Biotechnologia Acta

6-28

Authors: O. V. Matseliukh, L. D. Varbanets

In a review the literature data on protein inhibitors of peptidases synthesised by different types of microorganisms are systematized. It is shown that at the present time on the basis of amino acid sequence homology protein inhibitors are grouped into 77 families, 29 of which include inhibitors of microorganisms. The mechanism of inhibition of peptidases by proteins may be related to their catalytic mechanism of action or include unrelated blocking of the active site or its surroundings. The structural elements of the protein inhibitors are responsible for binding to the peptidases, mostly include the N- or C-terminal sequences, the unprotected polypeptide loops (chains), which are acting independently or in combination with other elements. The basic properties, structural features and, where it is established, the functions of the protein inhibitors of peptidases are considered. Since some of these proteins effectively inhibit such peptidases as subtilisin, chymotrypsin, pancreatic elastase, their practical use in the treatment of diseases such as emphysema, arthritis, pancreatitis, thrombosis, hypertension, muscular dystrophy, cancer. It is suggested that the role of a bacterial homologue of Escherichia coli alphaacroglobulin, which is a periplasmic protein, is to protect the periplasmic space from the action of bacteria own proteases. Based on the specific properties of alpha-2-macroglobulin to bind endopeptidases active molecules, they are used in biotechnology to isolate endopeptidases from crude biological preparations and titration of its active centers. Some free–living bacteria are able to synthesize protein inhibitors to protect from the effects of its own enzymes, while the presence of these proteins in pathogens may play a certain role both in the infectious process and in the protection of the host proteases.

43-57

Authors: L. P. Buchatsky

The latest progress in biotechnology on fish aquaculture and different modern methods of investigations for increasing of fish productivity in aquaculture are analyzed. Except for the applied aspect, the use of modern biotechnological methods of investigations opens new possibilities for fundamental researches of sex-determining mechanisms, polyploidy, distant hybridization, and developmental biology of bony fishes. Review contains examples of utilizing modern biotechnology methods to obtain transgenic fishes with accelerated growth and for designing surrogate fishes. Methods for receiving unisexual shoals of salmon and sturgeon female fishes with the view of obtaining a large quantity of caviar, as well as receiving sterile (triploid) fishes are analyzed. Great attention is given to androgenesis, particularly to disperm one, in connection with the problem of conserving rare and vanishing fish species using only sperm genetic material. Examples how distant hybrids may be obtained with the use of disperm androgenesis and alkylated DNA are given. Methods of obtaining fish primordium germ cells, recent developments in cultivation of fish stem cells and their use in biotechnology, as well as ones of transplantation of oogonium and spermatogonium to obtain surrogate fishes. The examples of successful experiments on spermatogonial xenotransplantation and characteristic of antifreezing fish proteins and also the prospect of their practical usage are given.

71-76

Authors: І. І. Grynyuk, S. V. Prylutska, N. S. Slobodyanik, О. Yu. Chunikhin, О. P. Matyshevska

The distribution of pristine C60-fullerene nanoparticles by volume and number in water solution without chemical dispersants with different salt and protein composition was evaluated using the correlation spectroscopy analysis. It was shown, that water colloid solution contains C60-fullerene particles with hydrodynamic diameter from 38 to 600 nm, and the main peak position is at 43 nm. In saline solution (0,9% NaCl) the particle size distribution range enlarges that may result from the C60 aggregation. Albumin introducing into the medium promotes dispersed state of C60 nanoparticles and prevents their spontaneous as well as NaCl-dependent agglomeration. No obvious aggregation was detected when C60-fullerene in concentration 10–5 М was added to RPMI medium containing 5% fetal bovine serum. The data show that stable homogenous water colloid solution of pristine C60 fullerene could be used for studding cell respon ses in cultural medium.

86-93

Authors: L. A. Safronova, S. I. Voychuk, B. G. Suhov, V. S. Pidgorskiy

Due to the widespread of the ecological disorders and diseases of the gastrointestinal tract of humans and animals there is an increased interest in probiotic products (probiotics, prebiotics and synbiotic), which showed its efficacy and safety in their treatment and prevention. Yeast Saccharomyces cerevisiae and cell culture of a human larynx cancer НЕр-2 were used to study cyto- and DNA-damage effects of several polysaccharides (arabinogalactan and nanobiocomposite of arabinogalactan with flavonoids, karraginan, galactomannan and their hydrolyzed derivatives), which are new perspective prebiotic substances. Cytotoxic potential of substances was determined from MTT-assay and DNA-damage effects were determined by morphological and structural features of cell nuclei. The synergic action of the polysaccharides with chemical triggers of cellular damages (hydrogen peroxide, acetic acid, nitrosomethylurea) was evaluated. The studied substances possessed no cyto- and DNA-damage action on the yeast and mammalian cells. It was shown that polysaccharides were able to enhance the effects of hydrogen peroxide, acetic acid and nitrosomethylurea. Nature of the marked effects remains unclear, but it may be assumed that the studied prebiotic substances can be used to enhance the effects of some chemical compounds and appear promising for use in integrated biomedical preparations of varied spectrum.

100-104

Authors: L. P. Kuznetsova, E. R. Nikitina, E. E. Sochilina

Butyrylcholinesterase preparations from horse blood serum widely use in the research purposes and as an analytical reagent for determination of biologically active substances. High sensitivity of butyrylcholinesterase to organophosphorous inhibitors which possess high toxicity for the warm-blooded is especially important. Influence of octanol on reactive capacity of horse serum butyrylcholinesterase to butyrylcholine and α-naphtylacetate and on its sensitivity to diisopropylfluorophosphate is investigated. Enzyme activity measured by a method of potentiometric titration in experiments with butyrylcholine and a fluorimetric method in experiments with α-naphtylacetate allowing to define speed of hydrolysis of small concentration of a substrate. It is shown, that octanol does not influence on the hydrolysis rate of butyrylcholine but activates the enzymatic hydrolysis of α-naphtylacetate and reduces the sensitivity of enzyme to inhibition by diisopropylfluorophosphate. The received results have the important practical value as octanol apply in some manufactures in the capacity of a defoamer. Our researches have shown that such standard procedures which used by manufacture of enzyme preparations as salting-out and desalting only partially delete octanol impurities. Complete separation of the enzyme from octanol and its sensitivity reduction to diisopropylfluorophosphate was possible by selective sorption of enzyme protein on the ion-exchange resin with the after-elution by a salt solution.

115-119

Authors: Bavol A. V., Dubrovna O. V., Morgun B. V.

The transgenic wheat cell lines were obtained via biolistic transformation of the callus cultures initiated from the 3-day-old sterile seedling shoot apexes. The pAHC25 vector construction used for 14- and 28-day-old callus cultures transformation carried the selective phosphinothricin-N-acetyltransferase (bar) gene and reporter ?-glucuronidase gene. The cell line selection was carried out on the media with phosphinothricin by means of graduated cell selection. The transgenic status of the obtained forms was proved by PCR-analysis. The presence of new relatively high molecular (more than 1 000 bp) amplicons were found out for three transformed lines by means of IRAP PCRanalysis with the primers coding for long termainal repeats sequences of SIRE 1 retrotransposon. This fact may prove transposition of this mobile genetic element. The new DNA fragments were detected for three of the seven analyzed lines but for the control callus. It is possible to assume at induction of SIRE 1 transposition bis probably caused by the genomic stress of foreign DNA inserting or associated with the transformation process (mechanical wounding, cultivation on selective media).

132-138

Authors: Musatova I. B., Prokopyuk O. S., Volina V. V., Prokopyuk V. Yu.

Phase transitions and vitrification of multicomponent cryoprotective media including NaCl, dimethyl sulfoxide, sucrose, polyvinylpyrrolidone, dextran and hydroxyethylic starch for cryopreservation of explants of placental tissue as well as its morphological integrity after cryopreservation in these media with the aim of adequate selection of the components were studied by differential scanning calorimetry. Analysis of the obtained thermograms has shown that in all multicomponent cryoprotective media, frozen with a high rate, the part of the solution transforms into a vitrified state during warming ice crystallization from amorphous phase and melting of solution area with eutectic composition are absent. Minimal intensity of endothermal effect of ice melting as well as the highest morphological integrity of placental tissue is observed in multicomponent solutions containing 5% dimethyl sulfoxide and 6,8% sucrose or 5% hydroxyethylic starch in physiological solution. These media should be considered as optimal ones for cryopreservation of placental tissue explants, and their composition is recommended to be used when designing complex cryoprotective media.