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.

9-18

Authors: V. I. Nazarenko, O. P. Demchenko

This review addresses the analysis of properties and applications of fluorescent nanodiamonds. They are carbon nanostructures with atomic arrangement of a diamond and carry all its properties, including record — high density, rigidity and refraction index. They are of almost spherical shape, and their small size (~4–10 nm) creates substantial surface area that can be used for absorption of different compounds including drugs. Their surface is formed by different chemical groups (hydroxyls, carboxyls, etc.) exhibits also chemical reactivity that allows different types of modifications. This opens innumerable possibilities for constructing different functional nanomaterials. The technologies have been developed for making these nanodiamonds fluorescent. Particularly, these properties are achieved by radioactive treatment with the formation of N–V impurities. These particles absorb and emit light in convenient for observation visible range of spectrum. They do not photobleach, which is very attractive for fluorescent microscopy of the cell. And, finally, these nanoparticles do not display toxicity on the cellular or whole — body level, and because of their biocompatibility they can be used in vivo as contrast agents and drug carriers. It is expected that future biotechnological applications of these nanoparticles will be connected with the creation of nanocomposites that combine multiple useful functions.

41-48

Authors: M. V. Grigoreva

Biodegradable polyurethanes attract interest of those developing composite materials for biomedical applications. One of their features is their ability to serve as carriers, or matrixes, for medicines and other bioactive compounds to produce a therapeutic effect in body through targeted and/or prolonged delivery of these compounds in the process of their controlled release from matrix. The review presents polyurethane composites as matrices for a number of drugs. The relation between structure of the composites and their degradability both in vitro and in vivo and the dependence of drug release kinetics on physicochemical properties of polyurethane matrix are highlighted. The release of drugs (cefazolin, naltrexone and piroxicam) from the composites based on cross-linked polyurethanes (synthesized from laprols, Mw between 1,500 and 2,000 Da and toluylene diisocyanate) demonstrated more or less the same pattern (about 10 days in vitro and three to five days in vivo). In contrast, the composites with dioxydine based on a linear polyurethanes (synthesized from oligotetramethilene glycol, Mw 1,000 Da, diphenylmethane-4,4’-diisocyanate and 1,4-butanediol) retained their antimicrobial activity at least 30 days. They also showed a significantly higher breaking strength as compared to that of the composites based on cross-linked polyurethanes.

62-78

Authors: V. D. Naumenko, B. V. Sorochinsky, V. I. Kolychev

Biological properties, chemical structures and biosynthesis pathways of plant isoflavones, especially soybean isoflavones (daidzein, genistein and glycitein) are reviewed. The structures of isoflavones, and their aglicone and glucosides (glycosides) forms as well as isoflavone biosynthesis pathways are described. General information about the advanced methods for the detection of isoflavones and their conjugates are considered. The importance of the profiling of isoflavones, flavonoids and their conjugates by means of analytical tools and methods to dissolve some questions in biology and medicine is discussed. The review provides data on the major isoflavone content in some vegetable crops and in the tissues of different soybean varieties. Health benefits and treatment or preventive properties of isoflavones for cancer, cardiovascular, endocrine diseases and metabolic disorders are highlighted. The mechanisms that may explain their positive biological effects are considered. The information on the application of advanced technologies to create new plant forms producing isoflavonoids with a predicted level of isoflavones, which is the most favorable for the treatment is given. The possibilities to use the metabolic engineering for the increasing of accumulation and synthesis of isoflavones at the non-legume crops such as tobacco, Arabidopsis and maize are considered. The examples how the plant tissues, which are not naturally produced of the isoflavones, can obtain potential for the synthesis of biologically active compounds via inducing of the activity of the introduced enzyme isoflavon synthase, are given. Specific biochemical pathways for increasing the synthesis of isoflavone genistein in Arabidopsis thaliana tissues are discussed. It is concluded that plant genetic engineering which is focused on modification of the secondary metabolites contain in plant tissues, enables to create the new crop varieties with improved agronomic properties and nutritional characteristics.

87-94

Authors: О. S. Gojster, W. Е. Kriwenchuk, G. O. Chmelnitsky, О. Н. Minchenko

The aim of this work was the development of T-2 toxin immunedetection method using biosensor approaches. For this aim the conjugates of T-2 toxin with proteins were obtained and used for antibody preparation. The specific properties of obtained antibodies were studied by immunochemical methods. It was shown that carrier enzyme plays important role in realization of immunogenic properties of T-2 hapten. The detection time of IgG fraction as well as antiserum binding activity by immunoenzyme and immunosensor analyses is 10–15 minutes. Two variants of T-2 toxin determination in competitive mode of immunochemical analysis were suggested, i. e. ELISA and immunosensor method (based on surface plasmon resonance phenomenon) with detection from 10 ng/ml and 1 ng/ml, correspondingly. These methods can be applied for detection of T-2 toxin in the food and fodder in concentration from 1 up to 500 ng/ml.

100-107

Authors: S. V. Malysheva, N. M. Bilko, J. Hescheler

The search for effective and safe methods to generate induced pluripotent stem cells is especially urgent. In the paper murine embryonic fibro blasts were reprogrammed towards actively proliferating colonies with typical induced pluripotent stem cells morphology by means of Sleeping beauty transposon-based vector system. The obtained clones were checked for the expression of various pluripotency markers: alkaline phosphatase, Oct4 and Sox2 genes, SSEA-1 expression in various clones was evaluated. Also the reactivation of endogenous pluripotency factors Nanog and Rex1 was indicated. The data obtained is analyzed and compared to the established pluripotent stem cell line. It is shown that somatic cells are reprogrammed towards pluripotency by means of Sleeping beauty transposon system. Therefore, the system is a new perspective biotechnological tool to generate pluripotent cells.

115-121

Authors: О. Yu. Rogulska, О. B. Revenko, Yu. O. Petrenko, H. Ehrlich, О. Yu. Petrenko

Development of the new types of tissue engineered structures is one of the promising trends of current biotechnology. The study was directed to the assessment of prospects for the application of chitin-based skeletons derived from marine sponges of Aplysinidae family (Aplysina fulva and Aplysina aerophoba) for creation of bioengineered constructs based on human mesenchymal stromal cells. After cleaning and demineralization procedures, sponge skeletons appeared as three-dimensional macroporous matrices formed by intersecting chitin fibrils. After seeding into chitin-based matrices the cells were attached to the surface of the fibrils and were able to spread and proliferate. Mesenchymal stromal cells within Aplysina fulva differentiated into osteogenic and adipogenic directions under the influence of appropriate inductors. Demineralized skeletons derived from marine sponges of Aplysinidae family could be used as scaffolds for mesenchymal stromal cells which provides new opportunities for the creation of adipose and bone tissue engineered structures.

131-136

Authors: O. M. Magomedov, A. F. Levytsky, D. V. Golovatiuk, A. N. Velikyy, T. A. Polishchuk

The results of the study of diagnostically important metabolism parameters in patients with bone cysts in different stages of the disease are presented. It is shown that an increase activity of protein banding collagenase, alkaline phosphatase and also of hydroxyproline, glycosaminoglycans contents due to lower levels of calcium and inorganic phosphate levels increase in blood serum are expressed in a stage osteolysis than the step of separating. Decreasing the amount of glycosaminoglycans and collagen in bone indicates an intensification of catabolic processes in the connective tissue matrix. Diagnostically important indicators of the degree of disturbance of bone metabolism are the level of collagen, proteoglycans and activity of marker enzymes — collagenase and alkaline phosphatase. Based on the evaluation of sensitivity, specificity and diagnostic efficiency of the obtained results, we can recommend the threshold values of the investigated parameters of basic organic components and mineral metabolism of bone for the differential diagnosis of stages of bone cysts in children, which will serve as a basis for the development of appropriate diagnostic tests.

143-149

Authors: О. V. Кyrychenkо, S. O. Gryniuk

The growth-regulatory activity of the complex algae extract from Spirogira sp. at pre-so wing treatment of soybean seeds was studied in green-house and field experiments. It was shown that phytoextract has stimulated seeds germination on 12%, plants growth on 11–37%, soybean productivity increased on 6–27% as well as activated the development and functional ability of rhizospheric nitrogen-fixing microorganisms. The quantity of oligoazotrophes increased in 1,5–6,3 times, nitrogenase activity in 1,5–1,7 times. The possibility aspects of growth-regulatory activity of the algae extract in both plants and rhizospheric microorganisms is under discussion. Our results have confirmed the perspectives of practical use of the biological activity substances from the algae at soybean growing in order to increase plants productivity and improve microbiological indexes of soil.

16-19

Authors: Michael Sela

History of developing synergy between monoclonal antibodies, anti-tumor activity of monoclonal antibodies against tyrosine-kinases receptors EGFR/ErbB-1 and HER2/ErbB-2 as well as growth factor VEGF in various combinations are considered in the article. There were proposed hypotheses about potential molecular mechanisms underlay synergy between monoclonal antibodies (for homo- and hetero combinations of antibodies appropriately specific for antigenic determinants on the same or different receptors). Future trends in researches necessary to deeper understanding causes of this phenomenon and perspectives for practical application of monoclonal antibodies acted synergistically as immunotherapeutic drugs for human tumors treatment are reviewed.

33-42

Authors: E. V. Lugovskoi, I. N. Kolesnikova, S. V. Komisarenko

It was shown by monoclonal antibodies that B?N-region of fibrin desA molecule (B?1-53) comprises the polymerization site including the peptide bond B?14-15. This site participates in the second stage of fibrin polymerization — lateral association of protofibrils. In the B?15-53 fragment was also found the site called «C», which together with the site «A» participate in the first stage of polymerization — the protofibrils formation. The model of the primary intermolecular interaction of fibrin was designed. It was found by monoclonal antibodies II-4d the site («c») in the N-terminal half of ? chain of the fibrin D-region. This site participates in the protofibrils formation and is complement to site «C» as we assume. We have discovered two neoantigenic determinants. One of these determinants exposes within the coiledcoil fragment B?126-135 of fibrin as a result of fibrinopeptide A splitting off from fibrinogen by thrombin. The structural rearrangements discovered in this site of the fibrin molecule are necessary for the following protofibrils lateral association. The second neoantigenic determinant is localized in the fragment B?134-190 of D-dimer formed after plasmin degradation of fibrin stabilized by FXIIIa. We have obtained the fibrin-specific monoclonal antibodie FnI-3C to the first determinant and D-dimer-specific mAb III-3b to the second one. Three monoclonal antibodies were obtained against the ?C-region of fibrin(ogen) molecule. It has been experimentally shown by of one of them that ?C-domains is connected with the fibrinopeptides B in fibrinogen and fibrin desA molecules, but removes from the core of the molecules after fibrinopeptides B splitting off by thrombin. Two other monoclonal antibodies specifically inhibit the fibrin polymerization by blocking two unknown polymerization sites within the ?C-region. The test-systems for the soluble fibrin and D-dimer quantification in human blood plasma were designed on the basis of monoclonal antibodies FnI-3C and III-3b as «catch»-antibodies and one II-4d as a «tag»-antibody, respectively. The clinical trials of the test-systems were carried out in Ukraine. It was shown that for the prediction of postoperative thrombotic complications and monitoring the efficiency of antithrombotic therapy the simultaneous quantification of soluble fibrin and D-dimer before the operation and at different time intervals after the operation is required. Only in this case it is possible to get information about the state of the balance between blood coagulation and fibrinolytic systems, and determine the degree of the threat of thrombosis.

68-77

Authors: A. Mikhailopulo, A. I. Miroshnikov

Some recent findings in the biotechnology of biologically important nucleosides will be discussed, viz., (i) a new strategy of the cascade one-pot transformation of D-pentoses into nucleosides based on the extension and deepening of the knowledge of the mechanism of functioning of the ribokinase, phosphopentomutase, and uridine, thymidine and purine nucleoside (PNP) phosphorylases, and the role of different factors (structural, electronic, stereochemical) in the glycoside bond formation, (ii) the modern chemistries of the chemo-enzymatic syntheses of nucleosides, (iii) the transglycosylation reaction using natural and sugar modified nucleosides as donors of carbohydrate residues and heterocyclic bases as acceptors catalyzed by nucleoside phosphorylases (NP).

86-93

Authors: А.M. Egorov, V. G. Grigorenko, I. P. Andreeva, M. YU. Ubtsova

The article deals with prospects of using recombinant horseradish peroxidase in analytical biochemistry and biotechnology. Problems of recombinant horseradish peroxidase cloning in different expression systems, possible approaches to their solution, advantages of recombinant recombinant horseradish peroxidase and recombinant horseradish peroxidase-fusion proteins for immunoassays are considered. Possibility for development of mediatorless bienzyme biosensor for peroxide and metabolites, yielding hydrogen peroxide during their transformations, based on co-adsorption of recombinant horseradish peroxidase and the appropriate oxidase was demonstrated. The possibility to produce a fully active recombinant conjugate of recombinant horseradish peroxidase with human heart-type fatty acid binding protein, which may be used in competitive immunoassay for clinical diagnosis of acute myocardial infarction, and recombinant conjugates (N- and C-terminus) of recombinant horseradish peroxidase with Fab-fragments of the antibody against atrazine, which may be applied for atrazine pesticides detection, are demonstra ted for the first time.

105-117

Authors: S. Souchelnytskyi

Cancer is a disease, which explicitly illustrates success, failures and challenges of the modern biomedical research. Technology development has been the driving force of improvements in the cancer treatment. Introduction into clinical practice of genomics, RNA profiling and proteomics technologies have provided a basis for development of novel diagnostic, drugs and treatments. In this chapter, contributions of OMICs technologies to personalization of cancer diagnostic and treatment are discussed. The focus is on technologies that showed capacity to deliver diagnostic that may be used in the clinic as routine tests. Three clinical cases are presented to illustrate already available individualized cancer diagnostic.

132-143

Authors: G. Kvesitadze

Elimination of contaminants from the environment by microorganisms of different taxonomic groups is an evolutionarily determined property, which have already been widely discussed. Until recently, plants still occupying above 40% of the world land, were considered as organisms having only a limited potential for contaminants conjugation and accumulation within cell organelles. Based on 40 years experience in this area author is making an attempts for the evaluation of different aspects of plants ecological potential from the modern understanding; to assume mechanism of inter replacement of enzymes participating in oxidative degradation of organic contaminants in higher plants; to stress the importance of phenoloxidase, enzyme hitherto unknown to participate actively in remediation processes (contaminants oxidative decomposition); to reveal the criterion for the evaluation under the action of contaminants of such precise indicator of plant detoxification potential as deviations in ultrastructural level of plant cells.

162-171

Authors: Christian Birr

SCTL is the fully synthetic correlate of an enzymatic partially hydrolyzed extract from calf thymus. To exclude completely the transmission of bovine spongiformic encephalitis by the bovine thymus product a fully synthetic correlate of the active principles in the thymus tissue hydrolysate has been developed, namely SCTL. This synthetic peptide library has meanwhile substituted calf thymus extract preparations in several cosmetics and drug products. The active principles of SCTL have been invented by the author but the application of the drug substance in cosmetic and pharmaceutical products has been exploited by others. For SCTL only limited pharmacological and toxicological data are available. Some interesting biological activities, though, have been shown for SCTL which might explain to some extend the modes of action and its clinical effectiveness.

182-189

Authors: A. A. Samoylenko, N. V. Byts, G. V. Pasichnyk, N. V. Kozlova, A. V. Bazalii1, D. S. Gerashchenko1, S. G. Shandrenko, A. V. Vorotnikov, T. Kietzmann, S. V. Komisarenko, L. B. Drobot

Ruk/CIN85 is an adaptor protein that plays important roles in the regulation of cellular processes such as cell death, proliferation and motility. It was recently shown that overexpression of Ruk/CIN85 increases the oncogenic potential of human breast adenocarcinoma MCF-7 cells. It was the aim of the present study to investigate whether inhibition of Ruk/CIN85 expression has an effect on the biological properties of the cells. In order to down-regulate Ruk/CIN85 expression of small interfering RNA-based approach was used. For down-regulation of Ruk/CIN85 lentiviral constructs encoding Ruk/CIN85-specific small hairpin RNA sequences were generated. By using the obtained recombinant lentiviruses it was shown that inhibition of Ruk/CIN85 expression influences biological properties (motility, proliferation, ABCG2 expression, and ROS generation) of various tumour cell types such as human breast adenocarcinoma MCF-7, human colorectal adenocarcinoma HT-29, and Lewis mouse lung carcinoma cells.

9-22

Authors: S. V. Isayenkov

The pivotal role of plant vacuoles in plant survival was discussed in the review. Particularly, the providing of cellular turgor, accumulation of inorganic osmolytes and nutrients are the primary tasks of these cellular organelles. The main mechanisms of tonoplast transport systems were described. The known transport pathways of minerals, heavy metals, vitamins and other organic compounds were classified and outlined. The main systems of membrane vacuolar transport were reviewed. The outline of the physiological functions and features of vacuolar membrane transport proteins were performed. The physiological role of transport of minerals, nutrients and other compounds into vacuoles were discussed. This article reviews the main types of plant vacuoles and their functional role in plant cell. Current state and progress in vacuolar transport research was outlined. The examples of application for rinciples and mechanisms of vacuolar membrane transport in plant biotechnology were iven. The perspectives and approaches in plant and food biotechnology concerning transport and physiology of vacuoles are discussed.

36-45

Authors: K. V. Avdiyuk, L. D. Varbanets

The ability of Aspergillus flavus var. oryzae 80428 and Bacillus subtilis 147 α-amylases to split different carbohydrate-containing substrates, such as maltose, sucrose, trehalose, dextrin, α- and β-cyclodextrin, amylose, amylopectin, glycogen, pullulan, soluble starch, insoluble starch, corn starch, wheat starch, dextran 500 has been studied. It was shown that investigated enzymes differ by substrate specificity. α-Amylase of A. flavus var. oryzae 80428 rapidly hydrolysed soluble potato and wheat starch, while the α-amylase of B. subtilis 147 — only wheat starch. Both enzymes don’t cleave maltose, α-cyclodextrin and dextran 500. A. flavus var. oryzae 80428 α-amylase display very small ability to hydrolyze pullulan, while α-amylase of B. subtilis 147 it does not act in general. The lowest values of Michaelis constant for both enzymes at splitting of glycogen have been obtained, indicating that enzymes have the greatest affinity to this substrate. The studies of influence of chemically active substances on activity of A. flavus var. oryzae 80428 and B. subtilis 147 ?-amylases show there are resistant to urea, deoxycholic acid, Tween-80, Triton X-100 and hydrogen peroxide. It’s indicate the enzymes tested may be competitive in compare with earlier described in literature enzymes. The obtained results give a possibility to propose in future usage these enzymes in different fields of industry, foremost in detergent industry.

53-62

Authors: N. M. Boiko, Yu. V. Senkiv, Ye. A. Shlyakhtina, O. V. Kluchivska, N. P. Skorohid, N. E. Mitina, T. V. Skorohoda, O. S. Zaichenko, R. S. Stoika

The main tasks of modern biopharmaceutics are focused at the development of new nanoscale carriers with low toxicity, given size, regulated response to local and remote effects, and capability of visualization of drug action and diagnostic results. A novel oligomeric carrier VAMANG-MP for delivery of the anticancer drug doxorubicin to tumor cells of different lines have been used. The synthesized oligomeric carrier was additionally functionalized by phosphatitylcholine. It was demonstrated that such delivery of doxorubicin to the target cells permits 10 times decreasing of its acting cytotoxic dose comparing with such dose of free doxorubicin, with preserving similar level of the antineoplastic effect. This dose-specific effect was demonstrated both in vitro towards various mammalian tumor cells, and in vivo towards mice with experimental NK/Ly lymphoma was shown. Action of immobilized doxorubicin was followed by more intense formation of vesicles on a surface of the target cells in vitro, and by their inter-nucleosomal DNA fragmentation as compared to action of free doxorubicin, as well as by appearance of a higher amount of the dead cells in the ascytic fluid of the treated NK/Ly lymphoma mice. Thus, the synthesized nanoscale olygoelectrolytic carrier is a perspective system for delivery of anticancer drugs to target cells.

69-74

Authors: V. O. Antonyuk, L. V. Panchak, M. O. Starykovych, K. S. Strutovskaya, R. S. Stoika

A new lectin with 6.5 mg/kg yield was purified from fresh bulb Hyacinthella аcutiloba K. Perss. by combination of affinity chromathography on yeast mannan and by ion exchange chromatography on DEAE-toyopearl. The best lectin inhibitor among tested mono- and disaccharides was D-turanose (Glcp ?1-3 Fruf). Very powerful inhibitor of lectin activity was yeast mannan. The lectin revealed weak affinity to ?-methyl-Dmannoside, D-fructose and 2-acetamido-D-galactopyranoside. The lectin interacted also with pig liver glycogen, starch and mannose-containing glycoproteins (ovoalbumin, ovomucoid and calf thyroglobulin), but don’t interacted with horsera dish peroxidase and calf intestine alkaline phosphatase. According to electrophoresis, in 20% SDS-PAAG containing SDS-Na the lectin consists with subunits of molecular weight 12 kDa. Molecular weight of the lectin is 48 kDa according to gelchromatography on Toyopearl HW-55. The lectin agglutinated best of all rabbit erythrocytes and worse agglutinated guinea-pig, very weak — rat erythrocytes and did not agglutinate human and goat erythrocytes. After dialysis against 1% EDTA sodium salt solution the lectin did not lose hemaglutinating activity and endured heating to +65 °C during one hour.

83-88

Authors: K. S. Maznik, N. A. Matvieieva

Dependence of efficiency of fructans extraction on soaking time, temperature and time of high temperature extraction was investigated. Dried and powdered chicory Cichorium intybus L. cv Pala rossa «hairy» roots obtained by Agrobacterium rhizogenes-mediated transformation with pCB161 vector were used for study. There were used low- and high-temperature extractions without heating at +22 °C during 0.5; 1 and 24 hours and with heating at +70 °C, +80 °C and +90 °C during 10, 20 and 30 minutes. Fructans fractionation was conducted by two ways: separation of high molecular weight fraction by crystallization at +4 °C and low molecular weight separation by extraction with 95% ethanol. To determine fructans concentration in the extracts McRary and Slattery method was used. Based on the experimental data, a mathematical model of fructan extraction process was created. Its adequacy was tested with the Fisher criterion and coefficient of determination. Optimal parameters of the extraction process chosen using the methods of linear programming were determined. Extraction for 30 minutes at 90 °C without soaking identified as the most tech nological one. It allowed to extract fructans general amount from transgenic roots (146 ±8,77 mg/g of root dry weight). Optimal regime of fructan obtaining from chicory «hairy» roots is extraction at +90 °C for 30 min. Preliminary soaking time does not affect any effectiveness for such extraction. The most effective mode of obtaining of low- and high molecular fractions of fructans from transgenic chicory roots is twostage extraction with 95% ethanol at +80 °C and water at +90 °C with the duration of each stage of 30 minutes.

89-94

Authors: T. E. Voloshko, О. V. Fedotov

The dynamics of growth and catalase activity of 57 strains of basidiomycetes were investigated. Glucose-peptone medium was used for surface cultivation of fungi. The objects of study were 57 strains, 5 of which belongs to 5 species of the order Polyporales, and others do to 7 species of the order Agaricales. The weight measurement to estimate accumulation of absolutely dry biomass was used to study growth rates. The spectrophotometric methods were used for determination of catalase activity and protein content in mycelium and culture filtrate. The specific catalase activity was calculated based on this data. The levels of biomass accumulation and catalase activity of the strains on the 9-th and 12-th days of cultivation and ability of the most fungi to synthesize mainly extracellular catalase were determined. Individual variability of the strains was shown. The results allowed selecting the strains — active producers of catalase, including F. velutipes F-2 and P. ostreatus R-208, which are perspective for use in biotechnology of enzyme preparations.

110-115

Authors: O. V. Khrobatenko, N. V. Prytulska, S. M. Shulga

Liposomal form of adenosine-5-triphosphate (ATP) for oral applying was studied. The methods of obtaining and determining the size of the liposomes, the definition of ATP percentage content in liposomes were proposed. The effect of liposomal form of ATP on physical performance of laboratory animals by swimming to the limit of exhaustion was studied. It was shown that using of the liposomal form as compared with free ATP (in a similar dose) promoted the increased efficiency of laboratory animals. Application of liposomal form occurring at single administration for 6 days at a dose of 30 mg/kg facilated a 62% increase of efficiency, and 60 mg/kg did 76%, while consumption of liposomal form of ATP for 12 days did 64% and 93%, respectively.

121-131

Authors: S. A. Starovoitova

Modern huge and world-wide known projects concerning studying of human microbial ecology and construction of probiotics, particularly: Society for Microbial Ecology and Disease, Probiotics & Health Targeted Initiative of International Science and Technology Center (TI PROBIO ISTC), Human Microbiome Project of National Institutes of Health, MetaHIT Project (Metagenomics of the Human Intestinal Tract) of European Commission, Human Metabolome Project of Canadian University of Alberta and some more else were characterized in the article. Brief historical information and reference to official sites of every discussed project were given. Main goals and tasks of every project were described. Short characteristic of discussed projects and also modern accessible results of researches were given. Importance of every examined project for widening scientific knowledge in the field of human microbial ecology and also for improvement and/or for construction of modern effective probiotics on basis of human normal intestinal microflora were paid attention. Close interaction of scientific data received by realization of every discussed project was shown.

9-21

Authors: S. M. Shulga, O. A. Tigunova, Y. B. Blume

Energy and environmental crisis facing the world force us to reconsider the effectiveness or find an alternative use of renewable natural resources, especially organic «waste» by using environmentally friendly technologies. Microbial conversion of renewable resources of biosphere to produce useful products, including biofuels, currently is an actual biotech problem. Anaerobic bacteria of Clostridiaceae family are known as butanol producers, but unfortunately, the microbiological synthesis is currently not economical one. In order to make cost-effective aceton-butanol-ethanol fermentation, solventproducing strains using available cheap raw materials, such as agricultural waste or plant biomass, are required. Opportunities and ways to obtaine economic and ecological processing of lignocellulosic wastes for biobutanol creation are described in the review .

33-42

Authors: O. A. Vasylchenko, V. V. Sologub, K. K. Vasylchenko

Nowadays, the value of bactericidal nanomaterials research increases at the increasing number of bacteria strains resistant to the most highly potent antibiotics. In the review the characteristic of nanoparticles and methods for their production are done. The scope of nanoparticles application is observed, special attention is focused on silver nanoparticles usage in medicine, in particular, as bactericidal products. It is indicated that nanoparticles may have toxic effects. Much attention is paid to nanoparticles application in the treatment of various diseases, for example, for targeted drug delivery, wound healing, bone regeneration, local heating of tumors in cancer pathology, immune system stimulation, for antibodies, viruses, bacteria detection, for liquids filtration. Penicillins and their producers — Penicillium sp. characteristic is done. The mechanism of penicillin antimicrobial action is estimated. It is revealed that silver nanoparticles usage in combination with antibiotics, particularly penicillin, leads to antibiotics antibacterial activity increasing against gram-positive and gram-negative microorganisms.

68-73

Authors: Е. А. Kiprianova, L. D. Varbanets, V. V. Shepelevich, S. I. Voichuk

Lipopolysaccharides and their fractions were obtained from strains Pseudomonas chlororaphis subsp. aureofaciens UCM В-111 and UCM В-306 — components of insectofungicidal gaupsin biopreparation; their activity against tobacco mosaic virus has been studied. Lipopolysaccharides of strains В-111 and В-306 proved to be highly active antiviral agents and inhibited tobacco mosaic virus infectivity for three species of indicating plants in concentrations 0,001–10 mg/ml. First lipopolysaccharides fraction (O-specific side chains) did not inhibit and often stimulated the virus reproduction. At the same time the core oligosaccharides (the second and the third lipopolysaccharides fractions) decreased to different extent the virus infection development. According to electron microscopy data the virions sticked together forming the sneafs at the direct lipopolysaccharides-virus contact in vitro whereas the single free virus particles were observed in the control. Evidence of direct interaction between lipopolysaccharides of P. chlororaphis subsp. aureofaciens strains and tobacco mosaic virus is provided.

80-84

Authors: E. A. Shesterenko, I. I. Romanovska, O. V. Sevastyanov, E. A. Semenishina V. I. Pavlovsky, S. A. Andronati

The synthesis of new series of 7-bromo-5-aryl-1,2-dihydro-3H-1,4-benzdiazepine-2-one derivatives, containing in the tree position phthalimidoacyl and hexylacyl fragments was accomplished. The structure of new compounds was proved by mass-spectrometry and PMRspectroscopy methods. For the first time, hydrolysis of the earlier synthesized 3-hydroxy-7-bromo-5-aryl-1,2-dihydro-3H-1,4-benzdiazepine-2-one esters, potential anxiolytic and hypnotic means, catalyzed by carboxyl esterase in composition of pig liver microsomal fraction was studied. The quantitative inhibition of pig liver microsomal fraction esterase activity in the presence of carboxyl esterase selective inhibitor di-(pnitrophenyl)-phosphate was shown. The nonlinear dependence both of hydrolysis degree with acyl moiety length in 3-acyloxy-7-bromo-5-aryl-1,2-dihydro-3H-1,4-benzdiazepine-2-ones and decreased substrate transformation degree after substituent introduction in the first position of molecule was established. For the derivatives with phthalimidoacyl and hexylacyl-mo ie ties in the molecule 3 position it was shown, that increasing of CH2-groups number in this substituents and incorporation of chlorine atom in o-position of phenyl ring bring to increasing of hydrolysis degree.

92-96

Authors: E. V. Greek, E. A. Krasulya, E. A. Tigunova

The main parameters of fermentation of whey-malt wort with the use of different strains of lactose-fermentation yeast was investigated experimentally. According to the findings of investigation of fermentive activity for different types of lactose-fermentation microorganisms in whey-malt wort it was found that the most active spirituous fermentation for all parameters was in wort fermented by microorganisms Zygosaccharomyces lactis 868-K and Saccharomyces lactis 95. High capacity for utilization of malt carbohydrates represented by easily metabolized carbohydrates of malt extract was determined. Also organoleptic analysis of fermented whey drinks derived from the renewed mixtures of dry whey and fermented malt and yeast Zygosaccharomyces lactis 868-K and Saccharomyces lactis 95 was carried out. It was found that the drink fermented with yeast Zygosaccharomyces lactis 868-K had intense refreshing flavor of rye bread with fruit tones. Intensity growth of aromatization for complex of sample with microorganisms Saccharomyces lactis 95, indicating high organoleptic indexes of the drink was observed.

107-114

Authors: К. G. Dreval, M. I. Boyko

Adsorption of cellulases on substrate taking place during the cultivation process was determined. Adsorbed enzymes can be eluted by buffer solution with high ionic strength, but for determine their activity they should be transferred into the aqueous solution. On the basis of the results a method for obtaining of cellulases preparations from cultural liquids of basidiomycetes was developed. This method is the elution of cellulases from the cultivation substrate of basidiomycetes. It was found that using of the last allows to obtain enzymatic preparations with a high degree of purification in 3 stages (salting out of proteins — dialysis — gelchromatography). Cellulase preparations received original products of basidiomycetes strains К-1, А-Дон-02, Д-1 Irpex lacteus and AnSc-1 Daedaleopsis confragosa f. confragosa were obtained. They contained different proteins, enzymes with specific peaks out of column and their activity. However, common to them was a distinct maximum of outing from the column by endoglucanases or cellobiases, which may indicate that the studied cellulolytic complexes of basidiomycetes do not contain multiple forms of cellulases with different molecular mass. This method allowed to obtain preparations with different degree of purification in comparing with the original culture filtrate 7,3 for endoglucanase and 33,3 for cellobiase of strain А-Дон-02 I. lacteus; 13,1 for endoglucanase and 25,5 for cellobiase of strain Д-1 I. lacteus; 29,9 for endoglucanase and 90,1 for cellobiase of strain К-1 I. lacteus; 2,1 for endoglucanase and 30,6 for cellobiase of strain AnSc-1 D. confragosa f. confragosa.

121-127

Authors: L. V. Gorbunov, I. V. Petrov

Optimal cryopreservation parameters were determined for the berry-like crop cuttings: blackcurrant (Dachnica, Yuviley, Sofiivska, Kitayskaya); redcurrant (Kitaivska, Djoker, Svyatkova); gooseberries (Krasen, Malachite, Kolobok); raspberries (Novost’ Kuz’mina, Struyka, Skromnica). This procedure allowed increasing viability of deconservation samples in 2–5 times compared to the existing methods of cryopreservation. Maximal values of cutting viability were obtained after their temperature adaptation at –10 °C during 14–60 days, stepwise cooling of the samples with 0.1–0.5 °C/hr rate down to –30 °C (exposure 3–7 days), direct plunging into liquid nitrogen, storage from 1 to 30 days and thawing rate of 70 °C/min. It was found that the in the region of allowable values for maximum viability of investigated birch (control) cuttings humidity values correspond 32±40%, for blackcurrant are 40±50%, and raspberries 37±40 % in affirmative temperature span and 14±28 %, 30±40 %, 20±23 % in below zero. Using dispersion analysis it has been established that the development probability of frozenthawed plant cuttings significantly depends on the selected cultivar and cryopreservation method. It is noted that the force of influence η2А, due to the individual properties of the studied cultivars of berries, is comparable to that of the force of influence η2В of cryopreservation procedures. The differences of average indexes of viability of the studied cultivars were showed within a single species. It was found that these rates are different for black currant by 92%, redcurrant — 54%, gooseberries — 48%, raspberries — 70%. Using paired Student’s t-test has reduced to 3 times the error of representativeness of the sample that enabled to improve the reliability of the significance of differences compared to the values of the P ≥0,99. Selection of the cuttings’ cultivars and cryopreservation method significantly affect the reproducibility of experimental results. The analysis of reproducibility of indexes of viability of the studied cuttings held by the coefficient of variation, showed that during the cryopreservation this value is changed to blackcurrant cultivars from 5 to 91%, redcurrant — 9–137%, gooseberries, 22–74%, raspberry 18–105%.