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2017
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CLUSTER ANALYSIS IN BIOTECHNOLOGY

O. M. Klyuchko

"Biotechnologia Acta" V. 10, No 5, 2017
https://doi.org/10.15407/biotech10.05.005

Abstract
Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of the National Academy of Sciences of Ukraine, Kyiv The goal of publication was the analysis of cluster methods and possibility of their application in biotechnology. The evidences found in scientific literature were summarized and analyzed. This article gives a brief description of cluster analysis — basic principles, some examples of their application are given for biotechnological problems. Results of the biotechnological studies that required application of cluster methods in combination with other mathematical approaches are considered. The conclusion contains an evaluation of the performed analysis as well as recommendations on the application of cluster analysis methods in biotechnology.

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INCREASING OF THE EXPRESSION OF RECOMBINANT scFv-ANTIBODIES EFFICIENCY

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

"Biotechnologia Acta" V. 10, No 5, 2017
https://doi.org/10.15407/biotech10.05.019

Abstract
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.

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TECHNOLOGY OF STERLET REPRODUCTION BY MEANS OF CRYOPRESERVED SPERM
L.P. Dragan, A.I. Mruk, V.M. Golian, L.P. Buchatsky

"Biotechnologia Acta" V. 10, No 5, 2017
https://doi.org/10.15407/biotech10.05.030

Abstract
The purpose of the work was to reproduce the sterlet of the Danube and Dnieper populations using cryopreserved sperm frozen in modified cryoprotective medium. Low-temperature cryopreservation of sterlet male reproductive cells was carried out in accordance with conventional procedures of cryobiology.

As a result of the performed work, it has been found that the applied cryoprotective environment, which includes methanol, enhances the cryoprotective effect and is technologically optimal for the process of the Danube and Dnieper populations of sterlet sperm storage and reproduction. Fish sperm cells retained viability after defrosting; their ability to fertilize the eggs was restored. The results of the conducted studies indicate that, despite a certain decrease in the quality properties of sperm after freezing/defrosting, its ability to fertilize did not significantly decrease. Revived in this way larvae from two populations of sterlet were used to reproduce repairing brood stock under conditions of fish farming.

Such a method of sterlet sperm cryopreservation could be recommended for use at commercial enterprises with sturgeon fish commodity cultivation.

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BIOBUTANOL ACCUMULATION USING ALTERNATIVE SUBSTRATES BY CULTIVATION OF Clostridium acetobutylicum STRAINS
О. О. Тigunova, H. S. Аndriiash, N. Ye. Beiko, І. V. Melnyk, S. М. Shulga

"Biotechnologia Acta" V. 10, No 5, 2017
https://doi.org/10.15407/biotech10.05.036

Абстракт
The aim of the research was to investigate the accumulation of biobutanol by strains C. acetobutylicum using alternative substrates (Jerusalem artichoke juice, technical glycerol, crude glycerol, shredded biomass of soy, rape, wheat and switchgrass). In order to increase the accumulation of butanol in the process of cultivation, the cells of C.acetobutylicum were statically immobilized on carriers (belting strips, ferrite rings and Raschig rings) by the method of adsorption immobilization. The cells were precipitated by centrifugation, the supernatant was distilled and then fermentation products were determined. Gas chromatography was used to determine the presence of solvents in the culture fluid. The biggest accumulation of  butanol (2 g/dm3) was at concentration of crude glycerol 16 g/dm3 in the medium, and complete inhibition of culture development ‒ at concentration of 25 g/dm3. The accumulation of butanol by the strain Clostridium acetobutylicum IMB B-7407 using fill and draw method depended on the amount of sequestered and infused medium. Immobilization of the culture using the Raschig rings allowed increasing the bioconversion to butanol twice. So it is shown the possibility to use non-traditional substrates for the production of biobutanol. Immobilization of C.acetobutylicum culture cells on carriers increased the accumulation of butanol. The use of Raschig rings, as carriers for immobilization, allowed increasing the accumulation of  butanol twice.

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INHIBITION OF THE EXPRESSION OF PHYSIOLOGICAL PRIONS WITH ANTISENS-OLIGONUCLEOTIDES
N. U. Susol, D. D.Ostapiv, V. V. Vlizlo

"Biotechnologia Acta" V. 10, No 5, 2017
https://doi.org/10.15407/biotech10.05.043

Abstract
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.

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УГНЕТЕНИЕ ЭКСПРЕССИИ ФИЗИОЛОГИЧЕСКИХ ПРИОНОВ

АНТИСЕНС-ОЛИГОНУКЛЕОТИДАМИ

Н. Ю. Сусол, Д. Д. Остапов, В. В. Влизло

 
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"Biotechnologia Acta" V. 10, No 5, 2017
https://doi.org/10.15407/biotech10.05.051

HYDRATION FEATURES OF COMPOSITE SYSTEMS BASED ON HIGH-DISPERSED SILICA AND SUCROSE IN DIFFERENT MEDIA

Turov V.V., Krupska T.V., Golovan А.P., Кartel М.Т.

Abstract

The state of water in the hydrated sucrose powder, in its melt, and composite systems containing 3 parts of silica (A-300 or AM1) and 1 part of sucrose in air, low-polar organic solvent-deuterochloroform and CDCl3 medium with addition of strong acids has been studied by low-temperature 1H NMR  spectroscopy. It was demonstrated that at the joint mechanoactivation of sucrose with nano-silica, its amorphization takes place. In this case, the adsorption of water in such a composite is not accompanied by the dissolution process in interfacial water of the sucrose immobilized on the SiO2 surface. It was found that the effect of sucrose solubility increasing accompanies the placement of the composite in a medium that does not dissolve water and sucrose, an organic solvent - deuterochloroform. It was suggested that it is due to an increase the contact area of adsorbed water clusters with the sucrose surface. The maximal concentration of sucrose, which dissolves in interfacial water, is about 30% by weight. The replacement of hydrophilic silica (A-300) by hydrophobic silica (AM1), while maintaining the concentration ratio of components and the amount of adsorbed water, reduces the free energy of water binding in the composite system. The obtained regularities can be used in the design of a new generation of enterosorbents.

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THE INFLUENCE OF CERIUM DIOXIDE NANOPARTICLES ON GERMINATION OF SEEDS AND PLASTIC EXCHANGE OF PINE SEEDLINGS (Pinus sylvestris L.)

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

"Biotechnologia Acta" V. 10, No 5, 2017
https://doi.org/10.15407/biotech10.05.063

Abstract

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.

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Ж-л "Biotechnologia Acta" Т. 10, № 4
https://doi.org/10.15407/biotech10.04.005

Abstract

APPLICATION OF ARTIFICIAL NEURAL NETWORKS METHOD IN BIOTECHNOLOGY O. M. Klyuchko

The aim of the work was to analyze the method of artificial neural networks and to examine its implementation in biotechnology. Nearly 300 publications are reviewed because this method is very widely used. The artificial neural networks are described and analyzed, and the examples of their application in biology and medicine are given. Solutions of complex problems, which required combining this method with other modern mathematical methods, are examined. Recommendations are presented for the application of this method in biotechnology.

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Ж-л "Biotechnologia Acta" Т. 10, № 4, 2017
https://doi.org/10.15407/biotech10.04.014

Abstract

ADJUVANT PROPERTIES OF NANOPARTICLES IMMOBILIZED RECOMBINANT DIPHTHERIA TOXOID FRAGMENT T. O. Chudina, A. Yu.  Labyntsev, D. V.  Kolybo1, 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.

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Ж-л "Biotechnologia Acta" Т. 10, № 4
https://doi.org/10.15407/biotech10.04.025

Abstract

INTENSIFICATION OF MICROBIAL EXOPOLYSACCHARIDE ETHAPOLAN BIOSYNTHESIS ON MIXTURE OF MOLASSES AND SUNFLOWER OIL T. P. Pirog A. A. Voronenko, M. O. Ivakhniuk

The purpose of the research was to establish Acinetobacter sp. IMB B-7005 cultivation conditions, which provide the maximal synthesis of microbial exopolysaccharide ethapolan on a mixture of molasses and sunflower oil, and to explore the possibility of replacing refined oil in a mixture with molasses for waste one. On the basis of theoretical calculations of energy consumption for the synthesis of ethapolan and biomass, it was determined that the optimal molar ratio of the concentrations of energy-deficient (sucrose) and energy-excessive (sunflower oil) substrates in the mixture was 1.0:0.9. Experiments have shown that the highest values of exopolysaccharide synthesis were observed at a molar ratio of monosubstrates in mixture 1.0:1.1, which is as close as possible to the theoretically calculated one. It was shown that increasing concentration of molasses and refined oil in mixture from 1.0 to 1.5% was accompanied by increase in amount of synthesized exopolysaccharide and its synthesizing capacity by 1.2 and 1.3 times, respectively. The possibility of replacing refined oil in a mixture with molasses for various types of waste (after frying potatoes, meat, vegetables and mixed) was established. The maximum parameters of exopolysaccharide synthesis (concentration 14 g/l, synthesizing capacity 3.5 g exopolysaccharide/g biomass) were observed when using mixed waste oil for both inoculum obtaining and EPS biosynthesis. The obtained results testify to the possibility of development of universal technology for obtaining microbal exopolysaccharide ethapolan on a mixture of waste (molasses and waste oil) independent of the type and provider of waste oil.

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Ж-л "Biotechnologia Acta" Т. 10, № 4
https://doi.org/10.15407/biotech10.04.034:

Abstract

IRE1 KNOCKDOWN MODIFIES THE EFFECT OF GLUTAMINE DEPRIVATION ON THE EXPRESSION OF A SUBSET OF PROTEASES IN U87 GLIOMA CELLS

O. V. Halkin,    O. O. Riabovol, D. O. Minchenko, A. Y. Kuznetsova,    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.

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