Biotechnologia Acta

26-35

Authors: N. A. Matvieieva

The review focused on the data concerning current state in the field of Compositae “hairy” roots and transgenic plants construction using A.tumefaciens- and A. rhizogenes-mediated transformation to obtain biologically active compounds, including recombinant proteins. The article presents data on the results of genetic transformation of Cichorium intybus, Lactuca sativa, Artemisia annua, Artemisia vulgaris, Calendula officinalis, Withania somnifera and other Compositae plants as well as studies on the artemisinin, flavonoids, polyphenols, fructans and other compounds accumulation in transgenic plants and roots. The data show that the use of biotechnological approaches for construction of "hairy" roots and transgenic plants with new features are of great interest. The possibility of increase in the accumulation of naturally synthesized bioactive compounds and recombinant proteins production via A. tumefaciens and A. rhizogenes-mediated transformation have been shown. In vitro cultivation of transgenic plants characterized by high level of bioactive compounds accumulation and synthesis of recombinant proteins makes it possible to obtain guaranteed pure raw material. Using of biotechnological approaches preserved natural populations of plants is particularly important for rare and endangered plant species.

52-57

Authors: A. M. Taranenko, L. O. Sakhno, B. V. Morgun, M. V. Kuchuk

The purpose of research was to analyze 10 developed at the Institute of Cell Biology and Genetic Engineering lines of rapeseed to confirm the presence and functionality of the transferred transgene CP4 epsps, as well as the differences among those lines from registered transformation events GT73 and GT200 (Monsanto). During the study extraction of total rapeseed DNA, PCR analysis, electrophoretic separation and visualization of amplicons in agarose gel were conducted, as well as testing of green plants for resistance to glyphosate in greenhouse. The structural difference among 7 transgenic lines from registered transformation events GT73 and GT200 was revealed. Plants showing the presence of synthetic CP4 epsps sequence were resistant to the herbicide in a closed soil. The uniqueness of the obtained transformation events was confirmed, as well as the prospect of using them in breeding.

69-77

Authors: S. V. Pykalo, M. О. Zinchenko, S. I. Voloshchuk, O. V. Dubrovna

The direct and step-type in vitro selection with application of selective system based on low molecular mannitol and the selection of callus lines of winter triticale being resistant to simulated water deficit were carried out. As a result, from line 38/1296 and variety Obriy respectively, 5 and 4 resistant callus lines were identified that had a high survival rate on the selective medium with 0.6 M mannitol and maintained morphogenetic potential. From the resistant lines plant regenerants were induced and their rearing, rooting and transfer to in vivo conditions were optimized and followed by testing to water deficit tolerance. During the testing under simulated 3-weeks drought it was revealed that plant regenerants derived from resistant lines were characterized with reliably higher relative water content and free proline content as compared with non-resistant plants. A step-type in vitro selection was more effective, because resulted from the selection more resistant callus forms were identified. Evaluation of plant regenerants of triticale obtained from resistant calli showed increased level of their tolerance to water deficit.

84-90

Authors: E. V. Маtseliukh, L. А. Safronova, L. D. Varbanets

Proteases from probiotic strains of the genus Bacillus, just like the antibiotics, bacteriocins and other hydrolytic enzymes, are one of the main factors that determine their biological activity. The aim of this work was to study the synthesis and biochemical properties of proteases from two strains Bacillus amyloliquefaciens subsp. plantarum UCM B-5139 and UCM B-5140 that included in the probiotic Endosporin. The cultivation of strains was carried out in flasks under rotating for two days. The influence of physico-chemical parameters of the reaction medium on proteolytic activity was studied on partially purified protease preparations. Lytic activity was determined by turbidimetric method. On the second day of cultivation B. amyloliquefaciens subsp. plantarum UCM В-5139 and UCM В-5140 synthesized the metal-dependent peptidase and serine protease, respectively. The optimum conditions of their action were the following: temperature 37–40 °C and pH 6.5–7.0. Isolated proteases are able to lyse the living cells of Staphylococcus aureus and Candida albicans. Thus we demonstrated that B. amyloliquefaciens subsp. plantarum UCM B-5140 and UCM B-5139, included in the probiotic veterinary preparation Endosporin, produced proteolytic enzymes that hydrolyze the native insoluble proteins (elastin, fibrin and collagen). These enzymes belong to the group of neutral metal-dependent and serine proteases. They are active under physiological conditions against gram-positive bacteria and yeasts. The application of these proteases in biotechnology is considered.

96-106

Authors: V. V. Turov, I. M. Todor, N. Yu. Lukyanova, T. V. Krupskaya, A. P. Ugnivenk, V. F. Chehun

The aim was to study the changes in the structure of intracellular water in the intact rat liver and liver with Guerin carcinoma. The state of water in lyophilized liver tissue of rats – intact and affected by Guerin carcinoma at different stages of tumor development was studied by low-temperature 1H NMR spectroscopy. It is shown that in progress of tumor the contribution from the water clusters whose size is 3–12 nm is grows. It was established that in intact tissue present 6 types of water clusters capable of dissolving different amounts of trifluoroacetic acid. In the liver with Guerin carcinoma clusters of strongly associated water more uniform, and their dissolving power with respect to trifluoroacetic acid and the electron donor molecules significantly decreased. These results are important for elucidating the mechanism of action of water on the development of neoplastic processes.

113-119

Authors: A. Yu. Galkin, 2, A. G. Komar, A. A. Grigorenko

In article we analyzed national and international regulations concerning the quality and safety of medical devices for in vitro diagnostics. We discussed the possibility of a partial application of the recommendations of the State Pharmacopoeia of Ukraine to this type of product. The main guiding regulatory documents establishing requirements for quality and safety tools for the serological diagnosis products are The technical regulation on medical devices for the diagnosis in vitro, DSTU ISO 13485 “Medical devices. Quality management system. Regulatory requirements”, and DSTU ISO/IEC 17025 “General requirements for the competence of testing and calibration laboratories”. Similar requirements of the State Pharmacopoeia of Ukraine which are used for drug standardization can not be directly applied to the medical devises for in vitro diagnostics due to a number of features, namely, the serological diagnosis products pre-designed to determine the unknown concentration of a particular analyte in a biological material, the diagnostic kits has to include the control samples (internal standard systems) that need to be calibrated. It was determined following parameters of bioanalytical standardization and validation characterization for of qualitative (semi quantitative) test-kits for serological diagnosis: precision (convergence, intralaboratory precision and reproducibility), diagnostic and analytical specificity, diagnostic sensitivity. It’s necessary to inspect additional parameters for quantitative test-kits such as accuracy (precision), linearity, analytical sensitivity and range.