treatment in comparison
with control lines with suppressed SOD under the such conditions. In our current experiments we
determined electrolyte leakage and SOD activity in leaf explants after incubation of plants at elevated
temperature (cultivation at 420C for 12 h and 36 h). Analysis of electrolyte leakage showed no significant
differences in control and experimental plants for both incubation periods. Activity of SOD in experimental
plants did not differ from that in the control lines after 12 h incubation at high temperature and increased
after 36 h while the level of SOD activity in the control remained constant. This may suggest higher
sensitivity of transgenic lines to increased temperatures and induction of intracellular mechanisms of stress
compensation.
The work has been carried out with support of grant of NASU UkrISTEI №0115U004171
Gerasymenko I.M. et al., Russian J. Plant Physiol. 2015, 62(3), 283-291.
38
MOLECULAR MECHANISMS OF OVERCOMING MULTIDRUG RESISTANCE OF TUMOR
CELLS BY ANTIBIOTICS OF LANDOMYCIN FAMILY
Liliya Lehka1, Rostyslav Panchuk1, Petra Heffeter2, Walter Berger2, Rostyslav Stoika1
1
Institute of Cell Biology National Academy of Science of Ukraine, Drahomanov Str 14/16, 79005, Lviv,
Ukraine;
2
Institute of Cancer Research, Vienna Medical University, Borschkegasse 8A, 1090 Vienna, Austria.
Email address: lilyalehka@gmail.com
Development of multidrug resistance (MDR) of tumor cells to chemotherapy treatment is one of the
most crucial problems in modern oncology. Landomycins (LS) belong to a novel family of angucycline
antibiotics possessing high anticancer potential that is comparable with such potential of clinically
approved drugs (e.g., doxorubicin).
The main aim of current project was to study in-depth molecular mechanisms of anticancer activity
of 7 members of landomycin (L) family (LA, 11-deoxyLA, LB, 11-deoxyLB, LE, LD, 11-deoxyLD).
Using carcinoma and leukemia cell lines possessing different mechanisms of multidrug resistance
(overexpressing of P-gp, MRP1, BCRP) we demonstrated that all studied compounds were able to
overcome MDR. Most active in this case was LA, which contains 6 sugar residues in its molecule.
Modulating reactive oxygen species (ROS) levels has been proposed as a therapeutic strategy to
selectively kill tumor cells. Treatment of cancer cells in vitro with LS resulted in the rapid generation of
reactive oxygen species (ROS). The inhibition of ROS formation under LS action by catalase, mannitol and
superoxide dismutase suggested the involvement in this process of Н2О2, ОН. and О2- radicals. Trypan blue
staining and cytomorphological investigation revealed that catalase was the most effective in inhibiting of
LS-induced cell death. Scavenger of OH. radicals mannitol also reduced cytotoxic activity of LA; however,
SOD had no significant inhibitory effect on LS-induced apoptosis; this might have resulted from the
enhanced formation of H2O2 probably the most potent of the ROS.
In-depth study of signaling apoptotic pathways under LA (as the most active compound) action on
human promyelocytic leukemia cells of HL-60 line and its resistant subline HL-60/vinc (overexpressing Pgp) was performed using Western-Blot analysis. The first event had been observed under LA treatment was
effector caspase-7 activation and cleavage of PARP-1 and DFF-45 proteins already at 1 h after treatment,
that indicated the upstream role of caspase-7 in mechanism of LA-induced apoptosis. This phenomenon
was tightly correlated with the maximal level of ROS production at the same time point. However, in drugresistant cells increase of ROS level under landomycins treatment was much higher and such ROS peak
was observed for much longer period of time, than in sensitive cells. We did not observed activation of
effector caspase-3 and very week activation of effector caspase-8 in HL-60/vinc cells. Other effector and
initiator caspases and their substrates cleavage took place considerably later than caspase-7. Not only to kill
tumor cells with MDR was LA able but also to induce apoptosis in these cells even in the absence of other
key enzymes (caspase-3 and 8) involved in apoptosis. Literary data indicate that different cancer types
including melanoma, breast, ovarian, prostate, and leukemia are expected to be particularly sensitive to
therapeutic strategies that induce cancer cell death through further ROS induction. Our finding that LS can
overcome MDR and are inducers of ROS highlights the potential of their usage as novel therapeutics for
the treatment of different types of cancer.
39
EFFECT OF NITRIC OXIDE DONOR ON MIGRATION AND
MORPHO-FUNCTIONAL PROPERTIES OF WM793 HUMAN MELANOMA CELLS UNDER
ARGININE DEPRIVATION
Mayevska O.M., Karatsai O.V, Chen O.I., Bobak Ya.P. , Barska M.L., Igumentseva N.I., Stasyk O.V.
Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Street 14/16, 79005
Lviv, Ukraine.
Email address: o mayevska@yahoo.com
Certain types of cancer, human melanoma in particular, are highly metastatic and are markedly
resistant to conventional therapies. Anticancer therapy based on arginine-degrading enzymes is one of the
promising strategies for treatment of these malignant neoplasms. This therapy can also include application
of nitric oxide releasing compounds for compensation of NO-deficiency, which results from arginine
starvation, and may cause cardiovascular collapse through high blood pressure and vasoconstriction. The
aim of this study was to elucidate morpho-functional and migration alterations in WM793 human
melanoma cells caused by NO-donor sodium nitroprusside (SNP) under arginine deprivation.
WM793 cell line belongs to vertical growth phase melanomas which spread into the subcutaneous
tissue and facilitate development of highly aggressive cancers in vivo. We observed that this cell line is
ASS-negative and sensitive to arginine deprivation. The ability to recover growth after arginine starvation
can correlate with colony formation capacity. The number of colonies detected by clonogenic assay in
arginine deficient medium was decreased relative to arginine-sufficient conditions. Single cells and clusters
were revealed under combination of SNP and arginine depletion, whereas many colonies were detected in
arginine-rich medium supplemented with SNP. The level of the cleaved form of PARP proteins, as an
apoptotic marker, increased in arginine-free medium supplemented with SNP and PARP cleavage was
detectable after 24-h incubation that is earlier as compared to arginine deprivation alone.
Tumor cell motility is one of important characteristic for monitoring metastatic potential of
malignant melanoma in vitro. To examine the alteration of WM793 migration properties under SNP
treatment (0,1mmol/L), a wound-healing assay serves as a screening test for directional cell migration in
vitro. SNP administration into arginine-rich medium did not affect the migratory activity and the complete
wound healing was achieved within 12 h. The cell ability to migrate into the scratch area decreased under
arginine starvation and wound-healing was not achieved even after 24 h of subsequent incubation. We
observed dramatic decrease in cell motility under SNP treatment in arginine-free medium compared to
arginine-rich medium (approximately 13-fold).
The process of cell migration is tightly linked with cell cytoskeleton organization. The staining of
actin filaments by TRITC-phalloidin revealed that SNP promoted the depolymerization of the F-actin
cytoskeleton in WM793 cells regardless of the presence of arginine in the medium. Of the note, the
combination of SNP and arginine deficiency triggered a defective disassembly of stress fibers which are
necessary for the formation and maintenance of cell-cell and cell-extracellular matrix adhesion.
The obtained results suggest that exogenous NO-donor at physiological low concentration did not
rescue viability or motility of human melanoma cells under arginine deprivation in vitro. Therefore NOdonor, such as SNP, may be potentially used as an adjuvant in combinational anti-melanoma therapy based
on arginine deprivation to decrease its systemic toxic effects related to NO limitation.
40
COMPARISON OF EXPRESSION of HUMAN INTERFERON ALPHA 2B GENE WITH
DIFFERENT SIGNAL SEQUENCES IN TRANSGENIC NICOTIANA TABACUM
Mariia Mazur1, Zoya Olevinska2, Mykola Spivak2, Mykola Kuchuk1
1
Institute of Cell Biology and Genetic Engineering, National Academy of Science of Ukraine,
Academika Zabolotnoho Street 148, 03143 Kyiv, Ukraine;
2
D. K. Zabolotny Institute of Microbiology and Virology, National Academy of Science of Ukraine,
Academika Zabolotnoho Street 154, 03680 Kyiv, Ukraine.
Email address: mariamazur17@gmail.com
Interferons are important glycoproteins of cytokines class. They are used in medicine as non-specific
drugs for treatment of a wide range of tumors and viral diseases. An increase of the interferon stability has
been suggested due to improvement of its transport to the apoplast using plant gene signal sequences which
facilitate to a limitation of the plant protease impacts upon this glycoprotein (Gils et al., 2005). The aim of
this study was to examine and compare the peculiarities of expression of both human interferon alpha 2b
gene with plant apoplastic signal sequence and one with human signal sequence in transgenic Nicotiana
tabacum plants. As a result of series of Agrobacterium-mediated transformation of tobacco, selection of
regenerants, following by multiplex PCR analyses of transformants (Gerasymenko et al., 2012), fifteen
transgenic lines were obtained. Six plant lines have expressed human interferon alpha 2b gene with the
human signal sequence, and nine lines - human interferon alpha 2b gene translationally fused with the
apoplast signal sequence of calreticulin gene from N. plumbagenifolia. Both plasmids contained selective
bar gene and target genes were under the control of cauliflower mosaic virus 35S promoter. The interferon
antiviral activity in transgenic plant extracts was determined due to interferon ability to protect cells from
the pathogenic viruses. We used microtitration method. Interferon-like activity was not detected in the
control untransformed plants of N. tabacum. Interferon activity in plants bearing interferon gene with
human signal was low. Functional heterogenecity of these transgenic lines was shown. Interferon activity
has reached up to 100 IU/g fresh weight, corresponding to approximately 16 IU/mg total soluble protein.
Interferon activity of plant extracts with interferon gene translationally fused with plant apoplast signal
sequence was significantly higher. Interferon activity reached up 400 IU/g of fresh weight, or 74 IU/mg
total soluble protein. The results obtained allow to conclude that plant apoplast signal sequence provides
higher activity of transgenic interferon than human one, probably because of higher influence on this
cytokine stability in apoplast.
Gils M. et al. Plant Biotechnol. J., 2005, 3:613-620.
Gerasymenko I. M. et al. Cytol. Genet., 2012, 46(4):3–8.
41
THE INFLUENCE OF MAGNETIC FIELD ON HUMAN BUCCAL EPITHELIUM CELLS
SURVIVAL
Miroshnik D.B.
Research Institute of Biology, Karazin's Kharkov National University 4, Svobody sq., Kharkov, Ukraine.
Email address: fildomik@mail.ru
The effects of low intensity static or low frequency magnetic field on living cells are under
consideration in connection with their possible health hazards. The acute problem is the investigation of
high intensity SMF used in NMR studies on human organism. These investigations induced limitations of
high intensity SMF usage in medical purposes. Many studies explore the effects of low intensity static
magnetic field (SMF) on cell. Biological effect of SMF in contradistinction to low frequency looks
paradoxical at first sight, because of there are no electromagnetic quanta, which energy can be compared
with the energy of biochemical conversions. As it is known, the static magnetic field has no thermal effect.
It seems plausible that effect of the static magnetic field use another mechanism and able to accumulate on
some biological level, bypassing the stage of primary physical oscillators.
Among typical cell answers to external factors we have chosen the assessment of viability of cells
after SMF exposure after Hoechst 33342 and ethidium bromide staining as a marker of magnetic field
effect.
The cells of human buccal epithelium of two donors (men): donor A (24 years) and donor B (23
years) were exposed to different doses of magnetic field. Cells were scraped from the inner surface of
donor’s cheek by spatula and suspended in buffer solution (3.03 mM phosphate buffer, pH 7.0
supplemented with 2.89 mM CaCl2. A static magnet generated magnetic field (induction 25 mT). Samples
of cells were exposed to magnetic field for different time (1, 3, 5, 10, 15, 20, 30 min). Samples of cells
were exposed to magnetic field for different time (1, 3, 5, 10, 15, 20, 30 min).The level of cells survival
was assessed after cell staining by Hoechst 33342 (10 mg/l) and ethidium bromide (5 mg/l). Hoechst 33342
is a vital dye and stains viable cells, ethidium bromide stains cells with depressed vitality.
To estimate cell survival 1200 cells in each variant of experiment were tested (400 cells in triple
experiment). In each experiment variant the percentage of stained only by Hoechst dye was calculated, the
damaged cells were stained by ethidium bromide.
The answer of cells of donor B to SMF is different. The decrease of viability is observed only at
exposure times 10 and 15 min. At 20 min SMF exposure cell viability even increased.
The data obtained show the decrease of buccal epithelium cell viability after SMF exposure in a
donor-dependent manner. If viability of cells of one donor decreases at short exposure time (1 min), such
cell answer in cells of other donor is observed only at more exposure time (10-15 min). Our present data
are in a good agreement with previous results indicating cell stress reaction and cell viability decrease
induced by 25 mT SMF. These data to some extent are in consistency with data on cell type specificity of
cell answer to SMF. Difference in reaction of cells of different donors to SMF is connected with individual
peculiarities of cells of different donors. We suppose that absence of viability decrease at longer exposure
times (20 and 30 min) is connected with the process of recovery of cell SMF-induced lesions.
Medium intensity SMF (25 mT) in isolated human buccal epithelium cells induces decrease of cell
viability in cells of buccal epithelium immediately after cell exposure 1, 10 and 15 min in a donordependent way. At a longer exposure time (20-30 min) no viability decrease is observed.
42
ISOLATION AND CHARACTERIZATION OF PEPTIDES FROM BLOOD SERUM OF
PATIENTS WITH MULTIPLE SCLEROSIS
Myronovkij S.1, Negrych N.2, Nehrych T.2, Stoika R.1, Kit Y.1
1
Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov St., 14/16, 79005,
Lviv, Ukraine;
2
Danylo Halytsky Lviv National Medical University, Pekarska St., 69, 79010, Lviv, Ukraine.
Email address: severyn92@gmail.com
MS, or "disseminated sclerosis", is an inflammatory disease in which the myelin sheaths around
axons of the brain and spinal cord are damaged, leading to demyelination and scarring, and as a result to a
wide range of signs and symptoms. MS is referred of autoimmune character, because of cellular and
humoral immune reactions towards brain tissue self-antigens, basically to myelin basic protein (MBP) is
main characteristic of this disease. The main feature of patients with MS is the formation of inflammatory
sites in the brain tissue, accompanied by the increasing the proteolytic activity followed destruction of
MBP and subsequent demyelination of axons. This activity should be accompanied with elevation of level
of different peptides in cerebrospinal fluid (CSF). There are data showing the rising of permeability of
blood-brain barrier (BBB) in MS patients. We believe that such peptides might penetrate into blood from
CSF where they could be detected, isolated and studied. Here we demonstrate the results of determination,
isolation and characterization of low-molecular weight peptides (oligopeptides) from blood serum of MS
patients and study of their biological activity toward tumor cells and microbial cells.
In order to find novel molecular markers of multiple sclerosis we developed the schema of
oligopeptides isolation including their extraction from blood serum with 10% trichloroacetic acid followed
precipitation of the soluble substances with acetone in ratio 6:1. Oligopeptides were dissolved in water and
their characteristic was determined by gel filtration under HPLC condition and thin layer chromatography.
Obtained data have shown that blood serum of MS patients contains two oligopolypeptides with average
molecular masses 300-500 Da. We also studied of biological activity of TCA-soluble peptides toward some
eukaryotic and prokaryotic. It was found that TCA-soluble peptides are capable to effectively inhibit of
HeLa cells proliferation while their inhibitory effect was on the Jurcat cells and was not detectable toward
of U373 cells. Neither antibacterial nor antifungal activities of these oligopeptides were not detected.
43
AGROBACTERIUM-MEDIATED TRANSFORMATION OF MAIZE OF UKRAINIAN
BREEDING GENOTYPES
Iryna Nitovska1, Ihor Komarnytsky1, Olha Аbraimova2, Tetiana Satarova2, Bogdan Мorgun1
1
Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine,
Akademika Zabolotnoho Street 148, 03143 Kyiv, Ukraine;
2
Institute of Agriculture for the Steppe Zone, National Academy of Agricultural Sciences of Ukraine,
Dzerzhynskoho Street 14, 49600 Dnipropetrovsk, Ukraine.
Email address: molgen@icbge.org.ua
Maize has an important value for human necessities and occupies the second place after wheat in the
export of grain-crops by Ukraine. Genetic transformation is a powerful tool for improvement of existent
cultivars to increase their economic attractiveness for production. Particle bombardment and
Agrobacterium-mediated transformation are two popular methods currently used to obtain transgenic
maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies
of the transgene and more predictable pattern of integration (Shou et al., 2004). However, vectors that are
efficient in dicots show relatively low activity in transgenic monocots. That is why for transformation of
cereals it is needed to create the adapted vectors with special promotors and regulator elements directed for
the increase of transgene expression. On the basis of two vectors – pICBV16 (Icon Genetics GmbH,
Germany) and pAHC25 (Christensen et al., 1996) – a binary vector pCB202 was created for monocot
transformation by means of Agrobacterium tumefaciens. It contains neomycin phosphotransferase II (nptII)
gene controlled by nopaline synthase (nos) promoter and β-glucuronidase (uidA) gene regulated by maize
ubiquitin (Ubi-1) promotor. The plasmid DNA was transferred into A. tumefaciens strain GV3101. Thus,
the aim of our research was to check the suitability of the vector pCB202 in A. tumefaciens GV3101 for
cereal transformation on the example of maize of Ukrainian breeding.
Agrobacterium-mediated transformation of maize immature embryos was conducted according to the
protocol (Sidorov et al., 2009) for five genotypes (inbred line DK232 and F1 hybrids DK959×DK232,
KP7×PRZh5, KS277×RS15, RS15×KS277). 350 embryos were processed. Selection and regeneration of
transgenic plants were performed on selective medium containing 100 mg/l paromomycin. The
regeneration of plantlets was observed for three maize genotypes (DK232, KS277×RS15 and
RS15×KS277). The frequency of regeneration of paromomycin resistant shoots varied from 15% for
DK232 and KS277×RS15 to 23% for RS15×KS277. Rooted regenerants were plated in soil. From 7 plants
(50% of all regenerants) transplanted in soil, 5 of them survived and grew under greenhouse conditions. All
greenhouse plants displayed the activity of β-glucuronidase enzyme. Most activity of β-glucuronidase was
observed in female generative organs. The presence of nptII gene in plant DNA of all 5 greenhouse plants
was shown by PCR analysis.
Thus, maize transgenic plants were obtained for three genotypes of Ukrainian breeding, namely
inbred line DK232 and F1 hybrids KS277×RS15 and RS15×KS277. The constructed binary vector pCB202
in A. tumefaciens GV3101 strain was shown to be functionally active and can be used to test various maize
genotypes for competence to Agrobacterium-mediated transformation.
Shou H. et al. Mol. Breed. 2004, 13:201-208.
Christensen A.H. and Quail P.H. Transgenic Research. 1996, 5: 213-218.
Sidorov V., Duncan D. Methods in Molecular Biology: Transgenic Maize. 2009:47-58.
44
CONCENTRATION OF URIC ACID IN BLOOD SERUM AS A PREDICTOR OF GOUT IN
ATHEROSCLEROSIS
Piskun R.P., Piskun A.O.
Department of medical biology of Vinnitsa National Medical University named by N.I. Pirogov
Pirogova st., 56, Vinnitsa, 21018, Ukraine.
Email address: Piskyn2006@mail.ru
Through the last couple years studies have shown, that frequency of coronary disease and
cerebrovascular accidents are higher in people with increased content of uric acid in blood. According to
this gout is considered as a risk factor of atherosclerosis. The causes of the relationship of these factors are
not identified yet. That's why it's interesting to hold on a study, that shows the dependence between blood
level of cholesterol and uric acid. It is also known, that one of foreign spasmolitic and coronarydilating
drug – benziodaron – has antigout effect. Benziodaron is a synthetic benzofuran derivative, and while
stydying native antianginal derivatives of this group (fonicaberan and metozaron), we made a goal to find
out whether they have the same effect. Uric acid was determined in rabbits before the beggining of the
experiment, and then in three months of feeding them cholesterol, and at the end of the experiment – after a
month of treatment with fenicaberan, metozaron, and for comparison, with anaprilin. Received results have
shown, that quarterly feeding of cholesterol leads to increase of blood serum level of uric acid almost in 15
times comparing to initial level. After a month treatment the level of uric acid decreases to 1480 ± 160
mkmol/l vs 1590 ± 140 mkmol/l before treatment, in a group of animals which received metozaron, and
1330 ± 130 mkmol/l vs 1650 ± 120 mkmol/l in group which received fonicaberan. Among rabbits, which
received anaprilin, a slight increase of uric acid is noticed (1550 ± 130 mkmol/l vs 1400 ± 160 mkmol/l,
which matches with the results of untreated animals – 1570 ± 140 mkmol/l vs 1460 ± 150 mkmol/l).
45
NANOLUMINESCENSE A SCANNING OPTICAL MICROSCOPE FOR RESEARCH OF
FUNCTIONING OF MICROORGANISMS UNDER INFLUENCE OF LOW TEMPERATURES
Ivan Prudyus1, Aleksandr Zaichenko2, Lubov Palianycia2, Аnatolij Pedan1, Volodymyr Shkliarskyi1
1
Institute of Telekommunications, Radionics and ElecthonicTechnics, Polytechnic National University,
S. Bandery Str., 12, Lviv, 79013, Ukraine;
2
Institute of Chemistry and Chemical Technology, Polytechnic National University, S. Bandery Str., 12,
Lviv, 79013, Ukraine.
Email address: shkliarskyi@ukr.net
At research of microorganisms in biology methods of freezing - warming there is a necessity of
preservation of object after carrying out of all expected procedures of researches without reception of its
irreversible damages to its structure and ability to live. For research of biological objects are used both
optical and electronic microscopes. Proceeding from a principle of the action optical microscopes has
restrictions of useful scale of increase and resolution. These restrictions are connected to the wave nature of
light. Electronic microscopes for the work demand presence of vacuum which is destroying for biological
objects. For today used methods of research of microorganisms do not allow to take their temperature
during freezing – warming. Authors of work suggest creating (NSOM), which will unite opportunities of
reception of useful scales of increase and resolution, commensurable with initial values of scanning
electronic microscopy and the natural environment of existence of the biological objects, inherent in optical
microscopy.
The NSОМ with controlled cryostats for definition of temperature of a fragment of a microorganism
is offered, by direct introduction in it functional luminescent tolerant nanoparticles. These nanoparticles
under influence of low dynamic temperatures at an irradiation their light probe change parameters of the
radiation depending on a degree of cooling. The analysis of changes of parameters of radiation biologically
tolerant nanoparticles realize during transformation of light radiations in an electric signal. In view of
preliminary calibration in a microscope it is possible to define temperature of separate fragments of a
researched microorganism during freezing – warming.
As a result of execution of work will be: it developed a new way of measurement of the current
temperature of a fragment of a microorganism during freezing; warming at simultaneous supervision of
structure of a microorganism; it is developed controlled cryostat for freezing - warming of microorganisms;
the problem of heat removal is solved at freezing - warming of microorganisms in NSОМ from small
volume on microscope a little table; parameters of a signal of the induced photoluminescence nanoparticles
which are suitable for use at measurement of temperature are determined; are developed new functionally
biologist polymer - mineral nanoparticles with the set sizes and distribution on the size, controllable
parameters of a luminescence of a nucleus and the set functionality of an environment; dependence of
parameters of a luminescence from the sizes nanocomposites, the nature of a mineral or organic nucleus,
the nature of a functional environment, length of a wave of excitation and temperature is determined.
During execution of work will be: 1) it is created NSОМ with controlled thermostat, which will
allow to use photoluminescence functional biologically tolerant nanoparticles for measurement of
temperature of fragments of a researched organism during freezing - warming; 2) dependences of
characteristics of a luminescence of colloid-chemical and biological properties nanoparticles by nature
luminophore, the sizes and functionality of particles are determined; 3) the problem of preservation of
proof morphology-structural and physiological properties is solved during various ways of storage.
46
PROTECTIVE EFFECTS OF CYP2E1 INHIBITORS ON METABOLIC SYNDROME-INDUCED
LIVER INJURY IN GUINEA PIGS
Volodymyr V. Rushchak1, Ganna M. Shayakhmetova2, Mykola O. Chashchyn1,3
1
Molecular Oncogenetics Department, Institute of Molecular Biology and Genetics, NAS of Ukraine,
Zabolotnogo Str. 150, Kyiv, Ukraine;
2
General Toxicology Department, SI “Institute of Pharmacology & Toxicology NAMS of Ukraine”,
Eugene Pottier Str. 14, Kyiv, Ukraine;
3
National Scientific Centre for Medical and Biotechnical Research,
Volodymyrska Str., 54 Kyiv, Ukraine.
Email address: biomed@nas.gov.ua
Aim. The present work studies the effects of cytochrome P450 2E1 (CYP2E1) inhibitors (quercetin,
4- methylpyrazole and disulfiram) on the pathological processes in the liver of guinea pigs with metabolic
syndrome (MS) induced by protamine sulfate repeated treatment.
Methods. The investigation of CYP2E1 inhibitors effects on hepatic CYP2E1 protein level and
activity changes was conducted. Simultaneously, the content of reactive oxygen species (ROS) and markers
of liver damage were determined in experimental animals’ blood. Also we investigated the link between
increased hepatic expression of CYP2E1, prominent ROS generation and liver damage in animals with MS.
Results. We have demonstrated that CYP2E1 protein level and activity in guinea pigs with MS rose
almost 3 times in comparison with intact animals. These events were accompanied by almost 4 times
increase in ROS generation, and the appearance of metabolic and liver disordes symptoms: serum glucose
and cholesterol contents increase (2 and 2.6 times respectively), activity elevation of alanine
aminotransferase (2.8 times), aspartate aminotransferase (6.4 times), and alkaline phosphatase (1.8 times).
Our investigation suggests that treatment of guinea pigs with MS by all three CYP2E1 inhibitors caused
decrease in CYP2E1 protein expression, as well as its enzymatic activity in liver. The content of blood
ROS was partially restored or normalized by quercetin, 4-methylpyrazole, and disulfiram treatment. At the
same time, suppression of CYP2E1 activity and ROS generation led to decrease in hepatic MS
manifestation.
Conclusions. It is apparent from the present observation that quercetin has the highest efficiency
among the investigated substances. Further studies on various quercetin doses and treatment regimens
could be important for elimination of MS-related liver injury.
47
THE IDENTIFICATION OF AUTO-ANTIBODIES IN BLOOD SERUM OF PATIENTS WITH
RECURRENT MISCARRIAGE USING WESTERN-BLOT ANALYSIS
Yuriy Kit1, Marina Starykovych,1 Danuta Zastavna2, Rostyslav Stoika1
1
Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine
2
Institute of Hereditary Pathology, National Academy of Medical Sciences of Ukraine, Lviv, Ukraine
Email address: marina.starikovich@gmail.com
Recurrent miscarriage (RM) is defined as the loss of three or more consecutive pregnancies. Classic
factors associated with RM include parental chromosome translocations, uterine malformations, endocrine
and autoimmune factors.
Our previous studies have shown that the biotinylated IgGs isolated from blood serum of RM women
recognized a distinct set of chorionic proteins which includes endoplasmin (92 kDa, Acc:
GANAB_HUMAN) and were subsequently identified by the MALDI-TOF MS ( Kit Y. et al., 2014). The
endoplasmin is also known as Heat Shock Protein 90 kDa Beta Member 1 (HSP90B1) may participate in
immune response in RM patients. HSP90B1 can serve as an endogenous activator of the dendritic cells.
Besides, there are data that anti-HSP90B1 auto-antibodies could be directly engaged in reproductive
dysfunction in women. Our studies are directed to investigation whether the appearance of anti-HSP90B1
auto-antibodies in blood serum of RM patients is linked to the overexpression of this stress-responsive
protein in human chorionic tissues.
We studied the presence of autoantibodies using Western-Blot analysis. Protein with molecular
weight of 90 kDa, which corresponds to HSP90B protein, was isolated from line U373 MG glioblastoma
cell line.
The obtained data indicate that autoantibodies patients, which bind to the 90 kDa protein in blood
serum in 4 from 8 RM. Clinical data on the severity of the pathological process of RM patients are
correlated with the efficiency autoantibodies of binding to the 90 kDa protein in blood serum of RM
patients.
Kit Y.et al. Croat Med J. 2014; 55(3): 259–264.
48
NEW MODELS OF PARKINSON'S DISEASE BASED ON THE THERMOTOLERANT YEAST
HANSENULA POLYMORPHA FOR STUDDING THE PROCESSES OF HUMAN ALPHASYNUCLEIN DEGRADATION
Denega I.O.1,2, Sybirna N.O.1,2, Romanyshyn O.R.1, Snegirenko O.O.1, Stasyk O.V.2, Stasyk O.G.1,2
Ivan Franko National University of Lviv, Biological Faculty, 4 Hrushevskyi Str., Lviv, 79005 Ukraine;
2
Institute of Cell Biology, NAS of Ukraine, 14/16 Drahomanov Str., Lviv, 79005 Ukraine.
Email address: olenastasyk@gmail.com
1
Around 60% of the yeast genes show sequence homology to a human orthologue, and of the human
disease-related genes, over 25% have a close homologue in yeast. Importantly, yeast and human cells share
fundamental aspects of eukaryotic cell biology. This allows a number of key processes, which are of
particular interest to PD pathology, to be efficiently investigated in the well-understood yeast model. It has
already been a decade since yeast was used for the first time as a model to study α-synuclein (α-syn)
toxicity. The α-syn, which is found broadly in the brain, has been implicated in several neurodegenerative
disorders. Patients with a rare familial form of Parkinson's disease (PD) had too many normal copies of the
α-synuclein gene, which resulted in a buildup of protein inside brain cells, causing the symptoms of PD.
Although some studies have suggested that the ubiquitine-proteasome system is the main for α-synuclein
degradation, it was shown that such degradation is mainly carried out by lysosomal pathway, and in
particular by macroautophagy and chaperone-mediated autophagy.
We utilized the recombinant strains of the thermotolerant methylotrophic yeast Hansenula
polymorpha for modeling the processes leading to PD in humans and for screening different factors that
potentially induce α-syn aggregation (such as metal ions, high or low concentration of glucose in growth
medium) and autophagic degradation (such as nutrient and amino acids deficiency). The fused ORF SNCAGFP under РMET25 promoter was multicopy integrated into the genome of H. polymorpha wild type strain
(NCYC495). We observed that human α-syn overexpression impaired growth of model strains. Our
preliminary data indicate that nutrient limitation (nitrogen starvation) up-induced α-syn degradation in
model yeast strains, whereas Mn2+ ions overexposition of these strains affected the process of α-syn
degradation at low and normal glucose concentrations in the culture medium. Moreover, ions Mn 2+ reduced
cytotoxicity of α-syn in yeast cells and, conversely, strains producing α-syn were more resistant to
manganese influence comparative to wild type strain. Consequently, we suggest that α-syn and Mn2+ can
interact in vitro and this may be important in the realization of different cellular processes in model yeast
strains.
49
BIOLOGICAL ACTIVITY OF ANTI-Hsp60 ANTIBODIES IN ESSENTIAL HYPERTENTION
Yakovenko L. 1, Smalyuk Y. 1, Chornyy S. 1, Tsisarenko A. 1, Kapustian L. 2, Pogrebnoy P. 3,
Granich V. 3, Sirenko Yu. 1, Sidorik L.
1
Institute of Molecular Biology and Genetics NAS of Ukraine, Kyiv, Ukraine
150, Zabolotnogo Str., Kyiv, Ukraine, 03680;
2
R. E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine, Kyiv,
Ukraine;
3
Natsionalnyy Scientific Centre "M. D. Strazhesko Institute of Cardiology” of NAMS of Ukraine, Kyiv,
Ukraine.
Email address: l.f.yakovenko@imbg.org.ua
The circulating levels of IgG and IgM antibodies are elevated in patients with essential hypertension
(EH). One of the possible explanations for autoantibody production in hypertension involves molecular
mimicry, where foreign or pathogen-derived antigens trigger an immune response against “self” peptides of
similar homology. Heat shock protein 60 (Hsp60) or molecular chaperone belongs to a group of
phylogenetically conserved proteins and involved in protein folding, protein degradation and protein
trafficking among subcellular compartments. Hsp60 has highly homologous sequences between different
species, from bacteria to humans. The levels of Hsp60 are increased in the cell and on the cellular
membrane in response to stress (including psycho-emotional stress, infections, oxidants, oxidized LDL,
smoking, elevated blood pressure etc). Anti-Hsp60 antibodies, especially cross-reactive antibodies against
microbial Hsp60, can cause autoimmune response directed against Hsp60 localized on the surface of
stressed cells. The results of study the anti-Hsp60 antibodies levels at EH are inconsistent, their role in
pathogenesis has not been establish yet. The aim of our work was to investigate of biological activity of
IgG anti-Hsp60 antibodies purified from highly immunoreactive sera of patients with EH.
Methods. The levels of antibodies against prokaryotic (GroEL Escherichia coli) and human Hsp60
(hHsp60) were estimated in sera of 27 patients with EH by ELISA and Western blotting. The control
included the donor’s sera with low reactivity to GroEL (n=83) and hHsp60 (n=66). Highly reactive to
Hsp60 sera of patients with EH were cleared with the method of affinity chromatography using Protein-G
sepharose.
Results. We observed that patients with elevated levels of anti-Hsp60 antibodies had the changes in
target organs (retinal hemorrage, acute ischemic stroke, cardiosclerosis, and nephrosclerosis). Western-blot
analysis revealed that of IgG anti-Hsp60 antibodies purified from highly reactive sera of patients with EH
recognized both prokaryotic (GroEL) and hHsp60 and also recognized the protein with a molecular mass of
about 60 kDa in vessels and heart lysates. Using MTT-assay we examined the effect of IgG anti-Hsp60
antibodies on primary cardiomyocyte culture. We identified decrease of dehydrogenase activity of primary
cardiomyocytes treated with IgG anti-Hsp60 antibodies purified from highly reactive sera of patients with
EH at 24 hour after incubation.
Conclusion. Anti-Hsp60 antibodies, especially antibodies against microbial Hsp60, may be involved
in the development of lesions target organs in patients with EH. The data obtained are the basis for new
diagnostic tool creation.
50
EFFECT OF CYTOSTATIC DRUG, 5-AMINO-4-(1,3-BENZOTHIAZOL-2-YL)-1-(3METHOXYPHENYL)-1,2-DIHYDRO-3-Н-PYROL-3-ONE,
ON RAT ASCENDING COLON MUCOSA
Yena М.S., Dzyubenko N.V.
Taras Shevchenko National University of Kyiv, Volodymyrska str. 64/13, Kyiv, 01601, Ukraine
Email address: yenamaryna.7@gmail.com
Target therapy is an alternative to the traditional cytostatic drugs due to its targeted effect on the
tumor cells and relatively low toxicity. However, a small number of the tested drugs of this class do not
meet the needs of the modern oncology. Membrane tyrosine kinase inhibitor, dihydropyrrole derivate,
exerts a significant antitumor activity in colorectal cancer and has low toxicity in the effective dose.
However, it is necessary to conduct the studies with the higher doses of this substance to evaluate its
therapeutic window.
The purpose of the work was to evaluate the effect of the high doses of tyrosine kinase inhibitor of
pyrol derivate, 5-amino-4-(1,3-benzothiazol-2-yl)-1-(3-methoxyphenyl)-1,2-dihydro-3Н-pyrol-3-one (D1).
The study was conducted on the white male rats with the average weight of 150 g. D1 was
administered in the oil solution per os, daily for 2 weeks at the doses that under complete absorption reach
the blood concentration of 10-4М (effective), 5х10-4М, 10х10-4М (2.3 mg/kg, 11.5 mg/kg and 23 mg/kg,
respectively). Evaluation of the rat ascending colon mucosa stained with hematoxylin and eosin at the lightoptical level was performed and morphometric measurements were carried out.
When D1 was used in the effective dose for 14 days, no pathological changes of the ascending colon
mucosa were observed. Morphometric parameters of the mucosa did not significantly differ from the
control values.
Effect of 5-fold D1 dose caused the insignificant infiltration of the proper mucous plate by the
lymphocytes and somewhere lymphoid accumulation of tissue in the submucous layer. In addition, blood
capillaries, small-bore arteries and veins were dilated. The mitotic index increased by 37% compared to the
control group. The morphometric changes are indicative of the intensity of proliferation processes.
The 10-fold D1 dose caused the insignificant lympho-infiltration in the mucosa. The changes of the
blood stream including the dilated capillaries, erythrostasis and parietal thrombi were also observed; these
signs may be indicative of the inflammation. The analysis of the morphometric data showed the significant
reduction of the crypt depth by 22%, which may be associated with the atrophic changes of the mucosa.
Therefore, the toxic effects of D1 show the dose-dependent increase. However, even used at 10-fold
effective dose, this compound does not cause the significant morphofunctional changes of the ascending
colon mucosa as the colon segment which is indicative of its relative safety to the gastro-intestinal tract.
51
PHYSIOLOGICAL ACTION OF NEW YEAST CHROMIUM BIOCOMPLEXES ON RATS
Daniel Broda1, Halyna I. Nechay2, Paulina Cynk1, Ewelina Gawel1, Anna Gorka1, Maciej Wnuk1,
Mykhailo V. Gonchar1,2
1
Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Sokolowska 26, 36-100
Kolbuszowa, Poland;
2
Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Street 14/16, 79005
Lviv, Ukraine.
Email address: gonchar@cellbiol.lviv.ua
Production and sale of dietary chromium had become multimillion-dollar industries. Supplements
containing Cr(III) are available in various forms for human consumption, including tablets, sports drinks,
cocktails and chewing gum, and are recommended for use in certain circumstances, and as additives in the
diet of cattle and pigs. The aim of this work was to study the physiological actions on animals of the new
yeast chromium (III) biocomplexes, as a source of biologically available chromium. Tested were plasma
concentrations of glucose, C-peptide and cholesterol, activities of antioxidant enzymes in liver and kidney,
and tissue accumulation of chromium. The isolated Cr(III) bio-complexes showed a positive biological
activity in physiological studies using rats as the test model: they induce a decrease of blood glucose level
without any toxic effect (as approved by ALT and AST transaminases’ assay test).
52
KINETIC CHARACTERIZATION OF MYCOPLASMA HOMINIS ARGININE DEIMINASE
OVEREXPRESSED IN ESCHERICHIA COLI
Lyubov Fayura1, Volodymyr Boretsky1, Yuriy Boretsky1,2, Andriy Sybirny1,3
1
Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Street 14/16, 79005
Lviv, Ukraine;
2
Department of Biochemistry and Hygiene, Lviv State University of Physical Culture,
Kosciuszko Street 11, 79000 Lviv, Ukraine;
3
Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, 35-601
Rzeszow, Poland.
Email address: fayural@gmail.com
Our studies aimed at evaluating arginine-degrading enzyme arginine deiminase (ADI - catalyzes the
imine hydrolysis of arginine to produce citrulline and ammonia) as a promising anticancer drug or a key
element for arginine biosensor construction. They are based on the previous findings that demonstrated
positive effect of arginine depletion on remission of certain types of cancer (mainly hepatocarcinomas and
melanomas) in vitro and in vivo. The reported procedure for production of Mycoplasma hominis ADI
expressed in Escherichia coli is cost consuming and therefore needs to be improved.
Recombinant strain of E. coli which overexpress M. hominis gene encoding ADI was constructed. A
protocol for high-level production of ADI in a cheap mineral medium was developed. The rADI was
accumulated as inclusion bodies. Effect of different additives to both denaturation and renaturation buffers
on ADI activity yield was studied. The main improvements include manipulation with dense suspensions of
E. coli, use α-lactose instead of isopropyl β-D-1-thiogalactopyranoside as an inducer and use of the cheaper
though not less efficient buffer for solubilization of arginine deiminase inclusion bodies. The renatured
enzyme was purified by anion-exchange chromatography using Q-Sepharose column and a linear gradient
of 0-1 M NaCl in 20 mM sodium phosphate buffer. The active fractions were pooled and NaCl was
adjusted to 2 M. The enzyme was applied to a Phenyl-Sepharose column, washed with 20 mM sodium
phosphate buffer containing 2M NaCl and eluted with the buffer containing 1M NaCl. As a result, stable
enzyme preparations with specific activity of 30-34 U/mg of protein were obtained.
The affinity for arginine (Km) and Kcat values of ADI were examined in phosphate buffer, pH 6,5 at
37°С. The Kcat and Km (S0.5) values were determined from initial velocity data measured as a function of
substrate concentration. All assays were performed in triplicate and the means of 3 separate experiments
are presented in Line Weaver Burk plot. The values of Vmax and Km for ADI were a 0.052±0.01
µM/min·ml and 300±20 µM respectively. The Kcat was calculated from the ratio Vmax and enzyme
concentration. The Kcat value was 50±4 c-1. The enzymatic efficiency (Kcat/Km) for ADI was 166±10
mM-1c-1 that is 3.2-fold higher compared to arginine degradating enzyme human arginase I overexpressed
in Hansenula polymorpha (51.9 mM-1c-1 , Zakalsky, FEMS 2011) and 1600-fold higher at plasma arginine
concentranion (100 µM) . The possible application of obtained ADI preparations will be discussed.
53
“GREEN” SYNTHESIS OF NANOPARTICLES BY NON-CONVENTIONAL YEASTS
Galina Gayda1, Helena Ksheminska1, Natalia Stasyuk1, Mykhailo Gonchar1,2
1
Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Street 14/16, 79005
Lviv, Ukraine;
2
Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Sokolowska Str. 26, 36100 Kolbuszowa, Poland.
Email address: galina.gayda@gmail.com
“Green” synthesis is the use of biological entities as reducing agents in nanoparticles (NPs) synthesis
to replace toxic chemicals. NPs that are used in environmental cleanup are classified into three categories:
carbon NPs, metal NPs (include both magnetic and non magnetic) and bioNPs. Metallic bioNPs are very
prospective in different branches of science, industry and medicine: as catalysts, spectrally selective films
for solar energy absorption; intercalation material for electrical batteries as optical receivers; biolabels in
clinical diagnostics and treatment. Many living organisms (plants, bacteria, fungi, yeast), polysaccharides,
DNA, RNA, proteins, and polypeptides are known to produce nanostructured mineral crystals and metallic
NPs with properties similar to chemically synthesized materials.
The green synthetic processes are rapid, eco-friendly, cost-effective and they can easily be scaled up.
The exact mechanism for the synthesis of NPs using biological agents has not yet been elucidated, but it
has been suggested that various biomolecules, including enzymes, are responsible for the synthesis of NPs.
In our study we demonstrate that non-conventional yeasts are able to reduce ions of noble and
transitional metals to the relevant nanomaterials by the yeast culture and by extra-cellular liquid. To
characterize the formed bioNPs the methods of transmission electron microscopy, scanning electron
microscopy, atomic force microscopy, fluorescent microscopy, UV-visible absorption spectroscopy were
used. Influence of various parameters, namely: temperature, pH, time, concentration of corresponding salts
on NPs formation was studied. The advantage of “green” synthesis technique is economic efficiency:
yeasts grow rapidly with a high yield of correspondent NPs not only inside, but outside the cell too. The
yeast Hansenula polymorpha were used for “green” synthesis of different types of corresponding
monometallic or bi-metallic NPs from Ag +, Au3+, Pt4+, Pd3+ and other ions. The yeast Pichia guilliermondii
was shown to be able to reduce chromate − over Cr(V) to Cr(III) − extra-cellularly with formation of stable
soluble green bio-complexes, containing NPs of chromium (III) oxide.
Our results will be promising for fundamental and applied biological sciences: for the study of
molecular mechanisms of cell’s protection from stress, caused particularly by exposure to toxic compounds
and for the development of methods for the synthesis of nano-sized labels, including fluorescent, for
clinical diagnostics and drug delivery.
54
NEW ENZYMO-CHEMICAL METHOD FOR METHANOL DETERMINATION
IN ALCOHOLIC BEVERAGES
Dorota Grabek-Lejko1, Władimir Sibirnyj2, Anna Górka3, Mykhailo Gonchar3,4
1
Department of Biotechnology and Microbiology, University of Rzeszow,
Zelwerowicza 4, 35-601 Rzeszow, Poland;
2
Department of Chemistry and Food Toxicology, University of Rzeszow,
Zelwerowicza 4, 35-601 Rzeszow, Poland;
3
Institute of Applied Biotechnology and Basic Science, University of Rzeszow,
Werynia 502, 36-100 Kolbuszowa, Poland;
4
Institute of Cell Biology, National Academy of Science of Ukraine,
Drahomanov Street 14/16, 79005 Lviv, Ukraine.
Email address: dorobek@o2.pl
Alcoholic beverages may contain the minimum allowable impurities of methanol; however, there are
cases of poisoning including fatal when consuming beverages, acquired in the trade network. Therefore, it
is important to define the content of methanol in alcoholic beverages. The aim of this study is to develop a
new enzymo-chemical method for determining methanol in alcoholic beverages.
In the proposed approach, methanol is analyzed by monitoring formaldehyde (FA), produced in
alcohol oxidase-catalyzed reaction, followed by formation of colored product in reaction of FA with
Purpald with previous masking of FA in reaction with MBTH. To avoid an interference of excessive
amounts of MBTH on methanol assay, the optimization of MBTH content was performed. It has been
shown that decreasing MBTH concentration in the reaction mixture results in a better sensitivity of the
assay, as well as in a wider linearity of the calibration curve. It was shown that MBTH concentration of
0.02 mg/ml is the optimal.
Using the developed method, methanol content was determined in a variety of strong alcoholic
beverages produced in Poland, Ukraine, Scotland, and the USA, like cognacs, whiskeys, vodkas and
“sliwowica”.
To evaluate the practical significance of the developed enzymo-chemical method, an internal
standard addition method based on a headspace gas chromatographic (GC) technique has been used.
Abbreviations: MBTH - 3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate; Purpald
(AHMT) - 4-Amino-3-hydrazino-5-mercapto-1,2,4-triazole.
Acknowledgement: This work was supported by the Cross-border Cooperation Programme Poland–
Belarus–Ukraine 2007-2013 (the project IPBU.03.01.00-18-452/11-00 "Scientific integration of the PolishUkrainian borderland area in the field of monitoring and detoxification of harmful substances in
environment") and by the Polish Ministry of Science and Higher Education funds for science in 2013-2015
for an international co-financed project (decision number 3020/PBU/0755/11/13/2014/2 - "Scientific
integration of the Polish-Ukrainian borderland area in the field of monitoring and detoxification of harmful
substances in environment»).
55
EFFECT OF ATG13 MUTATIONS ON XYLOSE ALCOHOLIC FERMENTATION
OF THERMOTOLERANT YEAST HANSENULA POLYMORPHA
AND SCHEFFERSOMYCES STIPITIS
Orest Hryniv1, Dorota Grabek-Lejko2, Kostyantyn Dmytruk1, Barbara Kshanovska1,
Andriy Sibirny1,2
1
Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine,
Drahomanov Street, 14/16, Lviv 79005 Ukraine;
2
Department of Biotechnology and Microbiology, University of Rzeszow,
Cwiklinskiej 2, Rzeszow 35-601, Poland.
Email address: orhryniv@gmail.com
Fuel ethanol production from the renewable raw material, such as plant biomass or lignocellulose,
has a great economic and ecological significance. However, the production of fuel ethanol from non-starch,
lignocellulosic materials is not developed yet. The main reason is the absence of robust microorganism
capable of efficient alcoholic fermentation of all main sugars of lignocellulose, most importantly, xylose.
Thermotolerant yeast Hansenula polymorpha is a promising organism which ferments glucose and xylose
even at 48°C. But for cost-effective ethanol production, molecular mechanisms of regulation of xylose
fermentation need to be studied and available strains need to be substantially improved.
Method for positive selection of ethanol overproducers from xylose based on the usage of 3bromopyruvate as selective agent has been developed. The method was combined with insertional
mutagenesis. One among other selected transformants revealed reproducible two fold increase of ethanol
accumulation during xylose fermentation. It was shown that insertion cassette disrupted gene ATG13. Gene
ATG13 together with ATG1 are responsible for initiation of autophagy process in other organisms.
Disruption of the ATG13 gene in mutant doesn’t influence on peroxisomal degradation. However, deletion
of the gene caused to the blocking of peroxisomal degradation. The ∆atg13 strain of H. polymorpha
produced elevated amount of ethanol from xylose, similar to insertional strain.
Among several extensively studied microorganisms, the non-conventional yeast Scheffersomyces
stipitis seems to be promising organisms in terms of xylose and glucose alcoholic fermentation. The
influence of Atg13 on xylose alcoholic fermentation in S. stipitis was studied. It was shown that ∆atg13
strain of S. stipitis possessed the same performance of xylose alcoholic fermentation as parental strain.
It could be conclude that the autophagy-related gene ATG13 is somehow involved in regulation of
xylose alcoholic fermentation in H. polymorpha as distinct from that in S. stipitis. The role of peroxisomes
in xylose alcoholic fermentation is discussed.
56
FLAVOCYTOCHROME b2-BOUND MAGNETIC MICROPARTICLES AND THEIR
APPLICATION IN L-LACTATE ANALYSIS
Maria Karkovska, Oleh Smutok
Department of Analytical Biotechnology, Institute of Cell Biology, NAS of Ukraine,
Drahomanov Street 14/16, Lviv 79005, Ukraine.
Email address: mariakarkovska@gmail.com
In recent decades, magnetic microparticles (FeMP) have received increasing attention in bio-related
research, especially in the field of biocatalysis, due to their several distinct properties: high surface area to
volume ratio, special magnetic behavior, high dispersibility, and various functionalities. Immobilization of
enzymes on functionalized FeMP results in the improvement of their catalytic activity, higher stability, and
easy recyclability under external magnetic field. The possibility of reusing the enzyme will reduce the costs
of analysis.
The aim of this investigation is approving possibility of recyclability of flavocytochrome b2-bounded
magnetic microparticles (FCb2-FeMPs) under external magnetic field in enzymatic assay of L-lactate. For
this, several tasks were carried out: isolation and purification of FC b2 from overproducing yeast strain
Hansenula polymorpha “tr1” (gcr1 catX/prAOX_CYB2), biofunctionalization of amino-containing FeMP
by purified FC b2, characterization of the obtained FCb2-FeMPs and assessment of reusing in the enzymatic
kit.
The purified FC b2 has been obtained with the specific activity up to 22.5 U•mg-1. The efficacy of the
isolation and purification stages, calculated as the final yield of enzyme activity, was about 94%.
FeMPs with the diameter of 1 micron were used for immobilization of FC b2 without any drop in
enzymatic activity. The FCb2-FeMPs were further used for L-lactate analysis by enzymatic kit “Lactatest”
(instead of free enzyme).
The FCb2-FeMPs were separated from reaction mixture in magnetic field after enzymatic stage of Llactate assay. Then to the supernatant there were added components necessary for a colored product
formation and its solubility. The separated and washed FCb2-FeMPs were repeatedly used for another
series of analysis. It was shown that FCb2-FeMPs can be reused at least for 6 independent assays of Llactate.
Acknowledgements: This research was supported in part by NAS of Ukraine in the frame of the
Scientific-Technical Program “Sensor systems for medical, ecological and industrial-technological needs:
metrological assurance and research exploitation” and Ukrainian-Lithuanian Project “Investigation of Land D-lactate: cytochrome с oxidoreductases isolated from the recombinant yeast Hansenula polymorpha
and their usage for construction of amperometric biosensors”.
57
ENGINEERING OF GLYCEROL UTILIZATION PATHWAY FOR ETHANOL PRODUCTION
BY HANSENULA POLYMORPHA
Iwona Kata1, Justyna Ruchała1, Marta Semkiv2, Olena Kurylenko2, Kostyantyn V. Dmytruk2,
Andriy A. Sibirny1, 2
1
Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, 35-601
Rzeszow, Poland;
2
Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Street 14/16, 79005
Lviv, Ukraine.
Email address: iwonakata.ik@gmail.com
Biodiesel is produced by a transesterification reaction using vegetable oils or animal fats and an
alkohol. This process inevitably generates large amounts of glycerol as a by-product. The availability of
crude glycerol is predicted to increase in the next years because of the tremendous growth in the production
of biodiesel worldwide. To maximize the full economic potential of the biodiesel process, it is important to
convert crude glycerol into useful chemicals, e.g. ethanol. Glucose fermentation is efficiently carried out by
microorganisms, at the same time there are few microorganisms that are capable of converting glycerol to
ethanol. Therefore, the tools of metabolic engineering along with classical selection should be used for
construction of the recombinant strains with improved of ethanol production (Yazdani et al., 2007).
Thermotolerant methylotrophic yeast Hansenula polymorpha is able to convert glycerol as well as
glucose and xylose, to ethanol. However, the yield of ethanol from glycerol by wild type strain is too low
and need substantial improvement before introduction of those strains for production of ethanol on
industrial scale.
The aim of this work was to improve production of ethanol from glycerol using thermotolerant
methylotrophic yeast H. polymorpha. According to published data, overexpression of PDC1 (encodes
pyruvate decarboxylase) gene improved the production of ethanol from glucose (Ishchuk et al., 2008). In
addition, it was shown that the strain with overexpressed ADH1 (encodes alcohol dehydrogenase) gene
produces slightly higher amount of ethanol from glycerol (Suwannarangsee et al., 2010). In this study,
vectors for multicopy integration of both PDC1 and ADH1 genes under the control of strong constitutive
promoter have been constructed. Recombinant H. polymorpha strains revealed increased specific activities
of both Pdc1 and Adh1. The newest data of glycerol conversion to ethanol by recombinant strains will be
provided and discussed.
Ishchuk O. et al., FEMS Yeast Res, 2008.
Suwannarangsee S. et al., AMB, 2010.
Yazdani S. et al., Curr. Opin. Biotechnol, 2007.
58
THE EFFECT OF DIFFERENT ANTIFOAMS ON RIBOFLAVIN ACCUMULATION BY THE
RIBOFLAVIN OVERPRODUCING STRAIN #91 OF THE YEASTS CANDIDA FAMATA
Maciej Kluz1, Rafał Kordiaka1, Kostyantyn Dmytruk2, Andriy Sibirny1, 2
1
University of Rzeszow, Department of Biotechnology and Microbiology,
Zelwerowicza 4, 35-601 Rzeszow, Poland,
2
Institute of Cell Biology, National Academy of Science of Ukraine,
Drahomanov Street 14/16, 79005 Lviv, Ukraine.
Email address: mkluz@univ.rzeszow.pl
Riboflavin, also known as vitamin B2 or additive E101, is an easily absorbed micronutrient with a
key role in maintaining health in humans and animals. It serves as biosynthetic precursor of flavin
nucleotides FMN and FAD used as coenzymes by flavoproteins. As such, vitamin B2 plays a key role in
energy metabolism, for metabolism of fats, ketone bodies, carbohydrates, and proteins. Recently, applying
approaches of random mutagenesis and metabolic engineering, a riboflavin overproducing strains of the
flavinogenic yeast Candida famata were constructed. The aim of this work was optimization of cultivation
conditions for maximal accumulation of riboflavin by the isolated recombinant strain C. famata #91. A
series of experiments were carried out to study the effects of different antifoams for the riboflavin
production by the C. famata #91 strain during fermenter cultivation. For this, foaming was suppressed by
addition of four sterile antifoams: Structol 673, Silicone Antifoam 30% (ROTH), Silicone Antifoam
(SIGMA), Antifoam 204 (SIGMA); each to the final concentration of 20 mg/L. The experiments were
conducted in the 1.3L laboratory glass fermenters (BioFlo®/CelliGen® 115, New Brunswick Scientific
Co.). The batch operation mode was adopted to obtain a high cell density and high concentration of
riboflavin. The inoculum in a concentration of 20% was added to 0.5L of optimized medium OP-5m. The
dissolved oxygen (DO) concentration was monitored using the fermenter control unit based on
polarographic DO probe. Air was used for culture aeration (1.1 vvm). The bioreactor was operated at 28°C
and 600 – 1100 rpm. The pH 5.5 of the medium was automatically adjusted (Mettler – Toledo pH
electrode) by 1 N HCl or 0.1 N NaOH. Samples were withdrawn twice a day, biomass and riboflavin
concentrations in the medium were determined. The following results on riboflavin accumulation were
obtained after 120 h cultivation using different antifoams used: Structol 673, 8.2 g/L, Silicone Antifoam
30% (ROTH), 7.18 g/L, Silicone Antifoam (SIGMA), 6.9 g/L, Antifoam 204 (SIGMA), 7.4 g/L. Thus the
antifoam Structol 673 appeared to be the most appropriate for maximal riboflavin accumulation. During
accumulation of 8.2 g of riboflavin per L with Structol 673, biomass production was achieved 60.4 mg/mL.
Thus, optimization of cultivation conditions led to a noticeable improvement in riboflavin production by
the recombinant C. famata strain #91 during fed-batch cultivation.
Dmytruk K. et al. J Biotechnol. 2014, 172:11-7.
Dmytruk KV, Sibirny AA. Yeast. 2012, (11):453-8.
59
BACTERICIDAL PROPERTIES AND CYTOTOXICITY EVALUATION OF GREEN
SYNTHESIZED SILVER NANOPARTICLES
Małgorzata Kus-Liśkiewicz1, Joanna Depciuch2, Angelika Myśliwiec2, Bartosz Fryc1, Andrzej Kowal3,
Magdalena Parlinska-Wojtan3
1
Biotechnology Centre for Applied and Fundamental Sciences, Department of Biotechnology, University
of Rzeszow, Sokołowska Street 26, 36-100 Kolbuszowa, Poland;
2
Faculty of Biology and Agriculture, University of Rzeszow, ul. Ćwiklińskiej 1, 35-601 Rzeszów, Poland;
3
Facility for Electron Microscopy & Sample Preparation, Center for Microelectronics and
Nanotechnology, Faculty of Mathematics and Natural Sciences, University of Rzeszow, ul. Pigonia 1,
35-959 Rzeszow, Poland.
Email address: mkus@ur.edu.pl
Nowadays, new nanomaterials with their unique biochemical and physical properties are of interest
in various applications. In the preparation of nanoparticles, nanobiotechnology is becoming very useful; it
provides a platform for eco-friendly green synthesis of nanoparticles. Such biosynthesis, using biological
sources as well as agricultural wastes, is less toxic and cost effective compared to physical or chemical
techniques. In this study, an attempt was made to synthesize silver nanoparticles (AgNPs) from aqueous
extracts of camomile and test their bactericidal activity and cytotoxicity effect.Various antimicrobial tests
using four bacteria strains: Staphylococcus aureus, Bacillus subtillis, Pseudomonas aeruginosa and
Escherichia coli were employed to evaluate the potential of antimicrobial activity of the nanomaterial.
Furthermore, the degree of cytotoxicity of green synthetized silver nanoparticles to human keratinocytes
has been also assessed. Physico-chemical characterizations with DLS measurements, FTIR or UV-Vis
spectra and STEM HAADF images were demonstrated.
Gogoi N. et al., Mater Sci Eng C Mater Biol Appl. 2015, 46:463-9.
Azizinezhad F. et al., European Journal of Experimental Biology. 2014, 4(2):124-127.
Kaviya S. et al., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2011, 79(3):594–
598.
60
GENETIC CONSTRUCTION OF THE SACCHAROMYCES CEREVISIAE STRAINS WITH
IMPROVED THERMO-, OSMO- AND ETHANOL-TOLERANCE FOR ETHANOL
PRODUCTION ON INDUSTRIAL MAIZE WORT
Kutsyaba V.1, Gonchar M.1, Nevo E.2, Sibirny A.1
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16,
Lviv 79005, Ukraine;
2
Institute of Evolution & International Graduate Center of Evolution, University of Haifa, Mount
Carmel, Haifa 31905, Israel.
Email address: vasylkutsiaba@yahoo.com
1
Bioethanol produced from renewable biomass (sugar, starch and lignocellulosic materials) is one of
the important alternative energy resources and the most promising biofuel today. Production of bioethanol
is one way to reduce both consumption of crude oil and environmental pollution.
Yeast Saccharomyces cerevisiae remains the most commonly used microorganism for ethanol
production by fermentation of carbohydrates at 30 – 32 0C. However, there is a need in isolation of the
strains with an improved ethanol yields at higher temperatures (34 – 37 0C) of fermentation, at 13 - 14%
ethanol content in ripe spirit brew and 28 - 30% initial concentration of dried substances in the wort.
Hybridization and selection of meiotic segregants is the method that can produce strains with recombinant
genotypes. Some of them can have significant improvement in thermo-, osmo-, ethanol-tolerance and other
quantitative traits. It is suggested that such improvements could lead to a higher efficiency of fermentation
and significant increase in the ethanol production capacity of constructed strains at higher temperatures of
fermentation.
The thermotolerant natural S. cerevisiae IMB Y-5053 strains (Kutsyaba et al., 2014) isolated from
the Evolution Canyon in Israel and commercial baker's yeast strain “Enzyme” were used for genetic
construction of the thermotolerant strains on industrial maize wort. By means of crossing of the
auxotrophic mutants the diploid D96/11 and more than 500 meiotic segregants were obtained. The strains
that had simultaneously improved growth at 37 0C, osmo-, ethanol-tolerance, ethanol productivity
compared to the strain IMB Y-5053, used as a control, were not revealed.
It was carried out the study of the selected thermotolerant segregants of diploid industrial ethanolic
strains "Angel", "Tega Yeast", "Distillers Yeast-T1" toward their efficiency of maize wort fermentation,
osmo- and ethanol-tolerance. Some of them are more thermotolerant during cultivation at 37 0C on the
maize wort than initial strains of industrial ethanolic yeasts. They accumulate approximately 8-10 % higher
biomass. At the same time their osmo-, ethanoltolerance, ethanol productivity are in the same levels. It is
interestingly to use isolated segregants for genetic construction of new recombinant strains with inproved
thermo-, osmo- and ethanol-tolerance and to test their ethanol production during fermentation of maize
wort at 37 0C.
This work is financially supported by the National Academy of Sciences `of Ukraine in the
framework of the Program “Biological resources and the newest technology of bioenergy conversion”.
Kutsyaba V. et al., Patent of Ukraine № 106336, publ. 11.08.2014.
61
THE ROLE OF THE CYTOPLASMIC C-TAIL OF THE PICHIA PASTORIS GSS1 PROTEIN IN
SIGNAL TRANSDUCTION
Kateryna Levkiv1, Andriy Sibirny1, 2
1
Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Street 14/16, 79005
Lviv, Ukraine;
2
Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, 35-601
Rzeszow, Poland.
Email address: sibirny@cellbiol.lviv.ua
Several signal transduction systems has been identified in the baker’s yeast Saccharomyces
cerevisiae: Gpr1/Gpa2, MAPK, cAMP, Snf3/Rgt2. Despite the identification of 40 autophagy-related ATG
genes, molecular mechanisms of glucose sensing which induce pexophagy still remains unknown. We have
found that defects of high (SNF3) and low (RGT2) affinity glucose sensors involved in glucose-dependent
induction of hexose transporters have only slight effect on glucose-induced degradation of peroxisomal
thiolase though simultaneous defects of both sensors, SNF3 and RGT2 strongly inhibit this process in S.
cerevisiae (Nazarko et al., 2008). At the same time, defects in ScSnf3 or ScRgt2 do not affect the glucose
catabolite repression.
Previously, it was described a newly identified glucose sensor homolog of ScSnf3 and ScRgt2 in the
methylotrophic yeast Pichia pastoris designated as Gss1. It was shown that deletion of GSS1 affects growth
on high and low glucose concentrations as well as pexophagy of both methanol and oleate peroxisomes
induced by glucose (Polupanov et al., 2012). However, P. pastoris Gss1 is involved in glucose catabolite
repression, unlike to S. cerevisiae Snf3/Rgt2 sensors.
In this work, we studied the effects of deletion of cytoplasmic C-tail of P. pastoris Gss1. The vectors
carrying different variations of C-tail deletion of Gss1 protein GSS1-ΔC50, GSS1-ΔC100, GSS1-ΔC200 alleles
were constructed. Constructed plasmids were verified with PCR and restriction enzyme analysis. To obtain
transformants P. pastoris, Δgss1 strain was transformed with one of the constructed plasmids linearized by
SalI. The transformants obtained were analyzed by the alcohol oxidase plate overlay assay, the growth test
of yeast strains on plates and in liquid medium, and also by the pexophagy and the catabolite repression
assays performed by Western blot analysis.
Partial C-terminal truncation (ΔC50, ΔC100) of the Gss1 protein restores growth of Δgss1 mutant on
high and low glucose concentrations and doesn’t affect catabolite repression and pexophagy. The complete
removal of the cytoplasmic tail of the P. pastoris Gss1 leads to the significant growth impairment in both
high and low glucose containing medium. Such removal also affects glucose catabolite repression and
micropexophagy (usually induced by presence of glucose).
Nazarko V. et al., Autophagy. 2008, 4:381-4.
Polupanov A. et al., Int. J. Biochem. Cell Biol. 2012, 44(11): 1906-1918.
62
HIGH TEMPERATURE FERMENTATION EFFICIENCY AND IMPROVING OF STRESS
RESPONSE IN YEAST SACCHAROMYCES CEREVISIAE
Luzhetskyi T.1, Semkiv M. 1, Dmytruk K.1, Sibirny A.1,2
1
Department of Molecular Genetics and Biotechnology, Institute of Cell Biology,
NAS of Ukraine, Lviv, Ukraine;
2
Department of Biotechnology and Microbiology, University of Rzeszow,
Rzeszow, Poland.
Email address: luzhetskyi@gmail.com
The main way of ethanol deriving is the alcoholic fermentation of sugary substrates by yeast
Saccharomyces cerevisiae. The hexoses conversion to ethanol by means of glycolysis is an exothermic
process which entails the release of energy that is partially released in the form of heat. That is why the
fermenters need to be cooled to the optimal temperature conditions (34-35 °C) for yeast during the alcohol
fermentation. Cooling of industrial vessels requires significant energy expenditures. Сonstruction of S.
cerevisiae yeast stains able to efficient alcohol fermentation at temperatures exceeding 35 °C provides
advantage during ethanol production by costs reduction for fermenters cooling, and decrease temperature
difference for the further distillation. At elevated temperature the productivity of alcohol fermentation
increases. Trehalose accumulation and activation of heat shock proteins were shown to be involved in
tolerance to elevated temperature in S. cerevisiae cells (Wiemken, 1990). Trehalose is synthesized through
two sequential steps. Trehalose-6-phosphate synthase (encoded by S. cerevisiae gene TPS1) catalyzes
trehalose-6-phosphate synthesis from glucose derivatives glucose-1-phosphate and UDP-glucose.
Trehalose-6-phosphate is further dephosphorylated by trehalose-6-phosphate phosphatase (encoded by
gene TPS2) to produce trehalose. A gene HSP104 is a stress tolerance factor that promotes the reactivation
of heat-damaged proteins. The vector for multicopy integration was constructed, in which ORFs of TPS1,
TPS2 and HSP104 genes were placed under the control of strong constitutive promoter ADH1. The
resulting vector was used for transformation S. cerevisiae industrial strains. Recombinant strains possessed
increased resistance to stress conditions and achieved higher ethanol yields during fermentation at elevated
temperature as compared to parental strain.
63
EXPRESSION OF SEF1 GENE, AS THE CENTRAL KEY REGULATOR OF RIBOFLAVIN
BIOSYNTHESIS IN THE FLAVINOGENIC YEAST CANDIDA FAMATA
Oleksii Lyzak1, Kostyantyn Dmytruk1, Andriy Sibirny1, 2
1
Institute of Cell Biology, National Academy of Science of Ukraine,
Drahomanov Street 14/16, 79005 Lviv, Ukraine;
2
Department of Biotechnology and Microbiology, University of Rzeszow,
Zelwerowicza 4, 35-601 Rzeszow, Poland.
Email address: sibirny@cellbiol.lviv.ua
Riboflavin (vitamin B2) is one of the most important vitamins for humans and animals. This vitamin
is a metabolic precursor of flavin nucleotides, FMN (flavin mononucleotide) and FAD (flavin adenine
dinucleotide) involved as coenzymes in numerous enzymatic reactions. This compound is manufactured on
a large scale for use in agriculture, medicine and food industry.
Although the sequence of reactions of riboflavin biosynthesis is well established, the regulation of
this pathway is poorly understood. Only one regulatory gene SEF1 was found identified till now in the
flavinogenic yeast Candida famata, which is involved in positive regulation of vitamin B2 biosynthesis.
In C. famata, riboflavin overproduction is observed in the absence of iron (II) ions in the medium.
To find out whether SEF1 transcription is iron-dependent, we used qRT-PCR. For this, total RNA was
isolated from C. famata and other two non-flavinogenic yeasts, Saccharomyces cerevisiae and Pichia
stipitis, grown in under iron-depleted or iron-supplemented conditions. The relative amount of SEF1
mRNA was calculated using ΔΔCt method with ACT1 as an internal control. The SEF1 expression in C.
famata was highly correlated with iron concentration in medium and showed a 3-fold induction in under
the iron-depleted conditions. P. stipitis, despite being a non-flavinogenic yeast with lower basal SEF1
expression than in C. famata, displayed similar trends, suggesting conserved SEF1 functions in these two
species of CTG clade. On the other hand, iron transcriptional regulation of SEF1 was not observed in S.
cerevisiae.
One-hybrid system basing on S. cerevisiae was used to identify DNA binding sites of Sef1. The
strains BY4742 of S. cerevisiae with plasmid harboring SEF1 gene of C. famata under the control of
galactose-inducible GAL1 promoter of S. cerevisiae and reporter plasmid bearing LAC4 gene of
Kluyveromyces lactis under control of RIB1 and SEF1 promoters of C. famata were constructed. Two
shortened sequences of promoter RIB1 lacking hypothetical Sef1 binding sites were used. β-Galactosidase
assay was used for detection of the Sef1 binding capacity of the target sequences. Developed one-hybrid
system is suitable for studying the interaction of Sef1 with targeted DNA sequences. Sef1 activates
transcription of RIB1 gene in heterologous host system. Truncation of RIB1 promoter substantially
decreases its activation. We also found that Sef1p directly interacts with own promoter providing evidence
for Sef1 autoregulation.
64
INCREASE IN GLYCEROL PRODUCTION IN SACCHAROMYCES CEREVISIAE BY
DEREPRESSION OF ILV2 ALONG WITH GPD1-GPP2 FUSED GENES
Lidiia R. Murashchenko1, Kostyantyn V. Dmytruk1, Andriy A. Sibirny1,2
1
Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine,
Drahomanov Street, 14/16, Lviv 79005 Ukraine;
2
Department of Biotechnology and Microbiology, University of Rzeszow,
Zelwerowicza 4, 35-601 Rzeszow, Poland.
Email address: mlidiia@gmail.com
Glycerol (1,2,3-propanetriol) has multipurpose uses. Currently, it is mainly recovered as a byproduct of biodiesel and soap manufacturing or produced from propylene and allyl alcohol. Alternatively,
glycerol can be produced by microbial fermentation, using sustainable carbohydrate feedstocks. There is
continuous interest in development of the improved microbial strains which effectively convert cheap
feedstocks (e.g. glucose) to glycerol. Very efficient processes of microbial glycerol synthesis based on the
use of osmotolerant yeasts, algae and some bacteria are known, however, all they are based on aerobic
organisms, so glycerol production demands air or oxygen purging which considerably increases production
costs. Therefore the development of the Saccharomyces cerevisiae yeast strains capable of efficient
glycerol production from glucose under anaerobic conditions is of great interest. Yeast S. cerevisiae is able
to produce substantial amounts of glycerol after adding sulfites, which trap acetaldehyde, to the medium or
during fermentation at alkaline pH. However, glycerol yields are low and in the case of sulfite process,
operation with large amounts of aldehyde-bisulfite adducts causes environmental concerns. Much more
efficient would be the metabolic engineering of S. cerevisiae strains producing glycerol as main product
under anaerobic conditions.
Native mitochondrial acetolactate synthase (Ilv2) catalyzes conversion of pyruvate to acetolactate. It
was assumed that cytosolic activity of Ilv2 could decrease intracellular pyruvate concentration and as a
result increase glycerol production. Truncated version of Ilv2 lacking mitochondrial targeting signal was
overproduced in S. cerevisiae. Constructed strain possessed increased both Ilv2 activity and glycerol
production. In S. cerevisiae, glycerol is synthesized from dihydroxyacetone phosphate by subsequent action
of glycerol-3-phosphate dehydrogenase (Gpd1) and glycerol-3-phosphate phosphatase (Gpp2). Multicopy
integration module was used for expression of hybrid gene GPD1-GPP2 encoding artificial fusion of Gpd1
and Gpp2 under the control of strong constitutive promoter of the alcohol dehydrogenase gene on the
background of strain overproducing cytosolic Ilv2. Glycerol production of the constructed strain showed 4fold increase as compared to the parental strain.
65
FERROMAGNETIC NANOPARTICLES AS THE CARRIERS FOR THE IMMOBILIZATION OF
YEAST RECOMBINANT ENZYMES
Tetyana Prokopiv1, Oksana Zakalska1, Andriy Zakalskiy1, Mariya Lavryk1, Oleh Smutok1,
Mykhailo Gonchar1,2
1
Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine
2
Institute of Applied Biotechnology and Basic Sciences,
Rzeszow University, Kolbuszowa
Email address: tetyanaprokopiv@gmail.com
In recent years, magnetic nano- and microparticles and composites have become key materials in
various fields (environmental rehabilitation, biomedicine, cells labeling, immunomagnetic separation,
magnetic resonance imaging, targeted drug delivery, etc.). This interest in magnetic nanoparticles (NPs) is
caused by the prospect of their widespread use because of low toxicity and high magnetization. It makes
possible to remove the magnetic NPs with immobilized biological components (e.g, enzymes) from the
reaction mixture, that facilitates analysis and allows reusing of bio-components.
For the immobilization of enzymes isolated from yeast recombinant strain cells we used
ferromagnetic Fe3O4-nanoparticles synthesized by hydrolysis of mixture of ferric (II) and (III) chlorides in
a solution of ammonium hydroxide. The surface of magnetite previously was functionalized by 3aminopropyltriethoxysilane (APTES) with the following modification by glutaraldehyde. The steps of
Fe3O4-NPs modification were controlled by Fourier transform infrared spectroscopy (FTIR). Magnetic
properties of NPs were characterized using a vibrating magnetometer. Purified preparations of arginase I
and flavocytochrome (FC) b2 (final concentration of 0.05 mg protein • ml-1) were immobilized on the
surface of functionalized magnetic NPs.
The unmodified and bio-functionalized ferromagnetic NPs were analyzed using scanning electron
microscopy (SEM) and atomic force microscopy (AFM). AFM analysis revealed spherical ferromagnetic
NPs with an average size of 30 nm. The purified FC b2 isolated from recombinant strain Hansenula
polymorpha «tr1», with the activity of 20.8 U • mL-1 was used for immobilization on the surface of NPs.
After immobilization FC b2 the size of bionanoparticles increased to 100 nm. The yield of immobilized FC
b2 was 35%.
The purified (His)6-tagged human arginase I isolated from Saccharomyces cerevisiae W303
recombinant strain with activity 181 U • mL-1 was also used for immobilization. The yield of immobilized
enzyme was 75%.
The effect of NPs on the storage stability of FC b2 was studied. After 13 days of storage
immobilized FC b2 kept 70% of activity compared to 40% for free enzyme as a control. Thus, immobilized
on Fe-NPs FC b2 is twice more stable compared to the free enzyme, what is essential for bioanalytical
practice.
The possibility of reusing of FC b2-bound commercial magnetic microparticles in bioanalysis was
investigated. The results confirm the possibility of ferromagnetic microparticles application to produce
enzymatic kits for repeated (up to 6 times) using of the enzyme.
66
SEARCH FOR NOVEL EFFICIENT PROMOTERS IN SHEWANELLA ONEIDENSIS MR-1
Nazarii Rachkevych1, Solomiya Boyko1, Kateryna Sybirna2, Yuriy Boretsky1,3, Andriy Sibirny1,4
1
Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine,
Drahomanov Street 14/16, 79005 Lviv, Ukraine;
2
IBiTec-S, SB2SM, LMB (UMR CNRS 8221), DSV, CEA, 91191 Gif-sur-Yvette, France;
3
Department of Biochemistry and Hygiene, Lviv State Universityof Physical Culture, Kosciuszko str. 11,
79000 Lviv, Ukraine;
4
Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, 35-601
Rzeszow, Poland.
Email address: n.o.rachkevych@gmail.com
Hydrogen is considered to be one of the best candidates for a future energy carrier since it has high
energy density and causes no environmental pollution. Conventional methods of getting H2 require the use
of fossil fuels and the investment of great amounts of energy. Microbial hydrogen production could be a
promising alternative. Shewanella oneidensis MR-1 is a facultative anaerobic metal reducing bacterium.
Due to its unique respiratory capabilities it has gained great interest as a biohydrogen producer and as a
bioremediation agent of heavy metals contaminated areas.
The intensive research of S. oneidensis demands the availability of molecular tools for its genetic
manipulations. In particular, efficient strong promoters are important to provide protein expression in this
host. In current work we used β-galactosidase reporter system to assess activity of S. oneidensis promoters.
We cloned several potentially active S. oneidensis promoters (promoters of transketolase (tkt), transaldolase
(tal), and phosphogluconate dehydrogenase (gnd) genes) and a native lac promoter of E. coli, a widely used
and well described bacterial promoter, to be applied as a reference. The highest rate of β-galactosidase
activity was achieved in S. oneidensis transformants bearing vector with β-galactosidase gene under control
of tkt promoter. The activity of lac promoter of E. coli was about 30% and that of tal promoter of S.
oneidensis was 20% of tkt activity, whereas gnd promoter demonstrated almost no activity at all. Also, we
constructed genomic library of S. oneidensis to search for efficient promoters. Primary screening for
positive clones was carried out in E. coli cells on LB plates supplemented with X-Gal where blue clones
were selected with subsequent β-galactosidase activity assay in S. oneidensis MR-1 cells. The most
efficient promoter selected was identified as a promoter of gamma-glutamylputrescine oxidoreductase
gene. However, β-galactosidase activity driven by this promoter appeared to be only about 50% of the
activity achieved under control of tkt promoter.
Also, we used SDS-PAGE to detect the protein expression of Pseudotrichonympha grassii HydA1
gene which encodes [Fe-Fe]-hydrogenase under control of tkt promoter in S. oneidensis MR-1 cells.
Unfortunately, we did not obtain any visible expression of HydA1. This result may indicate either low
expression level of HydA1 gene or the fault of genetic construction used. To overcome these obstacles we
constructed the c-Myc-tagged truncated version of HydA1 without N-terminal leader peptide and
translational fusion of truncated HydA1 with N-terminal leader sequence from cctA gene of S. oneidensis
MR-1 which encodes periplasmic small tetraheme cytochrome c. These constructions were
transcriptionally fused to tkt promoter of S. oneidensis. The applicability of newly obtained genetic
constructions is under investigation. Thus, several S. oneidensis promoters have been cloned, one of which
showed very high constitutive activity (tkt promoter). It cannot be excluded that even more efficient
promoters could be identified in future in the genome of S. oneidensis MR-1. However, cloned and
analyzed promoters permit different levels of the expression of desirable genes depending on the aims
assigned by researcher.
67
INVOLVEMENT OF CAT8 GENE IN REGULATION OF XYLOSE ALCOHOLIC
FERMENTATION IN THE THERMOTOLERANT METHYLOTROPHIC YEAST
HANSENULA POLYMORPHA
Justyna Ruchala1, Olena Kurylenko2, Kostyantyn Dmytruk1, Andriy Sibirny1,2
Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow 35601 Poland;
2
Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine,
Drahomanov Street, 14/16, Lviv 79005 Ukraine.
Email address: justynar87@gmail.com
1
Xylose is considered as semi-fermentative carbon source showing features of both fermentative and
respiratory substrates. Being important carbon source for alcoholic fermentation, search for approaches
which activate fermentative abilities of this pentose and simultaneously block its respiration is of great
interest. We pay attention to CAT8, the global transcriptional regulator involved in regulation of
gluconeogenesis and utilization of alternative to glucose carbon sources in Saccharomyces cerevisiae.
However, the functions of CAT8 homologue in the thermotolerant methylotrophic yeast Hansenula
polymorpha were not studied. It is known that H. polymorpha is promising organism for high-temperature
alcoholic fermentation of lignocellulosic sugars, such as glucose, cellobiose and xylose, however, ethanol
yield and productivity by the wild-type strains is very low (Ryabova et al., 2003). During last decade,
strains of H. polymorpha accumulating 15-20 times more ethanol from xylose have been constructed
(Kurylenko et al., 2014), however, parameters of xylose alcoholic fermentation have to be further improved
to meet requirements for feasible cost-effective process. Homologue of S. cerevisiae CAT8 gene was
isolated from the sequenced strain H. polymorpha NCYC495 and used for construction of the deletion
cassette. The strains with knock out in CAT8 gene were constructed on the background of the wild-type
strain and available the best ethanol producer from xylose. Both types of deletion strains have defect in
growth on gluconeogenic substrates (glycerol, ethanol) whereas growth on glucose and xylose was not
affected. The mutants Δcat8 isolated from the wild-type strain did not show changes in ethanol production
in glucose medium whereas accumulated 2-3 times more ethanol in the medium with xylose. The Δcat8
mutants isolated from the most advanced ethanol producer from xylose also did not show any differences in
ethanol production in glucose medium whereas accumulated 25-30% more ethanol in the medium with
xylose. Maximal accumulation from xylose reached 12.5 g of ethanol per Liter at 45 oC which exceeds
ethanol accumulation in the wild-type strain NCYC495 near 25 times. Data on the expression of number of
genes involved in xylose metabolism, glycolysis, gluconeogenesis, pentose phosphate pathway and
respiration as well as on the specific activities of the corresponding enzymes will be provided.
Summarizing, it could be concluded that the transcription regulator CAT8 is apparently involved in
repression of xylose alcoholic fermentation and consequently its damage strongly activates this process and
could be useful for construction of the industrial xylose fermenting strains.
Kurylenko O. O. et al., Microbial Cell Factories. 2014, 13:122.
Ryabova O.B. et al., FEMS Yeast Research 2003, 4(2):157–164.
68
L-LACTATE-SELECTIVE MICROBIAL SENSOR BASED ON
FLAVOCYTOCHROME b2-ENRICHED YEAST CELLS USING RECOMBINANT
AND NANOTECHNOLOGY APRROACHES
Oleh Smutok,1 Maria Karkovska1, Nataliya Stasyuk1, Mykhailo Gonchar1,2,*
Department of Analytical Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street
14/16, Lviv 79005, Ukraine;
2
Institute of Applied Biotechnology and Basic Sciences, Rzeszow University, Sokolowska Str. 26,
Kolbuszowa, Poland.
Email address: osmutok@cellbiol.lviv.ua
1
In the recent years, nanotechnology is the most developing branch due to a wide variety of potential
applications in biomedical, biotechnological and agriculture fields. The binding nanoparticles with various
biological molecules make them attractive candidates for using in sensors’ technologies. The particularly
actual is obtaining the bionanomembranes based on biocatalytic elements with improved sensing
characteristics.
The aim of this investigation is to study the properties of microbial L-lactate-selective sensor based
on using the recombinant Hansenula polymorpha “tr 1” (gcr1 catX/prAOX_CYB2) yeast cells
overproducing flavocytochrome b2 (FC b2), as well as additionally enriched by the enzyme bound with gold
nanoparticles (FC b2-nAu). Although the high permeability of the living cells to nanoparticles is being
intensively studied (mostly for delivery of drugs), the idea of using both recombinant technology and
nanotechnology to increase the amount of the target enzyme in the sensing cellsr is really novel.
The nAu were synthesized using inorganic reduction of HAuCl4 by sodium borohydride used as a
carrier for immobilization of FC b2 and for their transfer into living and permeabilized yeast cells. The
purified FC b2 with a specific activity of 22 U·mg-1, isolated from the recombinant yeast strain H.
polymorpha “tr 1”, was used for bio-functionalization of nAu. The catalytic activity and stability of the
obtained FCb2-nAu were studied. The permeability of the intact and permeabilized yeast cells toward FCb2nAu was analyzed using scanning electron and transmission electron microscopy.
The FC b2-nAu-enriched living and permeabilized yeast cells were used for construction of a
bioselective membrane of microbial L-lactate-selective amperometric biosensor. Phenazine methosulphate
was served as a free defusing electron transfer mediator which provides effective electron transfer from the
reduced form of the enzyme to the electrode surface. It was shown that the output to L-lactate of FC b2nAu-enriched permeabilized yeast cells is 2.5-fold higher when compared to the control cells.
The obtained results confirm that additional enrichment of the recombinant yeast cells by the
enzyme, bound with nanoparticles, improves the analytical parameters of microbial sensor.
Acknowledgements
This research was supported in part by NAS of Ukraine in the frame of the Scientific-Technical
Program “Sensor systems for medical, ecological and industrial-technological needs: metrological
assurance and research exploitation” and Ukrainian-Lithuanian Project “Investigation of L- and D-lactate:
cytochrome с oxidoreductases isolated from the recombinant yeast Hansenula polymorpha and their usage
for construction of amperometric biosensors”.
69
AN ENZYMATIC ASSAY OF MANGANESE IONS USING APOENZYME OF
MN2+-DEPENDENT RECOMBINANT ARGINASE I
Nataliya Stasyuk1, Andriy Zakalskiy1, Oksana Zakalska1, Galina Gayda1, Mykhailo Gonchar1,2
1
Institute of Cell Biology, NAS of Ukraine, Drahomanov Str. 14/16, 79005 Lviv, Ukraine;
2
University of Rzeszow, Sokolowska Str. 26, 36-100 Kolbuszowa, Poland.
Email address: stasuk_natalia@ukr.net
Manganese is considered as one of the most abundant element in the biosphere. It is widely
distributed in soil, sediment, water, and live organisms. Although manganese is essential for humans and
other species of the animal kingdom as well as for plants, it exhibits the high level of toxicity at higher
concentrations. In human, chronic manganese excess affects the central nervous system, with the symptoms
resembling those of Parkinson’s disease. It can also affect the ecosystem negatively, accumulating in the
food chain.
Arginase I is a manganese containing enzyme of the urea cycle. It catalyses the final cytosolic
reaction of urea formation in the mammallian liver – the conversion of arginine to ornithine and urea. We
suggested the possibility to assay manganese by recovering arginine-hydrolyzing activity after specific
binding of Mn2+ with apoenzyme of arginase I, because the active site of the enzyme contains Mn 2+ ions
responsible for the catalytic activity.
The (His)6-tagged human liver arginase I was purified in one step from the cell-free extract of
Saccharomyces cerevisiae recombinant strain by metal-affinity chromatography on Ni-NTA agarose. The
maximal specific activity of the 40-fold purified enzyme was 1600 µmol∙min-1∙mg-1 protein. The
apoenzyme of arginase I was used as a Mn2+-recognizing bioelement for the development of a new
enzymatic-fluorometric assay of Mn2+ ions. The new method is selective and rather simple. Linear
detection range was estimated to be from 0.010 nM to 1.5 nM and detection limit – 0.001 nM Mn2+. The
method was tested on the real samples of waste water from the copper and gold mines. It was shown that
the estimated Mn2+ content in tested samples was in a good correlation with the results of reference atomic
absorption method (R=0.998).
70
DEVELOPMENT OF TOOLS FOR OMICS PROFILING OF NON-CONVENTIONAL YEASTS
Ievgeniia A. Tiukova, Volkmar Passoth
Uppsala Biocenter, Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala,
Sweden.
Email address: ievgeniia.tiukova@slu.se
The advance of tools for genome and transcriptome analysis is essential for development of research
on non-conventional yeasts. Despite the rapid progress of Next Generation Sequencing technologies, tools
for assembly of genomes at chromosome resolution are scars. We developed efficient tool for de-novo
assembly of genomes of non-conventional yeasts. This method is based on multi technology approach with
application of short and long-read sequencing and optical mapping.
We performed genomics and transcriptomics analysis on non-conventional yeast Dekkera
bruxellensis in order to understand molecular basis of its competitiveness in ethanol production
environments. Previously, D. bruxellensis was isolated from several alcohol production plants with high
ethanol production efficiency, where it had replaced the initial inoculum of Saccharomyces cerevisiae
(Passoth et al., 2007). Global gene expression analysis indicated that low transcription activity of genes
involved in glycerol biosynthetic pathway and expression of NADH-ubiquinone reductase (complex I of
respiratory chain), which is missing in S. cerevisiae might provide basis for more energy efficient
metabolism of D. bruxellensis as compared to S. cerevisiae. Moreover, high expression of putative highaffinity sugar transporters might contribute to high affinity of D. bruxellensis to limiting glucose substrate.
Passoth et al., Applied and environmental microbiology. 2007, 73:4354-4356.
71
DEVELOPMENT OF A STRATEGY FOR PRODUCTION THE DROUGHT RESISTANT
CEREALS BY USAGE OF THE TREHALOSE BIOSYNTHESIS GENES FROM
SACCHAROMYCES CEREVISIAE
Yatsyshyn V.1, Luzhetskyi T.2, Dmytruk K.2, Yemets A.1, Sibirny A.2,3
Institute of Food Biotechnology and Genomics NAS of Ukraine, Оsipovskogo str., 2A, 04123 Кyiv,
Ukraine;
2
Institute of Cell Biology, NAS of Ukraine, Drahomanov str., 14/16, 79005 Lviv, Ukraine;
3
Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, 35-601
Rzeszow, Poland.
Email: yatsyshyn.v@gmail.com
1
Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of
organisms. It affects sugar metabolism as well as osmoprotection against several environmental stresses,
such as heat and desiccation. For several years, there have been attempts and developments in applying the
trehalose metabolism to engineer drought-and salt-tolerant crop plants using TPS1 or TPS2 genes
originating from yeast and bacteria. Also trehalose accumulation and activation of heat shock proteins were
shown to be involved in tolerance to elevated temperature in Saccharomyces cerevisiae cells.
Trehalose is synthesized through two sequential steps. Trehalose-6-phosphate synthase (encoded by
S. cerevisiae gene TPS1) catalyzes trehalose-6-phosphate synthesis from glucose derivatives glucose-1phosphate and UDP-glucose. Trehalose-6-phosphate is further dephosphorylated by trehalose-6-phosphate
phosphatase (encoded by gene TPS2) to produce trehalose.
In this study, we carried out the construction of recombinant plasmids coexpressing S. cerevisiae
genes for the trehalose synthesis: TPS1 and/or TPS2 under the control of the strong constitutive promoter.
The construction of vector for the enhancing of the expression of these genes in S. cerevisiae cells was
carried out in several stages. Basic pUC57-delta1_2 plasmid contained δ-sequences, which provides the
multicopy integration of expression modules in S. cerevisiae genome; promoter of ADH1 gene (encodes
alcohol dehydrogenase); CYC1 terminator (cytochrome c) and kanMX4 gene which provides resistance to
geneticin. The resulting vector was used for transformation of S. cerevisiae strains. Recombinant strains
were examined for resistance to stress conditions.
The TPS1 and/or TPS2 genes of S. cerevisiae will be introduced also into binary vectors under the
control of different constitutive or inducible promoters for Agrobacterium-mediated transformation of such
cereals as barley and wheat. The resulted transgenic plants expressing TPS1 and/or TPS2 genes will be
examined for their development and stress tolerance including drought resistance.
This work is supported by National Academy of Sciences of Ukraine (the target complex multidisciplinary
program of "Molecular and cellular biotechnology for medicine, industry and agriculture", Grant No.
21/15А, 2015-2019, Reg. No. 0115U005022).
72
MODIFICATION OF γGLUTAMYLCYSTEINE SYNTHETASE FOR CONSTRUCTION OF
GLUTATHIONE OVERPRODUCERS IN THE YEAST HANSENULA POLYMORPHA
Marianna Yurkiv1, Olena Kurylenko1, Roksolana Vasylyshyn1, Kostyantyn Dmytruk1 and
Andriy Sibirny1,2
1
Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine,
Drahomanov Street, 14/16, Lviv 79005, Ukraine;
2
Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4,
Rzeszow 35-601, Poland.
Email address: mariannayurkiv1@gmail.com
Glutathione (γ-L-glutamyl-L-cysteinyl-glycine; GSH) is a tripeptide with diverse physiological
functions in eukaryotic cells. Most of these functions have been related to its antioxidative properties
caused by the thiol group in the cysteine moiety. This tripeptide is an important natural compound used in
medicine, biotechnology, food and cosmetic industry. As an active ingredient of food, drugs and cosmetic
products, GSH could alleviate harmful oxidative processes, scavenge toxic compounds at different kinds of
human intoxications and strengthen whitening, skin repair antiaging effect. The accumulation of GSH in
yeast is tightly controlled at different levels. Thus, controlling impairment of the molecular regulatory
mechanisms which restrict GSH over-accumulation is apparently the major obstacle toward obtaining the
efficient GSH producers in microorganisms. Microbial GSH overproduction is limited by mechanisms of
feedback inhibition of γ-glutamylcysteine synthetase (GCS), the first and rate-limiting enzyme of GSH
biosynthesis, by the end product of the pathway, GSH. In addition, the expression of gene coding for GCS,
is repressed by GSH. The methylotrophic yeast Hansenula polymorpha is considered as a rich source of
GSH due to the role of this thiol in detoxifications of key intermediates of methanol metabolism,
formaldehyde and hydrogen peroxide.
In this work, the selection scheme providing generation of GCS insensitive to feedback inhibition
was developed. The modified versions of GSH2 gene obtained by error prone PCR were cloned under the
control of strong constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase in replicative
plasmid pYT3. Selected transformants were analyzed for their resistance to different prooxidant agents
(1,2,3-triazole, diethylmaleate, ethionine) and compared to the strains carrying unmodified GSH2 gene.
Strains providing more intensive growth on the selective medium revealed higher GSH accumulation as
compared to strains carrying unmodified GSH2 gene, assumed to possessed the reduction of Gsh2 feedback
inhibition. Sequencing of one of the isolated mutant GSH2 alleles enabled us to identify five amino acid
substitutions in the highly conserved Gsh2 domain. Detection of GSH2 gene mutations leading to the
elimination of negative regulatory mechanisms of GSH biosynthesis will create a competitive producer of
this tripeptide.
73
INTERACTION OF MYOSIN VI WITH DOCK7 PLAYS IMPORTANT ROLE IN PROTRUSION
FORMATION IN PC12 CELLS
Magdalena Sobczak1, Emilia Wojtera1, Łukasz Majewski1, Paweł Pomorski1, Jolanta Nowak1
Junji Yamauchi2,3 and Maria Jolanta Rędowicz1*
1
Laboratory of Molecular Basis of Cell Motility, Department of Biochemistry, Nencki Institute of
Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland
2
Tokyo Institute of Technology, Midori, Yokohama 226-8501, Japan
3
Department of Physiology, Kitasato University School of Medicine, Sagamihara,
Kanagawa 252-0374, Japan
Email address: j.redowicz@nencki.gov.pl
DOCK7 (dedicator of cytokinesis 7) is a guanidine nucleotide exchange factor (GEF) for Rac1
GTPase that is involved in neuronal polarity and axon generation as well in Schwann cell differentiation and
myelination. Recently, we identified DOCK7 as the binding partner of unconventional myosin VI (MVI) in
neuronal-lineage PC12 cells and postulated that this interaction could be important in vivo [Majewski e al.
(2012) Biochem Cell Biol., 90:565-574]. Herein, we found that MVI-DOCK7 interaction takes also place in
other cell lines and demonstrated that MVI cargo domain binds to DOCK7 C-terminal regions, M2 and
DHR2 (DOCK homology region 2) domains. In MVI knockdown cells, lower Rac1 activity and a decrease of
DOCK7 phosphorylation on Tyr1118 were observed, indicating that MVI could contribute to DOCK7
activity. MVI and DOCK 7 co-localization was maintained during NGF-stimulated PC12 cell differentiation
and observed also in the outgrowths, as revealed by immunofluorescence staining of native proteins or
overexpression of full length GFP-MVI as well as by proximity ligation assay. Also, during differentiation an
increase in DOCK7 and JNK kinase phosphorylation was detected. Interestingly, overexpression of GFPtagged MVI cargo domain (GFP-GT) impaired protrusion formation indicating that the motor and not the
cargo domain is important for this process. Moreover, overexpression of GFP-MVI but not GFP-GT caused
transient increase in Rac activity. These data indicate that MVI-DOCK7 interaction has functional
implications in the protrusion outgrowth and MVI motor activity seems to be important for delivery and
maintaining DOCK along the protrusions allowing to exert its GEF activity in the right destination.
The work has been supported by a grant 2012/05/B/NZ3/01996 from the National Science Centre.
74
OPTIMIZATION OF PAOX1 INDUCTION IN PICHIA PASTORIS USING A
METHANOL/SORBITOL CO-FEEDING STRATEGY
F. Carly1, H. Niu2, F. Delvigne3, P. Fickers1,3
1
Unité de Biotechnologies et Bioprocédés, Université libre de Bruxelles, Av Roosevelt 52, CP152/51, 1050
Bruxelles, Belgium
2
Centre for Process Systems Engineering, Department of Chemical Engineering, Imperial College
London, South Kensington Campus, London SW7 2AZ, England, UK
3
Microbial Processes and Interaction, Univerité de Liège – Gembloux AgroBio Tech, Passage des
Déportés, 2, 5030 Gembloux, Belgium
Email address: pfickers@ulg.ac.be
Using a Pichia pastoris Mut+ strain bearing a pAOX1-LacZ reporter system, we have previously
reported that methanol/sorbitol feeding at ratio 60/40 C-mol/C-mol could increase pAOX1 promoter
induction by 40% compared to pure feeding of methanol. Besides, it also allows to reduce oxygen
consumption by 30 %, alleviating challenging oxygen limitation issues in the P. pastoris fermentation. As a
follow-up, this work centers on an improved understanding of the pAOX1 regulation during
methanol/sorbitol feeding regarding carbon feeding rate and oxygen limitation.
Chemostat cultures with increasing total carbon concentration in the combined feed of
methanol/sorbitol 60/40 C-mol/C-mol were performed at a dilution rate of 0.03 h-1. Methanol and sorbitol
uptake rates, cell growth and pAOX induction level were determined in regard to carbon concentration and
oxygen limitation. Methanol accumulation and oxygen limitation were found to influence directly the level of
β-galactosidase activity and consequently, pAOX1 induction. For dissolved oxygen (DO) value > 0, an
increase of the carbon concentration in the feed yielded to an increase of pAOX1 induction. By contrast,
when DO was around 0, methanol accumulated, causing a 30% decrease of induction. Moreover, in these
conditions of low DO values, cells preferred to utilize sorbitol. This highlighted the ability of cells to adapt to
transient oxygen limitation that often occurs at industrial scale due to medium heterogeneity without loss of
cell viability and reduced negative effect on pAOX1 induction.
Accordingly, methanol/sorbitol feeding not only allows to increase pAOX1 induction level but also
contributes to process robustness. The optimal feeding rate of the methanol/sorbitol tested here is determined
to be 6.6 mmolC-1(DCW.h)-1 at an oxygen transfer rate (OTR) of 8.28 gO2(l.h)-1.
75
REAL TIME MONITORING OF YARROWIA LIPOLYTICA CULTURE GROWTH USING FLOW
CYTOMETRY TECHNIQUE
Hosni Sassi1, Franck Delvigne3, Tambi Karr3, Jean-Marc Nicaud2 and Patrick Fickers3
1
Unité de Biotechnologies et Bioprocédés, Université libre de Bruxelles, Belgium
2
Institut Micalis, INRA UMR1319 – AgroParisTech, Paris-Grignon, France
3
Microbial Processes and Interactions, University of Liège - Gembloux Agro-Bio Tech, Belgium
Email address: hsassi@ulb.ac.be, pfickers@ulg.ac.be
Key words: Yarrowia lipolytica, flow cytometry, bioprocess
The non-conventional yeast Yarrowia lipolytica is an interesting host organism witch found a great interest
with both academic and industrial applications Control of physiological parameters has become a challenge
to further setting an optimized culture process of heterologous protein production in this yeast. In this work,
Y. lipolytica growth in complex media supplemented with oleic acid has been monitored in real time during
culture on bioreactor coupled with flow cytometer. The use of this technology is getting more and more
attention for the control of the microbial culture process since it permits a rapid single cell analysis. Using
this technique, biomass production and substrate consumption (oleic acid) can be measured online using
forward scatter (FSC) and side scatter (SSC) parameters. In addition, the ovoid, pseudo-hyphae and hyphae
cells can be also detected during Y. lipolytica cell growth. In front of these results, this methodology
presented in this work can be used as tool to better control the physiological parameters of Y. lipolytcia
culture growth in presence on hydrophobic substrate such as oleic acid.
76
INTRACELLULAR LIPASES PRODUCTION BY Yarrowia lipolytica USING
RESIDUAL FRYING OIL
Patrícia Martins Botelho Nunes1,3, Priscilla Amaral1, Patrick Fickers2,3
Departamento de Engenharia Bioquímica, Escola de Química,Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil.
2
Microbial Processes and Interactions, University of Liège - Gembloux Agro-Bio Tech, Passage des
Déportés 2, 5030 Gelmbloux
3
Unité de Biotechnologies et Bioprocédés, Université libre de Bruxelles, Av Roosevelt 50, 1050 Bruxelles.
1
Lipases (glycerol ester hydrolases EC3.1.1.3.) are enzymes that catalyze the hydrolysis and synthesis of esters. Thus, it features a
wide use in industry and the interest in the application of these enzymes in industrial bioprocesses is growing because of its versatility
(Ribeiro et al., 2011). New technologies are being developed to circumvent the economic limitation and lipases with more specific
properties are being investigated (Ha et al., 2007). Among the microorganisms capable of producing lipase, Yarrowia lipolytica has
attracted great interest in the biotechnological area by having the ability to excrete various metabolites in large quantities (Barth and
Gaillardin, 1997). Many works show intracellular lipase production with olive oil as inducer. The frying oil used repeatedly in homes
and restaurants is usually discarded improperly in sewer system and it is a residue that accumulates and can clog the sewer tubes.
Researches seeking for wastes for lipases production are already being carried out (Haba et al., 2000; Rywińska et al., 2008). Aiming
the study of intracellular lipase stimulation in the presence of oils and to verify the potential of residual frying oil induction,
experiments in shaker were performed in OO medium (peptone 0.64% and yeast extract 1%) and inducer (olive oil or residual frying
oil 1 % (v/v)). Extracts were obtained through ultrasound, in ice bath, and lipase activities were quantifies using p-nitrophenyl laurate
and olive oil as substrate. The fatty acid composition of frying oil was determined by gas chromatography of methyl esters of fatty
acids (FAME) obtained after acid transesterification. Considering oleic acid is described on literature as the best inducer for lipase
production and it is present both in olive oil and waste frying oil, this fatty acid was used as a model for preliminary intracellular
lipase production kinetics by Yarrowia lipolytica IMUFRJ 50682 grown in microplate and the activity profiles obtained with the
different inducers were compared. Oleic acid concentration was quantified by HPLC. In 15 hours of growth, in presence of oleic acid,
maximum activity for intracellular fractions was obtained. At this point, half the oleic acid has been consumed and the yeast are at the
end of the exponential growth phase. Besides, were observed 2 extracellular activity peaks: one in 15 hours of growth and another one
in 40 hours of growth, when oleic acid was completely devoid from the growth medium. The profile presented in the presence of olive
oil is quite similar to that presented for oleic acid induction. However, in the presence of fatty acid, a high associated lipase activity is
not noted before 15 hours of growth. Therefore, both maximum extracellular and intracellular activity occur at the same time (15
hours). In the presence of residual frying oil (RFO) maximum activity values for intracellular fractions were detected in 15 hours of
growth and the activity profile for these fractions was quite similar to observed in oleic acid. Low extracellular activity was detected
in growths with RFO as inducer. In this report, residual frying oil stimulated lipase intracellular production by Y. lipolytica, but with
lower maximum lipolytic activity values than using olive oil. This can be due to lower oleic acid level present in RFO (26% of oleic
acid and 46% of linoleic acid). Olive oil and residual frying oil were able to stimulate intracellular lipase production by Yarrowia
lipolytica IMUFRJ 50682 in the absence of other carbon sources. RFO has fatty acids and triglycerides in its composition in an
amount sufficient to stimulate the production of intracellular lipases (maximum lipolytic activity 400 U/g); however it has proven less
efficient than olive oil (maximum lipolytic activity 600 U/g). Intracellular lipase production with residual frying oil had interesting
results according to this study. Conditions for lipase production in presence of the two inducers were the same and the inducers were
used by yeast for cell growth and lipase production. However, residual frying oil favoured the production of intracellular fractions,
reaching activity values close to those obtained with olive oil. Although the enzymatic extract from olive oil induction has shown a
slightly better lipolytic activity, the residual frying oil has potential to be used for production of intracellular lipases by Y. lipolytica.
The use of wastes for lipase production may contribute to decrease in the process costs and, moreover, has the benefit of reuse of this
industrial and commercial waste.





Ribeiro BD, De Castro AM, Coelho MAZ, Freire DMG (2011) Production and Use of Lipases in Bioenergy: A Review from the
Feedstocks to Biodiesel Production Enzyme Researche 2011
Rywińska A, Witkowska D, Juszczyk P, Rymowicz W, Kita A (2008) Waste Frying Oil as Substrate for Lipase Production by
Geotrichum candidum strains. Polish J. of Environ. Stud. 17:925-931.
Ha SH, Lan MN, Lee SH, Hwang SM, Koo YM (2007) Lipase catalyzed biodiesel production from soybean oil in ionic liquids.
Enzyme and Microbial Technology 41:480-483.
Haba E, Brescoa O, Ferrera C, Marque´S A, Busquets B M, Manresa A (2000) Isolation of lipase-secreting bacteria by deploying
used frying oil as selective substrate. Enz and Microbial Technol 26:40–44.
Barth G, Gaillardin C (1997) Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiol Rev 19:219237.
77
CHANGES IN THE EXPRESSION OF MIRNANAS, RESPONSIBLE FOR INVASIVE ACTIVITY,
IN BREAST CANCER CELL LINES WITH DIFFERENT MALIGNANCY LEVELS
Tetiana Borikun, Vasyl Chekhun
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine,
Vasylkivska str. 45, 03022 Kyiv, Ukraine
E-mail address: tborikun@gmail.com
Introduction. Invasiveness is the most common feature of malignant phenotype in breast cancer. At the same
time epigenetic mechanisms involved in this process today are explored insufficiently.
Purpose. Identify the features of miRNANA expression that are involved in the regulation of adhesion and
invasion of breast cancer cells with different malignancy level.
Materials and methods. RT-PCR was used to analyze the differential expressions of the microRNA between
breast cancer cell lines with different malignancy levels(184A1, MCF10A, MCF-7, T47D, MDA-MB-468,
MDA-MB-231).
Results. We established significant changes in expression of miRNA - 10b, miRNA-221 and miRNA - 200b,
aberrant expression of which is associated with increased invasiveness of breast cancer cells. Expression
profile between immortalised breast cells 184A1, MCF10A doesn’t differ, exept miRNA-10b – in 184A1
cells it was higher 1,4 times. Expression of miRNA-221 and miRNA-10b increased and level of miRNA200b decreased in most malignant cell lines MDA-MB-468 and MDA-MB-231. In cell line T47D level of
miRNA-221 was the lowest – 12,6 times less than in immortalised breast cells MCF10A. There were no
significant changes in miRNA-200b exppression in MCF7 cell line compared to control. The most significant
changes in miRNANA expression were observed in MDA-MB-231 cell line.
Conclusion. It was established that due to malignancy level there are observed changes of miRNA
expression profile, responsible for the invasion.
These data suggest increased invasive properties, as far as a target of miRNA-221 and miRNA-10b are MMP
inhibitors, tumor suppressors p27 and p57 and estrogen receptor, and target genes of miRNA-200b regulate
EMT, migration and invasion and changes of their expression are an indicator of adhesional properties and
invasiveness of breast cancer cells.
The results make it possible to regard miRNANA - 10b, miRNA-221 and miRNA - 200b as prognostic
markers of malignancy and invasive activity of breast cancer cells.
Express my gratitude Ph.D. Lukyanova N. Yu. head of Laboratory of cytology and molecular-biological
markers of tumor growth.
78
INFLUENCE OF COOLING SPEAD ON HSC RECOVERY DURING CORD BLOOD
CRYOPRESERVATION IN VIALS
Volodymyr Shаblii1, Hanna Svitinа1, Maria Kuchmа1,2, Yulia Prokopez1, Tetiana Indychenko1, Yulia
Shаblii1, Dmytro Lobyncev1, Oleksandr Karakatenko1, Lubov Lukаsh2,
Tetiana Bukreieva1, Galyna Lоbyntsevа1
1
Institute of Cell Therapy , Institute оf Mоlecular Biolоgy and Gеnetics National Acаdemy of Science of
Ukraine2, Kyiv, Ukraine
E-mail: v_shabliy@ukr.net
Freezing program extremely critical affect the viability of hematopoietic stem cells (HSCs). Cord
blood banking is widespread and integrated in many national health programs for treatment of inherent or
malignancy disorders. Cord blood units (CBUs) are cryopreserved by different methods using various
cryobags, vials, cryoprotectant compositions therefore appropriate freezing programs should be developed.
The speed of cooling on different steps of freezing process significantly influences the viability of HSCs.
Cord blood was collected in utero in the blood collection bags with CPDA, erythrocytes were
depleted by HES-sedimentation, and plasma was reduced by centrifugation, cord blood cells (CBCs) were
cryopreserved with DMSO/Dextrane 40 solution in vials using software based freezer. CFU analysis was
performed in MethoCult (StemCell Tech., Canada) culture medium. CFU number was calculated as number
of colonies per 105 seeded CBC, CFUs recovery was calculated as the ratio (%) of CFU number of thawed
CBCs to CFU number of pre-freeze CBCs. Data were represented as median and interquartile range. P values
less than 0,05 considered statistically significant.
CFU number of CBUs cryopreserved with 10% DMSO and freezed with slow cooling (-1 oC/min)
from seeding (crystal formation) to - 30 oC was significantly higher as compared to the fast cooling (-5
o
C/min) with meanings of 103 (88-115; n=13) and 54 (19-96; n=30) respectively. Similar variancewas
observed in case of CBUs cryopreservation with 5% DMSO, since CFU number of samples freezed with
slow cooling was 2.28-fold higher than those freezed with fast cooling [103 (94-150; n=10) vs 45 (12-96;
n=13)].
Furthermore the number of CFU of pre-freeze CBUs correlated with final CPDA concentration in the
blood collection bag (r=-0,54, n=18). Importantly, CFU recovery of CBUs depended on the shipping time of
CBUs followed by their cryopreservation with 5% (r=-0,57, n=13) and 10% (r=-0,63, n=29) DMSO by fast
cooling in contrast to slow cooling where no significant correlation was observed. CFU number of CBUs
cryopreserved with 5% and 10% DMSO and slow freezing did not significantly differ.
In summary, we showed that CBUs cryopreserved in vials by slow cooling from seeding to -30 oC
have had better viability. Controlled crystal initiation point combined with slow cooling are giving an
opportunity to decrease concentrarion of DMSO from 10% to 5%.
79
CD150 SPLICE ISOFORMS EXPRESSION AND TOPOLOGY IN CHRONIC
LYMPHOCYTIC LEUKEMIA
Inna Gordiienko, Larysa Shlapatska, Valeriia Kholodniuk, Lilia Sklyarenko and Svetlana Sidorenko
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine,
Vasylkivska str. 45, 03022 Kyiv, Ukraine
E-mail address: hordiyenko24@mail.ru
Chronic lymphocytic leukemia (CLL) is a most common leukemia in Western word. CLL is
characterised by accumulation of neoplastic monoclonal CD5+CD19+CD23+ B cells in peripheral blood, bone
marrow and secondary lymphoid organs. CD150 receptor is involved in regulation of signalling via antigen
receptors as co-stimulatory molecule in normal and malignant B cells. Recently it was shown upregulated cell
surface CD150 expression in mutated IGHV CLL (M-CLL) cases that associated with favourable clinical
outcome (Zucchetto A. et al., 2011, Cornet E. et al., 2015). However, its role in CLL pathogenesis and
contribution of CD150 prevalent expression in M-CLL to favourable prognosis still was not addressed. The
aim of the work was to study CD150 topology in CLL B-cells, answered the question which of CD150
isoforms is predominantly expressed in CLL, and compared basal levels of Akt, ERK1/2, JNK and
p38MAPK phosphorylation. Studies were performed on peripheral blood lymphocytes from patients with
confirmed diagnosis of CLL (n=42). Flow cytometry, western blot analysis, Q-PCR and confocal microscopy
were used.
According to the flow cytometry analysis, 50% of CLL samples expressed CD150 on the cell surface
(sCD150+ CLL cases). Unexpectedly, western blot analysis revealed CD150 expression on protein level both
in sCD150+ and sCD150- CLL cases. To clarify obtained result we used differential sCD150 and cytoplasmic
CD150 immunostaining approach in CLL samples followed by visualisation using confocal microscopy.
CD150 expression was revealed in cytoplasm of sCD150- CLL cases, but not on the cell surface. In sCD150+
CLL CD150 was expressed predominantly on the cell surface and only in low amount was detected in
cytoplasm. Furthermore, CD150 was colocalized on the CLL cell membrane with positive regulators of BCR
signalling (CD19, CD180), tetraspenin receptor CD37, but not with CD40 and negative regulator of BCR
(CD5). Expression of conventional CD150 transmembrane isoform (mCD150) and a novel splice isoform –
nCD150, with 83 bp insert between transmembrane and cytoplasmic domains, were studied by Q-PCR in
CLL samples in comparison with normal naïve IgD+ B cells. Expression of both isoforms was revealed in all
tested CLL samples with higher level of mCD150 expression in comparison with nCD150 isoform. However,
in CLL cells expression level of mCD150 isoform was significantly lower than in normal naïve B cells At the
same time, nCD150 level often was comparable with normal B cells and in 30% sCD150 cases exceeded
normal levels up to 18 times. Since CD150 is involved in regulation of PI3K/Akt and MAPK signaling
pathways in normal and malignant B cells, we examined basal Akt, ERK1/2, JNK and p38MAPK
phosphorylation in sCD150+ and sCD150- CLL cases. No correlation in basal phosphorylation level of Akt,
ERK1/2, JNK and p38MAPK and CD150 expression in CLL samples was determined.
Taken together, CLL is characterized by heterogeneity in CD150 isoforms expression. sCD150 - cases
of CLL also expressed CD150, but in cytoplasm. The biological significances of CD150 expression in CLL
will be further investigated.
80
ADAPTOR PROTEIN RUK/CIN85 OVEREXPRESSION LEADS TO INCREASED ADHESION,
MIGRATION AND INVASION OF MOUSE 4T1 BREAST ADENOCARCINOMA CELLS
Iryna Horak1,, Ganna Pasichnyk1, Denys Gerashchenko1, Dmytro Petukhov1, Nadia Shabas2,
Liudmyla Drobot1, Lucia Knopfova3, Lubor Borsig4
1
Palladin Institute of Biochemistry, NASU, Kyiv, Ukraine;
2
Kyiv National Taras Schevchenko University, Kyiv, Ukraine;
3
Masaryk University, Brno, Czech Republick;
4
University of Zurich, Zurich, Switzerland;
E-mail address: iryna.horak@gmail.com
Introduction. Adaptor protein Ruk/CIN85 was demonstrated to be involved in essential cell processes such
as intracellular signalling, cell death, proliferation, and motility. Previously, it was found that overexpression
of Ruk/CIN85 leads to the reduction in proliferative rate, increased resistance to anticancer drugs and
migration potential in human MCF-7 breast adenocarcinoma cell line. In order to study the role of
Ruk/CIN85 in carcinogenesis in vivo the syngeneic murine model was originated from mouse 4T1 breast
adenocarcinoma. The aim of the present study is to investigate the role of Ruk/CIN85 in 4T1 cells migration,
invasion and adhesion in vitro.
Methods. Ruk/CIN85-overexpressing 4T1 cells were generated by calcium phosphate transfection with
pRc/CMV-Ruk1 plasmid. Ruk/CIN85 expression in wild-type cells was supressed with Ruk/CIN85-specific
shRNA lentivirus. Stably transfected/infected cells were selected using specific drugs G418 and puromycin
respectively. Ruk/CIN85 expression level was determined both by Western blot analysis and Real Time RTPCR. Cell migration was estimated with scratch test. For invasion assay Boyden chambers coated with
different matrixes were used. Effectiveness of adhesion was assessed by the ability of cells to attach to
extracellular matrix.
Results. It was found that Ruk/CIN85-overexpressing 4T1 cells migrate faster than control ones. Also, these
cells are characterized by higher ability to invade through Matrigel, collagen 1 and fibronectin. Adhesion
assay demonstrated more effective attachment of Ruk/CIN85-overexpressing 4T1 cells to Matrigel and
fibronectin, but not to the collagen 1.
Conclusions. Our data suggest that adaptor protein Ruk/CIN85 is involved in the control of key processes in
tumor cells physiology such as adhesion, invasion and migration.
81
IMMUNOPHENOTYPE AND COLONY-FORMING POTENTIAL OF HEMATOPOIETIC
STEM/PROGENITOR CELLS FROM HUMAN PLACENTAL TISSUE, UMBILICAL CORD
BLOOD, AND FETAL LIVER
Maria Kuchma 1,2, Vitaliy Kyryk3, Hanna Svitina1,Yulia Shablii1, Lubov Lukash2,
Galina Lobyntseva1, Volodymyr Shablii1,2
1
Institute of Cell Therapy, Ukraine, 03680, Kyiv, Komarovaave., 3;
2
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine,
Ukraine, 03680, Kyiv, Zabolotnogo str., 150;
State Institute of Genetics and Regenerative Medicine, National Academy of Medical Sciences of Ukraine
3
, Ukraine, 04114, Kyiv, Vyshgorodska str., 67
e-mail: kuchma@gmx.com,
phone: +380442079206
We investigated the similarities and differences of HSPCs population from full-term placental tissue
(FTPT) compared to HSPCs from cord blood (CB) and fetal liver (FL). HSPCs of FTPT origin were obtained
by enzymatic method. Immunophenotyping was performed on BD FACSAria (USA). MethoCult (Canada)
was used for differentiation assays. FTPT was cryopreserved by special program in the software based
freezer. FTPT are characterized by higher phenotypic heterogeneity compared to CB and contained cell
subpopulations at different stages of differentiation as CD34+/lowCD45low/-, CD34++CD45low/-,
CD34+++CD45low/-, CD34+/lowCD45hi, and CD34++CD45hi. The content of “true” HSPCs
(CD34+CD45lowSSClow) among viable CD45+ cells from FTPT tissue was 0.56 % (0.39 – 0.76 %, n = 16).
HSPCs from FTPT expressed the CD90 at a significantly higher level in comparison with CB and the same
level of CD133 and CD31. FTPT contains significantly higher number of lineage committed HSPCs
compared
to
CB
(namely
CD34+CD45lowCD14+CD33+SSClow,
CD34+CD45lowCD14+SSClow,
+
low
+
low
CD34 CD45 CD235 SSC cells) but significantly lower than FL (namely CD34+CD45lowCD33+SSClow,
CD34+CD45lowCD7+SSClow, CD34+CD45lowCD19+SSClowcells). Similarly to CD14 and CD33, the CD7 and
CD19 expression increase was accompanied by the intensity decrease of CD34 expression on CD45 low/-cells
from the FTPT and the CB. Furthermore, the co-expression of CD14 and CD33 was increased on placental
CD34-positive cells with growing intensity of CD45 expression and decreasing intensity of CD34 expression.
HSPCs of FTPT origin gave rise to various type of colonies in vitro and their ratios did not significantly
differ compared to CB, in addition the ratios of myeloid and erythroid progenitors among the lineage
committed cells were the same for FTPT and CB. Differences and similarities of HSPCs from studied tissues
evidence about placental hematopoiesis and suggest FTPT is a valued source of HSPCs for medicine. We
obtained viable HSPCs from cryopreserved placental tissue fragments. Optimization of method of tissue
cryopreservation gives the possibility of banking and testing it for clinical use.
82
CHROMATIN CHANGES UNDER EXPOSURE TO NEUTRON RADIATION
Kuznetsov K.A.1, Kyzym P.S.2, Onishchenko G.M.2, Berezhnoy A.Y.2, Shckorbatov Y. G.1
1
Research Institute of Biology, V.N.Karazin Kharkiv National University, Kharkiv, Ukraine
School of Physics and Technology, V.N.Karazin Kharkiv National University, Kharkiv, Ukraine
Co-Smith@yandex.ru
It is known that neutron radiation can cause different biological effects on any level of organization.
High doses can induce chromosome damage (Olamide AF et al., 2013), affect p53- expression (Qing-Jie Liu
et al., 2014). Slow neutron radiation has lower biological efficiency than gamma-radiation (Lowy RJ et al.,
2001) but higher than X-rays (Barendsen GW, Broerse JJ, 1969).
The aims of our research were to investigate the effect of low intensity neutron radiation on the state of
chromatin in human cells and to assess difference between effects of slow and fast neutrons. The chromatin
in interphase cell nucleus is presented in two forms: euchromatin (decondensed, biologically active) and
heterochromatin (condensed, less biologically active). Therefore, by measuring the portion of
heterochromatin in interphase cell nucleus one can assess the level of functional activity of cell nucleus.
As experimental object used isolated human buccal epithelium cells of 24 years old donor collected
immediately before experiment. Cells were placed in the 3,03 mM phosphate buffer solution with addition of
2,89 mM CaCl2 (pH=7,0).
The sources of neutron radiation were 2 Pu-Be sources IBN-17 (Russia) (100 keV – 10 MeV, 5*107
n/sec., isotropic distribution). Neutrons thermalization (10% of the flux) was obtained by using 3.5 cm
paraffin barrier and section of fast neutrons – by using 2 mm Cd barrier. The emitted by the neutron source
gamma-radiation (59 keV) was blocked by 2 mm lead sheet. To assess the heterochromatin granules quantity
(HGQ) the cells were stained by 2% orcein solution in 45 acetic acid immediately after neutrons exposure
and 1, 2, 5 and 10 min after the cell exposure and examined at magnification 600x. The assessment of HGQ
in control samples were done two times – immediately after obtaining of cells and at the end of experiment,
approximately 2-3 h later (sham control). The quantity of heterochromatin granules was assessed in 30 cells
in 3 independent experiments (3x30)< the mean HGQ for cell sample and standard error of the mean was
calculated, the results were processed by Student's method.
The results of experiment showed the increase of HGQ after cell exposure for both slow and fast
neutron radiation. The highest value of HGQ was registered at 1 minute after cell exposure for both types of
neutron radiation (13% above control for partially thermalized neutrons and 16% for fast neutrons). With
increase of the period after exposure the HGQ decreased. Only at immediate staining of cells after exposure
to the fast neutrons radiation the HGQ was significantly lower than in control. Thus, our results show the
stress answer reaction of cells to neutron radiation exposure and the difference in cell answer to fast and
thermalized neutrons exposure.
83
ShRNA-MEDIATED KNOCKDOWN OF IL-6 EXPRESSION RESCUES TNFα-INHIBITED
OSTEOGENESIS IN MOUSE MESENCHYMAL PRECURSOR CELLS
Khrystyna Malysheva1,2, Karien de Rooij3, Clemens Löwik3, Dominique Baeten4, Rostyslav Stoika1,
Olexandr Korchynskyi1,3,4
1
Institute of Cell Biology, NAS of Ukraine, 14/16, Drahomanov St., Lviv 79005, Ukraine
2
Insitute of Animal Biology, NAAS of Ukraine, 38, V. Stus St., Lviv 79034, Ukraine
3
Leiden University Medical Center, 2, Albinusdreef, 2333 ZA Leiden, The Netherlands
4
Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
E-mail: khrystyna.malysheva@gmail.com.
Rheumatoid arthritis (RA) is a severe autoimmune inflammatory disorder associated with different
morbidities and socioeconomic expenses. The etiology of the disease remains unknown. It has been
demonstrated that interleukin-6 (IL-6), a tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β) target
gene, plays a crucial role in RA pathophysiology. The bone morphogenetic protein (BMP) and Wnt
regulatory pathways are key players in signaling mechanisms that induce and support cartilage and bone
formation and maintenance. Recently, we found that IL-6 inhibits activation of Wnt signaling in primary
human synoviocytes, and TNFα and IL-6 cooperatively inhibit the activation of Wnt response. Here we
performed in vitro evaluation of functional contribution of IL-6 and TNFα interaction to inhibition of bone
formation by using treatment with recombinant cytokines combined with blocking IL-6 expression by small
hairpin RNAs (shRNA) in mouse mesenchymal precursor cells of C2C12 and KS483 lines. These cells were
induced to differentiate into osteoblasts by different BMPs including BMP2 and BMP7 (Korchynskyi et al.,
2003; de Gorter et al., 2011).
We found that treatment of C2C12 cells with TNFα completely inhibited the myoblast differentiation,
as well as strongly inhibited BMP-induced osteogenesis evaluated by alkaline phosphatase (ALP) activity
that was detected spectrophotometrically using -nitrophenylphosphate as a substrate. Transient
overexpression of shRNA targeting IL-6 mRNA similarly to many other small interfering (siRNA) and
shRNA induced an off-target interferon response that inhibits BMP signals. At the same time, application of
shRNA constructs (IL6 shRNA-1) allowed to rescue partially the osteogenic differentiation from negative
effect of TNFα, or in case of using IL6 shRNA-2, even to convert TNFα from inhibitor of osteogenesis into
its potentiator. ALP is a widely used marker of early stages in osteoblast differentiation (Korchynskyi et al.,
2003; Katagiri et al., 1994). However, ALP cannot be applied as a marker of late stages of osteoblast
differentiation for which the bone mineral deposition and nodules formation are specific. Since C2C12 cells
cannot undergo late stages of osteoblast differentiation, we used KS483 cells that can efficiently follow late
stages of osteogenesis (van der Horst et al., 2003). Treatment of KS483 cells with hBMP2/7 strongly
intensified the late osteoblast differentiation, and overexpression of a combination of six versions of shRNA
constructs targeting IL-6 further potentiated osteoblast differentiation observed through nodules formation
and matrix mineralization compared with scrambled shRNA as a control. Histochemical examination of
mineral deposition by KS483 cells was conducted using a conventional staining with Alizarin Red, however,
we were not able to combine hBMP2/hBMP7 treatment with TNFα because of massive cells death induced in
KS483 cells by TNFα (data not shown). Similar effect was observed by other investigators with different
(pre)osteoblastic cell lines (Kitajima et al. 1996).
Thus, IL-6 is an important mediator in inhibition of the osteoblast differentiation by TNFα, and
knockdown of IL-6 partially rescues osteogenesis from negative control of inflammation. The antiosteoblastic effects of IL-6 are most likely mediated by its negative action in Wnt signaling pathway.
84
EXPRESSION OF HUMAN INTERFERON ALPHA 2B GENE WITH PLANT APOPLAST
SIGNAL SEQUENCE IN TRANSGENIC LINES OF NICOTIANA BENTHAMIANA
Mariia Mazur1, Yana Sindarovska1, Zoya Olevinska2, Mykola Spivak2, Mykola Kuchuk1
1
Institute of Cell Biology and Genetic Engineering, National Academy of Science of Ukraine, Academika
Zabolotnoho Street 148, 03143 Kyiv, Ukraine.
2
D. K. Zabolotny Institute of Microbiology and Virology, National Academy of Science of Ukraine,
Academika Zabolotnoho Street 154, 03680 Kyiv, Ukraine.
Email address: mariamazur17@gmail.com
Nicotiana benthamiana is the most widely used plant-based system for transient production of
heterologous proteins and rarely used for obtaining of stable transformed plants with target proteins.
Interferons are used in medicine as non-specific drugs for treatment of a wide range of tumors and viral
diseases. Earlier it was shown that targeting of pharmaceutically valuable proteins to apoplast resulted to
increasing levels of product (Gils et al., 2005). The aim of this study was to examine the character of
expression of human interferon alpha 2b (ifnα2b) gene translationally fused with the apoplast signal sequence
of calreticulin gene from N. plumbagenifolia in transgenic N. benthamiana plants. Series of Agrobacteriummediated transformation of N. benthamiana were carried out. The plasmid vector contained selective bar
gene, and ifnα2b gene was driven by the cauliflower mosaic virus 35S promoter. As a result, DNA of the
thirty five selected transformants was analyzed by multiplex PCR (Gerasymenko et al., 2012). The presence
of ifnα2b gene and the absence of Agrobacterium contamination were confirmed for the twenty nine lines.
The interferon antiviral activity in extracts from the transgenic plants was determined for eight lines by
microtitration method. Interferon-like activity was not detected in extracts from the control untransformed
plants of N. benthamiana. Analysis of transgenic lines showed the presence of a functional target protein in
all eight lines with ifnα2b gene. The antiviral activity of interferon was varied among the lines. Maximum
values of the interferon activity had reached up to 3200 IU/g fresh weight from most transgenic lines, and
corresponding to approximately from 219.2 to 888.9 IU/mg total soluble proteins of these lines. Average, the
functional activity of the lines transgenic plants was at 2800±740.7 IU/g fresh weight and 423.75±267.5
IU/mg total soluble proteins. Levels of interferon accumulation in transgenic plants of N. benthamiana were
comparable to those in N. excelsior where product accumulated after transient ifnα2b gene expression
(Sindarovska et al., 2010). In another study, we were examined and compared the peculiarities of expression
of both human ifnα2b gene with plant apoplastic signal sequence and one with human signal sequence in
transgenic N. tabacum plants. The levels of ifnα2b gene expression in transgenic lines of N. benthamiana
were higher than the levels of ifnα2b gene expression in transgenic N. tabacum pants. The reason for these
differences may be particularly species of plants. Here we showed that transgenic plants of N. benthamiana
produce human interferon alpha 2b with high functional activity, and thus demonstrate the possibility and
prospects of using this species as transgenic producer of heterologous proteins. Also, obtained lines can be
used as experimental model hosts for the studies of the plant-virus interaction.
Gils M. et al. Plant Biotechnol. J., 2005, 3:613-620.
Gerasymenko I. M. et al. Cytol. Genet., 2012, 46(4):3–8.
Sindarovska Y. R et al. Cytol. Genet., 2010, 44(5):60-64.
85
ESTIMATION OF CA2 + AND MG 2+ IONS INFLUENCE ON LACTOBACILLI
ADHESION TO MICE ENTEROCYTES
V.V. Ogurtsova, S.E.Kovalenko, I.F. Kovalenko, O.I.Gordiyenko
Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine,
Pereyaslavskaya str., Kharkov, 61015, Ukraine
Email address: o-vikulya@yandex.ru
Lactobacilli are widespread in nature and as useful symbionts are involved in the digestive,
biosynthetic, detoxifying and other functions of the normal human microbiota (Vancanneyt et al., 2006). It is
known that environmental factors, including its composition, have a significant effect on bacterial adhesion.
It is still unclear specific role of divalent ions Ca2+ and Mg2+ at the processes of bacterial cells adhesion.
Therefore the aim of this study was to determine the index of adhesion of lactobacilli to the mice intestine
enterocytes at the change of calcium and magnesium ions amount at the environment.
Lactobacillus Streptococcus salivarius subsp. Thermophilus were used as an object of study. Intestinal
cells of mice were isolated according to the procedure described by Carter (Carter et al., 1982). Portions of 65
mg Lactobacillus were incubated 35 minutes at 37C in solutions with addition of CaСl2 or MgCl2 (10 ml of
Hank's medium + CaСl2 or MgCl2 in a concentration of 10, 20, 30, 40, 50 and 60 mM). Then 300 µl of
isolated enterocytes were added to each solution and continued incubation for 40 minutes. In control
experiments were used the bacterial suspension in Hanks medium without adding bivalent cations. Adhesion
index (the number of adherent lactobacilli per enterocyte) was determined visually using microscope Axio
Observer Z1 (oil-immersion objective x63).
The experimental data showed that increasing of calcium ion concentration in the medium to 40 mM
significantly raised the number of adherent Lactobacilli cells per enterocyte (relative adhesion index is 1,8204
± 0,33 с ), and further concentration growth, on the contrary, reduces the level of adhesion (relative adhesion
indexes are 1,177 ± 0,25 с and 0,8794 ± 0,11 а for solutions with 50 and 60 mM of CaCl2 respectively).
Alteration of the number of adherent lactic acid bacteria to enterocytes in presence of the extracellular Mg2+
ions had extremal dependence with minimum at Mg2+ concentration 20mM (relative indexes of adhesion are
1,282051 ± 0,18 b, 0,60373 ± 0,11 с, 0,961538 ± 0,16 a, 1,294872 ± 0,31 b for solutions with 10, 20, 30 and 40
mM of MgCl2 respectively). Data significantly different from control: а - p << 0,05; b - p << 0,01; c - p <<
0,001.
J. H. Carter, H. Carter, J. Nussbaum, A. Eichhou Isolation Of Hamster Intestinal Epithelial Cells Using
Hypoosmotic Media And Pvp // Journal Of Cellular Physiology 111:55-67 (1982)
M. Vancanneyt, G. Huys, K. Lefebvre, V. Vankerckhoven, H. Goossens, J. Swings Intraspecific
Genotypic Characterization of Lactobacillus rhamn.osus Strains Intended for Probiotic Use and Isolates of
Human Origin // Applied And Environmental Microbiology.- 2006.- Vol. 72.- № 8.- P. 5376–5383.
86
SEED SURFACE STERILIZATION AS THE FIRST STEP IN ESTABLISHING ASEPTIC
CULTURES OF CRAMBE ASPERA M. BIEB
Pushkarova N.O., Belokurova V.B., Kuchuk M.V.
Institute of Cell Biology and Genetic Engineering NAS of Ukraine,
Zabolotnogo str. 148, Kiev 03680, Ukraine
Email address: pushkarovan@mail.ua
Plant biodiversity conservation is a global issue, concerning governments and communities
depending on the intrinsic value of biodiversity. Biodiversity plays a great role in delivery of a broad range of
benefits which can be considered vital for human life and welfare (such as regulation of soil and water
quality, waste recycling, moderation of toxic wastes and greenhouse gas emissions). At the same time it can
provide financial returns and economical growth. Despite this, the conversion of natural habitats for
agriculture, urbanization and other anthropogenic activities continues in many parts of the world leading to
impending extinction crisis and the increasing loss of species and habitats. Biotechnology methods, in vitro
techniques in particular, have proven to be useful in the process of plant biodiversity conservation
(Belokurova V.B, 2010).
Crambe aspera М. Bieb. is a threatened spaces listed in the Red data book of Ukraine. This species is
being used as a fodder crop, food and oil culture (Ya.P. Didukh 2009). The seeds of Cr. aspera which were
collected by the employees of The National Museum of Natural Sciences of Ukraine at Karadag massif in
Autonomous Republic of Crimea, were used for initializing in vitro culture.
Surface sterilization of the seeds is the first step in establishing aseptic cultures. Seeds were first
washed under running water for five minutes and then the hard external coverings of seeds were removed.
After that the explants were put into 70% ethanol for 60 seconds, and then into diocidum for 3 minutes. After
establishing aseptic conditions the explants were washed with sterile water for 5 minutes three times. The
prepared seeds were placed on hormone-free solid MS medium (Murashige T., Skoog F., 1962) at 24°С and
16-h photoperiod. In 2-4 days after the sterilization procedure aseptic seeds start to germinate. This method of
seed surface sterilization provides 90% of aseptic seeds material, 46% of which has sprouted. The increase of
diocidum sterilization time can provide 100% of aseptic seeds material but it leads to the decrease in the
number of the aseptic material that has germinated. Aseptic plantlets that were formed were cultivated on the
solid MS/2 medium with twice reduced sucrose and basal salts at 24°С and 16-h photoperiod and were
regularly subcultured by transferring apical or lateral buds to the fresh medium.
As a result this article suggests an effective way of Crambe aspera М. Bieb. seed sterilization of for
the further cultivation and study.
1.
Belokurova V.B. “Methods of Biotechnology in System of Efforts Aimed at Plant
Biodiversity Preservation”. Cytology and Genetics, 2010, Vol. 44, No. 3, pp. 174–185.
2.
2009.– 900 p.
Red data book of Ukraine. Vegetable kingdom ed. by Ya.P. Didukh — К.: Globalconsalting,
Murashige T., Skoog F. "A revised medium for rapid growth and bioassays with tobacco tissue
cultures". Physiol. Plant., 1962, v.15, p. 473-497.
87
BSA IMMUNIZATION EFFECTS THE OOCYTES AND FOLLICULAR CELLS,
THYMIC AND LYMPH NODES СELLS IN FEMALE MICE
Alina Lytvynenko, Valentyna Sribna, Natalia Grushka, Taras Blashkiv
Bogomoletz Institute of Physiology, National Academy of Science of Ukraine
Bogomoltsia Street 4, Kyiv, Ukraine, 01024
e-mail: valia-z@ukr.net
Increased cytogenetic instability could lead to infertility and to the development of cancer. Somatic
mutagenesis evaluation remains the most reasonable and promising approach to the formation of high
carcinogenic risk groups.
The effect of BSA immunization on the meiotic maturation of oocytes, viability of the ovarian
follicular cells (OF), thymic and lymph nodes (LN) cells and damage of the genome`s integrity of immune
cells in female mice was inestigated.
Immunization of mice was exercised by increasing doses of antigen - bovine serum albumin (BSA,
150-300 mg / kg of mouse, Sigma, USA) intravenously once a week for 6 weeks. Control mice were injected
with saline. On the seventh day after the last immunization animals were anesthetized and ovaries, inguinal
lymph nodes and thymus were removed.
A technique for in vitro culture of oocytes, double fluorescent vital assay and
DNA-comet assay (alkaline) were used. Comets were separated into 5 classes (0; 1; 2; 3; 4) depending on the
value of DNA in the "head" and "tail" comet.
Under the condition of BSA immunization it was observed: 1) reduction of the number of oocytes
that dissolved germinal vesicle in vitro (metaphase I) (p<0.05, n=8); 2) cell death growth: the number of
living OF cells surrounding oocytes is reduced (p<0.01, n=8); 3) increase in the number of OF cells
surrounding oocytes with morphological features of apoptosis (p<0.01, n=8), while the number of cells with
signs necrosis does not change significantly compared to the reference value; 4) reduction of the number of
living cells, which were isolated from the lymph nodes (p<0.001, n=6); 5) the increase in LN cell number
with signs of apoptosis and necrosis (p<0.01, n=6); 6) decrease in the number of living TDL cells (p<0.001,
n=6); 7) increase in the number of thymic cells with signs of apoptosis and necrosis (p<0.001, n=6); 8)
reduction of the number of thymic cells with 0/1 and 2 classes of nuclei to, respectively, 9,86±3,01% (p<0,05,
n=6) and 13 66±6,49% (p> 0,05, n=6) compared with control values, respectively 30,80±7,65% and
27,4±11,53%; increase the number of thymic cells with 3 and 4 classes of nuclei to, respectively
28,84±4,10% (p>0.05, n=6) and 47,64±4,99% (p<0.05, n=6) compared with control values, respectively
20,39±9,91% and 21,41±7,60%; 9) decrease in the number of the LN cells with 0/1 and 2 classes of nuclei to,
respectively 17,13±5,99% (p<0.05, n=6) and 12.92±3,03% (p<0.05, n=6) compared with control values,
respectively, 39,18±5,87% and 27,25±5,94%; increase in the number of the LN cells with 4 classes of nuclei
to, respectively, 47,03±5,38% (p<0,001, n=6) compared with control values 13,03±5,72%.
Thus, under the condition of BSA immunization oocytes damage, namely the suppression of meiotic
maturation, reduction of the number of living cells of lymph nodes, thymus and follicular cells surrounding
oocytes, nuclear DNA damage of the thymus and lymph nodes cells were observed.
On the bases of evaluation of integrated genome integrity, the ratio of DNA material in the "head" and
"tail" of the comet, as a feature of genome functioning thymic and lymph nodes cells, we suggest that BSA
immunization could modify the activity of gene expression associated with reparation displayed as singlestrand DNA break.
88
ENDOREDUPLICATION IN DROSOPHILA MELANOGASTER AFTER EXPOSURE
TO MICROWAVES AND MAGNETIC FIELD
Liliia Dyka, Lyubov Shakina, Volodymyr Strashnyuk, Yuriy Shckorbatov
Institute of Biology, V. N. Karazin Kharkiv National University,
Svoboda sq., 4, Kharkiv, 61022, Ukraine
Email address: vladimir.strashnyuk@mail.ru
The problem of the influence of electromagnetic fields on biological objects attracts now keen public
interest in connection to increase of “electromagnetic pollution” of environment (Shckorbatov, 2014). The
interaction of electromagnetic fields with living cell and living organism is rather complex and currently is
not fully investigated. It is therefore important to study the chromosomal level of this phenomenon. The
polyteny phenomenon deserves attention as one of the effective mechanisms for enhancing the expression of
eukaryotic genes (Lee et al., 2008).
The purpose of investigation was to study the effect of microwave irradiation and consistent action of
microwaves and static magnetic field on the function of giant chromosomes endoreduplication in Drosophila
melanogaster Meig.
Experiments were carried out on inbred wild-type line Canton-S. The degree of inbreeding was 77
generations. Flies developed at 24 ± 0,5оС. Exposure to microwaves and a magnetic field applied at the egg
stage after a 2-hour oviposition. Giant chromosomes were investigated in squashed acetoorcein preparations
of the salivary glands at the 0 hours prepupae stage. Differences in the level of polyteny was evaluated by the
cytomorphometric method (Strashnyuk et al., 1995).
Exposure to microwave irradiation (frequency – 36.64 GHz, power density – 1W/m2, exposure time –
30 seconds) on the stage of embryogenesis has a stimulating effect on endoreduplication in Drosophila
development. The polyteny level of giant chromosomes increased on average by 7.5%, the power of
influence was: h2 = 35,3%.
A similar effect occurred after the sequential action of microwaves and static magnetic field (intensity
25 mT, exposure time – 5 min): the polyteny level of chromosomes increased on average by 7.4%, the power
of influence was: h2 = 30,6%. Thus, the effect of microwave irradiation practically was not modified by the
action of magnetic field.
Shckorbatov Yu. J. Electr. Electron. Syst. 2014, 3 (2): 123. doi:10.4172/2332-0796.1000123.
Lee H.O., Davidson J.M., Duronio R.J. Genes and Development. 2008, 23 (21): 2461–2477.
Strashnyuk V. Yu., Nepeivoda S.N., Shakhbazov V.G. Rus. J. Genet. 1995, 31 (1):17–21.
89
CHARACTERIZATION OF RAT PLACENTA-DERIVED MULTIPOTENT CELLS AND
TRANSPLANTATION INTO RATS WITH DIMETHYLHYDRAZINE-INDUCED COLON
CARCINOGENESIS.
Hanna Svitina1,2,*, Vitaliy Kyryk3, Іnessa Skrypkina4, Maria Kuchma1,4, Yulia Shablii1, Halyna
Kuznietsova2, Yevheniy Denis2, Olena Dzhus2, Pavel Klymenko3, Volodymyr Zhezhera5, Volodymyr
Rybalchenko2, Liudmyla Garmanchuk2, Liudmyla Ostapchenko2, Galina Lobintseva1, Volodymyr Shablii1
1 Institute of Cell Therapy, Kosmonavta Komarova ave 3, Kyiv 03680, Ukraine
2 Educational and Scientific Centre “Institute of Biology”, Taras Shevchenko National University of Kyiv,
Prospekt Hlushkov 2, Kyiv 03187, Ukraine
3 State Institute of Genetics and Regenerative Medicine of the Academy of Medicine of Ukraine,
Vyshgorodska str. 67, Kyiv 04114, Ukraine
4 Institute of Molecular Biology and Genetics of the National Academy of Science of Ukraine,
Zabolotnogo str. 150, Kyiv 03680, Ukraine
5 National Children’s Specialized Hospital “Okhmatdyt” of the Ministry of Health of Ukraine,
Chornovola str. 28/1, Kyiv 01135, Ukraine
*Email address: anja.onishchenko@gmail.com
Recently, the placenta-derived cells have become a focus of interest as a potential source for cancer
treatment, because it has been concluded that the placenta is not a favorable niche for cancer cells. We
demonstrate the placenta-derived multipotent cells (PDMCs) have feathers of mesenchymal stem cells. Rat
PDMCs expressed CD90, CD29, vimentin, but were negative for CD44, CD45, pan-cytokeratin. Above all,
they had capacity to differentiate into adipogenic and osteogenic lineages at passage 4. In contrast, PDMCs
could potentially belong to trophoblast progenitors due to their expression of mRNA of CDX2 and ID2 and
lack of expression of TPBPA, PRL3B, NANOG. Colorectal cancer remains one of the most common type of
cancer. Therefore, finding of new approaches for colon cancer treatment remains an actual problem, since the
diagnosis is not discovered in the early stages. The effect of placenta-derived cells on the formation and
growth of tumors that are similar to developing human colon cancer in vivo have not been previously studied.
Experimental colon carcinogenesis was induced in male albino Wistar rats by injecting of dimethylhydrazine
at 20 mg/kg of body weight (b wt) once a week for 20 consecutive weeks. The administration of PDMCs was
performed when each rat exhibited at least one adenocarcinoma. In first pilot study the number and size of
colon lesions decreased, but not significantly. However, it was observed the correlation between dose of
injected cells and the number and size of tissue lesions (r=-0.691 for the number of lesions [p=0.013] and r=0.794 [p=0.002] for the size of tissue lesions, n=12). We assume that transplantation of more than 1.2×10 6
rPDMCs/kg b wt could halt subsequent tumor progression. In second study the dose of injected cells was
average 2.2×106 PDMCs/kg b wt (n=30), but no affect on tumor growth were observed. In addition, the
percentage of aberrant crypt foci (first step in malignant changes) among all lesions per rat decreased and
percentage of tumors increased after PDMCs administration, what might suggest about stimulation of
carcinogenesis. Furthermore, the survival rate of PDMC-treated group was lower than in control group. This
study identified that PDMCs did not effect on mid/late colon tumor growth.
90
NEURAL CREST-DERIVED MULTIPOTENT STEM CELLS FROM BULGE REGION OF HAIR
FOLLICLE: PROSPECTS OF USING IN REGENERATIVE MEDICINES
Vasyliev R.G.1,2, Demydchuk A.S.3, Lysenko O.S.3, Klimenko P.P.1, Rodnichenko A.E.1,2, Shamalo S.M.3,
Zubov D.1,2, Labunets I.F.1, Novikova S.M.1, Chaikovsky Y.B.3, Butenko G.M.1
1
State Institute of Genetic and Regenerative Medicine, National Academy of Medical Sciences of Ukraine,
Cell and Tissue Technologies Unit, 67, Vyshgorodska str., 04114, Kiev, Ukraine
2
Biotechnology laboratory ilaya.regeneration, Medical company ilaya®, 9, Ivana Kramskogo str.,
03115, Kiev, Ukraine
3
Bogomolets National Medical University, 34, Peremogy av., 02000 Kiev, Ukraine
rvasyliev.ilaya@gmail.com
The neural crest (NC), a transient structure during embryonic development, is unique to vertebrates.
In adult vertebrate organism, the NC derivatives are presented by neurons and glia of the peripheral nervous
system, melanocytes, odontoblasts, stromal and endothelial cells of cornea, osteoblasts and chondrocytes (in a
head and neck area). It is shown that many adult tissues and organs of mammals contain neural crest-derived
multipotent stem cells (NC-MSCs). One of the sites of NC-MSCs localization is a bulge region of the hair
follicle (BR HF) [Sieber-Blum M., 2004].
Aim. To obtain NC-MSCs culture and assess a potential of their use in regenerative medicine.
Material and methods: NC-MSCs were isolated by explant method from the BR HF of a whisker pad from
adult mice (FVB and C57BL/6 strains). The cells were cultured on fibronectin in DMEM:F12 medium with
10% FBS, 5 ng/ml bFGF, 1% B27 under 5% O2 and 5% СО2. The purity and identity of the obtained cell
cultures were examined using immunocytochemical analysis, flow cytometry and RT-PCR. The ability to
self-renew of NC-MSCs was investigated using serial subcloning of the clonal colonies. NC-MSCs
differentiation potential was determined by in vitro directed. The regenerative potential of NC-MSCs was
evaluated in models of sciatic nerve injury (transection) and the critical defects of calvarial bone. For the
treatment of damaged peripheral nerve 0.5×106 NC-MSCs were transplanted in 20 μl of a fibrin hydrogel
with its polymerization in a defect site. For transplantation in calvarial defects, a tissue-engineered bone
equivalent (TEBE) was pre-fabricated and consisted of 0.2×106 NC-MSCs, fibrin hydrogel and granules of
bone substitute material. Control - cell-free construct implantation and empty defect. The results have been
evaluated with use of functional assays, histological and morphometric analysis.
Results and conclusions: NC-MSCs expressed the markers of a neural crest (Sox10, p75 (LNGFR,
CD271) and CD117), as well as Sox2, nestin and Sca-1 (neural and stem cells markers); CD44, CD73, CD90
(MSCs markers). NC-MSCs were CD45 and cytokeratin negative. NC-MSCs had a significant clonogenic
potential (CFE = 72.58±12.35% at P1), the ability to self-renew in serial cloning and sphere-forming
capacity. The NC-MSCs culture contains a significant number of ALDHbright cells (30.4% avg). NC-MSCs
are capable of directed differentiation into adipocytes, osteoblasts, chondrocytes, melanocytes, neurons and
glial cells at a clonal level. NC-MSCs exhibit a good viability when cultured in collagen and fibrin hydrogels
and on xenogeneic bone and HA/TCP granules. NC-MSCs transplantation into the injured sciatic nerve
significantly stimulates its regeneration, leading to functional improvements and increase in nerve fiber
density in transection area (sham-operated 11024±628, NC-MSCs transplantation 10522±1044, cell-free
fibrin 8409±739/mm2 at day 28). Transplantation of NC-MSCs-based TEBE resulted in complete healing of
critical sized calvarial defect in a short time (1 month) with a formation of normal bone. Control defect and
defect with cell-free bone substitute material were only filled with connective tissue.
91
QUALITY CRITERIA FOR THE HUMAN CELL-BASED MEDICINAL
PRODUCTS: UKRAINIAN EXPERIENCE
Zubov D.A.1,2, Vasyliev R.G.1,2, Rodnichenko A.E.1,2, Zlatska A.V.1,2, Gubar O.S.3
1
State Institute of Genetic and Regenerative Medicine, National Academy of Medical Sciences of
Ukraine, Cell and Tissue Technologies Unit, 67, Vyshgorodska str., 04114, Kiev, Ukraine
2
Biotechnology laboratory ilaya.regeneration, Medical company ilaya®, 9, Ivana Kramskogo str.,
03115, Kiev, Ukraine
3
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150,
Akademika Zabolotnogo str., 03680, Kiev, Ukraine
e-mail: zoubov77@yahoo.com
At the moment, the most common somatic (adult) stem cells used in regenerative medicine,
are the multipotent mesenchymal stem/stromal cells (MSCs). Initially, they were discovered by
Alexander Friedenstein in the bone marrow [Friedenstein A.J. et al., 1968]. Their characteristic
feature is an ability to differentiate into mesenchymal cell types such as adipocytes, osteoblasts and
chondrocytes (orthodox differentiation directions), and they possess noticeable immunomodulatory
properties [Dominici M. et al., 2006]. In 2001 P. Zuk et al. have isolated MSCs from adipose tissue,
where their number was larger than in the bone marrow and tissue collection procedure is less
invasive and safer to the patient [Zuk P.A. et al., 2001]. According to ClinicalTrials.gov, about 4500
clinical trials involving MSCs are registered in the world today. In other words, different degreemanipulated stem cells, by European Medicines Agency (EMEA), are classified as “Human cellbased medicinal products” (CBMPs) being a part of Advanced-Therapy Medicinal Products
(ATMPs) [EMEA/CHMP/410869/2006]. The cells may be used for therapy alone or combined with
structural materials – scaffolds, and thus might be classified as combined ATMPs. Prior to wide
range application of cell therapies and ATMP Marketing Authorization the clinical trials to determine
the biosafety and effectiveness of their use must be conducted. As to any medicinal product intended
for application in humans, a number of stringent quality requirements are imposed to CBMPs.
The following criteria are implemented in the biotechnology laboratory ilaya.regeneration
for cultured mesenchymal cell types both for autologous and allogeneic use (e.g., human bone
marrow-derived MSCs, ADSCs, dermal fibroblasts, etc.):
1. Donor infection screening by peripheral blood.
2. Viability assay of cultured cells by trypan blue stain exclusion.
3. Cultured cells screening for absence of bacteria, fungi, viruses and mycoplasma.
4. Immunophenotyping of cultured cells for minimal set of MSCs positive markers (CD73+ CD105+
CD90+) and MSCs negative markers (CD34- CD45- HLA-DR-) to identify the cultured cell type.
5. The functional CFU-F assay and cell culture kinetics.
6. Functional assay for MSCs multipotency: directed multilineage differentiation into the adipogenic,
osteogenic and chondrogenic directions.
7. Assay for stress-induced cell culture senescence (SA-β-galactosidase assay).
8. Cytogenetic analysis of cultured cells for karyotype stability (GTG-banding).
9. Quality and cell viability assay for scaffolds seeded with cultured cells by combination stating with
FDA/PI, MTT-assay, and histology assay.
The results of assays performed are reflected in the CBMP Passport.
92
INTERACTION OF MYOSIN VI WITH DOCK7 PLAYS IMPORTANT ROLE IN PROTRUSION
FORMATION IN PC12 CELLS
Magdalena Sobczak1, Emilia Wojtera1, Łukasz Majewski1, Paweł Pomorski1, Jolanta Nowak1
Junji Yamauchi2,3 and Maria Jolanta Rędowicz1*
1
Laboratory of Molecular Basis of Cell Motility, Department of Biochemistry, Nencki Institute of
Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland
2
Tokyo Institute of Technology, Midori, Yokohama 226-8501, Japan
3
Department of Physiology, Kitasato University School of Medicine, Sagamihara,
Kanagawa 252-0374, Japan
Email address: j.redowicz@nencki.gov.pl
DOCK7 (dedicator of cytokinesis 7) is a guanidine nucleotide exchange factor (GEF) for Rac1
GTPase that is involved in neuronal polarity and axon generation as well in Schwann cell differentiation and
myelination. Recently, we identified DOCK7 as the binding partner of unconventional myosin VI (MVI) in
neuronal-lineage PC12 cells and postulated that this interaction could be important in vivo [Majewski e al.
(2012) Biochem Cell Biol., 90:565-574]. Herein, we found that MVI-DOCK7 interaction takes also place in
other cell lines and demonstrated that MVI cargo domain binds to DOCK7 C-terminal regions, M2 and
DHR2 (DOCK homology region 2) domains. In MVI knockdown cells, lower Rac1 activity and a decrease of
DOCK7 phosphorylation on Tyr1118 were observed, indicating that MVI could contribute to DOCK7
activity. MVI and DOCK 7 co-localization was maintained during NGF-stimulated PC12 cell differentiation
and observed also in the outgrowths, as revealed by immunofluorescence staining of native proteins or
overexpression of full length GFP-MVI as well as by proximity ligation assay. Also, during differentiation an
increase in DOCK7 and JNK kinase phosphorylation was detected. Interestingly, overexpression of GFPtagged MVI cargo domain (GFP-GT) impaired protrusion formation indicating that the motor and not the
cargo domain is important for this process. Moreover, overexpression of GFP-MVI but not GFP-GT caused
transient increase in Rac activity. These data indicate that MVI-DOCK7 interaction has functional
implications in the protrusion outgrowth and MVI motor activity seems to be important for delivery and
maintaining DOCK along the protrusions allowing to exert its GEF activity in the right destination.
The work has been supported by a grant 2012/05/B/NZ3/01996 from the National Science Centre.
93
OPTIMIZATION OF PAOX1 INDUCTION IN PICHIA PASTORIS USING A
METHANOL/SORBITOL CO-FEEDING STRATEGY
F. Carly1, H. Niu2, F. Delvigne3, P. Fickers1,3
1
Unité de Biotechnologies et Bioprocédés, Université libre de Bruxelles, Av Roosevelt 52, CP152/51, 1050
Bruxelles, Belgium
2
Centre for Process Systems Engineering, Department of Chemical Engineering, Imperial College
London, South Kensington Campus, London SW7 2AZ, England, UK
3
Microbial Processes and Interaction, Univerité de Liège – Gembloux AgroBio Tech, Passage des
Déportés, 2, 5030 Gembloux, Belgium
Email address: pfickers@ulg.ac.be
Using a Pichia pastoris Mut+ strain bearing a pAOX1-LacZ reporter system, we have previously
reported that methanol/sorbitol feeding at ratio 60/40 C-mol/C-mol could increase pAOX1 promoter
induction by 40% compared to pure feeding of methanol. Besides, it also allows to reduce oxygen
consumption by 30 %, alleviating challenging oxygen limitation issues in the P. pastoris fermentation. As a
follow-up, this work centers on an improved understanding of the pAOX1 regulation during
methanol/sorbitol feeding regarding carbon feeding rate and oxygen limitation.
Chemostat cultures with increasing total carbon concentration in the combined feed of
methanol/sorbitol 60/40 C-mol/C-mol were performed at a dilution rate of 0.03 h-1. Methanol and sorbitol
uptake rates, cell growth and pAOX induction level were determined in regard to carbon concentration and
oxygen limitation. Methanol accumulation and oxygen limitation were found to influence directly the level of
β-galactosidase activity and consequently, pAOX1 induction. For dissolved oxygen (DO) value > 0, an
increase of the carbon concentration in the feed yielded to an increase of pAOX1 induction. By contrast,
when DO was around 0, methanol accumulated, causing a 30% decrease of induction. Moreover, in these
conditions of low DO values, cells preferred to utilize sorbitol. This highlighted the ability of cells to adapt to
transient oxygen limitation that often occurs at industrial scale due to medium heterogeneity without loss of
cell viability and reduced negative effect on pAOX1 induction.
Accordingly, methanol/sorbitol feeding not only allows to increase pAOX1 induction level but also
contributes to process robustness. The optimal feeding rate of the methanol/sorbitol tested here is determined
to be 6.6 mmolC-1(DCW.h)-1 at an oxygen transfer rate (OTR) of 8.28 gO2(l.h)-1.
94
REAL TIME MONITORING OF YARROWIA LIPOLYTICA CULTURE GROWTH USING FLOW
CYTOMETRY TECHNIQUE
Hosni Sassi1, Franck Delvigne3, Tambi Karr3, Jean-Marc Nicaud2 and Patrick Fickers3
1
Unité de Biotechnologies et Bioprocédés, Université libre de Bruxelles, Belgium
2
Institut Micalis, INRA UMR1319 – AgroParisTech, Paris-Grignon, France
3
Microbial Processes and Interactions, University of Liège - Gembloux Agro-Bio Tech, Belgium
Email address: hsassi@ulb.ac.be, pfickers@ulg.ac.be
Key words: Yarrowia lipolytica, flow cytometry, bioprocess
The non-conventional yeast Yarrowia lipolytica is an interesting host organism witch found a great
interest with both academic and industrial applications Control of physiological parameters has become a
challenge to further setting an optimized culture process of heterologous protein production in this yeast. In
this work, Y. lipolytica growth in complex media supplemented with oleic acid has been monitored in real
time during culture on bioreactor coupled with flow cytometer. The use of this technology is getting more
and more attention for the control of the microbial culture process since it permits a rapid single cell analysis.
Using this technique, biomass production and substrate consumption (oleic acid) can be measured online
using forward scatter (FSC) and side scatter (SSC) parameters. In addition, the ovoid, pseudo-hyphae and
hyphae cells can be also detected during Y. lipolytica cell growth. In front of these results, this methodology
presented in this work can be used as tool to better control the physiological parameters of Y. lipolytcia
culture growth in presence on hydrophobic substrate such as oleic acid.
95
INTRACELLULAR LIPASES PRODUCTION BY Yarrowia lipolytica USING
RESIDUAL FRYING OIL
Patrícia Martins Botelho Nunes1,3, Priscilla Amaral1, Patrick Fickers2,3
Departamento de Engenharia Bioquímica, Escola de Química,Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil.
2
Microbial Processes and Interactions, University of Liège - Gembloux Agro-Bio Tech, Passage des
Déportés 2, 5030 Gelmbloux
3
Unité de Biotechnologies et Bioprocédés, Université libre de Bruxelles, Av Roosevelt 50, 1050 Bruxelles.
1
Lipases (glycerol ester hydrolases EC3.1.1.3.) are enzymes that catalyze the hydrolysis and synthesis of esters. Thus, it features a
wide use in industry and the interest in the application of these enzymes in industrial bioprocesses is growing because of its versatility
(Ribeiro et al., 2011). New technologies are being developed to circumvent the economic limitation and lipases with more specific
properties are being investigated (Ha et al., 2007). Among the microorganisms capable of producing lipase, Yarrowia lipolytica has
attracted great interest in the biotechnological area by having the ability to excrete various metabolites in large quantities (Barth and
Gaillardin, 1997). Many works show intracellular lipase production with olive oil as inducer. The frying oil used repeatedly in homes
and restaurants is usually discarded improperly in sewer system and it is a residue that accumulates and can clog the sewer tubes.
Researches seeking for wastes for lipases production are already being carried out (Haba et al., 2000; Rywińska et al., 2008). Aiming
the study of intracellular lipase stimulation in the presence of oils and to verify the potential of residual frying oil induction,
experiments in shaker were performed in OO medium (peptone 0.64% and yeast extract 1%) and inducer (olive oil or residual frying
oil 1 % (v/v)). Extracts were obtained through ultrasound, in ice bath, and lipase activities were quantifies using p-nitrophenyl laurate
and olive oil as substrate. The fatty acid composition of frying oil was determined by gas chromatography of methyl esters of fatty
acids (FAME) obtained after acid transesterification. Considering oleic acid is described on literature as the best inducer for lipase
production and it is present both in olive oil and waste frying oil, this fatty acid was used as a model for preliminary intracellular
lipase production kinetics by Yarrowia lipolytica IMUFRJ 50682 grown in microplate and the activity profiles obtained with the
different inducers were compared. Oleic acid concentration was quantified by HPLC. In 15 hours of growth, in presence of oleic acid,
maximum activity for intracellular fractions was obtained. At this point, half the oleic acid has been consumed and the yeast are at the
end of the exponential growth phase. Besides, were observed 2 extracellular activity peaks: one in 15 hours of growth and another one
in 40 hours of growth, when oleic acid was completely devoid from the growth medium. The profile presented in the presence of olive
oil is quite similar to that presented for oleic acid induction. However, in the presence of fatty acid, a high associated lipase activity is
not noted before 15 hours of growth. Therefore, both maximum extracellular and intracellular activity occur at the same time (15
hours). In the presence of residual frying oil (RFO) maximum activity values for intracellular fractions were detected in 15 hours of
growth and the activity profile for these fractions was quite similar to observed in oleic acid. Low extracellular activity was detected
in growths with RFO as inducer. In this report, residual frying oil stimulated lipase intracellular production by Y. lipolytica, but with
lower maximum lipolytic activity values than using olive oil. This can be due to lower oleic acid level present in RFO (26% of oleic
acid and 46% of linoleic acid). Olive oil and residual frying oil were able to stimulate intracellular lipase production by Yarrowia
lipolytica IMUFRJ 50682 in the absence of other carbon sources. RFO has fatty acids and triglycerides in its composition in an
amount sufficient to stimulate the production of intracellular lipases (maximum lipolytic activity 400 U/g); however it has proven less
efficient than olive oil (maximum lipolytic activity 600 U/g). Intracellular lipase production with residual frying oil had interesting
results according to this study. Conditions for lipase production in presence of the two inducers were the same and the inducers were
used by yeast for cell growth and lipase production. However, residual frying oil favoured the production of intracellular fractions,
reaching activity values close to those obtained with olive oil. Although the enzymatic extract from olive oil induction has shown a
slightly better lipolytic activity, the residual frying oil has potential to be used for production of intracellular lipases by Y. lipolytica.
The use of wastes for lipase production may contribute to decrease in the process costs and, moreover, has the benefit of reuse of this
industrial and commercial waste.


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Ribeiro BD, De Castro AM, Coelho MAZ, Freire DMG (2011) Production and Use of Lipases in Bioenergy: A Review from the
Feedstocks to Biodiesel Production Enzyme Researche 2011
Rywińska A, Witkowska D, Juszczyk P, Rymowicz W, Kita A (2008) Waste Frying Oil as Substrate for Lipase Production by
Geotrichum candidum strains. Polish J. of Environ. Stud. 17:925-931.
Ha SH, Lan MN, Lee SH, Hwang SM, Koo YM (2007) Lipase catalyzed biodiesel production from soybean oil in ionic liquids.
Enzyme and Microbial Technology 41:480-483.
Haba E, Brescoa O, Ferrera C, Marque´S A, Busquets B M, Manresa A (2000) Isolation of lipase-secreting bacteria by deploying
used frying oil as selective substrate. Enz and Microbial Technol 26:40–44.
Barth G, Gaillardin C (1997) Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiol Rev 19:219237.
96
Distant Participation
97
BIOLOGICAL PROPERTIES OF IMMOBILIZED CELLS OF BIFIDOBACTERIUM
BIFIDUM AND LACTOBACILLUS BULGARICUS AFTER CRYOPRESERVATION AND
STORAGE
Ananina G.Ye., Scheglov A.V., Vysekantsev I.P.
Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of
Ukraine, Pereyaslavskaya Street 23, 61015, Kharkiv, Ukraine.
Email address: anela.anna@mail.ru
Currently, the problem of dysbiosis and related intestinal dysfunctions are quite relevant due to the
combined causes of various origins, and is a well known to a wide range of the experts in clinical medicine.
According to the research data – up to 90% of the population suffers from one or other forms of dysbiosis
and intestinal dysfunctions. Disorders of intestinal biocenosis are associated with the pathogenesis of
diseases of gastrointestinal tract, cardiovascular system, allergic and autoimmune diseases etc. Due to
multiple functions of intestinal microbiota there is performed the correction of microbiocenosis with
probiotic drugs, the efficiency of those is demonstrated in some randomized controlled trials and Cochrane
meta-analysis. A significant number of probiotic drugs contains bifidobacteria and lactobacilli.
Recently the drugs of probiotics in different protective media and immobilized probiotics have
acquired special significance. Lyophilization and low temperatures are used to store the commercial drugs.
The effect of low temperature storage immobilized in gel carriers probiotics has been insufficiently studied.
As it was aimed the research was targeted to a comparative study of the effect of cryopreservation on
Bifidobacterium bifidum and Lactobacillus bulgaricus probiotics in liquid protective media as well as in
sodium alginate gel. As protective media there were used the Blaurock culture medium, sucrose-milklactose (SML) one (2% sucrose + 5% milk + 1% lactose) and 1% sodium alginate solution. The suspension
of cells in protective media was frozen with the cooling rate of 1, 5, 10, 15 deg/min and with a direct
immersion into liquid nitrogen (-196˚C).
It has been found that the least number of fatal injuries of B.bifidum cells in different media is
provided by slow cooling rate of 1 deg/min and quick immersion into liquid nitrogen. Number of viable
cells in 1% solution of sodium alginate was kept at the initial level after freezing with all the modes.
Analysis of the preservation rate after freezing of L.bulgaricus cells showed that after freezing with
all the modes the 100% viability of bacteria is provided by Blaurock and SML media. In 1% sodium
alginate gel the bacteria viability at initial level was preserved after cooling at 5, 15 deg/min.
After cryopreservation with the effective cooling programs in protective media and in 1% sodium
alginate as well as storage at low temperatures (-12, -80 and -196˚C) the bifidobacteria and lactobacilli
retained their cultural and tinctorial properties, adhesive activity, the one of acid formation.
98
STUDY OF BACULOVIRUS EXPRESSION VECTORS POTENTIAL FOR MULTI-TARGETED
CANCER GENE THERAPY IN MOUSE MODEL SYSTEM
Anopriyenko O.V.1, Vagyna I.N.1, Strokovskaya L.I.1, Chaschina L.I.1, Rudenko A.V.1, Kashuba V.I.1
1
Institute of molecular biology and genetics of NAS of Ukraine, Zabolotnogo Str., 150, Kyiv-680,
Ukraine, 03680.
Email address: o.v.anoprienko@imbg.org.ua
Background. Huge biological diversity and complexity of cancer make therapies that aimed at
targeting single signaling molecule or pathway of limited power. Strategic combination of agents targeted
against the most critical cancer alterations is needed. Baculovirus expression vectors (BEVs) capable of
efficient transduction of wide panel of mammalian cells, unable to replicate in them, have low cytotoxicity
and theoretically unlimited trans-gene capacity – features that provide framework for studying them as
multi-gene targeting vector in tumor therapy strategies (Airenne K.J. et al., 2013). One approach can use as
carrier or vector cells the fibroblasts or analogous cells, transduced in vitro by a multi-gene BEV and then
introduced in a model animal. A multi-gene vector could’ve comprised strategic combination of a cytokine
gene as major tumor-inhibitor and as having additional therapeutic benefits a factor, targeting signaling
pathways within the fibroblasts themselves being the components of tumor microenvironment.
Aim. The aim of this work is construction of multi-targeting BEV with interferon-β gene (Ifn-β) as
encoding antitumor cytokine with pluripotent action and shRNA, that could potentially enhance Ifn-βantitumor action, against bone morphogenetic protein antagonist gremlin 1 (Grem1) – a tumor-promoting
factor secreted by the fibroblast (Sneddon J.B. et al., 2006).
Methods. A series of BEVs was constructed on the basis of AcMNPV virus using Bac-to-Bac
expression system. The viruses preparations titre after amplification and concentration comprised 2–4х108
pfu/ml (Vagyna et al., 2008). Primary culture of fetal murine fibroblasts (C57Fb) was used as vector cells.
B16 mouse melanoma cell line was used for mouse tumor model in vitro and in vivo. BEV containing
reporter eGfp regulated by CMV promoter and mouse Ifn-b regulated by chicken b-actin promoter was
constructed and inhibiting action of Ifn-β in the composition of vector cells C57Fb-Ifn-β (C57Fb,
transduced by Ifn-β-expressing BEV) was assessed in vitro and in vivo. Three shRNA for different parts of
the Grem1 mRNA were synthesized by primer-extension method and cloned under the U6 promoter in the
pSIREN-RetroQ vector (Clontech). BEV constructions containing reporter (eGfp) or selection (puromycin)
genes in combination with anti-Grem and control shRNAs were composed.
Results. Co-culturing of melanoma B16 cells and C57Fb-Ifn-β, resulted in inhibition of tumor cell
proliferation in vitro. Using vector cells C57Fb-Ifn-β in vivo in mouse model lead to inhibition of
melanoma tumor growth and prolongation of lifespan of the experimental animals. These data will serve as
the control for assessment of multi-gene BEV action in the next step. Evaluation of more efficient shRNA
is proceeding. Conclusions. Further efforts should be made for the optimization of multi-gene Ifnβ/shRNA/gene-reporter cassette. Prolongation of the expression of trans-genes in the composition of BEV
can improve its therapeutic potential.
Airenne K.J. et al. Mol. Ther. 2013, Apr;21(4):739-49.
Vagyna I.N. et al. Biopolym. and Cell 2008, 24(6):508-512.
Sneddon J.B. et al. PNAS USA. 2006,103(40):14842-7.
99
ASSESSMENT OF APOPTOSIS OR NECROSIS STAGES OF HUMAN CORD BLOOD
NUCLEATED CELLS AFTER CRYOPRESERVATION BY DIFFERENT METHODS
Babiychuk L.A., Mykhailova O.O., Makashova O.Y., Zubov P.M., Ryazantsev V.V.
Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine.
Email address: mixolya@mail.ru
Recently during treatment of many diseases cryopreserved suspension of cord blood nucleated
cells (CB NCs, CD45+) has been used as a preparation containing hematopoietic progenitor/stem cells
(HPCs, CD34+). Efficiency of their application is mainly determined by the preservation of NCs structural
and functional integrity after thawing. In this context, the research aim was to investigate the apoptosis or
necrosis stages in various populations of human CB NCs after cryopreservation, depending on the method
of whole CB separation, and under the influence of cryoprotectants with different mechanism of action and
low temperatures.
The number of cells and their viability (7AAD) was determined by flow cytometry (FACS Calibur,
USA) using BD monoclonal antibodies (Becton Dickinson, USA). The apoptosis/necrosis stages were
evaluated using simultaneous staining of NCs with 7AAD and Annexin V. NCs were isolated from the
whole CB by three different method: in dextran solution, by two-step centrifugation and in ficoll-density
solution. Our previous studies revealed that for cryopreservation of NCs, which have been isolated by
different methods, the following combinations with cryoprotectants were more effective: cells isolated in
dextran solution were frozen with 5% of DMSO; cells isolated by two-step centrifugation were frozen with
10% of PEG-1500; cells isolated in ficoll-density solution were frozen with both 5% of DMSO or 10% of
PEG-1500.
The efficiency of the NCs isolation from the whole CB by the two-step centrifugation not involving
the use of chemicals and allowed to isolate 90,6±4,3% of NCs and 91,2±5,9% HPCs without loss of their
viability (93.2±0.8% and 94.4±1.2% of 7AAD‾-cells correspondingly) was shown. High efficiency of NCs
isolation was also provided by sedimentation in dextran solution: 82.9±1.4% of NCs and 81.3±4.6% HPCs
with high level of viability (97.1±0.4% and 98.4±0.8% of 7AAD‾-cells correspondingly). Ficoll-density
solution allowed isolate about half of all NCs from the whole CB.
It was established that NCs isolation in dextran solution and subsequent freezing under 5% DMSO
protection, as well as cells isolation by two-step centrifugation and subsequent freezing under 10% of PEG1500 protection, kept viable more than 80% CD45+- and 90% CD34+-cells. There was shown different
sensitivity of NC populations to the damaging effects of cryopreservation factors: lymphocytes and
monocytes were the most cryoresistant (more than 95 and 90% viable cells correspondingly). Thus, the
main decrease in cells amount and their viability was due to granulocytes.
The analysis of apoptosis or necrosis stages of CB NCs showed that isolation in dextran solution and
cryopreservation with 5% DMSO, as well as isolation by two-step centrifugation and cryopreservation with
10% PEG-1500, kept alive the majority of NCs: 79.3±1.2% and 72.9±2.4% of Annexin V‾7AAD‾-cells
respectively. At the same time, cryopreservation of ficoll-isolated cells, regardless of used cryoprotectant,
kept alive (Annexin V‾7AAD‾) about half of all NCs. Analysis of distribution of damaged cells between
apoptosis or necrosis stages showed that regardless of the method of cryopreservation cell damage occurred
primarily as necrosis pathway. However, a major percentage of injuries in any of the presented
cryopreservation technologies accounted for population of granulocytes, but lymphocytes and monocytes
were highly cryoresistant (more than 95 and 90% of living cell correspondingly).Thus obtained results
indicate that cryopreservation of CB NCs according to our method allows to preserve structural integrity
and viability of the cells at a sufficiently high level.
100
ESTIMATION OF ANTIOXIDANT PROPERTIES OF N-ACETYL-L-CYSTEINE DURING
CORD BLOOD NUCLEATED CELLS CRYOPRESERVATION WITH DMSO
Babiychuk L.O., Zubova O.L., Makashova O.E., Zubov P.M.
Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine,
Pereyaslavskaya Street, 23, 61015 Kharkiv, Ukraine.
Email address: pmzubov@mail.ru
Increasing frequency of clinical application of cord blood (CB) medicinal products stipulated
necessity to accumulate large amount of their doses. The solution of this problem is only possible if the
cells are stored in frozen state at the temperature of liquid nitrogen. However, freezing and thawing
processes can lead to the development of metabolic disorders in cells. One of the reasons of such disorders
may be associated with the accumulation of high concentrations of reactive oxygen species (ROS).
Prevention of the production of ROS may be due to adding of antioxidants into cryoprotective medium,
particularly N-acetyl-L-cysteine (AC).
The purpose of this work is the assessment of the influence of different concentrations of N-acetyl-Lcysteine on the amount of cord blood nucleated cells (NC) with excessive level of ROS, as well as their
safety and viability before and after cryopreservation with DMSO.
For the study the human CB after natural labors was used. CB NCs fraction was isolated by
sedimentation using 6% polyglucin with molecular weight 60000. Isolated cells were treated with 25%
DMSO to a final concentration of 7.5% in the sample. Samples were cryopreserved in «Cryoson» freezer
(Germany) with the cooling rate of 1-30C/min up to -800C, followed by immersion in liquid nitrogen (1960C). Antioxidant N-acetyl-L-cysteine («Sigma») was used in concentrations of 5; 10; 15 and 30 mM.
The cell amount was counted in the Goryaev chamber according to standard techniques. NCs viability was
evaluated by flow cytometry (FACS Calibur, Becton Dickinson, USA) using the monoclonal antibody
CD45FITC and fluorescent DNA stain 7-AAD (BD) according to ISHAGE protocol. The amount of cells
with excessive ROS content was determined by accumulation of 2',7'- dichlorofluorescein diacetate (DCF)
inside cells and evaluated with flow cytometry.
Estimation of CB NCs amount with excessive ROS content before and after cryopreservation
showed that the addition of DMSO shifts prooxidant-antioxidant balance in cells and characterizes by the
development of stress reaction such as the formation of excess ROS (10,3 ± 1,8% of cells). In consequence
of freezing and thawing an increase in ROS formation was more pronounced in CB NCs (20,6 ± 2,1) as
compared to those revealed before the cryopreservation. Safety and viability indexes upon the freezing of
cell suspension with 7.5% DMSO were 78,0 ± 2,1% and 78,9 ± 3,2%, respectively.
Adding of AC into the cryoprotective medium contributed to reducing in intracellular ROS content
inside cryopreserved cells as compared with data without adding antioxidant. The least CB NCs amount
with excessive ROS content was revealed upon cell cryopreservation in cryoprotective medium containing
10 or 15 mM AC (not more than 9% of the cells). It should also be noted that under such conditions the CB
NCs safety and viability indexes were higher by 7-10% as compared with samples without adding AC.
Thus, N-acetyl-L-cysteine can promote antioxidant processes in cells upon cryopreservation and
contributes to the prevention of developing of oxidative stress, reduction of ROS in cells and, consequently,
improves their safety and viability.
101
SIZE DEPENDENT INTERACTION OF HYDROPHOBIC NANOPARTICLES WITH
PLASMA MEMBRANE OF IMMUNE CELLS
Rostyslav Bilyy1,2,3, Tetiana Dumych1, Maxim M. Lutsyk3, Luis Munoz1, Martin Herrmann1
1
Institute of Cell Biology NAS of Ukraine, 79005, Lviv, Ukraine;
2
Department of Internal Medicine 3 and Institute for Clinical Immunology, Friedrich-Alexander
University of Erlangen-Nuremberg, 91054 Erlangen, Germany;
3
Danylo Halytsky Lviv National Medical University, Department of Histology, Cytology and
Embryology, 79010, Lviv, Ukraine.
Email address: r.bilyy@gmail.com
Although nanoparticles are widely used in biomedicine, their size-dependent effects on biological
systems and living organisms have not been fully investigated. Hydrophobic nanoparticles have a special
interest due to their ability to interact with hydrophobic components of cells plasma membrane. For some
types of hydrophobic nanoparticles, like nanodiamonds, there are no known natural ways for destruction in
human body, while our organism is in constant contact with such nanoparticles. Neutrophil granulocytes
are the most abundant type of white blood cells in mammals and constitute the first line and most essential
part of the body`s defence system. Employing hydrophobic nanoparticles of various sizes to challenge
living cells in several in vitro and in vivo conditions. We here demonstrate that very small hydrophobic
nanoparticles induce size-dependent damage of plasma membranes, including large vacuolarization,
instability of the lysosomal compartment, and inducing cell death accompanied by swelling and cell
blebbing. Interaction of small hydrophobic nanoparticles with neutrophils results in the formation of
neutrophil extracellular traps (NETs), whereas larger particles have only minor effects. No inflammatory
response was to be observed after natural exposure of hydrophobic nanoparticles to body surfaces. If
natural barriers are trespassed the innate immune system recognized micro- and nanoparticles, resulting in
local inflammation reaction accompanied by both cellular (delayed type hypersensitivity) and humoral
(production of antibodies) immune response. Granulocytes bound all kinds of particles. However, only the
small (10 to 40 nm) hydrophobic particles induced NETosis, a cellular pathway known to cause an
inflammatory reaction intrinsically endowed with its own resolution. NETs are shown to immobilize and
sequester nanoparticles thus isolating them from cell milieu.
102
CHANGES OF PIP2-AQUAPORIN GENES EXPRESSION IN SIUM LATIFOLIUM L. DURING
DAY-NIGHT CYCLE UNDER DIFFERENT WATER SUPPLY
Bliuma D.
M.G. Kholodny Institute of Botany NASU. 2, Tereschenkivska Str., Kyiv-01601, Ukraine.
Еmail: dbluma@ukr.net
Water transport through plant cell membranes is provided generally by aquaporins-membrane
proteins, that form pores in plasma membrane. Most numerous group of aquaporin family are plasma
membrane aquaporins (PIP). This group is devided to РІР1 та РІР2 subgroup. The РІР2 play important role
in water transport across plasma membrane of the cells. The regulation of aquaporin genes expression
level may be one of the mechanisms of regulation of the intracellular water balance during plant adaptation
to varying water supply. The suitable objects for studying the water deficit effect on the expression of
aquaporin genes are aerial-aquatic plants that capable to grow at terrestrial arias, close to ponds. Thus,
terrestrial forms of these plants are affected by some water deficit events in comparison to aerial-aquatic
plants during development. The aim of this paper was to investigate diurnal dynamics of PIP2-aquaporins
genes expression of aerial-aquatic and terrestrial plants of Sium latifolium L. using RT-PCR method. The
dynamics of PIP2 transcripts accumulation in S. latifolium leaves during twenty-four hours was
demonstrated The highest level of PIP2 expression observed during 6am to 12am in both aerial-aquatic and
terrestrial plants. After 12am amount of transcripts significantly decreased. In the period of 15pm to 3am
the PIP2 expression remained at low level. At 6am the significant increase of PIP2 expression was
observed again. We expect that there is a specific mechanism of diurnal regulation of aquaporin genes
expression and the increase of aquaporin genes expression at the first part of day is due to transpirational
demand and increased requirement of plants in water at this time, since the intensity of photosynthesis is
increased in the morning. Another suggestion that the aquaporin genes expression is regulated directly by
irradiation.
103
LOW TEMPERATURE PHASE TRANSITIONS IN FRACTIONS OF PLACENTAL EXTRACTS
EXPOSED TO ULTRASOUND
Bobrova О.M., Zinchenko О.V., Govorova Yu.S., Nardid Е.О.
Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of
Ukraine
23, Pereyaslavskaya Str., 61015 Kharkov, Ukraine.
Email address: helen.bobrova.77@gmail.ru
Human placental extracts have been widely used in various fields. Some reports showed that low
temperature storage of placental tissue allowed having raw materials for the extraction at any convenient
time. Ultrasound application allows to intensifying the extraction process and reducing the cost of extracted
substances, increase productivity. It is known that ultrasound also facilitates the release of substances used
in obtaining food and drug extracts. Accelerating the process of extract obtaining, the ultrasound treatment
of placenta can result in changes in the extracts’ composition and interaction with cells. These changes are
reflected in intermolecular interactions in the system and can be indirectly analyzed by the phase
transitions’ nature. The research aim was to study the ultrasonic effect and low temperature storage of
placenta on phase transitions in fractions of placental extracts, and red blood cell suspensions and
Saccaromyces cerevisiae supplemented with studied fractions.
Fractions of aqueous - saline extracts from human placenta were investigated in this paper. The
extracts were obtained by 12-hour exposure from fresh and cryopreserved placenta and also by applying
ultrasound with a frequency of 22 kHz for 4 minutes from fresh and cryopreserved placenta. The extracts’
factions were obtained by the method of gel chromatography. Low molecular weight (<4 kDa), medium
molecular weight (50-60 kDa) and high molecular weight fractions (> 150 kDa) were studied. The washed
out erythrocytes and S. serevisiae were mixed with the placental extract fractions in 1:1 ratio. Phase
transitions were studied with differential scanning calorimetry (DSC) within the temperature range of 150÷0°C. DSC thermograms were obtained when heating with the rate of 0.5°C/min after cooling to the
temperature of liquid nitrogen with the rate of 200°C/min.
In DSC thermograms of all the fractions a narrow exothermic peak of molecular inversion, an
intense endothermic peak of eutectic compositions’ melting and ice melting peak in the system were
recorded. Statistically significant difference between the intensity of inversion peaks in low molecular
weight fraction in comparison with high molecular weight one for extracts from fresh and cryopreserved
placenta and exposed to ultrasound were registered. We believe that the inversion is more typical for
molecules of peptide nature, which have a lower molecular weight, so the inversion process develops with
the emission of more heat in lower than 4 kDa fractions. Intermolecular interactions’ change after mixing
the fractions with cell suspensions as a result of competition between active centers of organic molecules
for joining water molecules and interactions of molecular fractions with cells leads to an increase in
inversion temperature by 4.2 ÷ 9.4°C and a decrease in the eutectic melting point by 3.8 ÷ 8.1°C. The
intensity of inversion peaks and eutectic melting peaks reduces in 1.5 ÷ 4.6 and 5.6 ÷ 18 times respectively.
It was found that the placenta storage at -196°C for 6 months and ultrasound treatment does not have
any effect on the phase transition temperature both in fractions of the extracts produced from this placenta,
and in the mixtures comprising these fractions and erythrocyte suspensions or S. cerevisiae.
104
EFFECT OF NANOPARTICLES ON PARAMETERS OF GAL-α-1,3-GAL IDENTIFICATION IN
PK-15 CELL LINE
Bohuslavskyi K. I.1, Bozhok G. A.1, Kavok N. S.2, Klochkov V. K.2, Bondarenko T. P.1,
Malyukin Yu. V.2
1
Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine, Pereyaslavska Str. 23,
Kharkiv, Ukraine 61015;
2
Institute for Scintillic Materials of the NAS of Ukraine, Lenina av. 60, Kharkiv, Ukraine, 61001.
Email address: bozhokgaru@gmail.com
Currently, porcine tissues are actively used as bioscaffolds, valve prostheses, tissue bioimplants.
However, one of the critical factors for the sustained function of bioimplants of swine origin is the
expression of xenoantigen Gal-α-1,3-Gal. This is due to the fact that human beings have a high titer of
natural antibodies to these antigens, and recognizing them by the human immune system leads to the
reaction of hyperacute rejection. It is known that nanoparticles can be used to modify cell surface antigens
(Thierry B. et al., 2003; Stephan M. et al., 2010).
The aim of the present study was to establish the impact of nanoparticles based on rare-earth
elements of different size and shape on the identification of Gal-α-1,3-Gal epitope in the line of epithelial
cells of pig kidney PK-15.
PK-15 cell line was maintained in the standard conditions using medium DMEM/F12 with
antibiotics and 10% FCS. For the experiment, cells were detached using a mixture of 0.25% trypsin and
Versen (1:1) solutions. Then cells were washed from the enzyme, resuspended in serum-free culture
medium and incubated with nanoparticles at room temperature for 2 hours with constant shaking. The
following nanoparticles were used: 1 – spherical GdYVO4:Eu3+ (1-2nm); 2 – spindle-shaped
GdVO4:Eu3+ (25×8nm); 3 – stick-shaped LaVO4:Eu3+ (57×8nm); 4 – spherical СеО2 (1-2nm); 5 –
spherical СеО2 (8-10nm). After washing the cells were incubated for 30 min with FITC-conjugated
isolectine BSI-B4 to detect Gal-α-1,3-Gal epitope. Number of labeled cells was analyzed by flow
cytofluorimeter BD FACSCalibur (BD Bioscience, USA) and softwares CellQuestPro and WinMDI 2.8.
It is known that PK-15 cell line has high expression of Gal-α-1,3-Gal epitope. In the control series of
experiments (without nanoparticles) the number of labeled cells was about 85,9±3,1%. Incubation with
nanoparticles led to a decrease in the percentage of labeled cells. Number of positive labeling was:
nanoparticles 1 – 40.3±4.2; nanoparticles 2 – 66.2±3.5; nanoparticles 3 – 63.0±3.2; nanoparticles 4 –
74.1±4.1; nanoparticles 5 – 68.6±2.8%. Strong dependence of labeling efficiency on the size or shape of the
particles hasn't been established.
Thus, treatment by nanoparticles can lead to masking of antigens on the cell surface and changing
parameters of their binding to specific antibodies. This should be considered when developing technologies
for the production of bioimplants of animal origin, which include the use of nanoparticles.
Thierry B. et al. J. Am. Chem. Soc. 2003,125(25):7494-7495.
Stephan M.T. et al. Nat Med. 2010, 16(9):1035-1041.
105
CELL SELECTION WITH HEAVY METAL IONS FOR OBTAINING GENETICALLY
MODIFIED PLANT FORMS
Sergeeva L.E., Bronnikova L.I.
Institute of Plant Physiology and Genetics National Academy of Sciences, Ukraine, Kyiv, 31/17,
Vasylkivska Str., 03022.
Email address: Zlenlo_lora@ukr.net
Cell cultures, especially plant cell cultures, have become a research priory subject. Plant in vivo is an organism
which growth and development is carried out by the limited number of undifferential meristem cells. At the same
time, the cell culture is a dynamic system that is entirely consists of dividing and growing cells. During discrete
passage of a culture a series of stages, each of which has its own characteristics, are undergoing. The first stage is a
lag-phase. In this period the genetic material there was doubling, however the total mass is stable. The second stage is
a stage of logarithmical growth. During this time cells divide. Biomass increases due to the maximum number of
mitoses. The development of this phase is provided by specialized reactions. After divisions completion cells enter
into stage of stationary growth. The total biomass is increasing due to the every unit extensibility. Stage is
characterized by maximum diversity of metabolism. Further, due to the resources exhaustion and accumulation of
toxic products, the cell culture grows old and dies. The transfer to fresh medium contributes to the development
restoration, counting from the first stage.The study of each stage of culture development will give the opportunity to
establish key processes that determine the success of cell expansion. This is especially true if cells challenge the
adverse environments. It is quite natural, that at stressful situation the resistant cells will have the advantage. Genetic
changes can be the reason for higher stability. Desirable mutation may occur in the plant, from which cell culture was
initiated, or it appears during in vitro cultivation. In any case this event is rare. The selection of genetically resistant
variant in the array of various units is very complicated procedure.
We propose to select cells with combined stress resistance via cell selection using lethal doses of heavy metal
ions (HMI). A method of cell selection, as such, involves the detection the single cell with required characteristic. The
lethal doses of stressors ensure the purveyance of only genetically modified objects. Single cell creates the resistant
cell line that is the basis of further manipulations.
It is known, that resistance can be the polygenic feature or arises from site-specific mutation. In first case
osmotolerance appears. In second case a modified form of a protein is constructed. A creative approach was required
to detect these unique variants. The HMI there were used. The selection frequency of both modified forms is not
exceeding 10-6 that is the indicator of a genetic cause of changes.
To obtain the first type variants barium, Ba2+ and cadmium Cd2+ cations were used. Tobacco and soybean cell
lines, obtained on selective media with the addition of lethal doses of these cations, have resistance to different
osmotic stresses. Tobacco regenerants, derived from such strains, grew under salinity or water deficit action. Their
resistance levels were comparable to those of natural halophytes or xerophytes.
Forms with site-specific mutation were selected on cultural media with the addition of lethal doses of tungsten
(WO42-) or vanadium (VO3-) oxyanions. Those compounds are known inhibitors of enzyme nitrate reductase (NR,
E.C. 1.6.6.1) NR is a first enzyme of the nitrogen fixation chain and catalyzes the reaction of nitrate reduction.
Resistant tobacco and soybean cell lines assimilated nitrates and grew under conditions of oxyanions pressure. The
NR activity (NRA) in tobacco plants, regenerated from resistant lines, is recorded during cultivation at normal
conditions and in presence of NR inhibitors. Moreover, NRA at normal conditions was higher than the rates,
registered in control plants. This indicates the emergence of a new type of enzyme. Thus, cell selection with HMI
guarantees the obtaining of genetically modified plant form.
106
EFFECT OF THE YKU70 DELETION ON THE STATE OF YEAST SACCHAROMYCES
CEREVISIAE CELL POPULATIONS
Chirko RA., Pronina O.V., Rushkovsky S.R
Taras Shevchenko Kyiv National University, 64/13, Volodymyrska Street, Kyiv, Ukraine, 01601.
Email address: rostislavchirko@gmail.com
YKU70p plays an important role in maintaining telomeric homeostasis throughout the cell cycle.
Due to the rapid reduction in telomere length yeast strains with yku70 deletion display G2/M cell cycle
arrest and apoptosis activated at 37 0C. The purpose of the present study was to investigate effects of the
yku70 deletion on the state of rho+ and rho0 (lack of mitochondrial DNA) cell populations of yeast
Saccharomyces cerevisiae.
The haploid strain DLY1412 Δyku70 (W303 line) and its rho0 clone were used in the study. Yeast
cell were cultivated in liquid nutrient medium YPD at 28 0C (optimum temperature) and 37 0C (nonpermissive temperature) for 1, 3 and 7 days. Average size of individual cells and their morphology were
evaluated. The sate of nuclear and mitochondrial genomes was assessed by DAPI in vivo staining.
Cultivation of the Δyku70 strain at 37 0C led to significant heterogeneity of the cell population size
(from 4.9 to 14.8 nm), as well as to increasing the number of dead cells compared to the population of cells
that were cultured at 28 0C. After the first day of the experiment, bimodal distribution of the mother cells
lengths was observed. After the third day of cultivation, the subpopulation of the giant cells (> 9.3 nm) was
not detected. Because of G2/M check-point activated at 37 0C the fraction of single cells was significantly
reduced (17.7 ± 3.5% compared with 69 ± 10.2% at 28 0C). During the cultivation of the rho+ strain at 37
0
C, viable cells are retained in population, but their clonogenic ability was significantly inhibited in 3 dayold cultures and lost after 7 day cultivation. In spite of apoptotic abnormalities lack of mitochondrial DNA
did not lead to significant recovery of clonogenic ability of yeast cells, but we observed that some rho 0 cells
were able to bypass G2/M check-point at 37 0C and form colonies.
107
THE EFFECT OF AGMATINE ON ENZYMATIC ANTIOXIDANT DEFENSE SYSTEM AND
FUNCTIONAL STATE OF RATS LEUKOCYTES UNDER ЕXPERIMENTAL DIABETES
MELLITUS
Datsyuk U., Burda V., Sabadashka M., Datsyuk L., Dzydzan O.
Ivan Franko National University of Lviv,
Hrushevsky Street, 4, 79005, Lviv, Ukraine.
Email address: datsyukulyana@gmail.com
Diabetes mellitus (DM) is an endocrine disease, in the pathogenesis of which hyperglycemia is the
leading element. The immune mechanisms of autoaggression activation is observed under type I DM that
ultimately leads to pancreatic β- cells death. The mobilization of physiological mechanisms of intra- and
extracellular glucose concentration decline (autoreduction, non-enzymatic glycosylation of proteins,
activation of the polyol pathway) results in an increase in advanced glycation end-products (AGEs).
AGEs play a causative role in the immunity disorder and induction of oxidative stress can result in
the activation of a number of signaling molecules expression or excretion.
The administration of agmatine promotes the insulin release by β-cells and increases the cellular
uptake of glucose and shows antiapoptotic and cytoprotective properties. Therefore, the study of its effect
under hyperglycemia is a perspective for ensure a preservation of antioxidant capacity and restore
functional activity of immune blood cells.
The aim of the work was to investigate the effect of agmatine on the activity of antioxidant system
enzymes (superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX) and glutathione reductase
(GSR)), to intensity the level of lipids peroxidation (TBA-RS) in rats peripheral blood leukocytes and to
assess neutrophilic granulocytes (NG) functional characteristics (phagocytosis and myeloperoxidase
(MPO) activity, NBT-test, the content of cationic proteins) under the experimental DM (EDM).
EDM was induced by streptozotocin injection at a dose of 60 mg / kg body weight. After the
development of hyperglycemia rats were administered agmatine (20 mg / kg of body weight) by
intramuscular injection for 14 days.
The administration of agmatine not cause the deviation of indicators in healthy animals compared to
control. The activity of SOD, GSR, catalase decreased and the content of TBA-RS increased compared to
control. Whereas agmatine treatment of animals caused an increase of SOD activity more than twofold,
GPX in 1,4 times, GSR in 1,7 times, normalization of catalase activity and decrease in TBA-RS content
compared to indicators of animals with EDM.
The deterioration of phagocytic activity of NG under EDM was found due to both slowing new cells
entry into phagocytosis and decreasing of a number of ingested yeast in comparison with control. In rats
with EDM was shown the bactericidal activity of NG decrease on the stage of superoxide anion generation
(NBT-test) and production of hypochlorite ions (MPO activity) and by the content of cationic proteins
decreasing. In the case of animals with hyperglycemia treatment, agmatine helps to continue the duration of
the phagocytosis process for another 90 minutes, increasing the number of ingested objects in phagocytosis,
superoxide anion generation acceleration, accompanied by increase of MPO activity and cationic protein
content in NG compared to EDM indicators, however not reached control values.
It was shown that agmatine treatment under hyperglycemia removed effective functioning of
antioxidant defense system and enhanced functional state of NG.
108
POLYMORPHISM OF MICROSATELLITE MARKERS IN CAMELINA SATIVA (L.) CRANTZ
Demkovich A. Ye., Bayer G. Ya., Pirko Ya.V., Yemets A.I., Blume Ya.B.
Institute of Food Biotechnology and Genomics, Nat. Acad. of Sci. of Ukraine,
Оsipovskogo str., 2A, Kyiv, 04123, Ukraine.
Email address: galabayer@ukr.net
Camelina sativa (false flax or gold of pleasure) is a relict oilseed crop of the Crucifer family
(Brassicaceae) with centers of origin in southeastern Europe and southwestern Asia. C. sativa was
cultivated in Europe as an important oilseed crop for many centuries before being displaced by higheryielding crops such as canola (Brassica napus L.) and wheat. C. sativa has several agronomic advantages
for production, including early maturity, low requirement for water and nutrients, adaptability to adverse
environmental conditions and resistance to common cruciferous pests and pathogens. C. sativa oil consists
of 90% unsaturated fatty acids that suitable for making jet fuel, biodiesel and high-value industrial
lubricants.
The importance of false flax as oilseeds potential donor of genes confirmed the recent sequencing of
the genome of this species that clearly confirmed its polyploid (hexaploid) nature. However, the problem of
analysis of genetic diversity and identification of false flax cultivars require simple, inexpensive and easily
controlled molecular genetic approaches, particularly based on the use of SSR markers. The aim of this
study was to evaluate the genetic diversity of C. sativa in Ukraine as one of the points of speciation
Camelina, based on previously developed microsatellite markers.
The 7 samples of a cultivar (Peremoga) and 7 for each of two samples in C. sativa (FEORZHYAF-4
FEORZHYAF-5), Ukrainian and one probably genetically distinct from domestic sample false flax seed
breeding Chinese (№509, BGI Tech, China) with 8 pairs of SSR primers (loci H2C2, P3H4, P4E6, P4C2,
LiB19, P3C3, P4B3, P6E4 (Manca et al. 2013) have been analyzed. Resulting amplicons were separated in
non-denaturing polyacrylamide gel and stained with silver nitrate. To assess the size of the amplicon on the
picture was used GelAnalyzer software. Statistical analysis of the results was performed in GenAlEx.
As results were obtained amplicons ranging in size from 171 bp to 303 bp. For any of the samples
fully monomorphic loci were not observed. This consistent with the results of similar studies for other
varieties of C. sativa. Two of eight analyzed loci (Р3С3, Р6Е4) were monomorphic in all analyzed samples.
Two alleles were revealed for loci H2C2, P4C2, LiB19, P4B3 three alleles - for loci P3H4 and P4E6.
Amplicon sizes generally were similar to previously identified during analysis of C. sativa, however P4B3
locus were polymorphic with alleles: 198 and 200 bp. The 205 bp amplicon were detected for P3H4 locus,
the allele 171 bp - for P3C3 locus. Established that analyzed microsatellite loci in C. sativa varieties of
Ukrainian selection possess a high level of genetic polymorphisms and may be used as molecular genetic
markers for evaluation of genetic diversity, comparative genetics and breeding of C. sativa.
This research supported by the project "Molecular genetic analysis of collection forms and varieties
of false flax in order to create highly productive genotypes for biodiesel" of comprehensive program of
scientific research of Ukraine "Biological resources and the newest technology for bioenergy conversion"
2013-2017.
109
TRANSGENIC ARTEMISIA DRACUNCULUS L.
“HAIRY” ROOT CULTURE CONSTRUCTION
Drobot K.O., Shakhovsky А.М., Matvieieva N.А.
Institute of Cell Biology and Genetic Engineering NAS of Ukraine
148, Academika Zabolotnoho St., 03143, Kyiv, Ukraine.
Email address: katyadrobot@gmail.com
Artemisia genus belongs to Asteraceae family and includes more than 300 plant species. Tarragon –
Artemisia dracunculus L. – is a well-known perennial plant, which grows in Ukrainian steppe and forest
steppe ecoregions. It is popular in cuisine as a spice and also is used for vinegar preparation. Antimicrobial,
antifungal and antioxidant activities of in vitro cultivated tarragon were studied. The investigations were
devoted to study of medicine compounds accumulation, especially to artemisinin synthesis which is used to
treat malaria (Obolskiy et al., 2011). Inspite of this there are only a few publications concerning in vitro
propagation of A. dracunculus. Genetic transformation of tarragon has not been carried out although A.
dracunculus “hairy” root culture produced artemisinin can be found as a source of this antimalarian
compound.
“Hairy” root culture could be obtained via Agrobacterium rhizogenes-mediated transformation. This
culture is characterized by unlimited and fast growth without exogenous growth regulators using; it is
unpretending to the growth conditions and doesn’t require lighting, so the roots can be cultivated in
fermenters. “Hairy” root culture is characterized by high genetic stability (Roychowdhury et al, 2013).
Concerning this the use of “hairy” root culture for biological active substances (BAS) production and study
of tarragon`s transformation possibility attracts particular attention.
Materials and methods. Leaves, cotyledons, stems and roots of 14-day tarragon seedlings were used as
the explants for genetic transformation. The transformation was performed using A. rhizogenes A4 wild
strain in accordance with the (Matvieieva N et al., 2011). After co-cultivating with bacteria, the explants were
grown on half-strength MS agar-solidified medium during two days. Then they were transferred to ½ MS
medium supplemented with 600 mg/l of cefotaxime. Roots were subcultured once in a week. The presence of rolB
genes was determined using PCR analysis. Extraction of DNA was carried out with CTAB-method.
Results. Roots formed on the 7-th day after genetic transformation only on the leaf explants. The root
formation frequency reached up to 20%. “Hairy” root lines (independent transformational events) differ in
length, growth rate and morphological peculiarities. PCR analysis proved the presence of rolB genes in
tarragon “hairy” roots obtained via A. rhizogenes-mediated transformation.
Conclusions. Thus, we firstly obtained the transgenic A. dracunculus “hairy” root culture using A.
rhizogenes A4-mediated transformation. It was shown that leaves of in vitro cultivated plants are the
optimal type of explant. The transgenic root formation frequency was up to 20%. Transgenic root lines
differed in phenotype and growth rate.
Obolskiy D. et al., J Agric Food Chem. 2011, 59(21):1367-1384.
Roychowdhury et al. Biotech Med Plants. 2013, 1:29-68.
Matvieieva N et al. Cytol and Genetics. 2011, 45(5): 277-281.
110
ANTIFUNGAL ACTIVITY OF 3-HETARYL-SUBSTITUTED COUMARINS
Kuziv Ia.B., Negrutska V.V., Dubey I.Ya.
Institute of Molecular Biology and Genetics, NAS of Ukraine, 150 Zabolotnogo str.,
03680 Kyiv, Ukraine.
Email address: dubey@imbg.org.ua
Due to the serious problem of bacterial and fungal resistance to classical antibiotics, the search for
novel efficient antimicrobial drugs has become an urgent task. Natural and synthetic coumarins and their
analogues have been found to exhibit a wide range of biological and pharmacological activities, including
antioxidant, anticoagulant, anti-inflammatory, antitumor and antibacterial properties. In addition to the
established low toxicity, coumarins are relatively cheap drugs. In the present work, the antifungal activity
of a series of new coumarin derivatives against Candida albicans was investigated.
A library of over 30 coumarins containing a substituted hetaryl (furyl or thiazolyl) fragment at C-3
position was synthesized and characterized. Aromatic or aliphatic substituents were introduced into the
hetaryl moiety, whereas the coumarin core contained 7-hydroxy, methoxy or acetoxy group. The structural
modifications were evaluated for in vitro antifungal activity to obtain data on structure-activity relationship.
The antifungal activity test was based on a standard agar diffusion method and carried out with C.
albicans strain АТСС10231. Water-insoluble coumarins were dissolved in dimethylsulfoxide and diluted
with distilled water (1:9) to obtain 1 mM stock solutions. Serial dilutions of tested compounds at
concentration range 25-400 µM were prepared in 10% aq. DMSO and applied into the wells. The control
contained 10% DMSO without the addition of test substances. The plates were then incubated at 35 oC for
48 h in the dark, and growth inhibition zones were visually inspected. All assays were performed in
duplicate.
Fungal susceptibility to test compounds was evaluated by determining the minimal inhibitory
concentration (MIC), i.e. the lowest drug concentration which resulted in total inhibition of the fungal
growth. A number of substances exhibited significant antifungal activity. Eleven coumarins fully
suppressed C. albicans with MIC value of 100 µM, and two compounds partially inhibited its growth at
100 µM concentration. Partial inhibition was observed for 6 compounds at 50 µM. Control solution did not
affect the growth of microorganisms.
In general, thiazolyl derivatives appeared to be more efficient than furyl-substituted coumarins. Only
carboxyfuryl-coumarin was active against C. albicans with a MIC of 100 µM. None of the most active
coumarins contained a free OH function at C-7 position. Perhaps non-ionisable hydrophobic group at this
position is preferable for biological activity. Aliphatic substituents at hetaryl fragment (Me, CH 2COOMe,
(CH2)3COOEt) were more efficient than aromatic ones: aryl-substituted compounds demonstrated
MIC>200 µM, except for methylenedioxaphenyl derivative (MIC 100 µM).
Our previous study of inhibitory activity of the same coumarins against bacterial pathogens, both
gram-positive (Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa), did not reveal
substances with significant activity. Such specificity is an obvious advantage for potential therapeutic use
of these compounds as antifungal agents.
111
DIFFERENTIAL RESPONSES OF ACTIVE TRANSPORT SYSTEMS IN THE MUSSELS ADAPTED
TO THERMAL AND CHEMICAL LOADING IN THEIR NATIVE SURROUNDING
Falfushynska H.I.1, Gnatyshyna L.L.1, Yurchak I.V.1,
Sokolova I.M.2, Stoliar O.B.1
1
Ternopil National Pedagogical University, 46027, Kryvonosa Str 2, Ternopil, Ukraine;
2
University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC, 28223, U.S.A.
Email address: Oksana.Stolyar@gmail.com
Global climate changing combined with the traditional pollution directly translates into the changes
of the rates of all biochemical and physiological reactions and can affect the organisms’ tolerance to other
stressors and diseases. The toxicity of novel pollutants, for instance nanocomposites may be enhanced by
elevated temperatures (Falfushynska et al., 2015). The aim of this study was to evaluate the active transport
systems and apoptotic consequences in the mussels from different biotopes by their subjecting to additional
thermal and/or chemical stress. Specimens of mussels from cooling ponds of two power plants (B- and Dgroups) were exposed for 14 days to elevated temperatures (T, 25oC), novel surface water contaminant
nano-scale Zinc Oxide (n-ZnO, 3.1 μM), Zn2+ (3.1 μM), ) or n-ZnO at 25oC (n-ZnO+T). Control groups
were held at 18oC for 14 days. Activity of multixenobiotic (MXR proteins) families of ABC transporters
was determined in isolated gill and digestive tissues of mussels measuring the rate of efflux of a fluorescent
substrate, rhodamine B in the presence or absence of MXR inhibitors verapamil and cyclosporine A. To
evaluate the vital consequences of exposures, the level of DNA fragmentation in digestive gland, nuclear
lesions in hemocytes and activity of apoptotic enzymes, caspase-3 and cathepsin D (lysosomal and free)
were also determined.
The MXR activities were higher in the mussels from the cooling ponds than in the specimens from
the pristine site. The exposures caused different responses in the mussels from two cooling ponds. In the Bgroup, particularly in the gills, exposures activated MXR proteins, whereas in the digestive gland of the Dgroup of mussels it was remarkable decreased (especially, in two-three times under the combine effect of
n-ZnO+T). Hence, the preference for MXR detoxification functions in B-group and its down-regulation in
the mussels originated from D-site was established. Additionally, exposures to the Zn, n-ZnO+T and T led
to a significant increase in DNA fragmentation and/or nuclear lesions in the D-group, but the changes in the
B-groups were less prominent or even opposite. Cathepsin D-related apoptotic activity was induced by
almost all exposures in the B-groups, while it was not sensitive or decreased (by thermal effect) in the Dgroups. The caspase-3 mediated cascade was induced prominently, particularly by heating in the B-groups
but decreased in the D-groups. Our study showed that long-term acclimation and/or adaptation of Unionid
mussels to elevated temperatures in the cooling pond from B-cite results in increased thermotolerance and
alleviate stress response to moderate temperature rise in the optimistic scenario whereas the mussels from
D-site demonstrate the narrowing of tolerance window in the worst scenario. The utilized model could be
explored for the estimation of the organism susceptibility to novel environmental challenges.
This work was supported by U.S. Civilian Research and Development Foundation (CRDF)
Cooperative Grants Program award (UKB1-7109-TE-13) and R&D project of MES of Ukraine #125B.
Falfushynska et al., International Journal of Medicine and Medical Research. 2015, 1:5-11.
112
EFFECT OF ANTIBIOTICS TIMENTIN AND CEFTRIAXONE ON ELIMINATION OF
AGROBACTERIUM TUMEFACIENS AND REGENERATION OF TRITICUM AESTIVUM
IN VITRO
Gorbatyuk I.R., Gnatyuk I.S., Bannikova M.O., Morgun B.V.
Institute of Cell Biology and Genetic Engineering, NAS of Ukraine
Ukraine, 03143, Kyiv, Akademika Zabolotnoho str., 148.
Email address: gorbatyuk.irina.90@gmail.com
The efficiency Agrobacterium-mediated transformation of plants in vitro depends mainly on
genotype of plant, type of explant, genetic vector, bacterial strain and composition of culture medium. The
success of transformation process is impossible without effective elimination of bacterial cells after
transformation. Antibiotics present in the culture medium usually have a negative effect on the regeneration
in vіtro (Si-Nae Hanet al., 2007). Cefotaxime has a broad spectrum of activity on Gram-positive and Gramnegative bacteria. It is normally used to inhibit growth of Agrobacteria tumefaciens (Rhizobium
radiobacter). But Cefotaxime at high concentrations reduces the effectiveness of shoot formation and
negatively affects regenerating explants. Thus, there is a need for antibiotic independent of pH and
regeneration medium components, stable, and without side or toxic effects on plant organisms. The aim of
work was to determine the concentration of antibiotics Ceftriaxone and Timentin, required for total
elimination of A. tumefaciens, and to study their impact on the wheat cv. Zymoyarka and Podolyanka
shoots regeneration in vitro.
The disc diffusion method (Priya A. Et al., 2012) was used to determine the antibiotics inhibitory
effect on A. tumefaciens strains ABI and GV3101. The 18-days old calli obtained from T. aestivum cv.
Zymoyarka and Podolianka apical meristem were used to show the effect of antibiotics selected
concentrations on morphogenesis and regeneration. Callus was cultivated on culture medium supplemented
with antibiotics at 24° C and 16 h photoperiod for 30 days.
The inhibitory effect of antibiotics Ceftriaxone and Timentin on the A. tumefaciens strains GV3101
and ABI cells was compared with inhibitory effect of Cefotaxime, which is usually used in the
Agrobacterium-mediated transformation.
It was established that the inhibition zone 12.5 mm in diameter for strain ABI was observed when
concentration of 350 mg/l Timentin and Ceftriaxone and of 500 mg/l Cefotaxime (which is common) were
used. The inhibition zone for strain GV3101 was 10.5 mm at 400 mg/l Timentin and Ceftriaxone and at 500
mg/l Cefotaxime. The highest shoot formation frequency for cv. Zymoyarka was observed on regenerative
medium supplemented with 350 mg/l Timentin (in comparison with other concentrations and control),
while cv. Podolyanka – 25 mg/l. The positive effect on the processes of morphogenesis and regeneration in
cv. Zymoyarka was observed when adding in regenerative medium 300 mg/l Ceftriaxone, but those levels
were somewhat lower compared to the control.
Since Timentin caused minimal phytotoxicity it can be successfully used to eliminate bacterial
contamination and to increase the shoot formation frequency in the Agrobacterium-mediated
transformation.
Si-Nae Hanet et al., Journal of Crop Science and Biotechnology. 2007, 10: 92–98.
Priya A. et al., Genet.Plant Breed. 2012, 48: 120–130.
113
mTOR-ASSOCIATED CYTOSKELETAL REMODELING AND 3D MOTILITY OF HeLa CELLS
UNDER PARACRINE INFLUENCE OF NIH 3T3 FIBROBLASTS IN VITRO
N. Ya. Gotsulyak, A. I. Khoruzhenko
Institute of Molecular Biology and Genetics, NASU, Kyiv, Ukraine.
Email address: nazariy.gotsulyak@gmail.com
Relevance. mTOR signaling network is one of the most important regulatory mechanisms of the
basic cell vital processes under normal and pathological conditions [Wataya-Kaneda M., 2015]. Special
interest in the recent studies is attracted to mTOR as to an important regulator of cellular motility and
metastasis [Zhou H., 2011], but the molecular mechanisms of this mTOR function is still not completely
clarified. Detailed knowledge about mTOR regulation of cell motility is necessary for its effective using as
one of the perspective targets in novel anticancer therapeutic strategies.
Aim. The aim of this study is to establish the role of mTOR signaling in 3D motility regulation by
fibroblasts paracrine action and associated with it cytokeratin cytoskeletal transformations.
Methods. Cell culture, soft agar colony formation, 2D/3D cell culture transformation, confocal
microscopy, immunoblotting, immunocytochemistry and Transwell test assay were used in this study.
Results. It was detected significant phosphorylation rate increasing by marker regulatory sites of the
key mTOR signaling network elements: mTOR kinase by Ser-2448, two isoforms of one of the main
mTOR substrates – p85-S6K1 kinase by Thr-389, p70-S6K1 kinase by Thr-389 and p70-S6K1 kinase by
Ser-371 in the lysates of HeLa cells that were cultivated in 20% NIH 3T3 conditioned medium during
24 hours in comparison with lysates of intact HeLa cells. Also, phosphorylation status of FAK kinase by
Tyr-925 as one of the molecular markers of cell migration activity and possible indirect mTOR signaling
network effector was increased. It was shown that these activations correlated with the terms of conditioned
medium action and its depletion – effect disappears after action during 48-72 hours. To compare effects of
mTOR pathway activation on cell motility and cytoskeletal structure and mTOR pathway inhibition was
applied Transwell assay using HeLa cells treating with 20% NIH 3T3 conditioned medium, 10 nM
rapamycin (specific mTOR kinase inhibitor) and combination of these factors. Preliminary result of tests
demonstrates that activation as well as inhibition of mTOR signaling network by investigated factors is
associated with significant motility decreasing under 3D conditions. Also, it was observed that these effects
is able to interfere and motility of cells treated by combination of factors was more reduced than motility of
cells treated by rapamycin and conditioned medium separately. Immunocytochemical analysis of HeLa
cells cultivated during 24 hours in 3D culture (spheroids) under influence of NIH 3T3 fibroblasts direct
action, treated with 10 nM rapamycin or combination of these factors and after that transferred on 2D
surface shown visible remodeling of cytokeratin cytoskeleton of part of cells. The cytoskeletal structure
suffered faint reduction of peripheral elements and decrease of filament branching under fibroblasts
influence as well as under the influence of rapamycin. This effect is maximally expressed in cells treated
with combination of factors namely it was observed some part of cells whose cytokeratin cytoskeleton is
presented only by granular perinuclear structure with minimized branching rate. In addition,
immunocytochemical analysis provides substantial grounds to believe that mTOR and cytokeratins have
significant rate of co-localization, and especially at the perinuclear zone.
Conclusion. Taking account all our data and literature review we suggests that mTOR signaling
network may be an important mediator of NIH 3T3 fibroblasts paracrine influence on HeLa cells motility
and liveness in particular through the regulation of cytokeratin cytoskeleton conformation.
114
INDIVIDUAL APPROACH FOR EVALUATION OF BIOCOMPATIBILITY OF BONE PLASTIC
MATERIALS USED IN PERIODONTAL SURGERY: EX VIVO TESTING OF PRIMARY
CULTURE OF CELLS OF PATIENTS’ GUMS BIOPTATE
1
Hodovana1 O.І., Klyuchivska2 O.Yu., Godovanyi1 O.V., Stoika2 R.S.
Danylo Halytskyi Lviv National Medical University, Pekarska Str. 69, 79010, Lviv, Ukraine;
2
Institute of Cell Biology, NAS of Ukraine. Drahomanov Str. 14/16,
79005, Lviv, Ukraine.
Email address: zorepad1775@gmail.com
In this paper we investigate the individual biocompatibility aspects of cultivated ex vivo fibroblast-like
cells of patients with intact periodontium, chronic catarrhal gingivitis (ChCG) and chronic generalized
periodontitis (GP) (of various degrees of severity) to osteoplastic materials, used in periodontal surgery.
Despite existing osteoplastic materials, the criteria of individual material choice for individual patient have
not been sufficiently substantiated. Ex vivo investigation of osteoplastic materials using primary cells,
obtained from the biopsy materials, might be an effective approach to solve this problem. Furthermore, in
GP cases with a persistent infection, it is expedient to test cells isolated from the locus of chronic infection
enabling evaluation of regenerative potential of tested ex vivo samples with conditions similar to the
clinical situation. The aim of this study was the elaboration of a new approach for ex vivo individual
biocompatibility determination of recipient bed to the osteoplastic materials used in the periodontal surgical
practice.
Design of research. To obtain biopsy material, after prior infiltration anesthesia using a scalpel we
separated interdental papilla fragment size of 2-4 mm3 and transferred it into a sterile tube with addition of
1 ml of DMEM medium containing 20 mg/ml of ciprofloxacin for 2-4 h. А piece of tissue was divided into
fragments and placed in Petri dishes containing 1 ml of culture medium, supplemented with 50 µg/ml of
gentamycin, for growing the biomass at 37° С in CO2-incubator IG150. Cells were grown to ~7,5*104 cells
per 1 sample of tested material and they were cultured in multidish, containing tested osteoplastic materials
had been prior placed and incubation was 3 weeks. The individual biocompatibility of the following
osteoplastic materials was estimated: 1) “Collapan-L®” – synthetic hydroxyapatite and scleral collagen
supplemented with addition of lincomycin hydrochloride; 2) “Osteoplast®” – material on the based on
demineralized and non-demineralized animal collagen, filled with sulphate glycosaminoglycans; 3)
“R.T.R.” – synthetic osteoconductive material of high degree of purification with β-tricalcium phosphate
crystalline structure; 4) “Easy-Graftтм” – synthetic osteoconductive material based on pure phase of βtricalcium phosphate with polylactide and polyglucoside; 5) “Calc-i-ossтм” – synthetic osteoconductive
material based on pure phase of β-tricalcium phosphate; 6) “Stimulus-Oss” – dental sponge containing
collagen, 2% chlorhexidine digluconate and hydroxyapatite. Cellular state was evaluated after staining with
trypan blue and acridine orange. Amount of transformed and intact cells was determined using light
inverted microscope Biolam-P and luminescent MIK MED-2. It was found that studied cells differ in
shape, state of cytoplasm, morphology of nuclei, and topology of stained substances inside the cell.
Indicator of number of alive cells had a reverse value to a response of cultured cells to tested osteoplastic
materials.
Conclusion. The “Osteoplast®” and “Easy-Graftтм” materials demonstrated the highest individual
biocompatibility in ex vivo conditions at GP of the II and III degree of severity. While high
biocompatibility of most materials tested in this series of studies, was detected in norm and at primary
stages of the pathological process (ChCG and GP of the I degree).
115
HISTONE ACETYLATION, ROS CONTENT AND ANTIOXIDANT ACTIVITY OF
АRABIDOPSIS THALIANA TISSUE CULTURE IN THE DEVELOPMENT OF ACUTE
OSMOTIC STRESS
Sergiy Jadko, Alexandr Syvash
N.G. Кholodny Institute of Botany, National Academy of Sciences of Ukraine,
Tereschenkivska Street 2, 01004, Kyiv, Ukraine.
Email address: ukrkiev55@mail.ru
In plants under influences of various stresses, changes in the histone acetylation and deacetylation by
the nuclear histone acetyltransferases (HAT) and deacetylases (HDA) take place. It is resulting in
significant changes in gene expression and formation of reactive oxygen species (ROS) and antioxidant
activity. The aim of this work was to study the interrelation between the HAT, HDA, ROS and antioxidant
activity. 12-14 days old tissue culture of A. thaliana, ecotype Columbia have been investigated. The tissue
culture was grown on the solid agar MS medium in the dark at 24°C. Acute osmotic stress was caused by
placing of the tissue culture into 25% polyethylene glycol-6000 (PEG) solution. After 1, 2 and 3 hours
HAT and HAD activities and intensity of spontaneous chemiluminescence (SChL)/ROS content, H2O2
content and antioxidant activities of ascorbate peroxidase (AP), catalase (CAT) and thioredoxin (TR) were
determined. Protein content was determined by the Bradford method. Experiments were repeated by 3-5
times independently. The obtained data were processed statistically. HAT and HDA activities increased
under influence of PEG. Acute osmotic stress also leads to an increase in the SChL intensity and H 2O2
content and AP, CAT and TR activities. Early increase in HAT and HDA activities associated with the
stress reorganization of the cell metabolism by epigenetic changes in gene expression. At that the HAT and
HDA activities also associated with the regulation of gene expression of proteins that responsible for the
production of ROS and activity of antioxidant enzymes. Increased level of ROS leads to ROS-dependent
increase in the activity of the studied antioxidant enzymes. At that an important role belongs to TR that
with H2O2 and peroxiredoxins can form stressful redox signals.
116
THE INFLUENCE OF АNTHOCYANIDINES EXTRACT FROM RED CABBAGE ON THE p 53
AND Bcl - 2 PROTEINS CONTENT OF LEUKOCYTES UNDER THE EXPERIMENTAL
DIABETES MELLITUS TYPE 1
Kanyuka O.1, Holovatska I.1 Naruto E.2, Buko V.2, Sybirna N1.
1
Ivan Franko National University of Lviv
Hrushevskogo St, 4, Lviv, 79005, Ukraine;
2
Institute of Biochemistry of Biologically Active Compounds,
Bulvar Leninskogo Komsomola, 50, 230030, Grodno, Republic of Belarus.
Email address: kanokaol@yahoo.com
Among myriad natural plants, red cabbage (RC) (Brassica oleracea var capitata) have been found to
have antioxidant, antihyperglycemic, anticancer and hypocholesterolemic properties. The principle
constituents of RC are isothiocyanates (glucosinolate), vitamins A, B, C and anthocyanins Anthocyanins, a
group of phenolic natural pigments present in RC, were found to have the strongest antioxidizing power of
150 flavonoids. Our objective was to investigate аnthocyanidines extract from red cabbage using
immunohistochemestry method quantitative redistribution of mononuclear leukocytes (MNL) of rat’s
peripheral blood. MNL contain proapoptotic protein p53 and antyapoptotic protein Bcl-2 under conditions
of experimental diabetes mellitus (EDM) type 1. Diabetes was induced in male Wistar rats using
streptozotocin (60 mg/kg body weight). Throughout the experimental period, diabetic rats exhibited many
symptoms including loss of body weight and hyperglycemia. Daily oral ingestion (0,6 g/kg body weight) of
B. oleracea extract for 28 days reversed the adverse effect of diabetes in rats. For detection and
visualization of intracellular proteins Bcl-2 and p53 was used indirect immunoperoxidase method. The
intensity of staining cells was divided into 3 groups: negative reaction, positive and sharply positive
reaction. Under EDM content MNL p53 protein in peripheral blood was increased as evidenced by the
growing number of cells with sharply positive reaction compared to the control. Against the background of
growing content proapoptotic protein in blood under EDM the amount of Bcl-2 was reduce. Application of
аnthocyanidines extract from red cabbage under EDM normalizes the ratio of mononuclear leukocytes
which contain the aforementioned protein-regulators of apoptosis. This indicates that the inhibitory effect
of this extract on programmed death of immunocompetent cells. The investigated anti-apoptotic effect of
the studied extract can be attributed to antioxidant action of bioactive substances in its composition.
117
THE YAKON EXTRACT INFLUENCE ON MORPHO-FUNCTIONAL STATE OF IMMUNE
COMPETENTIVE CELLS UNDER THE CONDITION OF DIABETES MELLITUS
Horbulinska A. V.1, Khokhla M. R.1, Mishchenko L. T.2, Sybirna N. O.1
1
Ivan Franko National University of Lviv, Hryshevskyi St. 4, Lviv 79005, Ukraine;
2
Taras Shevchenko National University of Kyiv, Volodymyrska Street 64/13, Kyiv 01601, Ukraine.
Email address: khmarija@gmail.com
The investigation of phagocytosis parameters under the condition of experimental diabetes mellitus
(EDM) is important due to the major role of this process, mainly in inflammation, wound healing and as a
factor of nonspecific immunity. Phagocytes are involved in elimination of immune complexes and
phagocytosis activity is closely connected with the activity of complement components, namely C3, IgGantibody concentration, the presence of other opsonisation factors.
The index of phagocytosis completeness (IFC), defined as the ratio between the average numbers of
phagocytized microbes after 30 minutes incubation to the average number of phagocytized microbes after
120 minutes of leukocytes incubation. The IFC indicates the phagocytosis digestion completion ability of
polymorphonuclear leukocytes.
In control animals IFC was 0,98 ± 0,03 and indicates the completeness of phagocytosis, whereas
animals with EDM investigating index 28% decreased compared with the control, and were equal to 0,78 ±
0,04. IFC less than 1,0 indicates the decrease of neutrophils killer function.
The administration of yakon extracts causes normalization of neutrophils phagocytic activity that
was indicated by IFC growth. This effect of yakons’ extracts may be caused by the presence of a large
amount of fructooligosaccharides. From literature data it is known, that daily consumption of this
biologically active substances cause the preservation of the inflammatory condition of phagocytic cells,
thereby preventing the possible risk of autoimmune and metabolic diseases.
Polymorphonuclear leukocytes are a connecting link between innate and adaptive immunity and
perform a major role in antibacterial defenses. The normalization of their functional competence by using
the yakons’ extracts under EDM can improve the complexity of the disease and next to its hypoglycemic
action may prevent the development and progression of diabetes complications.
118
PROBABILISTIC MODEL OF CARCINOGENESIS AND PROBLEMS IN ESTIMATING
MODEL PARAMETERS
Vladimir G. Knigavko, Olga V. Zaytseva, Marina A. Bondarenko,
Liliya V. Batyuk, Tatyana R. Kocharova
Kharkiv National Medical University, 4 Lenina ave., Kharkiv, 61022, Ukraine
Email address: vknig@mail.ru
It is assumed [1] that an individual’s genotype has a certain number (up to eight according to
different data) of genes, which in the event of damage (loss of function) of all such genes, the cell shall
undergo malignant change. In the following, such genes will be called а-genes.
The mutations that damage а-genes occur in a random manner. Hence, an individual’s genotype can
have a different number of such genes, and with time, this number decreases.
Study [2] has suggested a probabilistic mathematical model of cell malignant change. From this
model, it follows that the distribution function (F (t)) of the time of development of a cancer disease in an
individual is described by the following expression:
,
where
; is probability of а-gene damage during one cell cycle; t is time elapsed since embryo
formation, Т is duration of cell cycle, k is initial amount of а-genes in an individual’s genotype, and N is
number of cells of a given type in the body. Thereat the study uses a simplifying assumption that T does
not change with age.
In particular, from this model it follows that, at < 3, the average time of formation of a tumour is
less than a year, and at k = 7, this average time exceeds 100 years.
To analyse the model, it is necessary to estimate the probability of presence in an individual’s
genotype of a certain number of a-genes for the entire human population. More precisely, it is necessary to
estimate the distribution of the number of а-genes in the population. Knowing this distribution would
allow, for instance, to estimate the rate of growth of oncological diseases as a function of improving
treatment quality because such improvement of quality stipulates increasing survivability of individuals
with a small initial value of parameter k, thereby increasing the probability that such an individual will be
able to sire.
Such an estimate can be obtained using two methods. The first method assumes calculating the
probability of variation in the number of а-genes in gametes, with subsequent re-evaluation to obtain the
above distribution. This method involves solving nonlinear systems of equations.
The second method assumes using experimental (clinical) data on the dependence of oncological
morbidity on an individual’s age to solve the problem considered. The second approach is simpler;
however, it requires a big volume of statistical data, which is not yet available.
1. Гродзинський Д.М., Радіобіологія. К.: Либідь, 2000. 448 с.
2. Книгавко В.Г., Радзишевская Е.Б., Бондаренко М.А. Математическое
канцерогенеза // Біофізичний вісник.- 2010. Вип. 25 (2).- С. 93-100.
119
моделирование
MEIOTIC MUTANTS TOMATOES WITH DISTURBANCE OF CHROMATIN
CONDENSATION
Lisovska T.P., Kotsun L.O., Kuzmishyna I.I.
Lesia Ukrainka Estern European National University,
Ukraine, 43025, Lutsk, Voli prosp., 13
Emai address l: tlisovska@ukr.net
Reduction by half of the chromosomes number that occurs during sexual reproduction through meiotic division
requires coordination of all processes that occurs during meiosis. To date, important for synapsis and equal
distribution of homologous chromosomes and sister chromatids provide regular chromatin condensation and cohesion
of sister chromatids.
In mitosis and meiosis, condensin and cohesin complexes play essential role in maintaining of the structure of
chromosomes and sister chromatids cohesion. Cohesion of sister chromatids occurs in S-phase and stored in the area
of the centromere to anaphase during mitosis and to anaphase II during meiosis. Cohesion sister chromatids arms in
meiosis disappear in anaphase I that facilitate the separation of homologs in places chiasmata, but in the area of the
centromere, where cohesins protected by protein - shugoshin, remains to anaphase II. In establishing the genetic
mechanisms of chromatin condensation during meiotic prophase provide a significant contribution to the study of
meiotic mutations that control the condensation of chromosomes during meiosis.
In the laboratory of genetics and breeding of Biology Faculty Lesia Ukrainka EENU created a collection of
meiotic mutants of tomato, which consists of two meiotic mutants with impaired chromatin condensation. Both
mutants are spontaneous and have different origins: as4 mutant was isolated by sterility in a variety of San Marzano S.
Soost (Soost, 1951), sti mutant was isolated moldavian scientists (Lisovska et al., 1994).
Mutations are very similar cytological expression during male meiosis. In homozygous for the mutation as4
and sti plants, beginning of prophase I of meiosis is no apparent abnormalities. Thus, in late leptotene can see specific
clusters of chromosomes in the nuclei meiocyte as a "bouquet". In pachytene observed irregularly thickened
homologous chromosomes. Starting with dyplotene stage, chromosomes are characterized by indistinct contours,
irregular condensation of chromosomes, weave chromatin between bivalents, non-homologous chromosomes and
univalents that are difficult to separate. In metaphase I observed chromosomes locations outside the equatorial plane.
In anaphase I found numerous strands of chromatin between homologous and non-homologous chromosomes, which
went to the poles and fragmentation of chromosomes. Due to the numerous violations during the first meiotic division
to second division becomes a small part of the cells. Meiosis II also occurs with disorders such binding chromatin
bridges and fragmentation of chromosomes.
As a result of these violations during meiosis, the sporades stage formed mainly dyads, dyads and triads with
micronuclei. Mutant plants exhibit significant pollen sterility (1-3% fertile pollen) and a sharp decline in the number
of seeds in the fruit. Thus, mutations are an expression both in microsporogenesis and in megasporogenesis.Mutations
as4 and sti are recessive monogenic inheritance. According to the results test for alelizm these mutations is nonallelic.
Our data obtained on a similar phenotypic expression of as4 and sti nonallelic tomato mutations to predict the
presence of at least two paralogs cohesin with a similar function. Further studies of tomato mutants will detail the
mechanisms of structural maintenance of chromosomes in plant meiosis.
Soost R. K., Genetics. 1951, 36:410-434.
Lisovska T. P. et al., Conf. I Nationale. Chisineu. 1994, 37–38.
120
REDUCING OF C6 GLIOMA CELLS PROLIFERATIVE ACTIVITY UNDER THE INFLUENCE OF
FETAL NEURAL CELLS SUPERNATANT
Liubich L.D., Semenova V.M., Malysheva T.A., Stayno L.P., Vaslovych V.V.
SI “Acad.A.P.Romodanov Institute of Neurosurgery NAMS of Ukraine”
04050, Kiev, Platona Mayborody str., 32
Email address: lyubichld@gmail.com
In neurooncology primary and dissociated cell cultures derived from different tumors are widely
used in the study of proliferative potential and mechanisms of carcinogenesis, as well as for the initial
testing of new approaches to the treatment of tumors. Among CNS tumors the tumors of glial histogenesis
make up the largest group that are difficult to treat because of invasiveness and high frequency of
prolonged growth. One of the alternative approaches to solve this problem is to use neurogenic stem cells
and progenitor cells (Kim S.U., 2011; Ahmed A.U. et al., 2012; Bovenberg M.S. et al., 2013).
The aim of this study is to evaluate the impact of rat neurogenic fetal cells supernatant on
proliferative activity of cultured rat C6 glioma cells.
Меthods. The cells of rat brain glioma (cell line C6, n = 16) served as the material for culturing. Rat
neurogenic fetal cells supernatant (RNFCS) was obtained from suspensions of neurogenic rat brain cells on
14th (E14) day of gestation. For study the C6 glioma cultures with uniform growth zone (5-6 th day) were
taken, to the cultures RNFCS (0.10 mg / ml) was added and incubated for 48 hours. Immunocytochemical
staining for the proliferation marker Ki-67 was performed using rabbit monoclonal antibodies to Ki-67
(Thermo Scientific, USA) and detection system Ultra Vision Quanto Detection System HRP DAB
Chromogen (Thermo Scientific, USA). In cytological specimens the changes in cellular composition were
analyzed, the mitotic index (MI) and the number of Ki-67 immunopositive cells were determined.
Rezults. In control cultures the rat brain C6 glioma cells showed a high proliferative activity with
prevalence of a cell population of undifferentiated phenotype. The middle MI of cultured tumor cells
amounted to (4,90 + 0,09) %. Among the cells in the state of mitotic division the cytological features of
mitosis pathology encountered that is inherent for malignant tumors. The proportion of tumor cells
imunopositive for Ki-67, amounted to (27,86 + 2,91)%. At the same time, some tumor cells showed signs
of genetically determined histotypical astrocytic differentiation.
After 48 h incubation of C6 glioma cultures with RNFCS in a concentration of 0.10 mg / ml in the
growth zone of studied cultures the degenerative and necrobiotic modified tumor cells with rounded
cytoplasmic bodies and reduction of processes appeared. The majority of the cells was becoming the form
of naked nuclei (cell shade) due to degradation of the cytoplasm. MI was reduced to (1,10 + 0,04)% (UMann-Whitney test, p = 0.009 compared to control). The proportion of tumor cells imunopositive for Ki67, under the influence of RNFCS high statistically decreased to (10,47 + 0,91)%, (U-Mann-Whitney test,
p = 0.0015 compared to control).
Conclusions. The results of testing of cultured C6 glioma tumor cells sensitivity to the influence of
RNFCS (E14) at a concentration of 0.10 mg / ml for 48 hours suggest the antiproliferative effect of
RNFCS, which can become the theoretical basis for further development of optimized treatment regimens
of these tumors using fetal biopreparations.
121
EFFECT OF ALKALOID-FREE FRACTION FROM GALEGA OFFICINALIS L. EXTRACT ON THE
RATS LEUKOCYTES PRECURSORS PROLIFERATION AND DIFFERENTIATION UNDER
EXPERIMENTAL DIABETES MELLITUS
M. Lupak, O. Gryhorchyk, G. Hachkova, Ya. Chajka, M. Skybitska, N. Sybirna
Ivan Franko National Universiy of Lviv, Hrushevskogo St. 4, Lviv 79005, Ukraine.
Email address: marjanalupak@gmail.com
Inflammatory processes caused by high blood glucose levels under diabetes mellitus conditions,
primarily induce blood cells reaction, especially leukocytes. The blood system reaction on the
inflammation development contains stimulation of hematopoiesis, white blood cells outgate from bone
marrow reserve pool into the blood and leukocyte migration from the blood to the center of inflammation.
Science-based approaches and differentiated selection of phytotherapeutic drugs in the treatment of
diabetes are significantly improved outcomes at all stages of pathology and could reduce antidiabetic
medicines load and prevent tissue lesions. The aim of our work was to study the processes of white cells
precursors proliferation and differentiation using indicator of thymidine analogue including and
morphological analysis of rats bone marrow white blood cells precursors under the experimental diabetes
mellitus (EDM) and in the case of the introduction of alkaloid-free fraction from Galega officinalis L.
extract (AFGOE).
Assessing the myelogram, were revealed that under EDM conditions there are increased
lymphoblasts number by 44%, and reduced eosinophilic and neutrophilic myeloblasts number by 36% and
31% compared with the control, which may indicate a white blood cells strengthening emergency, reduced
immune system activity, development of autointoxication processes and chronic infections.
Using ELISA method, which allows to quantify level of thymidine analogue – 5-bromo-2'deoxyuridine (BrdU) inclusion in proliferating cells, were set to grow by 3.9 times compared to the control,
which may indicate an intensification of leukopoiesis.
These data can be caused by increased levels of tumor necrosis factor alpha (TNF-α) in plasma of
EDM animals by 46%. It is known that this cytokine have an influence on the proliferation of bone marrow
cells, activating nuclear transcription factor NF-kB, which may result a reduction of apoptosis and
increased proliferation of hematopoietic cells. AFGOE introduction contributed to normalization of
myelogram indicators, reduction of TNF-α concentration and proliferative activity of white blood cells
precursors, which may testify its positive effects on the immune system affected by inflammatory process
under the diabetes conditions.
122
INFLUENCE OF CHLORIDE SALINITY ON Ri-TRANSFORMANTS
FOXGLOVE DIGITALIS PURPUREA L.
Lyoshyna L.G., Bulko О.V.
Institute of Cell Biology and Genetic Engineering NASU,
148 Academika Zabolotnoho St., 03143, Kyiv, Ukraine.
Email address: llioshina@ukr.net
Foxglove (Digitalis purpurea L.) was transformed with soil bacteria A.rhizogenes, strain 15834.
Нairy roots and regenerated plants (Ri-transformants) were obtained. A changed in their morphological and
biochemical parameters were studied. The difference is shown in comparison with intact plants.
Further investigation of the impact of abiotic factors, such as chloride salinity on regenerated plants.
Plants and hairy roots were grown on MS medium supplemented with NaCl (50-200mМ). After 30 days,
the physiological state was measured: weight, length of plant, the size of the leaf plates, number and length
of roots and content of photosynthetic pigments and the amount of osmotic potential of the cell contents
was measured as well.
200 mM NaCl was the damaging dose and caused a decrease in the stability of the injury and death
of plants and cell cultures. 100 mM NaCl chloride salinity suppressed meristematic growth point,
development of leaf plates, reduced crude and dry weight in plants regenerated and intact plants. However,
the control plants had visible changes as xerophytes (reducing the size of the leaves and root length,
increasing increase venation, cuticle callosity) and 2-fold reduction of photosynthetic pigments. In
regenerated plants observed a complete lack of roots, slight changes in the content of chlorophyll and
carotenoids and water potential.
Culture of hairy roots have lost the ability to form roots in the salinity of 100 mM, but could forms a
callus. There was A decrease in the percentage of water in the cells. 50 mM NaCl caused a thickening of
the roots without changing their growth. 200 mM sodium chloride in the medium induced death of the cell
culture.
We have shown that the reaction on the chloride salinity in Ri-transformant plants is different from
the response of intact plants. Presumably this is due to the influence of rol-genes.
123
PRODUCTION AND CHARACTERIZATION OF POLYCLONAL
ANTIBODIES AGAINST 25OHD3
Anna Mazanova, Olha Lisakovska, Ihor Shymanskyy
Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine (NASU),
Leontovicha Street 9, 01601, Kyiv, Ukraine
Email address: ann.mazanova@gmail.com
Vitamin D3 deficiency and insufficiency have been reported worldwide and evidence of their
deleterious effects on health has been accumulating. Therefore, an important task of biotechnology is to
develop methods that will allow rapidly and reliably determine the level of vitamin D 3 in the blood serum.
One of these methods is enzyme immunoassay measuring of 25OHD3, which was proved to be a reliable
marker of vitamin D3 bioavailability. A key problem in developing of such immunoassay test systems is the
obtaining of antibodies to 25OHD3, because it is not immunogenic compound with molecular size
insufficient to be recognized by the immune cells. In light of these considerations, the aim of the present
study was to improve the existing approaches for obtaining polyclonal antibodies capable of recognizing
25OHD3 for their subsequent characterization and application in immunochemical test systems to
determine blood serum 25OHD3. For gaining polyclonal antibodies against 25OHD3, the rabbits were
immunized with 25OHD3-KLH (keyhole limpet hemocyanin) conjugate, which was synthesized using
modified carbodiimide method. After five booster immunizations antiserum was obtained and the titer of
polyclonal antibodies by indirect ELISA was characterized. It was demonstrated that the anti-25OHD3
antibodies titer lies in the range 1:1000-1:10000.
Conclusions: Our results suggest that conjugates synthesized as described herein successfully may be
used in the generation of antibodies targeting small hydrophobic 25OHD3 molecules. Immunochemical test
systems can be developed on the basis of obtained polyclonal anti-25OHD3 antibodies for screening studies
of 25OHD3 content in human blood serum, as a marker of vitamin D3 availability.
124
DECREASE OF CONNECTIVE TISSUE USING PRP TREATMENT IN CASE OF LIVER
FIBROSIS, EXPERIMENTAL STUDY
Iegor Maznichenko, Olena Kholodkova, Ievgeniia Pokora
Department of Human Anatomy, Odessa National Medical University, Valikhovsky lane, 2, Odessa
65082, Ukraine.
Email address: iegormaznichenko@gmail.com
Liver fibrosis is severe disease which characterized by lack of full recovery and often lead to
multiple organ failure. Platelet-rich plasma (PRP) is modern kind of biotechnological medicine. It is well
known fact that platelets contain a large number of protein molecules, that regulate regenerative process.
PRP is a platelet concentrate in a small volume of plasma, wherein the concentration of growth factors is
involved in wound healing processes ten times higher than in the peripheral blood.
In connection with the above, the aim of experimental study was investigation of the effectiveness of
the PRP treatment on liver regeneration after fibrosis.
Methodology: the experimental study was performed on mature male Wistar rats; for modeling of
liver fibrosis was used carbon tetrachloride solution in a 50% concentration in a dose of 0.1 ml and was
administered it intraperitoneally every other day during 3 month. To obtain PRP from whole blood the unit
SmartPrep (Harvester Corp, USA) was used. The animals were divided into three experimental groups: 1 animals with simulated fibrosis; 2 - animals with created liver fibrosis and followed doubly intrahepatic
PRP injections; 3 - animals with created liver fibrosis and subsequent introduction of saline at the same
concentration and within the same time frame as in the PRP group 2 animals. A separate group of animals
served as intact control. Animals were sacrificed on the 21th and 42th days from the final injection of saline
or PRP (respectively to groups 2 and 3), and for animals in group 1 - from the end of administration of
carbon tetrachloride. Pathomorphologic studies of the liver were performed with staining with
hematoxylin-eosin, by Van Gieson, Sudan-III and using the PAS-reaction.
Histological results of 2d group was shown reduces the amount of connective tissue and collagen
fibers at the places of hepatocytes necrosis after experimental liver fibrosis. PRP administration leads to the
rapid regeneration of the hepatic tissue with restoration of its normal structure. Animals from 1d and 3d
groups was shown high level of concentration of connective tissue and other pathological changes.
Tangkijvanich P., et. al., The European Journal of Surgery. Supplement. 2002, 587:100-112.
125
CHITIN-BASED SCAFFOLDS FROM SKELETONS OF MARINE DEMOSPONGES
IANTHELLA BASTA AND APLYSINA AEROPHOBA: BIOCOMPATIBILITY STUDY WITH
HUMAN MESENCHYMAL STROMAL CELLS
Mutsenko V.V. 1, Rogulska O.Yu.1, Tarusin D.N. 1, Bazhenov V. 2, Schütz K.3, Brueggemeier S.3, Gossla
E.3, Akkineni R.3, Lode A.3, Gelinsky M.3, Petrenko A.Yu.1
1
Institute for Problems of Cryobiology and Cryomedicine of National Academy of Sciences of Ukraine,
Kharkiv, Ukraine
2
Institute of Experimental Physics, TU Bergakademie, Freiberg, Germany
3
Centre for Translational Bone, Joint and Soft Tissue Research, Technische Universität Dresden,
Germany
Email address: v.v.muzenko@gmail.com
The rapidly evolving field of tissue engineering requires biomaterials that exhibit favourable
properties for cell attachment, proliferation and differentiation to promote the regeneration of the diseased
or damaged tissue. With this respect chitin is established as a very attractive biomaterial for tissue
engineering and stem cell-based technologies (Ehrlich H. et al., 2007).
Recently, for the first time chitin has been discovered in Verongida sponge skeletons in the form of
frameworks with evolutionarily unique design (Wan A. C et al, 2013). In our study, we have investigated
the biocompatibility of chitin scaffolds derived from two structurally attractive marine sponges, Ianthella
basta and Aplysina aerophoba, with human mesenchymal stromal cells for tissue engineering purposes.
Scaffolds were extracted from the sponges by an alkali-based treatment procedure and evaluated by
SEM and histology. Mesenchymal stromal cells from bone marrow and skin derma of adult donors were
seeded onto scaffolds using a semidynamic technique. Cell attachment and viability was analyzed by
DAPI/Phalloidin and FDA/EB staining, respectively. Cell colonization of scaffolds was monitored by light
microscopy for two weeks. The multilineage differentiation of the cells after respective stimulation was
evaluated by analysis of the gene expression of typical markers (RT-PCR) and by staining with
corresponding dyes.
The scaffolds obtained after alkali-based treatment presented different morphological compositions.
Positive staining by Calcofluore White revealed chitin nature of the scaffolds. In both cases, cells adhered
to and spread well on the fibers of scaffolds showing high degree of viability on day 1 and 7. In the course
of two weeks the cells proliferated completely covering the scaffold surface and subsequently exhibited the
capability of growing in the form of cell sheets within scaffold pores.
Following adipogenic induction, cells growing in the scaffolds from Ianthella basta accumulated
lipid droplets (Nile Red staining) and expressed adipogenesis-related genes (PCR). The cells cultured
within scaffolds from Aplysina aerophoba were able to differentiate into the chondrogenic (PCR),
osteogenic (Alizarin red staining) as well as adipogenic (Nile Red staining) lineage.
In summary, chitin scaffolds derived from marine sponges studied are biocompatible with human
mesenchymal stromal cells and could find use in stem cell-based tissue engineering and regenerative
medicine.
Ehrlich H. et al., Journal of Experimental Zoology Part B: Molecular and Developmental Evolution. 2007,
308B:347–356.
Wan A. C. et al., Biotechnology Advances. 2013, 31(8):1776–85.
126
CYTOGENETIC STUDIES OF DESCHAMPSIA ANTARCTICA DESV.
TISSUE CULTURE
Navrotska D.O., Тwardovska М.О., Andreev I.O., Кunakh V.А.
Іnstitute of Molecular Biology and Genetics NAS of Ukraine,
Ukraine, 03143, Kyiv, Academica Zabolotnogo street, 150
Email address: navrotska.daria@gmail.com
The plant cells in the culture in vitro accumulate genomic changes that can be revealed at the
chromosomal level as the alterations in the chromosome number and morphology, structural
rearrangements etc. To data, there are no published reports about cytogenetic studies of tissue culture of
Deschampsia antarctica Desv. – one of the two species of vascular plants of Antarctica. Cytology analysis
conducted previously showed that the cells of D. antarctica plants contain 13 pairs of chromosomes
(2n = 26) in their karyotype. However, recently obtained data revealed the chromosomal variation, as
manifested by the occurrence of mixoploidy (2n = 13-27) and additional B-chromosomes (2n = 26 + 1-3В)
in D. antarctica plants from Darboux Island. Therefore, the purpose of our research was to study the
cytogenetic structure of D. antarctica tissue culture at the early passages of culture.
Callus tissues used for the study were produced from the roots of D. antarctica plants collected on
the Darboux Island. For cytological analysis, callus tissues were sampled at the 7-th day of the 2-nd, 3-rd
and 4-th passages. Collected tissue was fixed in ethanol: acetic acid (3:1) during 24 hours. The fixed
material was stored in 70° ethanol. Samples were stained with 1% acetoorsein and then used to make
squashed preparations. The number of chromosomes was counted in 100 metaphase plates. Preparations
were examined under a NU-2E Carl Zeiss microscope.
Cytogenetic analysis of D. antarctica tissue culture demonstrated a wide variation in chromosome
number, which ranged from 18 to 52 with on average 25.8 chromosomes per metaphase. Modal class of
callus cells at all growth stages were composed of diploid cells that amounted 48 % in the second, 56 % in
the third and 43 % in the fourth passages of cultivation. A significant number of cells with aneuploidy were
observed. The highest percentage of aneuploid cells was found at the 2-nd passage of cultivation (48 %),
and the lowest was at the 3-nd passage (41 %). The percentage of polyploid cells was relatively low, only
3% of the studied cells at each passage belonged to the triploid. Furthermore, the level of chromosomal
aberrations, which were presented by single bridges in anaphase, was very low and amounted to only 1%.
The largest variations in chromosome number were observed at the fourth passage of cultivation.
Therefore, in vitro tissue culture of D. antarctica resulted in genetic changes displayed as a
variability in the chromosome number. Cytogenetic analysis of callus tissue showed mixoploidy with
prevalence of diploid cells in the proliferative pool. The level of genetic rearrangements was low at the
early stages of cultivation, whereas the increase in culture duration was accompanied with enhance in
number of chromosomal abnormalities.
127
FID ASSAY IN THE STUDY OF INTERACTION OF NOVEL BIOACTIVE ACRIDINE
DERIVATIVES WITH QUADRUPLEX DNA
Saraieva I.V.2, Negrutska V.V.1, Kostina V.G.1, Lysenko N.A.1, Alexeeva I.V.1, Dubey I.Ya.1
1
Institute of Molecular Biology and Genetics, NAS of Ukraine, 150 Zabolotnogo str.,
03680 Kyiv, Ukraine
2
Taras Shevchenko National University of Kyiv, 64/13 Volodymyrska str., 01601 Kyiv, Ukraine
Email address: v.v.negrutska@imbg.org.ua
FID (Fluorescent Intercalator Displacement) is a simple and fast method that allows to evaluate the
DNA binding affinity and selectivity of low-molecular compounds. This assay is based on the loss of
fluorescence of some dyes, usually Thiazole Orange (TO), upon their displacement from complexes with
DNA by a ligand. TO is virtually non-fluorescent in free staste, whereas its complexes with DNA are
highly fluorescent. Both duplex (Tse W.C. et al., 2004) and quadruplex (Largy E et al., 2011) DNA can be
used as a target. G-quadruplex DNA (G4-DNA) is a specific tetra-stranded structure formed in telomeres,
and its stabilization with small molecules may lead to the inhibition of telomerase resulting in antitumor
activity.
We have discovered novel trisubstituted actridine derivatives that efficiently inhibit telomerase in
vitro at low micromolar concentrations (IC50 values of the most active inhibitors determined in TRAP
asasay were about 2.5 µM). Telomerase inhibition may result from the binding of these drugs to
quadruplex DNA. In this work we studied the interaction of bioactive acridines with G4-DNA. A series of
about 25 compounds with aryl- or alkylamide substituents at C-4 and basic -N(CH2)nNAlk2 group (n=2-4)
at C-9 position of acridine ring were investigated using a FID approach. The strucures were based on
acridine core known to bind to guanine quartets of G4-DNA and contained additional basic/cationic
fragments able to interact with phosphate groups via the electrostatic mechnism.
The assay was performed in 96-well microplates in semi-automatic mode with BioTek Synergy HT
analyzer. The yellowish-green fluorescence of DNA-acridine compexes was observed in the same spectral
region where TO-DNA complexes emit. This makes impossible to apply TO as a FID probe wth the studied
compounds. So we have used ethidium bromide (EthBr) whose absorption and fluorescence maxima are
significantly red-shifted as compared to those of TO and acridines. Like TO, EthBr has the properties of a
light-up probe for quadruplex DNA. This label enabled to study acridines with various substutients and
allowed to determine their binding affinity towards G4-DN.
Model oligonucleotide d[AGGG(TTAGGG)3], a fragment of human telomeric DNA, was folded in
the presence of Na+ cations into G-quadruplex of antiparallel type (Tel22, PDB access code 143D).
We have determined DC50 values (concentration of a ligand reslting in 50% of dye substitution
showing the ligand efficiency) and binding constants for the compounds. These values correlate with
biological activity of acridines as telomerase inhibitors (IC50). The most efficient inhibitors appeared to be
also the most affine G4-DNA binders, with binding constants Kb of 1.6x106 and 1.0x106 M-1. Analysis of
structure-activity relationship in the series of acridines allowed to find structural elements ensuring high
binding affinity and biologial activity of compounds.
Thus, the studied acridine derivatives, active telomerase inhibitors, have strong binding affinity for
quadruplex DNA. In additin, our study demonstrates that FID assay offers the possibility to test a variety of
DNA binding compounds. It is a powerful tool to quickly identify high-affinity G4-DNA ligands, potential
antitumor agents.
Tse W.C., Boger D.L. Acc. Chem. Res. 2004, 37:61-69.
Largy E., Hamon F., Teulade-Fichou M.-P. Anal. Bioanal. Chem. 2011, 400:3419-3427.
128
MAMMALIAN ERYTHROCYTE SENSITIVITY TO HYPERTONIC SHOCK UNDER
CONDITIONS OF PRE-INCUBATION OF CELLS IN SUCROSE MEDIA
Shpakova N.M., Orlova N.V., Nipot O.Ye., Iershov S.S., Iershova N.A.
Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of
Ukraine, Kharkiv 61015, 23 Pereyaslavskaya str
Email address: nipotel@gmail.com
Study of sensitivity of mammalian cells to changed environmental conditions is one of the most
exciting and informative research areas of cell biology. This is especially important during long-term
storage of cells. That is, when freezing a cell suspension there is found a sharp increase of osmoticity of
extracellular solutions, which is a major damaging factor under these conditions. Pre-treatment of cells
using the compounds with a cryoprotective effect reduces a cell damage at freezing stage.
In our research as the object of study there were selected the mammalian erythrocytes (human, equine,
rabbit, rat), which are exposed to short-term hypertonic shock, i.e. incubation in 4 M NaCl (5 min).
Preliminarily the cells were incubated in sucrose solution (0.2 - 1 M) for 2 min to remove osmotically
active water. The research
was performed at 37 °C. The share of damaged cells was
spectrophotometrically determined at 543 nm wavelength.
The dependences of cell hypertonic hemolysis on sucrose concentration in the medium prior
incubation were obtained. For human, equine and rat erythrocytes there was observed a change in
sensitivity to hypertonic shock after incubation in sucrose medium. An exception is the rabbit erythrocytes,
that as we believe is due to generally low level of hypertonic hemolysis of its cells. It should be noted that
the shape of hemolytic curves are species-specific, and each of them has a minimum at determined sucrose
concentration. Rat’s erythrocytes show a minimal sensitivity to hypertonic shock after pre-incubation in
0.3- 0.6 M, the equine ones do in 0.4 - 0.6 M, and human erythrocytes in 0.6 M sucrose. The deepest
minimum is inherent to human erythrocytes: due to conditions of pre-incubation in sucrose media their
hypertonic injury reduces from 85 to 15%. For rat’s erythrocytes under these conditions a decrease from 40
to 10% is observed, for equine erythrocytes it is found from 50 to 30%.
Thus it could be concluded that pre-incubation of mammalian erythrocytes in sucrose medium is able
of significant altering their sensitivity to hypertonic effect. Certain degree of cell dehydration caused by
hyperosmotic concentration of sucrose probably affect positively the cell resistance to further influence of
osmotic shock. The difference between in erythrocyte sensitivity of different mammalian species to
hypertonic shock and pre-incubation in the media which contain the certain sucrose concentration can be
stipulated with various content of intracellular water.
129
SOME PROPERTIES OF CYTOSPHERES, WHICH ARE FORMED IN THE ADRENAL
PRIMARY CELL CULTURES OF NEONATAL PIGLETS
Plaksina K.M., Sidorenko O.S., Legach E.I., Bondarenko T.P., Bozhok G.A.
Institute for problems of cryobiology and cryomedicine of the of Ukraine, st. Pereyaslavskaya 23, 61015,
Kharkiv, Ukraine.
Email address: ekarinamih@gmail.com
In the absence of specific markers the identification of stem/progenitor cells can be made, in some
approximation, basing on their abilities to the cytospheres formation (Pastrana E. et al., 2011). Sphereforming culture conditions promote isolating a quite rare population of stem cells as well as increasing their
number. Adrenal cell cytospheres formation was shown in the primary cultures derived from adrenal glands
of fetal (Zhou H. et al., 2006) and adult (Santana M. et al., 2012) human, adult bull (Chung K. et al., 2009)
and newborn mice (Saxena S. et al., 2013).
The aim of our work was to obtain cytospheres from adrenal glands of neonatal piglets and study
some of their properties.
The adrenal cells were isolated by enzymatic method. Cells were seeded at a concentration of 15
2x10 cells/ml and cultured at 37°C in the atmosphere of 5% CO2 in the different culture conditions: case I
− in the flasks with normal adhesive surface in the medium with 10% fetal calf serum (FCS); case II − in
the Petri dishes with low adhesion surface in the medium with 10% FCS; case III − in the Petri dishes with
low adhesion surface in the medium supplemented with 2% B27 and growth factors (20 ng/ml EGF, 20
ng/ml bFGF, 10 U/ml heparin). The basic culture medium was DMEM/F12 with antibiotics. Replacement
of half the medium was carried out every 4-5 days.
The formation of cytospheres in the cultures of neonatal piglets adrenal glands has been established.
Type, size and number of cytospheres were different depending on culture conditions. In the culture
conditions performed according with case I, cytospheres were attached to the monolayer of fibroblast-like
cells, while in the cases II and III they were floating in the medium.
Number of cytospheres increased during time of culture in all studied cases. On the 13th day of
culture in the case I the number of counted objects was about 80±45 cytospheres/1 million seeded cells,
while in cases II and III − 72±61 and 20±11 cytospheres/1 million seeded cells, respectively.
Size of cytospheres also increased during culture. In the case II the maximal size of cytospheres was about
764±187 μm. In the case I the size of cytospheres was 240±69 μm, while in the case III − 519±59 μm.
When floated cytospheres, which were obtained in the case II, were transferred on the plates with
normal adhesion on the 11th day, the attachment and spreading of spheres was observed, after which the
cells with neuroblast-like morphology began to migrate out from them. This indicated that in the primary
cell culture of adrenal glands the sympatho-adrenal progenitor cells are present, which in certain
circumstances can differentiate into neuronal direction. In the case of passaging of the floated cytospheres,
which were formed under conditions III, neuroblast-like cells were not observed.
Thus, the composition of the culture medium and the degree of surface adhesion are the factors that
determine the proliferative and differential potential of cytospheres in primary cell cultures of neonatal
piglets adrenal glands.
130
HUMAN MESENCHYMAL STEM CELLS PROLIFERATION
IN THE MODIFIED CONDITIONS
Plotnikova L, Yanko R, Berezovskii V.
National Academy of Sciences of Ukraine A.A.Bogomoletz Institute of Physiology,
Bogomoletz Street 4, 01024 Kiev, Ukraine.
Email: lidiianik@ukr.net
Mesenchymal stem cells (MSC) presently find more wide use in regenerative medicine,
transplantology and tissue engineering. It is conditioned by their capacity for support, to the presence of
high proliferate activity and multilineage of differential potential. MSC can be derived from fetus and
adults tissues. In modern cell culture is usually carried out in a standard atmosphere (SA; 20,96 % O 2),
which corresponds to a oxygen partial pressure (Ро2) of 159 mm Hg with the addition of 5-6 % CO2.
However, cells in humans or laboratory animals operating under Ро2 at 20-80 mm Hg. Our study aims to
establish the hypoxic gas mixture effects on proliferation of human mesenchymal stem cells.
Materials and methods. Cell line 4BL − multipotent MSC derived from the human peripheral blood.
The culture obtained in the Department of Human Genetics, Institute of Molecular Biology and Genetics
NAS of Ukraine (L.L. Lukash). For the growth of MSC we used DMEM culture medium with addition of
10 % fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin, at 37 °C and 5 % CO 2 in a
humidified atmosphere. Gas mixtures with low (relative to air) Ро2 was served in two modes: I –
continuous (24 h) of Ро2 23, 38 or 76 mm Hg; II – intermittent (8 h/day, 3 days) of Ро2 38 or 76 mm Hg
and then transferred to research groups in CO2-incubator without changing the culture medium. To
determine the proliferative activity of the cell culture scattered by 50 thousand cells in Petri dishes (d = 35
mm). Their number counted in the chamber Goryaeva after 72 and 96 hours.
Results. The studies in three repeat samples showed that incubation of culture in SA and gas
mixtures with close to physiological levels of Ро2 significantly different. The proliferative potential of cells
exposed at Ро2 23 mm Hg (24 h) on the third day of cultivation was reduced in 2 times. Incubation of
studied cells in gas medium with Ро2 38 and 76 mm Hg (I mode) did not reduce cell proliferation compared
with control. Our results showed that in all embodiments of incubation (II mode) the proliferation of MSC
on the third day was not different from control values. On the fourth day of cultivation, after exposure Po 2
38 mm Hg, the number of cells was significantly (p<0,05) increases and was 462 000 cells/ml compared to
the control (363 000 cells/ml). The same increase in proliferative activity occurred and at intermittent Po 2
76 mm Hg – 528 000 cells/ml. These data allow to conclude that lowering the oxygen concentration in the
gaseous medium before incubation Po2 38 and 76 mm Hg (II mode) when cultured investigated line MSC
increases their proliferation by 27 % and 45 %, respectively, compared with the control. The maximum
effect was obtained at intermittent Po2 76 mm Hg.
131
INFLUECE OF NANOSILVER AT DIFFERENT CONCENTRATIONS ON THE GROWTH AND
THE GRAIN PRODUCTIVITY OF THE OAT PLANT CULTIVAR NEZLAMNY
Anatoly Potopalsky1,2, Larysa Yurkevich1,2, Valentyna Katsan1
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine,
2
Institute of health improvement and rebirth of the peoples of Ukraine,
Academika Zabolotnogo Street 150, 03680 Kyiv, Ukraine
Email address: val.katsan@gmail.com
1
Nanosilver is capable of to evoke the broad spectrum of biological effects, mediated by oxidative
stress induction and depend on of the shape and dimensions of its particles, surface-bound charge,
solvation, concentration, on the duration and the other conditions of treatment. Development of such effects
caused also by the organism state and by the nature of the cells and molecules, turned its targets. The
peculiarity of to influence on the stress signaling pathways make nanosilver good looking for use to plant
adaptability improvement. In the present study the nanosilver preparation SS1000 (S) was use, elaborated
in Institute for Superhard materials V.M. Bakul NAS of Ukraine and kindly given to us. The aim of this
study is the investigations of the influence of nanosilver SS1000 on the growth and the grain productivity
of the oat cultivar Nezlamny, obtained in our laboratory. The oat seeds were treated by the water solutions
of S in the wide range of concentrations (10÷60 mg/l and 600 mg/l) over the time of 24 hours, the plants
were grown in the field on the little plots of land. The plant growth intensity was estimated by the stem
length in booting (Ls). The stem length at the completion of development (Ls-2) and the grain productivity
compounds (the primary panicle length, L; the grains number in the primary panicle (G); the grains weight
from the primary panicle, W; the weight of 1000 grains, W1000) was performed after the gather in the
harvest.It is found the growth stimulation in booting: for the maximal S concentration (Ls increased on 8.8
%; P<0.001) and in the one-half fraction of the variants with its less concentration (Ls increased on 4.3–5.6
%; P<0.02; P=0.05). The stem length at the completion of development of treated plants was less: on 12.8
% (P<0.001) with using of the maximal S concentration; on 9.3–11.3 % (P<0.001) – in the half of the
variants with its less concentration, and the tendency of Ls-2 decrease was observed more in the one of
them. The highest S concentration caused the significant reducing of the primary panicle length, the grains
number in the primary panicle and the grains weight from the primary panicle: L decreased on 12.3 %
(P<0.01); G – on 35.8 % (P<0.001); W – on 42.8 % (P<0.001). The suppression of the primary panicle
length is not observed at the lesser S concentrations. At the same time the tendency of decrease G was
revealed in the one third of such variants, and the statistically significant G decrement (on 19.4 %; P<0.05)
– more in the one of them; the reliable W reduction (on 21.2–26.8 %; P<0.01; P<0.05) – in the half of
them, and the trend of W diminution – in more in third of them. Neither of the used S concentrations
influenced on 1000W parameter. These results indicate that the reduction of the grain number in panicles
likely to be responsible for the decrease of the grain crop per panicles has been mentioned above.The
findings of this study leads us to conclude that the nanosilver SS1000, when it has been used for seed
treatment before sowing, is capable of to mediate the stimulation of the oat growth in booting. Such
stimulating effect show less stable in the range of S concentrations, to be 10-60 times less than that its
maximal concentration, chosen for the study. Of no small importance fact is the truncated oat stem at the
termination of development that is favorable for resistance to the lodging. Same time the nanosilver
SS1000 may influence negatively on the corn crop, owing to the reduction of the panicles length and the
grains number in them and such unfavorable impact depend on it content in solutions, shown less stable,
when the content decreased 10-60 times. It is likely, that the appearance of the biological effects of
nanosilver, when the plants are growing in the field, has been effected also by the complex of the
environment stressors to be in existence, especially the weather impact, as far as in the study, had been
performed before in the more favorable summer, the one of S concentrations, has been used in the present
study, forwarded to the improvement the oat grain productivity.
132
INFLUECE OF NANOSILVER AT DIFFERENT CONCENTRATIONS ON THE
PHOTOSYNTHETIC PIGMENTS CONTENT IN THE LEAVES OF THE OAT PLANT
CULTIVAR NEZLAMNY IN THE ONCET OF PANICLE FORMATION
Anatoly Potopalsky1,2, Larysa Yurkevich1,2, Valentyna Katsan1
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine,
2
Institute of health improvement and rebirth of the peoples of Ukraine,
Academika Zabolotnogo Street 150, 03680 Kyiv, Ukraine
Email address: val.katsan@gmail.com
1
Nanosilver is capable of to evoke the broad spectrum of biological effects, mediated by oxidative
stress induction and depend on of the shape and dimensions of its particles, surface-bound charge,
solvation, concentration, on the duration and the other conditions of treatment. Development of such effects
caused also by the organism state and by the nature of the cells and molecules, turned its targets. The
peculiarity of to influence on the stress signaling pathways make nanosilver good looking for use to plant
adaptability improvement. In the present study the nanosilver preparation SS1000 (S) was use, elaborated
in Institute for Superhard materials V.M. Bakul NAS of Ukraine and kindly given to us. The aim of this
study is the investigations of the influence of nanosilver SS1000 on the photosynthetic pigments contents in
the leaves of the oat cultivar Nezlamny in the beginning of crop formation.
The oat seeds were treated by the water solutions of S in the wide range of concentrations (10÷60
mg/l and 600 mg/l) over the time of 24 hours, the plants were grown in the field on the little plots of land.
The chlorophylls a (Ca) and b (Cb) content, their sum (Ca+b), the carotenoids sum content (Ccar) and the
ratios Ca:Cb, Ca+b:Ccar were estimated in the even-aged leaves in the onset of panicles formation. As has
been reported in one more our paper, the treatment of the oat seeds by the solutions of the preparation
SS1000 caused the changes of the oat growth and the elements of its grain productivity. It is found a lot of
changes of chlorophylls and carotenoids content and the ratios content of some pigments and their
assemblages in the leaves in the onset of panicles formation.
The maximal S concentration (600 mg/l) conditioned the tendency of carotenoids content fall and the
significant decrease of the chlorophylls content in the leaves: Ca – on 17.1 % (P<0.02); Cb – on 18.4 %
(P<0.001); Ca+b – on 17.1 % (P<0.01). The decrement of the chlorophylls content was found in two thirds
of the variants with less S concentration: Ca – on 16.6–19.9 % (P<0.01; P<0.001); Cb – on 15.1–19.5 %
(P<0.001; P<0.01); Ca+b – on 15.6–19.9 % (P<0.02; P<0.01; P<0.001). The reduction of Ccar was observed
in 83.3 % of the variants with less S concentration – on 17.0–19.1 % (P<0.01; P<0.001). The highest S
concentration caused the diminution of the chlorophylls a and b portion in the pool of photosynthetic
pigments on 9.0 % (P<0.01), while the one of the less concentrations – the increasing that parameter on
13.6 % (P<0.001).
Of a special attention is the variation of chlorophylls a/b ratio, when S was used in the concentrations
range 60÷10 mg/l: significant increase of the chlorophyll b portion in the third of such variants on 3.05–
4.97 % (P<0.01; P=0.05) and the trend to increasing – in more the one of them.
These findings suggest that the nanosilver SS1000, when it has been used for seed treatment before
sowing, is capable of inducing of the changes both the content of some photosynthetic pigments and the
ratios of its contents in the oat leaves in the onset of panicles formation.
The most suppression of the chlorophylls content was observed for the maximal S concentration. The
negative S effect was revealed only in the two thirds of the experiment variants, when such S concentration
was decreased 10÷60 times, and such effect was less expressed.
At the same time such lower S concentrations is capable of inducing the chlorophyll b portion
increase among the main it forms, and this finding may be the evidence of the LHCII augmentation in the
photosystems of such plants.
133
TOXICITY AND GENOTOXICITY OF QUATUM DOTS OBTAINED BY BIOLOGICAL
SYNTHESIS
O.V. Protsenko,1 A.N. Tolstanova1, I.A. Kozeretskaya1, M. Inamystova1, M. Borovaya2, Ya.V. Pirko2,
A.I. Yemets2
1
Department of general and molecular genetics, Taras Shevchenko’ Kyiv National University,
Volodymyrska Street 64, 01033 Kyiv, Ukraine
2
Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine,
Osipovskogo Street 2a, 04123 Kyiv, Ukraine
Email address: mizgirevka@rambler.ru
Semiconductor nanocrystals, or quantum Dots (QDs), have gained considerable attention due to their
unique size-dependent optical and electronic properties that make them attractive for a wide range of
applications, including biology and nanomedicine. Their widespread use, however, poses urgent questions
about potential toxicity, especially because of their heavy metal composition that could causes harmful
effects to human health and environment. In our study we used Drosophila melanogaster as in vivo model
organism to detect the toxic and genotoxic activity of CdS quantum dots obtained by biological synthesis.
For successful synthesis of such semiconductor QDs, two different organisms – fungus Pleurotus ostreatus
and bacteria Escherichia coli were used as matrices (Borovaya et al., 2015; Borovaya et al., 2014). Their
toxicity was determined by comparing the numbers of imago which were grown in a medium with
biologically synthetized CdS QDs and with CdS salt as a control. Genotoxicity was evaluated by the wingspot assay.
It was established that CdS induces toxic effects in D. melanogaster, whereas CdS QDs are
significantly less toxic in vivo. Thus, on the medium with CdS mortality of flies was 100%, while in the
case with usage of CdS QDs some experimental variants remained alive. The obtained results demonstrate
that biologically synthetized CdS nanoparticles do not induce significant genotoxic or mutagenic effects in
D. melanogaster.
Borovaya M. et al. Biotechnology & Biotechnological Equipment. 2015, 30:1-8,
Borovaya M. M. et al, Reports of the National Academy of Sciences of Ukraine. 2014. 7:145-151.
Acknowledgements: This work was supported by the Specialized Training Department of Kyiv National
Taras Shevchenko University at National Academy of Sciences of Ukraine [grant number 3/28
(2014-2015)].
134
CALLUS FORMATION AND PLANT REGENERATION FROM SHOOT APICAL MERISTEMS,
IMMATURE EMBRYOS AND INFLORESCENCES OF TRITICALE
Pykalo S.V.1, Luzhans'kyj D.V.2
1
The V.M. Remeslo Myronivka Institute of Wheat, National Academy of Sciences of Ukraine,
Ukraine, 08853, v. Tsentral’ne, Myronivka district, Kyiv region
2
Vinnytsia National Agrarian University,
Soniachna Sreet. 3, 21008 Vinnytsia, Ukraine
Email address: pykserg@ukr.net
Triticale (×Triticosecale Wittmack), as a new crop, is becoming more demanded by grain producers
and grain industry. Biotechnological methods of in vitro culture of different explants currently are widely
used to solve applied problems of breeding agricultural crops, in particular, triticale. Today the question of
biotechnologically optimal explants and improving ways to implement their morphogenetic potential is far
from the final solution. The aim of this study was to study processes of callus formation and plant
regeneration of winter triticale genotypes in culture of 3 types of explants (shoot apical meristems,
immature embryos and immature inflorescences).
Four winter triticale genotypes, Baltico, Lider, Isomer and Kvazar, were tested in our experiments.
Callus tissue was obtained from shoot apex explants of 3-day-old sterile seedlings, immature embryos and
inflorescences. For callus induction MS medium supplemented with 2,4-D with a concentration of 2.0 mg/l
was used. Explants were cultured at 26оC in the dark; 40 days later, the callus induction rate was scored.
Induced calli were then transferred to MS medium supplemented with 1 mg/l 6-BAP and 0.5 mg/l IAA. In
3 weeks they were transferred to a modified medium for rooting. After 30 days of culture at 26оC under
3,000 lux of light (16-h photoperiod), the plant regeneration rate was studied. Regenerated plants were kept
at room temperature for 3 to 5 days before transfer into soil. Rooted plants were transferred to in vivo
condition.
The compact, organized tissue was formed from each shoot apical dome after further subculturing.
The immature embryos used as initial explants had a coleoptile, one or two leaf primordia, and a primary
root at an early stage of development. The same explant could produce both a friable and a compact callus,
but only the compact callus was selected routinely for subculture. The young inflorescence segments
callused only after an extended lag period. The compact inflorescence callus contained both surface and
internal meristematic zones. It was established that regeneration frequency of Isomer and Kvazar varieties
from calli derived from shoot apical meristems was the highest (26.3 % and 14.3 %, respectively). In
Baltico and Lider varieties maximum regeneration (30.0 % and 25.1 %, respectively) was recorded on
variants of immature embryos. It was ascertained that for the forms studied frequency of calli formation
and shoot regeneration along with genotypical dependence was also conditioned by explant type. During
regeneration from 1 to 6 shoots were formed per one callus for the varieties tested. Analysis of variance
showed a significant effect (p<0.05) for all factors of this experiment: genotype, explant type and their
interaction on the triticale morphogenesis. Effect of genotype was the highest on the stage of calli
formation. The highest effect of interactions genotype×explant type was observed at the stage of formation
of morphogenic callus. Effect of explant type was the highest at the stage of shoot regeneration.
Thus, the processes of callus formation and plant regeneration for four winter triticale varieties in
shoot apical meristems, immature embryos and immature inflorescences culture were investigated.
Optimized procedure of vigorous plant regenerant production of triticale in the in vitro callus cultures can
be used in cell selection and genetic engineering experiments.
135
THE EVALUATION OF THE CARCINOGENIC RISK UNDER COMBINED EFFECT OF LOW
DOSES OF IONIZING RADIATION AND DRUGS WITH CO-MUTAGENIC PROPERTIES IN
HUMAN CELL
Domina E.A., Pylypchuk O.P.
R.E. Kavetsky Experimental Pathology, Oncology and Radiobiology Institute, National Academy of
Sciences of Ukraine, Vasylkivska Street 45, 03022 Kyiv, Ukraine
Email address: lena.pylypchuk@ukr.net
The problem of tumours radiation genesis, at first sight detached from the attention of experts large
latent period of the disease and compared low doses of radiation of large population cohorts significant
territories polluted with radionuclides due to the Chernobyl accident. Current place in solving/overcoming
this problem should take determination co-mutagenic properties of some drugs that may increase the level
of radiation-induced damage by inhibition of repair processes. But until now in this direction is not
scientific-practical base. This is because the co-mutagens are not detected in genetic screening, as their own
do not cause damage.
The peculiarities the formation of genotoxic effects combined effects of low exposure doses and
medicines (antioxidant - ascorbic acid, calcium antagonists - verapamil) with identified co-mutagenic
properties at the genetic level T lymphocytes of the peripheral blood that are responsible for anti-tumor
defenses. Established that the modification (increase) radiation-induced cytogenetic effects in the culture of
human peripheral blood lymphocytes under the influence of co-mutagens depend on the concentration
medicines and radiosensitivity of cells. High concentrations of potential co-damaging mutagens enhance
the effect of low doses of ionizing radiation, raising the level of aberrant cells. Attention is paid to the fact
that the accumulation of chromosomal aberrations in immune cells is valid biological marker of cancer risk.
Considering the long-term radiological crisis Chernobyl the suggested approach has extraordinary
practical importance, given the carcinogenic dangers low doses of radiation, which may be enhanced by
additional action of drugs co-mutagens. In our view, the implementation in practice of the proposed method
of evaluation of the carcinogenic risk under the given conditions of irradiation and actions of co-mutagens
should be a component of radiogenic cancer prevention.
136
THROMBOPLASTIC ACTIVITY OF FROZEN ERYTHROCYTES
V.V. Ramazanov, Ye. L. Volovelskaya, V.A. Bondarenko
Institute for Problems of Cryobiology and Cryomedicine, National Academy of
Sciences of Ukraine, Pereyaslavskaya Street 23, 61015 Kharkiv, Ukraine
Email address: ramazanov.viktor@mail.ru
Development of transfusion protocols with taking into account the hemostatic potential of blood of
various patients at different diseases has been always remained an actual and unsolved task. In this
connection the taking into consideration and modification of thromboplastic properties of erythrocytes prior
to transfusion could be effective approach for their directed effect on general hemostasis.
Erythrocytes contain intra-erythrocyte and extra-erythrocyte hemostatic factors related to certain cell
structures. It is known that during transfusion of frozen erythrocytes there is inevitable partial hemolysis of
erythrocytes and release into a blood flow of erythrocyte factors of blood clotting, such as ADP and
thromboplastic factor of erythrocytes, as well as anticoagulants. Hemolysis of erythrocytes leads to
exposure of phosphatidylserine molecules on an inner surface of membranes, which activate the clotting
factors, causing an additional activation of thrombinase. In patients with different hemorrhagic diseases
there was found a rise in the percentage of erythrocyte hemolysis in blood which has a compensatory
adaptive character, diminishing the manifestation of hemorrhagic syndrome under conditions of
insufficiency of platelet-vascular hemostasis, since the hemolysate possesses higher thromoplastic activity
if compared with the intact erythrocytes.
In the work there was studied the time of plasma coagulation under the effect of erythroctes frozen in
a liquid nitrogen (-196°C) in combined media with non-penetrating (PEG-1500, dextran) and penetrating
(DMSO, 1,2-propane diol) cryoprotectants. It has been established that frozen erythrocytes washed with
physiological solution as well as their hemolysates reduce the plasma coagulation time in a greater extent
than intact cells.
Thus, during freeze-thawing of erythrocytes in the combined media with penetrating and
non-penetrating cryoprotectants and during washing-out likely occurs the activation of cell factors of blood
clotting as a result the time of plasma coagulation is reduced under the effect of frozen cells and their
hemolysates. In prospects the application of frozen erythrocytes will allow the designing of hemostatic cell
drugs to use them in clinic to improve the blood clotting activity.
137
A NOVEL TISSUE-ENGINEERING MODEL FOR THE EVALUATION OF CELL MIGRATION
AND PROLIFERATION
Rogulska O.Y.1, Petrenko Y.A.1, Podorozhko E.A.2, Lozinsky V.I.2, Petrenko A.Y.1
1
Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine;
2
A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences
Email address: rogulskaya.elena@gmail.com
The use of human cell-based in vitro models instead of animals provides obvious ethical and cost
advantages for systematic, repetitive and quantitative investigation of medications and chemicals generally.
Traditionally, the most of in vitro studies is carried out in 2D environment. However, cells in vivo exist in
3D environment with intricate cell-cell and cell-matrix interactions. Therefore, the development of
appropriate 3D models that more closely mimic native tissue seems to be important for in vitro diagnostics
and drug screening applications as well as for transplantation. Basic fibroblast growth factor (FGF-2) is
well-known regulator of cell migration, survival, division and angiogenesis. The aim of the study was to
design tissue-engineering model based on mesenchymal stromal cells and macroporous collagen scaffolds
for the evaluation of cell migration and proliferation capacity in 3D environment under the influence of
FGF-2.
Mesenchymal stromal cells (MSCs) were isolated from human adult bone marrow and expanded
in vitro until passage 4. Сollagen spongy disks with diameter 6±0.01 mm and thickness 2 mm with
interconnected macropores, prepared by the method of cryotropic gelation, were used as 3D scaffolds. To
prepare the 3D model, the smaller disk (d=3±0.01 mm) was cut out from the middle of the scaffold using
Biopsy punch. Then MSCs were seeded into obtained 3D collagen torus structures by perfusion technique
(2×105 cells/scaffold). Human recombinant FGF-2 (Sigma-Aldrich) in concentrations 3 ng/ml and 15 ng/ml
were used as chemoattractant. To provide the prolonged and site-specific release of factor during 3D
culture FGF-2 was encapsulated into alginate microspheres (d=100±0.2 µm). Alginate microspheres were
injected into clipped collagen core and this construct was placed in the center of torus scaffolds. Empty
collagen core without FGF-2 served as control. After 7 days of 3D culture (37°C, 95% humidity, 5% CO2)
obtained constructs were fixed and analyzed. Cell morphology within scaffolds was estimated by FDA
staining. Cell localization and distribution was assessed using MTT-test. DNA concentration in collagen
cores was determined using the Quant-iT PicoGreen dsDNA assay (Invitrogen).
Seeded into collagen torus scaffolds MSCs attached to pore surfaces and proliferated. When the
inner part of torus scaffolds was filled by 3D collagen-based inserts MSCs started to migrate into the core.
The quantitative evaluation of DNA content on the 7th day of culture revealed that the presence of FGF-2contiaining alginate microspheres within the 3D spongy inserts significantly improved cell migration
capacity. The average number of MSCs within the cores supplemented with 3 ng/ml of FGF-2 was 42%
higher than in control group. FGF-2 in concentration 15 ng/ml was more effective and increased cell
number in collagen-based 3D cores in more than 2 times. The obtained data were further confirmed by
analysis of cell distribution within the collagen scaffolds using MTT-test.
Our results demonstrate that migration and proliferation of MSCs directly dependent on the
concentration of FGF-2 in tissue-engineered 3D environment. Proposed simple 3D model could be used for
evaluating migratory mechanisms, for testing attractants or other chemicals and for revealing of drug
toxicity.
138
USING ELECTROCHEMICAL IMMUNOSENSORS FOR DETECTION OF
IMMUNOGLOBULIN M
Y. Ruban1, K. Shavanova1, N. Shpyrka1, V. Khranovskyy2, R. Yakimova2, N. Starodub1
1
National University of Life and Environmental Sciences of Ukraine,
Heroyiv Oborony Street 15, 03041 Kyiv, Ukraine
2
Linköping University, Sweden
Email address: yuliyaruban24@gmail.com
Immunoglobulin M (IgM) is a class of immunoglobulins, mostly providing primary immune answer.
They are first produced in response to acute infection and appear in the bloodstream providing primary
immunity. The primary immune response is associated mainly with lgM-antibodies (in contrast to the
secondary, which involves mainly antibodies of class IgG). IgM most efficiently bind with complement (to
trigger the mechanism sufficient antigen binding only one molecule of IgM), bacteria cause agglutination,
neutralization of viruses. Considerable increase of IgM in the blood is observed at a number of infections,
in both adults and in newborns. They play an important role in the activation of phagocytosis and pathogen
elimination from the bloodstream. IgM produced by the fetus and has engaged in anti-infection protection.
In utero IgM mothers do not cross the placenta into the baby's blood due to high molecular weight. High
levels of IgM in cord blood - a diagnostic criterion of intrauterine infection of the fetus. Also increase or
decrease the level of lgM in human blood can be a sign of the presence of serious disease or early stage of
cancer.
Standard methods for detection level of lgM in human blood at nowadays is Enzyme-Linked
Immunosorbent Assay (ELISA) and agar gel immunodiffusion (AGID), high-performance liquid
chromatography (HPLC) and turbidimetric/nephelometric analysis, but these assays are themselves
extremely time-consuming and expensive. Also they need a prior treatment of the sample for analysis
needed substance. Alternative to these methods is that various types of biosensors. They are highly
selective and do not require a prior preparation of the sample. Also they can detect the level needed
substances in real-time.
The goal of our work was obtaining electrochemical biosensors for detection level of lgM in samples
and their application in health monitoring using new types of transducers based on ZnO nanoparticles. ZnO
nanostructured materials, such as films and nanoparticles, could provide a suitable platform for
development of high performance biosensors due to their unique fundamental material properties. For
example, ZnO is interesting since it may be functionalized with biomolecules without any oxidation of the
surface and several sensing principles are possible.
The obtained results shown that immunosensor can be used in further experiments for creation novel
electrochemical biosensors. It was demonstrated the sensitivity of analysis by the new developed immune
biosensor is fully meets the practice requirements. In additional to the simplicity of the proposed
approaches for the biosensor creation, its cost and simplicity of the analysis fulfillment there is possibility
to recommend it to the medical practice at the accomplishment of the biochemical diagnostics of the early
stage of infection or disease. In particular, there is very important to mention that all needed components
could be prepared preliminary in stationary laboratory and analysis itself can be carried out directly at
home.
139
THE CONTENT OF 3`-NITROTYROSINE MODIFIED PROTEINS IN RATS`
LEUCOCYTES UNDER EXPERIMENTAL DIABETES MELLITUS
Sabadashka M., Fito A., Sybirna N.
Ivan Franko National University of Lviv,
Hrushevskyi Street 4, 79005 Lviv, Ukraine
Email address: m.sabadashka@meta.ua
The content of 3`-nitrotyrosine modified proteins is a marker of the oxidative-nitrative stress level in
leucocytes and shows the progress of pathological processes, among them diabetes mellitus. In the work,
for the 3`-nitrotyrosine modified proteins level assessment we used the affinity chromatography method
using Cyanogen bromide-activated-Sepharose covalently linked with specific Anti-Nitrotyrosine antibody.
It was found the increase of 3`-nitrotyrosine content in leucocyte lysates under diabetes mellitus, as
evidenced by the growth of protein concentration when eluted by acetic acid. The ratio of proteins with
different molecular mass in fractions, eluted from the column, was shown using SDS-PAGE separation.
The presence of a dominant protein with a molecular mass of about 40 kDa in peripheral blood
leukocytes lysates was observed. Under the experimental diabetes the dominant protein level decreased in
all fractions obtained after chromatographic separation, compared to control. Reduced protein nitration may
indicate the abuse of protein functioning, because of involving of protein nitration to the regulation of their
activity.
The revealed changes in the nitrated protein amount indicate the oxidative-nitrative stress
development in case of diabetes mellitus. The changes undergoes the total 3`-nitrotyrosine modifeied
proteins content and the level of the predominant band, corresponding to a cytoskeleton protein – actin. The
research of proteins nitration targets is urgent and significant problem. In the case of our experiments, the
change in intensity of actin posttranslation modification is a prerequisite of violation of immune cells
locomotor activity, cells` death and may become the target of new diagnostic or therapeutic approaches.
140
LEAF ANTIOXIDANT ACTIVITY OF BIOTECHNOLOGICAL CANOLA (BRASSICA NAPUS
L.) PLANTS RESISTANT TO BOTH GLYPHOSATE AND GLUFOSINATE HERBICIDES
Sakhno L.O., Lystvan K.V., Kuchuk M.V.
Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine,
Zabolotnogo Street 148, 03680 Kyiv, Ukraine
Email address: sakhno@icbge.org.ua
Herbicide-resistant crops have consistently occupied the largest planting area of biotechnological
ones since 1996, a starting year of their use. In 2014 both glyphosinate- and glyphosate-resistant rapeseed
cultivars were grown on 9 million hectars. It was 25 % of this crop total planting area in the world [1].
The tendency of new approach to herbicide-resistant variety creation is observed. It is the
combination of resistance to several herbicides simultaneously [2]. Thus, Triazine resistant Roundup Ready
canola plants were obtained by Monsanto Company (event MONO0894). They possess resistance to
glyphosate and triazins [3].
We have obtained several canola (Brassica napus L.) lines bearing and expressing both synthetic
epsps gene responsible for resistance to glyphosate herbicides and bar gene conferring glufosinate, or
phosphinotricine, resistance. Two spring canola varieties registered in Ukraine were genetically
transformed using Agrobacterium tumefaciens-mediated approach. In order to study of intended
biochemical peculiarities of these plants, total soluble protein content, total radical scavenging activity, and
superoxide dismutase activity were investigated using Bradford’s, DPPH (2,2-diphenyl-1-picrylhydrazyl
radical), and nitroblue tetrazolium assays, respectively. Plants were grown under in vitro conditions. It was
shown that leaf extracts from untransformed plants have no sufficient differences in parameters analyzed in
comparison with ones from herbicide-resistant lines of first and second generations. Biotechnological
plants of second generation were homozygous for genes for herbicide-resistance. Sufficient decrease in
total radical scavenging activity was detected for initial transgenic plants. It may be due to hemizygocity
for heterologous genes. At the same time, no differences were determined for these plants in both total
soluble protein content and superoxide dismutase activity.
In summary, biotechnological homozygous canola line of second generation expressing herbicide
resistance genes, namely synthetic epsps gene and bar gene, has demonstrated no sufficient differences in
leaf antioxidant activity compared with initial plants.
1. http://www.isaaa.org/resources/publications/pocketk/16
2. Vencill W.K. et al., Weed Science. 2012, 2–30.
3. http://roundupreadycanola.com.au/wp-content/uploads/2014/03/What-is-Triazine-Tolerant-RoundupReady-canola1.pdf
141
BACTERIAL EXPRESSION AND ISOTOPE LABELING OF AIMP1/P43 CODOSOME PROTEIN
FOR STRUCTURAL STUDIES BY MULTIDIMENSIONAL NMR SPECTROSCOPY
Vorobyova N. V.1, 2, Kornelyuk A. I. 1, 2
1
Institute of Molecular Biology and Genetics, , National Academy of Sciences of Ukraine,
Akademika Zabolotnoho Street 150, 03680 Kyiv, Ukraine
2
Institute of High Technologies, Taras Shevchenko National University of Kyiv
Pr. Akademika Hlushkova 2, korp.5, 03022 Kyiv, Ukraine,
Email address: vorobyova_natali_0307@ukr.net
AIMP1/p43 protein is a structural component of multisynthetase complex (codosome) in eukaryotes,
which reveals both tRNA binding and cytokine activities. The high-resolution 3D structure of full-length
AIMP1/p43 has not been established yet in either monomer or dimer state.
The goal of the present work is the optimization of protocols of expression and purification of
AIMP1/p43 in Escherichia coli to achieve maximal quantity of the uniformly 15N or / and 13C,15N isotopelabeled recombinant construct. Recombinant protein AIMP1/p43 was obtained from 600 ml of bacterial
suspension on M9 minimal medium with 15NH4Cl and 30 μg/ml kanamycin. The culture was grown at
37оC to an optical density of A600 = 0.5 and synthesis of target protein was induced by adding of 1 mM
IPTG followed by incubation for 4.0 h at 30оC. The cells were harvested by centrifugation. Then the cell
pellet was suspended in 48 ml of cell lysis buffer (50 mM sodium phosphate buffer, pH 8.0, 500 mM NaCl,
10 mM imidazole, 5 mM β-mercaptoethanol, 5 mM DTT, 5 % glycerol, 1 mM lysozyme, 1 mM EDTA,
1 mM PMSF). Ultrasonic lysis of cells was carried out by 6 cycles of 20 s and 20 s interval. The sonicated
cells were clarified by centrifugation at 13,000 rpm for 30 min to micro-centrifuge Sigma 1-13. The
supernatant was applied to the equilibrated Ni-NTA-agarose column, washed with 20 ml of wash buffer
(50 mM sodium phosphate buffer, pH 8.0, 500 mM NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol).
The target protein was eluted with 5 ml of elution buffer: 50 mM sodium phosphate buffer, pH 8.0, 150
mM NaCl, 200 mM imidazole, 5 mM β-mercaptoethanol and collected in 6 fractions of 1 ml stored at 4оC.
The purity of the obtained p43 protein was checked by electrophoresis under denaturing conditions in the
presence of sodium dodecyl sulfate 12 % polyacrylamide gel according Lemmli. Finally yield of
recombinant uniformly 15N-labeled AIMP1/p43 protein is about 8 mg from 800 ml of E. coli cell
suspension.
The prepared sample was used to acquire preliminary heteronuclear NMR spectra and to inspect the
dispersion of NMR resonances. Additionally, we controlled the experimental conditions (buffer,
temperature, pH) from the point of view of long-term protein stability in solution, requested to record the
multidimensional NMR data sets. This study is an initial step to evaluate the high-resolution 3D
AIMP1/p43 structure using modern techniques recently developed in multidimensional NMR spectroscopy.
Two-dimensional 1H-15N HSQC spectra of the 15N-labeled AIMP1/p43 protein demonstrated a good
dispersion of NMR signals coming from amide groups of the polypeptide chain. A comparison with the
previously recorded NMR data for EMAP II protein which constitutes the C-terminal module of
AIMP1/p43 exhibited very good correlation. This strongly supports an idea that the 3D structure of the
C-terminal part of the AIMP1/p43 protein is very similar to the previously determined 3D structure of the
EMAP II protein. The presented NMR data for AIMP1/p43 protein indicated also the presence of some
additional signals in comparison to EMAP II protein. The additional signals detected were well separated
from the signals coming from the C-terminal part and characterized by high amplitude and small linewidth,
and more probably correspond to the amino acids located in α-helical structure and unstructured part of p43
protein. In this regard it is important to note that our secondary structure prediction of AIMP1/p43
(unpublished data) suggested the presence of α-helical fragment at the N-terminal part connected with the
C-terminal domain by long mobile disordered fragment.
142
DEVELOPMENT OF COBALT-CONTAINING RECOMBINANT HUMAN ARGINASE I
EXPRESSION SYSTEM IN THE YEAST HANSENULA POLYMORPHA
Vovk O.I., Senchuk O.Yu., Pereverzeva G.G., Stasyk O.V.
Institute of Cell Biology, National Academy of Sciences of Ukraine,
Drahomanov Street 14/16, 79005 Lviv, Ukraine
Email address: vovk@cellbiol.lviv.ua
Numerous previous researches demonstrated that amino acid deprivation, in particular based on
using of arginine-degrading enzyme recombinant human liver arginase I (rhARG1) which is currently
under clinical trials, is one of the promising approaches of efficient, selective and relatively non-toxic
anticancer therapy against L-Arg auxotrophic tumors. The methylotrophic yeast Hansenula polymorpha as
a useful alternative for bacterial expression systems was utilized by us for secretory overexpression of
(His)6-tagged form of rhARG1. Creating the strains that harbor multiple copies of ARG1 expression
cassettes and designing optimal cultivation conditions allowed us to achieve considerably high yield (up to
15 mg of protein per liter of cultural medium) of the affinity purified His-rhARGI, which was successfully
tested in vitro as a tumor suppressor.
To overcome limitations of rhARG1 non-optimal pH range, recently the substitution of Mn2+ to Co2+
in rhARG1 active center that lowers pH optimum and also stabilizes enzyme at physiological pH was
reported. Introduction of certain point mutations into the second-shell metal ligands of rhARG1 led to a
remarkable optimization of its characteristics for in vivo use (Stone et al., 2010; 2012). The aim of our
work was to obtain overproducer of secreted rhARG1 on the base of yeast Hansenula polymorpha with
replaced Mn2+ for Co2+ in the active center.
Producer of secretory form of rhARG1 was pre-grown on YPD for 24 hours, and then transferred to
induction medium YP, containing 2% glycerol and 100 mM Tris-HCl buffer (pH 6.0) with initial
OD600=0.5. After incubation for 48 hours, methanol has been added to the final concentration of 1% along
with 2 mM CoCl2 and the cells were further incubated for 48 hours. The (His) 6-tagged rhARG1 was
purified in one step from both culture medium and cell-free extract of the recombinant strain by metalaffinity chromatography with Ni–NTA agarose. According to our preliminary data, we observed that
secreted rhARG1 co-incubated with Co2+ exhibits relatively higher catalytic activity as compared to Mn2+containing enzyme at physiological pH, which is important for its therapeutic use.
Our data suggest that Co2+-containing rhARG1 can be effectively produced in yeast in secreted
tagged form suitable for downstream one-step affinity purification.
Stone E.M. et al., ACS ChemBiol. 2010, 5(3):333-342.
Stone E.M. et al., J Control Release 2012, 158(1):171-179.
143
MULTICELLULAR TUMOR SPHEROIDS AS TEST-MODEL FOR FUNCTIONALIZED
CARBON NANOMATERIALS
Yakymchuk O.M.1, Elena M. Perepelytsina1, Mychailo V. Sydorenko1, Olga N.Bakalinska2,
Francesco Bloisi3, Valeria Califano3, Luciano Rosario Maria Vicari3
1
Department of biotechnical problems of diagnostic IPCC, Nauky Street 42/1, 03028 Kiev, Ukraine
2
Chuiko Institute of Surface Chemistry, National Academy of Sciences of Ukraine,
General Naumov Street 17, 03164 Kiev, Ukraine
3
Universita degli studi di Napoli Federico II, Sede di Ingegneria,
Piazzale Teccnio 80, 80125 Napoly, Italy
Email address: ElenaYakymchuk13@gmail.com
The goal of our work was to test the antitumor activity of carbon-protein constructs on multicellular
tumor spheroids model. As carbon part of antitumor agent were used ultra dispersed diamonds, UDDs or
onion like carbon, OLC. It was functionalized with specific antibodies to the tumor receptors (EGFR) and
metabolic drug (Doxorubicin, DOX).
Antitumor properties of UDD-DOX-anti-EGFR construct we determined by culturing it with HT29
cell line in 2D and 3D culture. Cell proliferate activity was defined by counting live and dead cells stained
with trypan blue. Mitochondria's redox potentials and cell survival was determined by MTT-test [Mosmann
T., 1983]. For analyze 3D culture program for Stemi2000 (Zeiss) was used with a conversion formula of
Bijercvig.
It was found that carbon nanomaterials - fullerenes C60, onion-like carbon and ultra dispersed
diamonds impact on cells adhesion and cohesion in culture and formation of multicellular tumor
aggregates. OLCs and UDDs reduce cell adhesion to the substrate and cohesion between cells and stimulate
increasing the number of small cell spheroids. Also we demonstrated that the UDD-DOX construct had
dose-dependent cytotoxic effect on tumor cells. Simultaneously, both antibodies after MAPLE deposition
maintained from 75 to 83% of the functional activity and specificity.
Thus, we can conclude about the prospects of selected methods and approaches for creating an
antitumor agent with capabilities targeted delivery of drugs.These conclusions are supported by statistical
calculations of Pearson`s correlation coefficient between size of MTS and concentration of nanomaterials
and carbon-proteine constructs. It was -0.638 for OLCs, -0.512 for UDDs and -0.99 for UDD-DOX, -0.67
for OLC-DOX, -0.88 for DOX. Obviously, through modification of the physical and chemical properties of
nanomaterials, we have possibility to influence on cell growth, depending on the tasks of biotechnology.
Thus, we can conclude about the prospects of selected biotechnology methods and approaches for creating
and testing an antitumor agent with capabilities targeted delivery of drugs.
144
INFLUENCE OF L-ARGININE AND L-NAME ON THE LEVEL OF OXIDATIVE
STRESS INDEXES IN PLATELETS UNDER EXPERIMENTAL CHRONIC ALCOHOL
INTOXICATION
N.V. Yefimenko, K.P. Dudok
Department of Biochemistry, Ivan Franko Lviv National University,
Hrushevsky Street 4, 79005 Lviv, Ukraine
Email address:nataliya_yefimenko@mail.ru
Hemostasis disturbance is one of the most frequent complications of chronic alcoholism
(Parkhomenko Y. M., 2007). It may be caused by endothelial dysfunction, the upset of thromboformation,
changes in rheological properties of blood etc. The development of pathological conditions is usually
accompanied by shifts in equilibrium between generation systems of reactive oxygen and nitrogen species
(ROS, RNS) and systems of their neutralization.
Thrombocytes fulfill a crucial function in the processes. They contain Ca2+- dependent NO-synthase
that is activated either by its contact with damaged blood vessels or when affected by xenobiotics (ethanol,
acetaldehyde and products of their oxidation), biologically active substances. The process is accompanied
by increased induction of nitric oxide (NO) synthesis from L-arginine. However, in pathological conditions
with hypoxia or ischemia, the level of NO-synthase pathway activity may decrease, whereas the level of
nitrate reductase systems activity, which provides NO formation in stable metabolite (such as NO2- and
NO3-) restoration reactions, increases (Reutov V. P., 2007).
In this case, during the interaction between NO and О2-, peroxynitrite (ONOO-) is formed, which is a
highly reaction cytotoxic powerful pro-oxidant that induces lipid membrane damage and inhibits the
activity of a number of enzymes. The process is called nitrosative stress.
The aim of the research has been: to determine the activity levels of catalase, superoxide dismutase,
as well as the level of lipid peroxidation secondary products (TBA-PP) and NO final metabolite products
under chronic alcohol intoxication, to correct oxidative-nitrosative stress in rat blood thrombocytes by
administering L-arginine, NOS main substrate, or L-NAME (Nω–nitro-L-arginine methylester), NOS
inhibitor.
The model of experimental chronic alcohol intoxication (ECAI) in rats treated with L-arginine and
L-NAME was made according to the described technique (Yefimenko 2007; 2014). The findings suggest
that under ECAI the activity of the enzymes is significantly lowered: catalase – by 29% and SOD – by 40%
compared with the control group. On the other hand, the content of lipid peroxidation products increased.
The content of TBA-PP increased by 75.9%. The total number of nitric oxide metabolites increased by
27.7%. The administration of L-NAME to rats under ECAI caused insignificant normalization of SOD and
catalase activity, as well as TBA-PP and NO2- + NO3- contents. More marked changes occurred when the
rats consumed L-arginine. In ECAI + L-arginine conditions, the content of catalase was higher by 32% and
SOD – by 18% compared with indexes of the group under ECAI without any additional factors.
Our findings led to the conclusion that oxidative stress is relieved when rats under ECAI are treated
with L-arginine.
145
CULTIVATION OF MESENCHYMAL STROMAL CELLS IN ALGINATE MICROSPHERES
MODIFIED WITH DIFFERENT PROTEIN COMPONENTS
Zaykov V.S., Petrenko Yu.A., Pravdyuk A.I., Petrenko A.Yu.
Institute for Problems of Cryobiology and Cryomedicine, Department of Biochemistry,
Pereyaslavskaya Street 23, 61015 Kharkiv, Ukraine
Email address: vedenii.zaikov@gmail.com
Encapsulation of mesenchymal stromal cells (MSC) in alginate spherical carriers is a promising area
of biotechnology, tissue engineering and transplantology. MSCs during cultivation within alginate saved
spherical morphology and didn’t represent proliferating activity due to weak adhesive properties of
alginate. Modification of alginate, that can provide adhesion and proliferation of cells, will contribute to
more widespread use of encapsulated MSCs.
In this study the effect of the inclusion with different protein components and preseeded gelatin
microbeads into the alginate spherical carriers on viability, metabolic activity and morphology of
encapsulated MSCs were investigated.
For encapsulation suspension of MSCs were mixed with the 1.2% sodium alginate solution and
dropwisely added into the 100 mM CaCl2 solution for future polymerization. For modification of alginate
carrier solution of human blood plasma (BP), platelet lysate (PL), gelatin in concentration 10-50% were
added into alginate(-cells) solution before polymerization. For gelatin microbeads preseeding suspension of
MSCs were mixed with 250-300 beads and placed into the plastic syringe filed with 2 ml of cultivation
medium for 24 h. Preseeded microbeads were encapsulated with and without 25% BP as described
previously.
It was shown that the optimal modification concentration of the BP, PL and gelatin is near 25-30%.
Protein components are uniformly distributed throughout the whole volume of the carrier according to
Amido Black staining. Encapsulated MSC’s were cultivated in standard conditions. At 1, 3, 7 and 14 day of
cultivation viability by MTT-assay and metabolic activity by AB-assay were measured. In addition, general
morphology by light and fluorescent microscopy was determined.
It was shown that encapsulated MSC’s and preseeded gelatin microbeads saved high viability during
14 days of cultivation. Metabolic activity of MSC’s, encapsulated into non-modified alginate and alginate
(control group)/gelatin spheres, didn’t change during cultivation. MSC’s within PL modified spheres
represented rapid increasing of metabolic activity at the day 3 and decreasing near to control level at the
day 7. No morphology changes of cells were observed. MSC’s within BP modified spheres and preseeded
gelatin microbeads represents stable increasing of metabolic activity level during cultivation. This could be
an evidence of proliferative activity of encapsulated cells. Some morphology changes were observed: large
number of cell saved spherical morphology, but 15-30% of cells change spherical shape to fibroblastic
form. In addition threadlike formations of cells were observed.
Modification of alginate with proteins can be considered as a new approach for usage of
encapsulated MSCs in regenerative medicine.
146
STRUCTURAL-AND-FUNCTIONAL CONDITION OF BLOOD CELL MEMBRANES IN
PATIENTS WITH RECTAL CANCER
Olga V. Zaytseva, Vladimir G. Knigavko, Liliya V. Batyuk, Marina A. Bondarenko
Kharkiv National Medical University, Lenina Street 4, 61022 Kharkiv, Ukraine
Email address: liliya-batyuk@mail.ru
A topical problem is proof of the existence of a definite focus of the protective influence of the
antioxidant system on the membranes of blood cells. The objective of the study was investigating the
features of the structural-and-functional condition of the membranes of blood cells of cancer patients for
possible use in prognosticating disease severity and effectiveness of adequate treatment.
The structural-and-functional condition of the membranes of red blood cells and lymphocytes
(micro-setting polarity, fluidity and embedment in the lipid matrix) has been investigated in patients of 35
to 78 years old (n=68) with rectal cancer (RC) in different stages of tumour process development confirmed
by clinical and histomorphological methods. The fluidity of plasmatic membranes of lymphocytes and red
blood cells was determined by the pyrene excimerisation coefficient, being the ratio of the number of
pyrene excimers and that of its monomers. This coefficient, which changes in proportion to fluidity, was
determined in the zone of protein-lipid contacts in the lipid bilayer. The fluorescence intensity of 1,8-AHC,
changing inversely proportional to the surface charge of lymphocyte membranes, was studied using the
LIUMAM-I3 microscope. The polarity of the probe micro-setting in the membrane was evaluated by the
probe fluorescence ratio at λtest=372 nm and fluorescence at λtest=393 nm. The degree of protein embedding
in the lipid bilayer was determined by the quenching of fluorescence of aromatic amino acids (tyrosine and
tryptophan) due to the radiationless transfer of energy to the pyrene molecule at λexcit=282 nm.
A significant reduction in membrane fluidity was identified in the zones of protein-lipid contacts and
in the lipid bilayer of RC patients as compared to that of the reference group of conventionally healthy
persons, thus reflecting the nonspecific tumour growth component. Analysis of changes in the structural
parameters of membranes is indicative of an increase in their stiffness, rigidity and negative charge on the
surface of the plasmatic membrane, thereby preventing adsorption thereon of the anion probe 1,8-AHC.
The change in the physico-chemical condition of the lipid matrix is a possible cause of cell transfer to a
new metabolism level, thus reflecting immune system imperfection in cancer patients, which is one of the
distinctions of the given pathology. The established increase in viscosity of plasmatic membranes is
indicative of the possible disturbance of links between cells. This results in development of
microcirculatory and immune destruction, and aggravates the condition of oncology patients.
147
CHANGES IN ERYTHROCYTE SURFACE MARKER CD44 UPON
TEMPERATURE PRESERVATION WITH DIFFERENT CRYOPROTECTANTS
LOW
Nina Zemlianskykh, Lubov Babijchuk
Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine,
Pereyaslavskaya Street, 23, 61015 Kharkiv, Ukraine
Email address: nzemliansky@gmail.com
Changes in surface markers associated with decreased stability and viability of cells reveal during
aging in vivo and reflect structural and functional adjustments of plasma membranes. In stressful conditions
related to low temperature storage of cells changes in surface markers can reflect rearrangements of
protein-protein and protein-lipid interactions in the membranes, which exert the stability of cells in extreme
conditions. It is known that in erythrocytes stability and mechanical and elastic properties of the membrane
are largely dependent on cytoskeletal proteins associated with point contacts to individual integral proteins
or protein complexes, including adhesion molecules CD44. In this regard, the study of changes in surface
structures of plasma membranes caused by stress factors can contribute to a better understanding of the role
of membrane structural and function rearrangements in mechanisms of cell stabilization or injury upon the
extreme conditions.
The aim of this study was investigation of changes in the parameters of CD44 marker in erythrocytes
exposed and cryopreserved with intra- and extracellular cryoprotectants (glycerol and PEG-1500).
Erythrocytes were incubated with cryoprotective solutions, which included 20% glycerol and PEG-1500.
Samples were frozen by immersion in liquid nitrogen (-196oC) and warmed in a water bath at 42oC.
Assessment of CD44 marker parameters in erythrocyte suspension was performed on the base of high
affinity binding CD44-FITC by erythrocytes with the following flow cytometry measurement (FACS
Calibur Becton Dickenson, USA). Data was analyzed using the software WinMDI 2.8.
It was revealed that only a prolonged exposure (20 h) to cryoprotectants glycerol or PEG-1500
induced changes in erythrocyte membrane–cytoskeleton complex followed by a decrease in the amount of
CD44-positive cells. Glycerol but not PEG-1500 also caused a slight but significant decrease in the CD44
expression. In contrast, after freeze-thawing a short exposure of cryopreserved erythrocytes to
physiological temperature led to a significant decrease in the marker expression and in the amount of
CD44-positive cells. In cryopreserved erythrocytes these changes were more pronounced in the presence of
PEG-1500 than in the presence of glycerol. Differences in the time of the appearance of changes in the
CD44 parameters in cryopreserved erythrocytes and erythrocytes exposed to cryoprotectants suggest that
these changes could be caused by different reasons. Removal of the cryoprotectants, accompanied by the
loss of damaged cells during washing, resulted in a recovery of the CD44 parameters in cryopreserved
erythrocytes. These results indicate that the observed changes concern only a part of the cells in suspension
and are associated with instability of erythrocyte population with altered СD44 characteristics.
Eventually the study of the adhesive properties of cryopreserved erythrocyte stipulated by the
presence of adhesion molecule CD44 will allow characterizing the safety of these cells for the patients
upon transfusion in order to avoid the risk of vaso-occlusive processes.
148
INFLUENCE OF HYPOXIA-INDUCED FACTOR HIF-1 α ON THE MYELIN STATE IN
THE CENTRAL NERVOUS SYSTEM
Zhdankin A. Ye.
Oles’ Hochar Dnipropetrovsk National University, Gagarin Street 72, 49010 Dnipropetrovsk, Ukraine
Email address: science_mbp@mail.ru
The myelin is a mandatory component of the nervous tissue, a key feature of which is the nervous
excitement integration. Due to the presence of specific hydrophobic proteins, myelin sheath stabilizes the
axons, adjusting the spread of an action potential with collateral (first neuron's axon → linker → second
neuron's axon) and orthodromic (catfish neuron dendrites → cell's somma → axon → effector cells)
mechanisms.
In the context of focal ischemia vascular endothelial cells synthesize HIF-1 α (hypoxia-inducible
factor 1-alpha, a factor induced by hypoxia) in high concentrations. Using polymerase chain reaction (PCR)
established that HIF-1 α is encoded by the gene hif1A, located on the 14th human chromosome and in
rodents – on 12th chromosome (Zhu T. et al., 2014; Wang G.L. et al., 1995). Hypoxia-induced factor at low
concentrations increases the resistance of cells to hypoxia, but it reduces the excess metabolic potential of
the latter, which leads to ischemic tissue necrosis (Wang G.L. et al., 1995).
Oligodendrocytes forming the myelin sheath around axons, in contrast to other types of glial cells are
most sensitive to the action of HIF-1 α, because they haven't the relevant defense mechanisms to maintain
the level of adenosine triphosphate (ATP) and other macroergs, causing their death can occur by necrosis,
at least – apoptosis. A significant loss of this cell type is considered one of the important reasons of
demyelination. At low concentrations of HIF-1 α, on the contrary, it increases the oligodendroglial
resistance to ischemia via activation of p38MAPK/MSK-1, which launches MAP kinase signaling pathway
(Zhu T. et al., 2014).
HIF-1 α at concentrations greater than 10 times the norm, irreversibly blocks oligodendroglial
livelihoods, which in turn stops the myelination. Sphingomyelinase activated by HIF-1 α, destroy the
sphingomyelin frame of myelin sheaths. An activity of this group of enzymes is much higher in the central
nervous system (CNS) than in the PNS. From this we see that the HIF-1 α has an indirect effect on the
nerve fibers' demyelination (Sim J., Johnson R.S., 2014). Thus demyelination can be regarded as a
pathological response in the nervous system at the level of "glial-neuronal-myelin complex" in response to
ischemia or other endo- and exogenous damaging factors (Wunderlich M.T., et al., 2004).
149
POSSIBILITY OF INTERACTION BETWEEN SIGNAL SYSTEMS WHEN INDUCING
CELL RESISTANCE TO COLD STRESS
Zinchenko V.D., Goriacha I.P., Golovina K.M., Volovelskaya E.L.
Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine,
Pereyaslavskaya Street 23, 61015 Kharkov, Ukraine
Email address: vd_zin@mail.ru
Recent studies have demonstrated the reactive oxygen species (ROS), being metabolic products of
living systems, to take an active part in different biological processes, including normal cell growth,
induction and transformation of transition states, ageing and programmed cell death. The ozone as the ROS
can adversely affect the living systems at high doses, but at low ones it reveals a positive effect on
physiological functions both at the level of certain cells and the whole body as well. We used here a weak
oxidative stress, caused by means of low-dose ozone for inducing cell resistance to impact of temperatures
lower than physiological ones. The low-dose ozone effect on the Saccharomyces cerevisiae yeast cell
resistance before freeze-thawing (-196 – 30°C), as well as the capability of sheep red blood cells to form
rosettes with human lymphocytes after storage under hypothermic (2 – 4°C) conditions were investigated.
The freeze-thawing (-196 – 30°C) of Saccharomyces cerevisiae cells in physiological saline leads to
membrane damaging in 65.6±1.6% of cells. If cells were treated with ozone in a dose of 29 pmol O 3/106
cells before freezing, the membrane damage after freeze-thawing was observed in 16.1±0.8% of cells. In
this experiment the Saccharomyces cerevisiae cells acquired an induced resistance to freeze-thawing in a
state of adaptive response to oxidative stress. The effect pronouncement depended on the degree of
stress-factor impact, i. e. homeostasis disorder extent in living system.
Sheep red blood cells were treated with ozonated physiological saline with ozone content of
0.16 mg/l and stored for 12 weeks under hypothermic conditions at 2 – 4°C in different media: 1) 0.9%
sodium chloride; 2) 7% sucrose solution; 3) glucose-citrate Alsever’s solution; 4) 5% mannitol solution; 5)
10% dextran one. After storing we studied the red blood cell osmotic resistance by measuring their osmotic
fragility, as well as the capability of rosette formation with human lymphocytes. The ozone-treated red
blood cells lose an osmotic resistance in a less degree than the non-treated ones. Those ozone treated cells
in the medium (4) preserve their ability to form rosettes with human lymphocytes after 12 weeks of storage.
Our findings with both red blood cells and yeast are explained by the existence of general mechanisms of
perception and a rapid body’s response to external effect: any organism has a set of defence reactions. Both
non-specific and specific reactions typical for this stress effect occur as a response to the effect of
unfavourable environmental factors. Namely non-specific defence reactions are the most economic and
universal way to preserve the balance of biosystem with external environment, ensuring the reliability of
their existence under rapidly changing conditions. At this stage of response the living system, affected by
one stress-factor may augment its capability to resist other adverse factors: a cross adaptation. A weak
oxidative stress is an alerting signal for adaptive systems of a cell, the response on which foresees a
genetically determined protection against adverse environmental factors.
150
CORRECTION OF GUT DYSFUNCTION IN CHRONIC HIV-1 INFECTION
K. Gorobchenko
Sumy State University, Medical Institute
Email address: horobchenko2013@yandex.ru
HIV-1 infection results in marked immunologic insults, including pronounced CD4+ T-cell loss
from gut lymphoid tissue, and structural damage to the intestinal mucosa following dysfunction of the
gastrointestinal system, including compromised barrier function. Increased intestinal permeability and
microbial translocation promote systemic immune activation, which is implicated in disease progression.
While the development of highly active antiretroviral therapy (HAART) has been a major advancement in
the treatment of HIV-1 infection, the need for novel complementary interventions to help repair intestinal
structural and functional integrity remains unmet. Known properties of pre-, pro-, and synbiotics suggest
that they may be useful tools in restoring normal intestinal flora. Our study goal was to determine whether
the use of a synbiotic, probiotics or a prebiotic can recover normal intestinal flora in chronic HIV-1
infection.
A randomized, double-blind placebo-controlled study was performed. 34 аntiretroviral-treated HIVinfected subjects were subgrouped and assigned to receive a synbiotic (probiotics, prebiotic) or a placebo
throughout 6 weeks, depending on changes of intestinal microflora.
We had no reports of serious adverse events. From baseline to 6 week, the synbiotic formulation
(probiotic and synbiotic groups) showed an increase in total bacterial load in stool (p=0.05). The probiotic
interventions resulted in significantly elevated levels of beneficial bacteria load (such as Bifidobacterium;
p=0.05) and a decrease in harmful bacteria load (such as Clostridium; p=0.063).
Synbiotic (probiotic) treatment for 6 weeks can successfully augment the levels of probiotic species
in the gut during chronic HIV-1 infection. These findings may help inform future studies aimed at testing
pre- and probiotic approaches to improve gut function and mucosal immunity in chronic HIV-1 infection.
151
EFFECT OF COOL STRESS ON PLANTS
EXPRESING HYBRID GENE -9-ACYL-LIPID DESATURASES
CYANOBACTERIA
Kyrpa-Nesmiian T.М.,
Institute of Cell Biology and Genetic Engineering NAS of Ukraine,
Zabolotnogo str. 148, Kiev 03680, Ukraine
Email address: t-kirpa@ukr.net
During recent decades, we see significant climate changes that negatively affect the crop capacity of
many kinds of plants. First of all, plant resistance to fluctuations in temperature depends on the adaptive
capacity of membrane lipids. At normal physiological temperature polar glycerolipid contains only saturated
fatty acids and do not form bilayer, the basic structure of biological membranes. Desaturases are enzymes
that contribute to the formation of double relations in fatty acids of membrane lipids thus increasing their
viscosity and enhance the fluidity, which increases the adaptive capacity of membranes lipids. The study of
biochemistry and molecular biology of desaturases opens wide possibilities for biotechnology and
agriculture, because it makes it possible to genetically engineer and design the varieties and species of plants
which could endure temperature limits of a certain climate zone. In our laboratory we constructed the vector
containing the gene for acyl-lipid desaturases desC (Δ9) Synechococcus vulcanus, whose sequence was fused
with the sequence of the lihenases thermostable Clostridium thermocellum licVM3 reporter gene controlled
by 35S promoter . Genetic transformation of Nicotiana tabacum (cv. Wisconsin) with this consrtution was
held and plants which, as it has been proved, had the expression of transgenes. In these plants analysis of the
spectrum of fatty acids was studied and an increase in the proportion of linoleic acid was detected. Also,
obtained plants were previously tested for resistance to hypothermia and showed an increase of superoxide
dismutase enzyme activity compared to control plants. As a control wild type Nicotiana tabacum plants and
transform Nicotiana tabacum, carrying the gene gfp: licBM3 were used.
In this paper the level of electrolyte yield has been tested as an indicator of membrane damage. Electrolytes
ensures the sustainability of the osmotic pressure of body fluids, enzymatic reaction is very dependent on the
ion-water balance (transition ATP ADP and vice versa, enolazy activation, oxidative phosphorylation in the
mitochondria, etc.). The yield of electrolytes in plants was measured after exposure to hypothermic stress (20
min. at + 5C after 20 min. at 0 C and 20 min at – 20 C). In plants expressing the hybrid gene desaturases the
tendency of the reduction of the yield of electrolytes compared to control - the wild-type Nicotiana tabacum
and transform Nicotiana tabacum, carrying the gene gfp: licBM3 plants.
The work was executed with support of grant of NASU UkrISTEI №0115U004171
1.Gerasymenko I.M., Sakhno L.O., Kyrpa T.М., Ostapchuk A.N., Khadjiev T.A., GoldenkovaPavlova I.V., Sheludko Y.V. (2015) Characterization of Nicotiana tabacum plants expressing hybrid genes
of cyanobacterial Δ9 or Δ12 acyl–lipid desaturases and thermostable lichenase. Russian J. Plant Physiol.
62(3), 283-291.
152
SIGNALLING FUNCTION OF β-CATENIN DURING CARDIOGENESIS
AND UDALT HEART ADAPTATION
Piven О. О., Palchevska O.L., Balatskii V.V., Маcewicz L.L., Lukash L.L.
Іnstituteof Molecular Biology and Genetic, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
E-mail: o.o.piven@imbg.org.ua
Heart failure is a major concern in modern society. According to the report by the European Heart
Network and European Society of Cardiology in 2012, cardiovascular diseases cause more than 4 million
deaths every year, 1.9 million of these are within the European Union (EU) countries (European Heart
Network and European Society of Cardiology; 2012). Unfortunately, the death rate from heart disease and
circulatory system in Ukraine is 2-4 times higher than in the EU and the world, in our country die from these
diseases happens more often and at early age. According to the State Statistics Service of Ukraine in 2013,
about half of all deaths were due namely cardiovascular diseases.
The spread of cardiovascular diseases, their threat to the health and socio-economic weighty burden
cause considerable interest of scientists to solve this problem. Recently, relevant researches are not only new
methods of diagnosis and treatment of cardiovascular disease, but also clarify the mechanisms of their
occurrence and course. Adapting adult myocardium to physical and physiological (such as high blood
pressure) loads as well as to age-related changes accompanied by significant morphological rearrangements
of heart tissue and changes at the molecular level. All of these adaptive changes may lead to the hearts
disease and hearts function violation. Interestingly, while the hypertrophy is considered as one of the major
risk factors of heart disease, along with aging, obesity, unhealthy lifestyles, diabetes, anemia and high blood
pressure. Activation of multiple signalling pathways is required for effective adaptation of adult myocardium
to the physical or physiological stress.
The aim of our work is to study the regulation of pathological adult heart remodeling by β-catenin.
Such studies are very important not only for basic biology of the heart but also for using this knowledge in
medicine. New data on the signalling role of β-catenin in the early or late stages of pathological remodeling
of the heart (fibrosis, hypertrophy, cardiomyopathy and other) is absolutely necessary for the development of
new methods of heart diseases diagnosis, prevention and treatment.
With conditional knockout mice using we showed that complete loss of β-catenin gene in embryonic
heart is lethal in late embryogenesis or early after birth. Deficiency of β-catenin gene leads to disruption of
fetal (hypertrophic) genes expression even in the absence of stress in the aged 1, 3 and 6 months. Also, we
have shown that β-catenin haploinsufficiency causes delays of heart in adult animals but also inhibits
canonical Wnt-signaling. Interestingly we found that activation of the canonical Wnt-signaling in adult
myocardium in adapting to long-term physical activity and high blood pressure. Our finding indicates the
importance of the signaling function of β-catenin in adult myocardial adaptation to stress.
153
PLANTS NICOTIANA TABACUM, EXPRESSING Δ12-ACYL-LIPID CYANOBACTERIA
DESATURASE DURING OSMOTIC STRESS
Kyrpa-Nesmiian T.,
Institute of Cell Biology and Genetic Engineering NAS of Ukraine, Zabolotnogo str. 148,
Kiev 03680, Ukraine
Email address: t-kirpa@ukr.net
Correlation of saturated and unsaturated fatty acids affects the physical properties of the cell membrane
that can increase plant resistance to low temperature and osmotic stress. Desaturases are enzymes that
contribute to the formation of double ties in fatty acids. The reaction of desaturation of fatty acids in
glycerolipids is one of the most important and necessary to support the physical properties of membrane
lipids. Under normal conditions polar glycerolipids which contains only saturated fatty acids do not form
bilayer, the basic structure of biological membranes, but the increase of proportion of unsaturated fatty acids
contributes to plant resistance to low temperature and to osmotic stress. The study of biochemistry and
molecular biology of desaturases opens wide prospects in biotechnology and agriculture because it makes the
construction of genetically engineered sorts and species of plants capable of withstanding a temperature
restriction and osmotic stress of a climatic zone possible. In this work we used acyl lipid desaturase desA
(Δ12) gene of Synechocystis sp. PCC 6803, reporter gene fused with thermostable Clostridium thermocellum
lihenase licBM3 under the control of the constitutive 35S promoter.
The genetic transformation of Nicotiana tabacum (cv. Winsconsin) using this constraction was done and
plants with shown and proven presence and expression of transgenes were obtained. The control plants were
of the same line planted on MS medium. For analysis of the resistance of plants to osmotic stress MS medium
with the addition of 200 mM concentration and 100 mM mannitol was used. Plants were weighed then
planted on the meddium and in 3-4 weeks were analyzed: biomass growth and activity of the enzyme
superoxide dismutase, which is responsible for neutralizing free radicals that occur when membranes are
damaged, was measured. The results showed that the plants biomass growth significantly decreased when the
plants were grown on medium with 200 mM mannitol, but the activity of the enzyme superoxide dismutase
increased compared to control. Cultivation on the medium with 100 mM mannitol showed a tendency for
increase of biomass growth compared to plants grown in medium containing mannitol 200 mm but compared
to control plants there was less biomass increase. When growing plants on MS with the addition of 100 mM a
slight decrease in SOD activity was observed, control plants had the smallest activity of the enzyme. These
data suggest that growth on medium with the addition of 200 mM mannitol inhibits the vital functions of all
studied transgenic plants, although not leading to fatal damage of cell membranes. Growing plants in an
environment with 100 mM mannitol showed a tendency to plant biomass increase and SOD activity
reduction.
The work was executed with support of grant of NASU UkrISTEI №0115U004171
154
IN VITRO ANTIPROLIFERATIVE ACTIVITY OF NOVEL
BENZIMIDAZOTHIAZOLE DERIVATIVES
Kostenko O.M., Kryvorotenko D.V., Negrutska V.V., Alexeeva I.V., Dubey I.Ya.
Institute of Molecular Biology and Genetics, NAS of Ukraine, 150 Zabolotnogo str.,
03680 Kyiv, Ukraine
e-mail: dubey@imbg.org.ua
Many anticancer drugs target the system of nucleic acids biosynthesis. Usually compounds of this type
are heteroaromatic molecules able to bind to nucleic acids, enzymes like DNA or RNA polymerases,
topoisomerases, etc., or to replication, transcription or DNA relaxation complexes. We have previously found
that 2-arylidene-substituted benzimidazo[2,1-b]thiazole derivatives are highly efficient inhibitors of
transcription in vitro active at low micromolar concentrations: 6 compounds demonstrated IC50 values in the
range 1.6–27 µM [1]. Transcription is a key process required for cellular growth and replication, and its
specific inhibition is a way to suppress viruses, bacterial and cancer cells. In the present work we studied a
series of the most efficient transcription inhibitors of this class as potential antitumor agents. These planar
molecules contain tricyclic benzimidazothiazol-3-one core and aryl fragment with varied substituents which
can ensure the efficient ligand binding to its biological target.
Antiproliferative activity of compounds was tested in vitro in HeLa and MCF-7 (human breast
carcinoma) tumor cell cultures. The cells were cultivated in DMEM medium containing 10% Fetal Calf
Serum. Tested compounds were dissolved in dimethylsulfoxide and applied in the concentration range 400
pM – 40 µM using a serial dilution approach. The cultures were then incubated at 37 oC for 3 days. Cell
cultures containing DMSO were used as controls. Quantitative analysis was performed using a standard
colorimetric MTT assay. Cell inhibition levels were plotted vs. drug concentrations, and EC50 value (i.e.
concentration resulting in 50% cell inhibition) was determined for each tested compound with Origin 6.1
software. Two independent experiments were performed for each tested compound.
Four derivatives were found to exhibit antiproliferative activity in vitro at low micromolar
concentrations. Their EC50 values were in the range 7-12 µM for HeLa cell line. MCF-7 cells were more
susceptible to tested substances: in this culture three compounds demonstrated EC50 ranged from 3.7 to 4.3
µM.
Active antitumor derivatives demonstrated medium to high level of transcription inhibition, although
there was no direct correlation between IC50 values obtained in the transcription assay and antiproliferative
activity. It is also worth mentioning that tested compounds did not exhibit significant in vitro inhibitory
activity against some other key enzymes of nucleic acids biosynthesis, including topoisomerase I and
telomerase.
1. Palchykovska L.G. et al. Biopolym. Cell, 2010, 26, 508-511.
155
BINDING OF CATIONIC PORPHYRIN-BASED TELOMERASE INHIBITORS
TO DUPLEX AND QUADRUPLEX DNA: A FID STUDY
Didan Yu.V.1,2, Dubey L.V.1, Negrutska V.V.1, Dubey I.Ya.1
1
Institute of Molecular Biology and Genetics, NAS of Ukraine, 150 Zabolotnogo str.,
03680 Kyiv, Ukraine
2
Radboud University Nijmegen, 4 Comeniuslaan, 6525 HP, Nijmegen, The Netherlands
e-mail: v.v.negrutska@imbg.org.ua
G-quadruplexes (G4) are specific four-stranded DNA structures formed by some guanine-rich
sequences. The sequences able to fold into G4 are predominant in telomeric DNA, although they also have
been found in a number of proto-oncogene promoters. Stabilization of these structures with small molecules
can inhibit telomerase, an enzyme responsible for telomere elongation upon cellular division. It is highly
active in a majority of tumor cells, in contrast to normal ones, so G4-DNA and telomerase are now
considered as promising targets for the development of novel anticancer drugs.
Most specific G4 ligands contain large heteroaromatic systems allowing the efficient π-π-interaction
with guanine quartets. Tetracationic porphyrin TMPyP is one of the most efficient G4 ligands and telomerase
inhibitors with anticancer properties. We have used its tricationic analog TMP3 to attach additional functions
potentially able to enhance its binding to G4 due to intercalation or electrostatic interactions. We have
synthesized TMP3 derivative with cationic trimethylaminopropyl group (TMP3-TMAP), conjugate of TMP3
with intercalating agent imidazo[4,5-b]phenazine (TMP3-ImPhz), and their Zn(II) and Mn(III) metal
complexes. These porphyrin compounds demonstrated antiproliferative activity and efficient telomerase
inhibition in vitro at 2-10 µM concentration [1].
In this work we studied the binding of porphyrin derivatives to double-stranded DNA and Gquadruplex formed by model oligonucleotide d[AGGG(TTAGGG)3], a fragment of human telomeric DNA
(Tel22, PDB code 143D). Their binding affinity to G4 and dsDNA was determined using FID (Fluorescent
Intercalator Displacement) assay based on the competitive displacement of a light-up probe like Thiazole
Orange from its complexes with DNA by tested ligands resulted in concentration-dependent fluorescence
decrease [2].
All compounds showed strong affinity to G4-DNA. Dissociation constants Kd were in the range (0.7–
46)x10-6 M. The most efficient G4 binders were TMP3-TMAP, TMP3 and (Mn)TMP3-ImPhz (Kd 0.7, 1.4
and 3.8 µM, respectively). Porphyrin complexes with dsDNA were much less stable, with dissociation
constants ranged from 12 to 360 µM. All ligands bind to G4-DNA much stronger than to duplex DNA, i.e.
they have a good selectivity for quadruplex over duplex DNA. This selectivity is required for highly specific
telomerase inhibition. For example, the ratio between dsDNA and G4-DNA constants is 5 for TMP3-ImPhz
and 8 and 11 for its Zn and Mn complexes. TMP3 and metal complexes of TMP3-ImPhz conjugate are more
selective to G4 than classic TMPyP, although their binding affinity to quadruplex DNA is lower. The most
selective G4 ligand is TMP3-TMAP (duplex/quadruplex Kd ratio 40) which at the sae time is the most affine
among the studied G4 ligands. Both G4 affinity and selectivity, as well as in vitro telomerase inhibition
activity of this porphyrin derivative, are higher than those observed for TMPyP.
Thus, the studied cationic porphyrins have strong binding affinity and selectivity for quadruplex DNA.
Due to G4-DNA binding they are efficient telomerase inhibitors and demonstrate good antiproliferative
activity. So these compounds are promising agents for antitumor therapy.
1. Negrutska V.V., Dubey L.V., Ilchenko M.M., Dubey I.Ya. Biopolym. Cell, 2013, 29, 169-176.
2. Monchaud D., Teulade-Fichou M.-P. Meth. Mol. Biol. 2010, 608, 257-271.
156
FLUORESCENT LABELING OF BIOMOLECULES WITH NOVEL
3-HETARYLCOUMARIN DERIVATIVES
Kuziv Ia.B., Dubey I.Ya.
Institute of Molecular Biology and Genetics, NAS of Ukraine, 150 Zabolotnogo str.,
03680 Kyiv, Ukraine
e-mail: dubey@imbg.org.ua
Fluorescence-based techniques employing appropriate fluorescent reporter groups belong to the most
informative approaches to detect biomolecules and study their cellular transport or interactions with other
biological and synthetic molecules. There is a wide variety of the dyes with intense green or red fluorescence.
However, the number of labels emitting in blue spectral region is quite limited. Most of them are based on
coumarin core.
Like other non-radioactive labels, coumarins can be attached to proteins, oligonucleotides or sugars via
the reaction of corresponding active derivatives with amino, carboxyl or thiol functions present (or
chemically or enzymatically introduced) in biomolecules. Carboxy-modified dyes are perhaps the most
suitable reagents for conjugation with amines via the formation of amide bond.
Many hetarylcoumarins are known to exhibit bright fluorescence. We synthesized a series of new 3thiazolyl- and 3-furylcoumarins bearing a COOH group on aliphatic linkers of various lengths (1-3 atoms)
attached to hetaryl moieties. In addition, coumarin core of the reagents contained at C-7 position
fluorescence-enhancing substituents – hydroxy, methoxy or acetoxy group.
These reagents were successfully used to introduce fluorescent labels with intense blue emission into a
number of biomolecules Carboxyl functions of coumarin derivatives were first activated with dicyclohexyl
carbodiimide and N-hydroxybenzotriazole (HOBT) in dry dimethylformamide (DMF). Resulting HOBTactivated esters smoothly reacted with aliphatic amines in aqueous DMF (pH 8-8.5) at room temperature with
good yields.
We prepared and characterized hetarylcoumarin-labeled model peptides (Gly-Gly, Leu-Leu-Leu),
aminosugar (glucosamine), nucleosides (thymidine, deoxycytidine) and oligonucleotides – triadenylate and
pentadecathymidylate dT15. Peptides and glucosamine contain reactive amino groups, whereas aminohexyl
linker was introduced at the 5'-end of oligonucleotides during their chemical synthesis to allow the selective
coupling with activated esters.
Unprotected deoxycytidine selectively reacted with activated esters at exocyclic amino group. At the
same time, primary 5'-OH group of nucleosides which is sufficiently nucleophilic was also able to react with
HOBT esters of the dyes at 40-45oC. This result suggests the possibility of direct labeling of nucleosides
without their preliminary functionalization with reactive groups.
Labeled products were purified by chromatographic methods (adsorption, reverse-phase or ionexchange chromatography, depending on the biomolecule). dT15 conjugate was additionally purified by gel
filtration on Sephadex G-25.
The fluorescence spectra of all dye conjugates contain intense peaks of 3-hetarylcoumarin
chromophores with emission maxima in the range 420-450 nm.
Thus, we synthesized new coumarin-based reagents and developed efficient coupling conditions for the
preparation of fluorescently labeled biomolecules. The efficiency of these reagents was fully confirmed by
the synthesis of fluorescent conjugates of a variety of low- and high-molecular natural and synthetic
compounds. The proposed derivatives may be useful reagents for biotechnology and biomedical research.
157
HIGHLY SENSITIVE VISUALIZATION OF NUCLEIC ACIDS IN ELECTROPHORETIC GELS
WITH NEW CYANINE REAGENTS
Didan Yu.V.1,2, Negrutska V.V.1, Kryvorotenko D.V.1, Dubey I.Ya.1
1
Institute of Molecular Biology and Genetics, NAS of Ukraine, 150 Zabolotnogo str.,
03680 Kyiv, Ukraine
2
Radboud University Nijmegen, 4 Comeniuslaan, 6525 HP, Nijmegen, The Netherlands
e-mail: v.v.negrutska@imbg.org.ua
Fluorescent dyes are widely used in life sciences and biotechnology in numerous applications based on
non-radioactive labeling and homo- or heterogeneous detection of nucleic acids, proteins and other
biomolecules. The majority of dyes currently used for nucleic acids visualization belong to the cyanine
family. In this work we have studied new cyanine dyes LO (Lepidine Orange derivatives) as highly sensitive
fluorescent stains for the detection of various nucleic acids in electrophoretic gels.
The asymmetric monomethine cyanines LO are structural analogues of well known commercial dyes
Thiazole Orange and SYBR Green I containing N,N-dialkyl- or arylalkylamino substituents at C-2 position of
the quinoline ring. We have first synthesized these compounds as DNA topoisomerase I inhibitors and found
them to be active in vitro at low micromolar concentrations.
We have discovered that these virtually non-fluorescent dyes strongly interact with nucleic acids
forming highly fluorescent and stable complexes thus allowing sensitive DNA and RNA visualization in the
gels. We have identified a number of LO dyes suitable for the detection of double-stranded DNA (dsDNA),
G-quadruplex DNA (G4-DNA), and RNA. The dyes display high affinity for nucleic acids, strong absorption
in visible spectral region and sharp (up to 1000-fold) increase of fluorescence upon the interaction with
various forms of DNA and RNA.
LO-3, LO-7 and LO-8 dyes are the best choice for the visualization of duplex DNA. As little as 50 pg
of dsDNA can be detected in agarose gel post-stained with LO-8 using a laser scanner, whereas the detection
limit for all 3 dyes with common 365 nm UV-transilluminator is about 0.15 ng. This sensitivity is several
times higher than can be achieved with classic ethidium bromide and close or even exceeds that of SYBR
Green I.
LO-1 and LO-2 are exceptionally efficient RNA stains allowing the detection of 2 ng of RNA in
denaturing polyacrylamide gel with UV-transilluminator. This remarkable sensitivity is twice as high as that
of SYBR Green II, a current standard for RNA detection. Moreover, it is comparable to the silver staining
detection limit. The linear fluorescence-quantity dependence for RNA quantification with LO-1 dye is
observed in the range 4–100 ng of RNA per band.
G-quadruplex (G4) DNA is a specific tetra-stranded type of DNA formed by certain guanine-rich
sequences present e.g. at the ends of the telomeres. G4-DNA and its ligands are intensely studied in the
search for novel antitumor drugs. Several dyes demonstrated a picomolar level of sensitivity to quadruplex
DNA, most efficient being LO-1 and LO-8. These dyes are more sensitive fluorescent G4-DNA stains than
SYBR Green I standard. The minimum quantity of G4-DNA that can be detected with LO-1 and UVtransilluminator in non-denaturing PAAG is 0.2 pmol (1.5 ng). The fluorescence-quantity dependence for this
dye is linear in the range from 0.2 to 5 pmol (1.5–35 ng) of G4-DNA.
Thus, the reported LO dyes belong to the most efficient fluorescent reagents for duplex and quadruplex
DNA and RNA visualization. So they can be used in advanced bioanalytical applications which require both
high level of sensitivity and a wide range of fluorescence linearity necessary for the accurate DNA/RNA
quantification.
158
INDEX OF AUTHORS
159
Аbraimova O. 44
Akkineni R. 126
Alexeeva I. 128, 155
Amaral P. 77, 96
Ananina G. 98
Andreev I. 127
Anopriyenko O. 99
Antonyuk V. 7
Areshkov P. 19
Babiychuk L. 100, 101, 148
Baeten D. 84
Bakalinska O. 144
Balatskii V. 153
Bannikova M. 113
Barska M. 40
Batyuk L. 147
Bayer G. 109
Bazalii A. 30
Bazhenov V. 126
Belokurova V. 87
Berezhnoy A. 83
Berezka K. 31
Berezovskii V. 131
Berger W. 18, 39
Bilyy R. 17, 102
Blashkiv T. 88
Bliuma D. 103
Bloisi F. 144
Blomqvist J. 31
Blume Ya. 109
Bobak Ya. 8, 40
Bobrova О. 104
Bohuslavskyi K. 105
Bondarenko M. 119, 147
Bondarenko T. 105, 130
Bondarenko V. 137
Boretsky V. 53
Boretsky Yu. 53, 67
Borikun T. 78
Borovaya M. 134
Borsig L. 81
Boychuk A. 35
Boyko S. 67
Bozhok G. 105, 130
Broda D. 52
Bronnikova L. 106
Brueggemeier S. 126
Buko V. 117
Bukreieva T. 19, 79
Bulbotka N. 32
Bulko О. 123
Burda V. 108
Butenko G. 91
Califano V. 144
Carlsson M. 31
Carly F. 25, 75, 94
Chaikovsky Y. 91
Chajka Ya. 122
Chaschina L. 99
Chashchyn M. 47
Chekhun V. 78
Chen O. 40
Chirko R. 107
Chornyy S. 50
Chumak V. 33
Cynk P. 52
Datsyuk L. 108
Datsyuk U. 108
Delvigne F. 75, 76, 94, 95
Demkovich A. 109
Demydchuk A. 91
Denega I. 49
Denis Ye. 90
Depciuch J. 60
Didan Yu. 156, 158
Dmytruk K. 24, 26, 27, 31, 56, 58, 59, 63, 64, 65, 68,
72, 73
Dmytruk O. 32
Domina E. 136
Drobot L. 30, 81 110
Dubey I. 111, 128, 155, 156, 157, 158
Dubey L. 156
Dudok K. 145
Dumych T. 102
Dyka L. 89
Dzhus O. 90
Dzydzan O. 108
Dzyubenko N. 51
Ehrlich H. 12
Falfushynska H. 112
160
Fayura L. 53
Fedorovych D. 21
Fickers P. 25, 75, 76, 77, 94, 95, 96
Fil M. 33
Finiuk N. 34
Fito A. 140
Fryc B. 60
Garmanchuk L. 90
Gawel E. 52
Gayda G. 54, 70
Gelinsky M. 126
Gerashchenko D. 81
Gnatyshyna L. 113
Gnatyuk I. 113
Godovanyi O. 115
Golovina K. 150
Gonchar M. 52, 54, 55, 61, 66, 69, 70
Gorbatyuk I. 113
Gordiienko I. 80
Gordiyenko O. 86
Goriacha I. 150
Gorka A. 52,55
Gorobchenko K. 151
Gossla E. 126
Gotsulyak N. 114
Govorova Yu. 104
Grabek-Lejko D. 55, 56
Granich V. 50
Grushka N. 88
Gryhorchyk O. 122
Gubar O. 92
Gurtovyy V. 15
Hachkova G. 122
Havrylyuk D. 33
Heffeter P. 18, 39
Herrmann M. 102
Hodovana O. 115
Holovatska I. 117
Horak I. 81
Horbulinska A. 118
Hryniv O. 31, 56
Iershov S. 129
Iershova N. 129
Igumentseva N. 40
Inamystova M. 134
Indychenko T. 79
Ivanenko G. 28
Jadko S. 116
Kaminskyy D. 34
Kanyuka O. 117
Kapustian L. 50
Karakatenko O. 79
Karatsai O. 40
Karkovska M. 57, 69
Karolczak J. 10
Karr T. 76, 95
Kashuba V. 99
Kata I. 58
Katsan V. 132, 133
Kavok N. 105
Khokhla M. 118
Kholodkova O. 125
Kholodniuk V. 80
Khoruzhenko A. 114
Khranovskyy V. 139
Kirieieva S. 35, 36
Kit Y. 16, 43, 48
Klimenko P. 90, 91
Klochkov V. 105
Kluz M. 37, 59
Klyuchivska O. 115
Knigavko V. 119, 147
Knopfova L. 81
Kolchigin N. 20
Komarnytsky I. 44
Korchynskyi O. 9, 84
Kordiaka R. 37, 59
Kordyum E. 28
Kornelyuk A. 142
Korolchuk O. 35
Kostenko O. 155
Kostina V. 128
Kotsun L. 120
Kovalenko I. 86
Kovalenko S. 86
Kowal A. 60
Kozeko L. 28
Kozeretskaya I. 134
Krasovska O. 26
Kroupskaya I. 15
Krupak V.9
Kryvorotenko D. 155, 158
161
Kshanovska B. 24, 56
Ksheminska H. 54
Kuchma M. 19, 79, 82, 90
Kuchuk M. 41, 85, 87, 141
Кunakh V. 127
Kunz-Schughart L. 8
Kurlishchuk Yu. 8
Kurylenko O. 24, 26, 58, 68, 73
Kus-Liśkiewicz M. 60
Kutsyaba V. 61
Kuziv Ia. 111, 157
Kuzmishyna I. 120
Kuznetsov K. 83
Kuznietsova H. 90
Kyrpa-Nesmiian T. 38, 152, 154
Kyryk V. 19, 82, 90
Kyzym P. 83
Labunets I. 91
Lavryk M. 66
Legach E. 130
Lehka L. 18, 39
Lenartowska M. 10
Lenartowski R. 10
Lesyk R. 33, 34
Levkiv K. 32, 62
Linder T. 31
Lisakovska O. 124
Lisovska T. 120
Liubich L. 121
Lobintseva G. 19, 82, 90
Lobyncev D. 79
Lode A. 126
Lootsik M. 17
Löwik C. 9, 84
Lozinsky V. 138
Lukash L. 79, 82
Lupak M. 122
Lutsyk M. 17, 102
Luzhans'kyj D. 135
Luzhetskyi T. 63, 71
Lyoshyna L. 123
Lysenko N. 128
Lysenko O. 91
Lystvan K. 141
Lytvynenko A. 88
Lyzak O. 64
Lоbyntsevа G. 79
Маcewicz L. 153
Majewski L. 10, 74, 93
Makashova O. 100, 101
Malysheva K. 9, 84
Malysheva T. 121
Malyukin Yu. 105
Manko N. 33
Markelova O. 15
Matvieieva N. 110
Mayevska O. 40
Mazanova A. 124
Maznichenko Ieg. 125
Mazur M. 41, 85
Mazur S. 12
Miroshnik D. 42
Mishchenko L. 118
Morgun B. 113
Munoz L. 102
Murashchenko L. 65
Mutsenko V. 126
Muzenko V. 12
Mykhailova O. 100
Myronovkij S. 16, 43
Myśliwiec A. 60
Nardid Е. 104, 117
Navrotska D. 127
Nechay H. 52
Negrutska V. 111, 128, 155, 156, 158
Negrych N. 16, 43
Nehrych T. 16, 43
Nevo E. 61
Nicaud J. 25, 76, 95
Nipot O. 129
Nitovska I. 44
Niu H. 75, 94
Novikova S. 91
Nowak J. 10, 74, 93
Nunes P. 77, 96
Ogurtsova V. 86
Olevinska Z. 41, 85
Onishchenko G. 83
Orlova N. 129
Ostapchenko L. 90
Palchevska O. 153
Palianycia L. 46
162
Panchuk R 18, 33, 39
Parlinska-Wojtan M. 60
Pasichnyk G. 81
Passoth V. 23, 31, 71
Pedan A. 46
Perepelytsina M. 144
Pereverzeva G. 143
Pershko N. 35, 36
Petrenko A. 12, 138, 146
Petrenko Yu. 12, 126, 138, 146
Petukhov D. 81
Pirko Ya. 109, 134
Piskun A. 45
Piskun R. 45
Piven О.153
Plaksina K. 130
Plotnikova L. 131
Podorozhko E. 138
Pogrebnoy P. 50
Pogribnyy P. 15
Pokora Iev. 125
Pomorski P. 74, 93
Potopalsky A. 132, 133
Pravdyuk A. 146
Prokopez Yu. 79
Prokopiv T. 66
Pronina O. 108
Protsenko O. 134
Protsyk V. 35
Prudyus I. 46
Pushkarova N. 87
Pykalo S. 135
Pylypchuk O. 136
Rachkevych N. 67
Ramazanov V. 137
Redowicz M-J. 8, 10, 74, 93
Rodnichenko A. 91, 92
Rogulska E. 12
Rogulska O. 126, 138
Rohr J. 18
Romanyshyn O. 49
Rooij K. 9, 84
Ruban Y. 139
Ruchala J. 26, 31, 58, 68
Rudenko A. 99
Rushchak V. 47
Rushkovsky S. 108
Ryazantsev V. 100
Rybalchenko V. 90
Sabadashka M. 108, 140
Sadowska-Bartosz I. 13
Sakhno L. 141
Saraieva I. 128
Sassi H. 76, 95
Satarova T. 44
Scheglov A. 98
Schütz K. 126
Semenova V. 121
Semkiv M. 27, 58, 63
Senchuk O. 143
Senkiv J. 34
Sergeeva L. 106
Shabas N. 81
Shablii V. 19, 82, 90
Shablii Yu. 19, 82, 90
Shakhovsky А. 110
Shakina L. 20, 89
Shamalo S. 91
Shavanova K. 139
Shayakhmetova G. 47
Shckorbatov Yu. 20, 83, 89
Sheludko Y 38
Shkliarskyi V. 46
Shlapatska L. 80
Shpakova N. 129
Shpyrka N. 139
Shymanskyy I. 124
Shаblii V. 79
Shаblii Yu. 79
Sibirny А. 5, 21, 24, 26, 27, 31, 32, 37, 53, 56, 58,
59, 61, 62, 63, 64, 65, 67, 68, 72, 73
Sibirnyj W. 55
Sidorenko M. 35, 36
Sidorenko O. 130
Sidorenko S. 80
Sidorik L. 15, 50
Sindarovska Ya. 85
Sirenko Yu. 50
Sklyarenko L. 80
Skowronek K. 10
Skrypkina I. 19, 90
Skybitska V. 122
163
Smalyuk Y. 50
Smutok O. 57, 66, 69
Snegirenko O. 49
Sobczak M. 10, 74, 93
Sokolova I. 112
Souchelnytskyi S. 16
Spivak M. 41, 85
Sribna V. 88
Starodub N. 139
Starykovych M. 48
Stasyk Oleh 40, 49, 143
Stasyk Olena 8, 49
Stasyuk N. 54, 69, 70
Stayno L. 121
Stoika R. 9, 11, 16, 17, 18, 33, 34, 39, 43, 48, 84, 115
Stoliar O. 113
Strashnyuk V. 89
Strokovskaya L. 99
Svitina G. 19
Svitina H. 79. 82, 90
Sybirna K. 67
Sybirna N. 49, 117, 118, 122, 140
Sydorenko M. 144
Syvash A. 116
Tarusin D. 12, 126
Tiukova Iev. 71
Tolstanova A. 134
Tsisarenko A. 50
Tykhonkova I. 15
Тwardovska М. 127
Usenko V. 15
Vagyna I. 99
Vaslovych V. 121
Vasyliev R. 91, 92
Vasylyshyn R. 73
Vicari L. 144
Volovelskaya E. 150
Volovelskaya Ye. 137
Vorobyova N. 142
Vovk O. 143
Vynnytska-Myronovska B. 8
Vysekantsev I. 98
Wnuk M. 52
Wojtera E. 74, 93
Yakimova R. 139
Yakovenko L. 15, 50
164
Yakymchuk O. 144
Yamauchi J. 74, 93
Yanko R. 131
Yatsyshyn V. 72
Yefimenko N. 145
Yemets A. 72, 109, 134
Yena М. 51
Yurchak I. 113
Yurchenko N. 35, 36
Yurkevich L. 132, 133
Yurkiv M. 73
Zaichenko A. 46
Zaikov V. 12
Zakalska O. 66, 70
Zakalskiy A. 66, 70
Zastavna D. 48
Zaykov V. 146
Zaytseva O. 119, 147
Zemlianskykh N. 148
Zhdankin A. 149
Zhezhera V. 90
Zinchenko V. 150
Zinchenko О. 104
Zlatska A. 92
Zubov D. 91, 92
Zubov P. 100, 101
Zubova O. 101
NOTES
165
NOTES
166
NOTES
167
NOTES
168