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BIOTECHNOLOGIESInstitute of Molecular Biology and Genetics, 150, Zabolotnogo Str., DNA markers and test-systemsTest systems for gene diagnostics of severe hereditary diseases and hereditary susceptibility to mass pathologies (Ludmila A. Livshits, Professor, Dr. Sci. (biol.)).The elaboration of diagnostic methods and individual components of the kits for DNA analysis of mutant genes that cause the development of the most widespread monogenic hereditary diseases in Ukraine (cystic fibrosis, phenylketonuria, spinal muscular atrophy, Duchenne muscular dystrophy, Martin-Bell syndrome (fragile X), hemophilia A, Huntingtion’s disease) hereditary chromatosis, Charcot-Marie-Tooth neuropathy (CMT), hereditary corneal dystrophies, Stargardt's macular dystrophy), hereditary susceptibility (phase II detoxification system genes) to mass pathologies (oncological, cardiovascular, endocrine pathologies) and genetical forms of men (azoospermia, oligospermia) and women (Premature ovarian failure (POF) ) infertility is being held. These diagnostic kits can be used for genetic testing, that is held during prenatal diagnostic, presymptomatic diagnostic, specification of the diagnosis, selective and mass screening for heterozygous carriers in the high risk groups and in the general population. The results of the genetic testing are the necessary requirements for prevention and precise treatment for the improvement of the gene pool state in Ukraine and the demographic setting. The list of the institutions, where the elaborated methods of the DNA-analysis for the clinical diagnostics of 1100 patients with cystic fibrosis, Martin-Bell syndrome (fragile X), genetical forms of men infertility (azoospermia, oligospermia), hereditary chromatosis and secondary pathologies connected with it were used in: Institute of Pediatrics, Obstetrics & Gynecology of The National Academy of Medical Science of Ukraine, Scientific Center of Radiation Medicine of The National Academy of Medical Science of Ukraine, Institute of Neurosurgery of The National Academy of Medical Science of Ukraine, The Filatov Institute of Eye Deseases (Diseases) and Tissue Therapy of The National Academy of Medical Science of Ukraine, Institute of Occupational Medicine of The National Academy of Medical Science of Ukraine, Institute of Hereditary Pathology (Lviv) of The National Academy of Medical Science of Ukraine, Crimean Republican Center of Medical Genetics and Human Reproduction (Symferopil), Ukrainian Center of Diagnostics and Treatment of Cystic Fibrosis (Odesa), Inter-regional medicogenetic centers in: Kyiv, Donetsk, Kharkiv, Odesa, Kryvyi Rig, Regional medicogenetic consultations: Ternopilska, Rivnenska, Poltavska, Khmelnytska, Vinnytska, Volynska, Ivano-Frankivska, Chernigivska, Zakarpatska, Luganska ;”Isida-IVF” clinic (Kyiv). The development of complex molecular diagnostics of chronic myelogenous and acute lymphoblastic leukemias using PCR and specific polyclonal antibodies (Stanislav S. Maliuta, Professor, Dr. Sci. (biol.), Corresponding Member of NASU).The method was tested on more than 200 patients from different regions of Ukraine after the corresponding assignment from The Institute of Hematology and Transfusiology of the Ministry of Public Health of Ukraine and from regional hematological departments. Entire correlation with other diagnostic tests and high sensivity (10-4–10-5 of malignant cells) were obtained. This method gives the possibility to detect the type of translocation as well as to exclude false results completely. It is very important to estimate prognosis and therapy protocols. Large-scale search for biomarkers of epithelial tumors (Alla V. Rynditch, Professor, Dr. Sci. (biol.), Corresponding Member of NASU).The creation of different diagnostic and prognostic tools for cancer research. For this purpose we use unique NotI-microarrays that permit to determine genetic and epigenetic changes in genome and patterns of gene expression on different stages of tumor development. NotI-microarrays consist of clones, which preferentially contain genes and could be used for the detection of deletions and amplifications in DNA derived from tumors as well as determination of loss of heterozygosity and changes in DNA methylation. We perform analysis of 300 DNA samples derived from epithelial tumors by hybridization with NotI-microarrays. The identification of tumour-associated antigens for the development of modern immunological approaches of cancer diagnostics and treatment (Valeriy V. Filonenko, Dr.Sci.(biol.), Senior Staff Scientist).The identification of novel tumour-associated antigens (TAA) is a prerequisite for the creation of modern anti-tumour vaccines and specific antibodies against TAA. While using SEREX methodology (serological identification of antigens by recombinant expression cloning) more than 60 tumour-associated antigens of melanoma, colon cancer, cancer of thyroid gland and breast cancer have been identified. The listed antigens could be used in future for the generation of humanized antibodies with the following application in clinics for cancer treatment. Up-to-date a number of mouse monoclonal antibodies have been generated against TAA and signalling molecules that undergo alteration at carcinogenesis. These are antibodies to S6K1, S6K2, TSC1, TSC2, Ki67, PTEN, mTOR, CK2. These antibodies could be used for detailed diagnosis and prognosis of oncological pathologies by immunohystochemical and serological approaches. Drug designNew anticancer and antiviral medications (Anatoliy I. Potopalsky, Ph.D., Senior Staff Scientist)Medications amitozyn and izatizon with antiviral, antitumor and immunoregulating activity have been elaborated. Amitozyn manifests antitumoral action and antimetastasis activity, but in comparison with existing medications it does not depress the hematosis processes, also it does not cause allergic reactions and has low toxicity. Amitozyn has undergone clinical tests in the system of the Ministry of Health of Ukraine. Izatizon has been approved in veterinary institutions and is studied in medicine. By using elaborated medications the technologies of diagnostics, prevention and treatment of plants agrobacterial cancer, non-vaccine prevention and treatment of mass virus diseases, increase in productivity of plants, mushrooms, useful insects (such as bees, silkworms, and oak silkworms), fish, poultry and animals have been worked out. In terms of a new biotechnology of the accelerated obtaining of new plant forms and varieties using nucleic acids preparations, such sorts as the feed lupine "Industrialny", pumpkin "Kavbuz Zdorovyaga", purple echinacea "Poliska Krasunya", rye "Drevlyanske", potato "Dzvin", tomato "Ukrainski" were obtained and registered in the State Register for the Plant Varieties of Ukraine. The elaborations do the significant social and economic effect. The molecular modeling and drug design: development of protein kinase inhibitors (Sergiy M. Yarmoluk, Dr.Sci., (chem.), Senior Staff Scientist).Nowadays, drug development starts from identification of biological macromolecule target (DNA, or, in most cases, enzyme) responsible for certain disorder. By altering target activity with drug-like organic compounds it is possible to prevent or treat target-associated disorder(s). Among human proteins, protein kinase family have one of the greatest medico-biologic meanings. Protein kinases constitute at least 2% of the human genome and represent one of the most fundamental intracellular signalling mechanisms. Imbalances in kinase activity are a major factor in many disease states, especially those involving inflammatory and proliferative responses. Hence, protein kinases are attractive targets for drug research. The goal of proposed project is the development of protein kinase inhibitors. The approach used to develop protein kinase inhibitors is based on combined application of computer modeling techniques, directed organic synthesis and biological in vitro screening. The main points of the project are:
We have been developing the inhibitors of Casein Kinase 2 (CK 2) for about a year. At the time we have founded 3 lead compounds with IC50 up to 10-7M and now perform their chemical optimization. The structural-based drug discovery against pathogenic bacteria (Mykhaylo A. Tukalo, Professor, Dr. Sci. (biol.)).The key role of Aminoacyl-tRNA synthetases in protein synthesis and other essential cellular activities has distinguished this family as an ideal antibiotic target (Schimmel et al., 1998; Kim et al., 2003). We are using the differences between human and prokaryotic prolyl- tyrosyl- and leucyl-tRNA synthetases for the development of the inhibitors as potential drugs against Mycobacterium tuberculosis, Enterococcus faecalis and Streptococcus pneumonia. Our strategy is based on the combination of several approaches: an X-ray crystallography, a small molecular docking (virtual screening) and a target-specific chemical screening. Recently we have obtained crystals of E. faecalis prolyl-tRNA synthetase (ProRS) and in collaboration with Dr. S.Cusack (EMBL Grenoble Outstation, France) have determined structure of enzyme in complex with substrates (Cusack, Tukalo and Yaremchuk, unpublished result). This structure reveals the differences in the active sites of ProRS from pathogenic bacteria compared to human, which we are exploiting for novel antibiotic design. The production of a novel antiangiogenic cytokine cendomap – C-module of tyrosyl-tRNA synthetase (Olexander I. Kornelyuk, Professor, Dr. Sci. (biol.)).A novel biotechnological product – cendomap cytokine has been obtained and tested in vitro. The bacterial system for the expression and purification of cendomap cytokine permits to acquire a recombinant protein at preparative scale of about 10 mg from 1 liter of bacterial culture. Cendomap cytokine may be widely used as a mediator of procoagulant and antiangiogenic action, an inductor of apoptosis and, in perspective, as a putative antitumour medication for clinical oncology. The baculovirus expression system for obtaining of recombinant proteins (Olexander P. Solomko, Professor, Dr. Sci. (biol.)).The system for an expression of recombinant proteins on the basis of a virus of nuclear polyhedrosis Malacosoma neustria (Mane) and cultures of insect cells was designed in the department of biochemical genetics. The designed system is the first baculovirus system of an expression of recombinant proteins in Ukraine. At present the similar systems find wide application in the world for obtaining recombinant proteins, including for effective antiviral vaccines. The laboratory procedure of obtaining biologically active recombinant human Prolactin with our system and commercial system was designed in cooperation with the Institute of biological chemistry and biophysics (Poland). The method is covered by the patent of Poland. Human Prolactin is a multifunctional hormon, which participates in many physiological processes. Recombinant Prolactin is necessary for effective tests - systems for detection of some pathologies (ischemia, renal failure, brain tumour). The protein can be used in therapy. Biotechnology of the plant secondary metabolites production (Stanislav S. Maliuta, Professor, Dr. Sci. (biol.), Corresponding Member of NASU).An efficient method for the increase of Glycyrrhizin (Gl) and flavonoids production from Ri-plasmids transformed licorice Glycyrrhiza sp. cells and hairy roots was developed. An endophyte mycorrhizal fungus was isolated from intact roots of the medical plant Potentila alba L. as a potential elicitor to stimulate the total biosynthesis of the secondary metabolites in transformed licorice cells and roots in 1.5 fold. Glycyrrhizin (triterpen saponin) production in the biotechnological way is very perspective as a source of the natural sweetener for the diabetes patients, and its aglycone Glycyrrhizic acid has shown the inhibiting II-β-dehydrohynase (hepaprotective effect in liver and kindneys tissues in vitro as well as in vivo). Flavonoids, 18-dehydroglycyrrhizic acid and Glycyrrhizinic acid have shown antioxidation effect. It is their anti-inflammatory effect by which one can explain their positive therapeutic effect on lungs and liver irradiated impact pathology, and note the anti-sclerotic, anti-inflammatory, anti-cancer, and anti-ulcer activity of licorice metabolites. The application of lectins from medicinal plants for biomedical research (Iryna S. Karpova, Ph.D., (biol.), Senior Staff Scientist).The lectins is a new group of biologically active proteins, which can specifically recognize receptors exposed on cellular membranes in all types of living organisms. In humans endogenic lectins play their role in fertilization, immune response, antiviral and antibacterial protection, clearance, apoptosis etc. Plant exogenic lectins have been widely used as a tool in the investigation of animal membranes which differ in their surface sugars: in the case of immuno-haematological and endocrine diseases, developing infection or neoplastic process and other pathological changes. In the United States some lectins are commercially used for purging of bone marrow for transplantations in leukemia patients. An original technology of medicinal plant lectins isolation and purification has been developed. The new approach to receptor diagnostics of different pathologies (such as acute radiation sickness and thyroid gland diseases) using a panel of lectins isolated from medicinal plants in reaction of haemagglutination with data processing has been proposed. The method is based on the unique properties of lectins to indicate the deviations in membrane structure. The endogenous misbalance of lectins occurs in a pathological state and the lectino-test as its indicator has been proposed. The application of herbal lectins in diagnostics and individual approaches to therapy is being developed. Contemporary research is devoted to the further studying of peculiarities of medicinal plants lectins: their adhesive, immunomodulating and antitumour activity, and possible usage for stem cell identification and separation. The production of Rauwolfia serpentina B. cultured cells biomass with high content of ajmaline used as a source of antiarrythmic & hypotensive medications (Viktor A. Kunakh, Professor, Dr. Sci. (biol.), Corresponding Member of NASU).Highly productive callus and suspension cell lines of R.serpentina accumulating about 1.5% of ajmaline have been generated. The quantity of ajmaline is 8 to 12 times as much as in plant roots. Rauwolfia serpentina cell biomass is produced in uninterrupted technological regime with the cell maintenance on artificial nutrient medium in the controlled conditions preventing the environment pollution. Cell culture growth cycle is 20 to 30 days for a suspension culture and 50 to 60 days for a callus culture. The technology of the production of biomass with ajmaline content of about 1.0% and ajmaline recovery from it was tested in the industrial conditions. Cell biotechnology of Ungernia victoris (Viktor A. Kunakh, Professor, Dr. Sci. (biol.), Corresponding Member of NASU).Ungernia victoris, endemic plant of Pamyr, provides a source of galantamine and lycorine. Galantamine-based medications are used for myasthenia, progressive muscle dystrophy (myopathia), radiculitis. Lycorine derivatives are useful during chronic and acute inflammation in lungs and bronchial asthma. An environmentally friendly technology of U.victoris cell biomass production and accelerated clonal propagation of plants have been developed. The production of Panax ginseng cell biomass (Viktor A. Kunakh, Professor, Dr. Sci. (biol.), Corresponding Member of NASU).Ginseng preparations offer the highly effective tonic remedies and are advised in the treatment of hypotony, overstrain, neurasthenia, atherosclerosis, leucocytosis, effects of ionizing radiation. We have developed an environmentally friendly technology of the production of P.ginseng cell biomass, which can be used as a raw material for manufacturing of medicinal form (“Biozhen’shen”), nutrient additives and cosmetics. Techniques to control the biosynthesis of ginsenozides, their amount and qualitative composition within the cell biomass were elaborated. Antimycobacterial agents (Anatoly D. Shved, Dr.Sci.(biol.), Senior Staff Scientist).Sharp increase in human morbidity and mortality caused by tuberculosis becomes a worldwide problem. It is mainly associated with the advent of Mycobacterium tuberculosis strains, which are resistant to one or more agents commonly used in the treatment. We have synthesized a new class of derivatives of phenazine-1-carboxylic acid as potential antimycobacterial agents capable to inhibit in vitro RNA synthesis. Their antimycobacterial activity has been investigated by Italian researchers headed by Prof. A. Delogou. A range of the chemicals have shown antimycobacterial activity at the concentrations, which are equal or even lower than those for well-known medications isoniazid, rifampicin, and ethambutol. Moreover, some derivatives have shown high activity against clinical isolates with multiple-drug resistance. We plan to study the most promising chemicals in vivo using laboratory animals. Gene therapy and stem cells technologiesThe development of skin equivalents on the base of human stem cells cultivated on special carriers in vitro or treatment of massive burns (Lubov L. Lukash, Dr.Sci.(biol.), Senior Staff Scientist).The development of biotechnology of skin equivalents with the usage of adult human stem cells isolated from blood (mesenchymal stem cells) has been started. Particularly the database of characterized cell lines originated from samples of embryonic and adult human tissues and compositions of cultural mediums, carriers and hydrogel substrates applied at the production and optimization of skin transplantants is in progress. There is also the process of creating a cellalur bank, which will be connected with Euro Skin Bank as a central provider of biomaterials for treatment of burns in the USA and Europe, and the international organization Bone Marrow Donor Worldwide which supplies the transplantations of bone marrow. The joint activity with the Center of Thermal Burns and Plastic Surgery of the Ukrainian Ministry of Health has been initiated on the introduction of the developed by the Institute skin equivalents, based on human fibroblasts, into medical practice. Gene and Cell Therapy (Vitalii A. Kordium, Professor, Dr. Sci. (biol.), Corresponding Member of NASU, Full Member of AMSU).The main directions of the department’s activity are the study of fundamental basis of aged and sick organism renewal, the development and usage of gene and cell technologies for damaged cells and hereditary material replacement, as well as biotechnological obtaining of therapeutic recombinant proteins. Scientific approaches for gene therapy technologies of multifactor diseases have been developed. They are based on the introduction of recombinant genetic material into an organism with the aim of pathologies correction. Effective non-viral systems for target therapeutic genes delivery to the cells and organs were created. Positive results of gene therapy for pathologies, namely artificially induced insulin – dependent diabetes mellitus and atherosclerosis - were obtained in laboratory animals. Significant success was achieved in gene-engineering biotechnology. The scientists of the department developed the unique method of recombinant proteins obtaining in E.coli with the use of λ phage. This method has no world analogues, it provides for target product obtaining in high concentration in active soluble form directly in culture medium. Its effectiveness for obtaining of both pro- and eukaryotic origin proteins was shown. Industrial technologies for such products as β-galactosidase, human α-2a and α-2b interferon, human growth hormone, etc. obtaining were developed according to this method. Preparation of recombinant human α-2a interferon, under the trademark “Laferon” is being produced in Ukraine for more than 10 years, and is successfully used in medical practice. Theoretical basis of damaged hereditary material substitution due to exchange of genetic material between organism cells, including stem cells, is created. Now it is under experimental examination. Analytical biotechnologiesThe development and creation of electrochemical and optical biosensors for medicine (Anna V. Elska, Professor, Dr. Sci., Full Member of NASU, Olexiy P. Soldatkin, Professor, Dr. Sci.)
The development and creation of analytical devices based on electrochemical mono- and multibiosensors for environment monitoring (Anna V. Elska, Professor, Dr. Sci., Full Member of NASU, Sergei Dzyadevych, Dr. Sci.)
The development and creation of electrochemical biosensors for biotechnological processes and foodstuff control (Olexiy P. Soldatkin, Professor, Dr. Sci., Sergei Dzyadevych, Dr. Sci.)
Plant biotechnologiesBacterial inoculant KLEPS® (Nataliya O.Kozyrovska, Ph.D., Senior Scientist)The first developed and registered in Ukraine bacterial inoculant KLEPS® provides the plant with biological nitrogen, phytohormones, and induces systemic plant resistance to diseases. The inoculant is recommended for pre-treatment of seeds before sowing. KLEPS® improves the yield of cereals and vegetables to 10-40 %, strengthens the seedling vigor, enhances root branching, provides earlier flowering and ripeness of the crop, increases protein content, reduces plant diseases. Using KLEPS®, farmers reduce application of mineral nitrogenous fertilizers and pesticides by 70%, preserve their health and protect the environment. Express-method of the authentication of Klebsiella oxytoca IMBG26 on the PCR basis (Nataliya O.Kozyrovska, Ph.D., Senior Scientist)The uniqueness of sequence of the pehX gene of industrial culture of bacterium of Klebsiella oxytoca IMBG26 cloned by us, enabled to use this sequence for the development of the method of authentication of this bacterium by the PCR. The rapid, sensitive and specific test is recommended for the discrimination of K. oxytoca from other Klebsiella under the establishment of à precise taxonomic position of bacteria and for ecological monitoring K. oxytoca. Recommendations of inoculant deployment for the potato in vitro adaptation to ex vitro conditions and plant protection within field potato growing (Nataliya O.Kozyrovska, Ph.D., Senior Scientist)Principles of the inoculant deployment based on beneficial for a plant bacteria for potato in vitro adaptation to ex vitro conditions and plant protection within field potato growing have been substantiated and verified experimentally. Recommendations addressed clonal potato micropropagation, sanitation, and growing healthy crops. Technology of bacteria-assisted phytostabilization of heavy metals (Nataliya O.Kozyrovska, Ph.D., Senior Scientist)To implement the plant-microbe system to a variety of polluted environments for a remediation, a flexible and hardy plant-bacteria system is designed. A rationally assembled bacterial consortium for a multipurpose application at different level of heavy metal (HM) pollution in the soil is proven in field model and natural environments. In the case of aerogenic HM pollution (transport and industrial off-gases) bacteria promote growing clean crops. On the other hand, the consortium provides environmentally appropriate technology for HM phytostabilization at moderate and extremely polluted lands near industrial enterprises and mines. A concept of low-cost technology of growing and utilizing pioneer plants in a lunar garden (Nataliya O.Kozyrovska, Ph.D., Senior Scientist)On the base of model system Tagetes patula L. – anorthosite (analog of lunar soil) – a consortium of microorganisms a concept of low-cost technology of growing and utilizing pioneer plants in a lunar garden has been developed for the first time. The putative scenario anticipates using a local material – regolith – and a consortium of a rationally assembled microorganisms (biomobilizing plant-essential elements, plant growth promoting, plant protection from abiotic stressors etc). | ||||||
| Institute of molecular biology and genetics 2004 | ||||||
