Biotechnologia Acta

18-27

Authors: A. P. Kharkova, Y. A. Garmash, O. S. Hnatiuk, O. Y. Luzina, S. V. Danilovskyi, A.Y. Kuznetsova, O. H. Minchenko

To study effect of glutamine deprivation on the expression of genes encoding the key proliferation associated factors on a relation to inhibition of inositol requiring enzyme-1 IRE1 in U87 glioma cells was the aim of the research. It was shown that glutamine withdrawal down-regulated the expression of DEK, BRCA1, LIF, and COL6A1 genes in control glioma cells (transfected by empty vector), up-regulated ADGRE5 gene expression, and did not significantly change the expression of TPD52 and GNPDA. Inhibition of ІRE1 signaling enzyme activity modified the effect of glutamine deprivation on the expression of TPD52, BRCA1, LIF, DEK, ADGRE5, and COL6A1 genes: introduces the effect of glutamine deprivation on TPD52 and GNPDA1, reduced – on COL6A1, and enhanced – on ADGRE5, DEK, and BRCA1 in U87 glioma cells. Therefore, glutamine deprivation affect the expression level of most studied genes in U87 glioma cells in relation to the functional activity of IRE1 signaling enzyme, which is responsible for control of cell proliferation and glioma growth.

35-44

Authors: V. M. Minarchenko, R. V. Kutsyk, N. P. Kovalska, B. O. Movchan, O. V. Hornostai, O. M. Strumensk, L. M. Makhynia

The aim of our research was to investigate the influence of silver nanoparticles on the physical and chemical features of plant oils of dogrose, flax, cedar, amaranth and watermelon and their antimicrobial activity. Plant oils were saturated with silver nanoparticles using electron-beam technology for depositing a molecular stream of metal in a vacuum. To characterize the rancidity of plant oils, the acid, iodine, peroxide, ester and saponification values were determined. A sharp drop in the iodine number and an increase in the peroxide number in oils saturated with silver nanoparticles were observed, as compared to pure oils, indicating a decrease in the number of unsaturated bonds in fatty acids and the formation of peroxides in oils. All pure plant oils and a separate sample of silver nanoparticles suppressed the growth of only E. faecalis colonies. Plant oils that were saturated with silver nanoparticles delayed the growth of S. aureus, S. epidermidis, E. faecalis, E. coli, P. aeruginosa, and C. albicans; the greatest delay in the growth of colonies was caused by flaxseed oil. Thus, the features of the plant oils under study essentially changed after they are aturated with silver nanoparticles. It can be assumed that the metal acted as a catalyst for peroxide oxidation of lipids in the investigated plant oil samples, the products of which caused toxic effects on cultures of bacteria and fungi in the experiment.

53-60

Authors: N. Ya. Levchyck, A. V. Liubinska, N. V. Skrypchenko, O. I. Dziuba, N. M. Kachalova, O. L. Mironov

The aim of the research was to detect the biological activity of aqueous solutions of various samples of amber from the Trancedniestria province, and to characterize their effect on the physiological processes of higher plants. The allelopathic and cytostatic effects of amber solutions were studied using biotesting methods in open soil conditions and in culture in vitro. The test-object was the seedlings of cucumber Cucumis sativus L. It was shown that the activity and nature of the action of the samples depend on the region of origin of the amber, the degree of shredding and concentration of the solution. According to the results, fine fractions of amber show biostimulating effects on plants. Thus, amber and its aqueous solutions can be effectively used for cultivating agricultural plants in vivo and in vitro conditions. For stimulation of the increasing of the plant stem mass it is recommended to use amber B1, to increase the mass of roots - amber B4, and amber B2 - to stimulate stem growth in length.

19-29

Authors: O.V. Galkin, T.O. Chudina, A.A. Siromolot, O.S. Oliynyk

Obtaining single-chain variable fragments (scFv) of recombinant antibodies in E. coli cells is often associated with numerous problems causing low yields or inactive conformation of the product. The aim of this work was to study the influence of staphylococcal protein A fragment fused with scFv antibodies (SpA-tag) on the efficiency of expression of final product. Examination of scFv antibodies of different origin and specificity has shown that in similar expression systems fused scFv is synthesized in much higher quantities than free scFv. Furthermore, the scFv antibodies in fused form retained their antigen-binding properties and the SpA fragment the ability to bind other immunoglobulins. Thus, the proposed strategy can be considered effective in improving the efficiency of scFv-antibodies production in E. coli cells.

41-50

Authors: N. U. Susol, D. D.Ostapiv, V. V.Vlizlo

The aim of the research was to investigate the ability of new complexes as specific single-stranded oligonucleotides ‒ ODN ‒ with dimethylaminomethylmethacrylate ‒ DMAEM ‒ based polymer carriers to inhibit the expression of the physiological prion. It has been established that the introduction of complexes of newly synthesized carriers based on dimethylaminomethyl methacrylate - PEG-DMAEM-MP-27 (magnetic particles) (MP-27), PEG- DMAEM-MP-2 (MP-2), PEG- DMAEM-MP-3 (MP-3) with as ODH into the organism of rats leads to a decrease in the physiological prion content in the tissues of the spleen and small intestine. The influence of complexes of newly synthesized carriers MP-27, MP-2 and MP-3 with as specific single-stranded oligonucleotides ODH on hematological and biochemical blood parameters of Wistar rats was also studied.

63-71

Authors: A. P. Pinchuk, A. F. Likhanov, L. P. Babenko, M. V. Kryvtsova, O. A. Demchenko, O. B. Sherbakov, L. M. Lazarenko, M. Ya. Spivak

The aim of this work was to study the effect of cerium dioxide nanoparticles on the germination of Scots pine (Pinus sylvestris) seeds and the subsequent physiological changes in plastid pigments and phenolic compounds accumulation in the seedlings tissues. Brief soaking of pine seeds in solutions of cerium dioxide nanoparticles (2-4 nm) increases the germinating power and seed germination by 1.3 times. In pine seedlings, germinated from six month old seeds, reduction in chlorophyll b, phenols and phenylalanine content is observed in 14 days after treatment. Simultaneously, concentration of carotenoids and flavonoids increased. This indicates the overall decrease in phenylpropanoid synthesis. Hence, it is reasonable to assume that cerium dioxide nanoparticles, having entered the cells of Scots pine seeds, are engaged in the regulation of phenol and terpenoid synthesis in seedlings, which greatly affects the intensity of their growth and development.

14-24

Authors: T. O. Chudina, A. Yu. Labyntsev1, D. V. Kolybo, S V. Komisarenko

The aim of the research was to compare the characteristics of nanoparticles with different chemical structure and size (colloidal gold Gold 1 and Gold 2, calcium phosphate CaP and poly(lactideco-glykolid) PLGA 1 and 2) to find the most efficient carriers of antigen — recombinant diphtheria toxoid for per os immunization. According to the MTT test, all studied particles show no significant cytotoxic impact on the studied cells in vitro, with the exception of CaP nanoparticles, which in high concentrations have cytotoxic effect on the U937 cells, and Gold nanoparticles 1 and 2, that are able to inhibit growth of the L929 cells. The most effective phagocytosis by macrophage-like cells J774 is observed for PLGA nanoparticles 1 and 2 with the immobilized antigen, while Gold nanoparticles 1 and 2 with antigen can interact with the surface of these cells without being phagocytated by them. In BALB/c mice immunized per os with antigen immobilized on PLGA 1 and 2 as well as Gold 2 carriers, the concentration of specific IgA antibodies in blood significantly increases after the second immunization, compared with controls. In the group of mice treated with PLGA 2 conjugated antigen, the concentration of specific IgG in blood after the third immunization also increases. These results show the promise of nanoparticles PLGA 1 and 2 as adjuvant for immunization per os.

34-43

Authors: O. V. Halkin, O. O. Riabovol, D. O. Minchenko, A. Y. Kuznetsova, O. O. Ratushna, O. H. Minchenko

The aim of this research was to study the effect of glutamine deprivation on the expression of genes encoding for HTRA1/PRSS11, LONP1/PRSS15, and some cathepsins in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme-1). It was shown that in control glioma cells (transfected by empty vector) glutamine deprivation up-regulated the expression of LONP1, CTSD, CTSF, CTSO, and CTSS genes, down-regulated HTRA1, CTSC, and CTSK gene expressions, and did not significantly change the expression of CTSA, CTSB, and CTSL genes. Inhibition of ІRE1 signaling enzyme function in U87 glioma cells modified the effect of glutamine deprivation on the expression of HTRA1, LONP1, CTSD, CTSL, CTSO, and CTSS genes: removed the effect of glutamine deprivation on HTRA1 and CTSO genes, introduces on CTSL gene, reduced — on CTSD gene, and enhanced — on LONP1 and CTSS genes. Therefore, glutamine deprivation affect the expression level of most studied genes in relation to the functional activity of IRE1 enzyme, a central mediator of endoplasmic reticulum stress, which responsible for control of cell proliferation and tumor growth.

44-52

Authors: I. A. Blaydа, N. Yu. Vasylieva, T. V. Vasylieva, L. I. Sliusarenko

The aim of the work was to optimize the process of germanium bioleaching from the dumps after coal beneficiation, namely, to determine the optimal composition of the new nutrient medium for acidophilic chemolithotrophic bacteria ensuring the maximum recovery of valuable metal in minimum time. We optimized the method of mathematical planning adapted to the plan in Greek-Latin squares. The calculations in this approach are based on the analysis of variance. The formal design of experiments has been carried out with four operating factors at four levels. The calculations were performed in Excel. The significance of the factor levels were analyzed using the Duncan’s multiple range test, the uniformity of the variances was examined the Cochran test, and the significance of the factors was tested by the Fisher criterion for each day of the experiment. The obtained results were interpreted mathematically and biologically. The following combination of factors and their levels was recommended as optimum nutrient medium, g/dm3: KH2PO4 — 1.0; (NH4)2SO4 — 2.0; KCl — 0.1; MgSO4 — 0.5; NH4Cl — 0.5; Na2S2O3 — 5.0. The proposed composition allows the more than 90% quick extraction of germanium into the solution (in four days), which was previously impossible.

59-67

Authors: E. N. Vlasenko, J. V. Stepnevskaya, O. V. Kuznetsova

The aim of the study was to determine the intensity of synthesis of volatile aroma compounds by Pleurotus ostreatus (oyster mushroom) on sunflower husks and barley straw using sensory profile analysis and UV spectroscopy. The main cultural and morphological characteristics of the mycelial growth and development of fruiting bodies are determined: the period of mycelial development on the substrate, the time of primordial formation, the number of mushroom bunches per unit volume of substrate, the morphology of carpophores. Characteristic attributes of the aroma of dried fruiting bodies (mushroom, woody, sweet, herbaceous, fish, meat, floral, earthy, acidic, putrescent) are established and their aroma profiles are built. Sensory profile analysis of flavor of dried samples showed that the mushroom flavor of fungi cultivated on the sunflower husk is more pronounced than of those grown on barley straw. The light absorption maxima are recorded in the ranges 204–210 and 250–290 nm according to UV absorption spectra. Optimal conditions for extracting aromatics from dried fungi samples are the extraction time of 20–35 min at the boiling point of the solvent. Analysis of the UV spectra of fungal alcohol and hexane extracts showed that the intensity of the synthesis of volatile compounds is higher for strains cultivated on sunflower husks than for samples obtained on barley straw.

20-30

Authors: Syvash O. O., Zolotareva O. K.

The purpose of the review was to analyze the basic biochemical processes leading to the chlorophyll degradation and ways to control this process in plant product storage. First of all, this is a complex of enzymatic reactions starting with the hydrolysis of chlorophyll with the formation of acyclic diterpene phytol and water-soluble chlorophyllide. An alternative primary reaction is the removal of magnesium from the chlorophyll tetrapyrrole ring to form pheophytin with the participation of Mg2+-dechelatase and/or low-molecular Mg2+-dechelating substances. The chlorophyll breakdown can also be caused by free radicals formed in the peroxidase-catalyzed reaction of Н2О2 with phenolic compounds or fatty acids. The unstable product of chlorophyll peroxidation, C132 –hydroxychlorophyll a decomposes to colorless low-molecular compounds. Expression of the genes of chlorophyll catabolism enzymes is controlled by phytohormones. Methods for controlling the pigment decomposition during storage of plant products are associated with the use of activators and inhibitors of chlorophyll decomposition. The best known inductor of the synthesis of catabolic enzymes is ethylene, widely used to accelerate fruit ripening. Gibberellins, cytokinins and nitric oxide, on the contrary, slow down the loss of chlorophyll.

41-49

Authors: Kapustianenko L. G.

The aim of the research was to obtain the polyclonal antibodies against a fragment of human plasminogen kringle 5 and to study their immunochemical properties. The following approaches were used: immunization of rabbits with plasminogen kringle 5, receiving of high-titer immune serum, synthesis of kringle 5-based affinity sorbent for selection of monospecific antibodies, chromatography on synthesized K 5-Sepharose, ELISA immunoblot assay. The obtained polyclonal antibodies reacted in ELISA with a plasminogen K 5 fragment, and to a much lesser extent with mini-plasminogen, Lys-plasminogen, K 1-3 and K 4 plasminogen fragments, Glu-plasminogen, in descending order. Based on the dissociation constant determination it was found that these antibodies had high affinity to their epitopes within K 5 fragment (3.89・10–10 M), mini-plasminogen (5.46・10–9 М) and Lys-plasminogen (2.54・10–9 М), and insignificant affinity to K1-3, K4 and Glu-plasminogen. These antibodies reacted in immunoblotting assay with an isolated K 5 fragment of human plasminogen, Lys-Pg and mini-Pg, and did not react with K 5 in Glu-Pg and with K 1-3 or K 4 fragments. Thus, the obtained polyclonal antibodies were monospecific and had a high affinity to kringle 5. These antibodies can be used for the development immuno chemical and immunosensory methods for the quantitative determination of angiostatin K 5 in biological materials.

57-64

Authors: Vodka M. V.

The aim of the work was to confirm the possibility of structural changes of Spinacea olearacea L. chloroplasts usage as biomarkers for assessing of environmental pollution by acid rain and heavy metals. Chloroplasts ultrastructural changes were recorded by transmission electron microscopy. Data on changes in the structure of chloroplasts under the influence of these factors are obtained, in particular the heterogeneity of thylakoid grana packing, the membranes thickness, the starch grains presence, and the lumen space increase as compared with the control. These structural changes can be applied as markers of abiotic stresses influence, notably acid rain and heavy metals, and for the creation of new sustainable high-tech varieties of agricultural crops.

22-33

Authors: T. P. Pirog, M. O. Shulyakova, L. V. Nikituk, S. I. Antonuk, I. V. Elperin

The aim of the work is to realize an alternative processing of toxic industrial waste into surfactants by strains Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii IMV B-7405 for remediation of environment. The studied strains were grown in liquid media containing such sources of carbon as waste (fried) sunflower oil, technical glycerol (by-product of biodiesel production), and aromatic compounds. The synthesis of surfactants was evaluated by emulsification index, conditional concentration of surfactants and concentration of extracellular surfactants, which was determined gravimetrically after their extraction from supernatant by the mixture of methanol and chloroform. The concentration of oil in water and soil was analyzed by gravimetric method after extraction with hexane. It was shown that with increasing concentration of the inoculum up to 10−15% and two times increase of nitrogen source content in medium containing 7−8% (v/v) of crude glycerol, concentration of surfactants synthesized by R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B 7241 and N. vaccinii IMV B-7405 was 3.4; 5.0 and 5.3 g/l, respectively, that is 1.6−1.7 times higher as compared with values on basal medium with the same content of substrate. The maximum concentration (3.9−4.3 g/l) of surfactants synthesized by A. calcoaceticus IMV B-7241 on fried sunflower oil (4%) was achieved by using the inoculum grown on refined oil. The ability of R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-7241 and N. vaccinii IMV B-7405 to decompose aromatic compounds (phenol, naphthalene, toluene, hexachlorobenzene, benzoic and N-phenylanthranilic acid) with simultaneous synthesis of extracellular metabolites with surface-active and emulsifying properties was established. In the presence of surfactants in the form of culture liquid (5−10%), the degree of degradation of complex oil with heavy metal (Cu2+, Cd2+, Pb2+, 0.01−0.5 mmol) pollution in water (3−6 g/l ) and soil (20 g/kg) after 20 days was 82−92%. Bioconversion of industrial waste into surfactants for environmental technology allows recycling toxic waste, reducing costs of microbial surfactants and provides double effect of environmental purification, which is achieved in the production and use of microbial surfactants.

40-56

Authors: V. V. Turov, T. V. Krupska, А. P. Golovan, L. S. Аndriyko, М. Т. Кartel

The aim of the study was to identify the influence of hydrodensified nanosilica particles on the binding of water by milled plant raw materials in neutral and acidic media. Flowers of Hibiscus sabdariffa and Calendula officinalis were used as the model materials. According to the microphotographs and low temperature 1H NMR spectroscopy data, the silica film forms on the surface of the milled plant particles, and it can significantly influence their hydration. According to the suggested scheme, some of the water from the inner cavities of plant raw materials moves (as evidenced by the decreasing radius of water-filled pores) to the zone of contact of the composite components (the radius of clusters of adsorbed water increases). In studies of desorption of active substances from milled medicinal herbs and their composites by the initial and hydrodensified nanosilica, it has been shown that the formation of a composite significantly reduces the rate of desorption. Minimal desorption is observed in composites containing hydrodensified nanosilica. The studied composite systems are promising for biomedical researches.

67-74

Authors: N. O. Ryzhenko, V. M. Kavetsky

The aim of investigation was to develop a new approach in Cd, Zn, Cu, Pb phytotoxicity assessing. This approach provided the probit analysis using. Methods included probit analysis, thin layer chromatography, statistical methods (calculation of the least significant difference, correlation analysis). We applied «doze-effect» model to obtain the ranking of the metals according to their phytotoxicity in spring barley field. We offered to estimate the phytotoxicity by PhLD50 index. Research results were: graphic formalization of "dose-effect" dependence and calculation of phytotoxic doses (PhLD50 and PhLD95) for Cd, Cu, Pb, Zn in polluted soil. According to PhLD50 value we conducted a comparative assessment of Cd, Cu, Pb, Zn phytotoxicity relatively to spring barley. According to PhLD50 value metals could be ranked: Cd> Cu> Pb> Zn. The most toxic metal was Cd. PhLD values, on which the estimation of metals phytotoxicity, were: Cd – 50, Cu –129, Pb –537, Zn –603 mg / kg mobile forms in turf-podzol sandy loam soil. Our findings are relevant to estimating the metal hazard and controlling the condition of the crop growth.