Biotechnologia Acta

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%.

9-21

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

The classification, structure, and mechanism of catalytic action of carboxylesterase of different origin are presented in the review. The prospects of carboxylesterases application for metabolism and both several drugs and prodrugs activation investigation in vitro are shown. The enzyme usage as biocatalyst of stereoselective hydrolysis and synthesis of a wide range of acyclic, carbocyclic and heterocyclic compounds — esters are also urgent. It was established that enantiomers obtainable with the help of carboxylesterase are characterized by high chemical yields and optical purity; immobilization on different supports stabilizes the enzyme and allows the repeated usage of obtained biocatalysts. The own studies conducted and the enzymatic hydrolysis features of news 3-acylhydroxy-1,4-benzodiasepin-2-ones — potential anxiolytic and hypnotic means, with a help of pig liver microsomal fraction carboxylesterase have been established. For the first time the enantioselective hydrolysis of 3-acetoxy-7-bromo-1-methyl-5-phenyl-1,2-dihydro-3H-1,4-benzdiazepine-2-one was accomplished using free and immobilized in phyllophorine and alginate, stabilized by Ca2+ microsomal fraction. The S-enantiomer of substrate was isolated, which suggests the increased specificity of pig liver microsomal fraction carboxylesterase to its R-enantiomer.

34-45

Authors: S. А. Starovoitova, O. I. Skrotska

Modern tendencies of recombinant microorganisms creation for obtaining on their basis a new effective biopreparations (probiotics) with wider spectrum of biological and therapeutic properties were considered. A lot of attention was focused on the main genera of perspective bacteria for creation of recombinant probiotics particularly: Lactococcus, Bifidobac terium,Bacillus, Escherichia. The main created Ukrainian and foreign gene-modified strains, that are widely used today in creation of effective recombinant biopreparations were characterized. Some fundamental directions and methods of gene-modified strains obtaining, which are used in getting effective biopreparations that used for therapy and prophylactic illness were reported, under which this group of pharmaceutical drugs were not used earlier. The safety matters of probiotics using on basis of genemodified strains were examined. Medical and veterinary biopreparations on basis of recombinant microorganisms could be used directly and effectively for therapy and prophylaxis of different illness, beginning from disbacteriosis up to cardiovascular diseases. It is related with some probiotic microorganisms ability for lowering of serum cholesterol at the host organism.

73-80

Authors: L. D. Varbanets, О. V. Matselyukh, N. А. Nidyalkova, Е. V. Аvdiyuk, А. V. Gudzenko, I. I. Seifullina, G. N. Маsаnоvets, N. V. Khitrich

Chloride, bromide and isothiocyanate complexes of cobalt(II) with N-substituted thiocarbamoyl-N?-pentamethylenesulfenamides (1)–(12), and also complexes of cobalt(II, Ш) with derivatives of morpholine-4-carbodithioic acid (13)–(18) have been used as modificators of enzymes of hydrolytic action — Bacillus thurin-giensis ІМВ В-7324 peptidases, Bacillus subtilis 147 and Aspergillus flavus var. oryzae 80428 amylases, Eupenicillium erubescens 248 and Cryptococcus albidus 1001 rhamnosidases. It was shown that cobalt (II, Ш) compounds influence differently on the activity of enzymes tested, exerted both inhibitory and stimulatory action. It gives a possibility to expect that manifestation of activity by complex molecule depends on ligand and anion presence — Cl–, Br– or NCS–. The high activating action of cobalt(II) complexes with N-substituted thiocarbamoyl-N?-pentamethylenesulphenamides (1)–(12) on elastase and fibrinolytic activity of peptidases compared to tris(4-morpholinecarbodithioato)cobalt(ІІІ) (14) and products of its interaction with halogens (15)–(17), causes inhibitory effect that is probably due to presence of a weekly S–N link, which is easy subjected to homolytic breaking. The studies of influences of cobalt(II) complexes on activity of C. аlbidus and E. еrubescens ?-Lrhamnosidases showed, that majority of compounds inhibits of its activity, at that the most inhibitory effect exerts to C. аlbidus enzyme.To sum up, it is possible to state that character of influence of cobalt(II) complexes with N-substituted thiocarbamoyl-N?-pentamethylenesulphenamides, and also cobalt(II, Ш) complexes with derivatives of morpholine-4-carbodithioic acid varies depending on both strain producer and enzyme tested. The difference in complex effects on enzymes tested are due to peculiarities of building and functional groups of their active centers, which are also responsible for binding with modificators.

86-96

Authors: A. A. Tykhomyrov, E. I. Yusova, S. I. Diordieva, V. V. Corsa, T. V. Grinenko

Components of plasminogen/plasmin system play crucial role in fibrinolytic rocesses. They are also involved in regulation of cell activity in normalcy and various pathological conditions. In particular, kringle-containing proteolityc plasminogen fragments, which are denoted as angiostatins, participate in processes during neovascularization, metastasis, tumor growth, inflammation. Angiostatins are considered to be potential markers of diseases associated with vascular pathologies. Therefore, elaboration of specific and sensitive methods for their detection is still important area of inquiry. In the present study, approaches for obtaining of plasminogen fragments through its limited proteolysis by pancreatic elastase and further purification of proteolytic derivates by means of gel filtration and affinity chromatography on Lys-Sepharose are described. Polyclonal antibodies raised to fragment K1-3 have been produced, and their principal immunochemical properties have been studied. It has been found that antibodies purified on immunoaffine sorbent demonstrate different affinity toward plasminogen and its fragment, as follows: Glu-Pg > Lys-Pg > К1-3 > mini-Pg > К4. Based on the data obtained, immunological features of plasminogen kringle domains are discussed. Antibodies against fragment K1-3 could be applied in immunochemical analysis, in particular, Western blot, for detection of angiostatins as markers of tumor growth, metastasis, cardiovascular diseases and inflammation processes, and used as molecular tool for investigation of plasminogen/plasmin system functioning in health and disease.

115-118

Authors: Sergeeva L. E., Komisarenko A. G., Bronnikova L. I., Mikhalska S. I., Tishchenko O. M.

Low frequency of in vitro plant regeneration limits significantly wide expansion of biotechnology methods. The screening of explants with stable and high level of the morphogenesis is an open problem. It is known, that free proline fluctuations correlate with processes of plant growth and development in vivo and in vitro. Sunflower mature seeds were germinated in vitro. The contents of free proline at somatic tissues with different regenerational abilities were measured. Following explants were estimated: cotyledons from 1- or 4-days cultivating shoots; primary explants prepared for the regeneration procedure (segments of 4-day cultivating shoots); primary explants with and without initial morphogenesis, 6 days on the regeneration medium; shoots and certain explants at the moment of their mutual separation; explants, 10 days after shoots release. The top levels of free proline were observed in tissues with fulfiled organogenesis. The hypothesis about proline involvement to the process of sunflower morphogenesis in vitro is discussed. During primary screenings of explants the level of free proline can be appropriate index of in vitro sunflower organogenesis.

9-26

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

The review is concern with α-L-rhamnosidases that hydrolytically cleave the terminal unrecovered α-1, 2-, α-1, 4- and α-1, 6-linked rhamnose residues in α-L-rhamnosides. It was considered the methods of determination of α-L-rhamnosidase activity both with natural and synthetic substrates. These enzymes were found in plants, animals, yeasts, fungi and bacteria. It was shown that the most active biosynthetics are micromycetes. Methods of extraction and purification of α-L-rhamnosidases of microbial origin, which include the precipitation of enzyme from solutions by organic solvents and neutral salts, dialysis, ultrafiltration, gel-filtration and ionexchange chromatography on columns, are described. The data of physical and chemical properties (thermal and pH optima and molecular weight), substrate specificity of α-L-rhamnosidases were generalized. Enzymatic preparations that were obtained from the same producer during cultivation at the presence of different inductors showed different substrate specificity. α-L-Rhamnosidases refer to glycosyl-hydrolases with catalytic domen (α/α)6-barrel. Biotechnological properties of enzyme are directed to usage in food, pharmaceutical and chemical industries. In food industries, α-L-rhamnosidases are used for increasing of drinks quality (lowering of bitterness and improvement of wine aroma) and production of food additives. In pharmaceutical industry α-L-rhamnosidases could be used for obtaining of drugs and their precursors. In chemical one — as precusor for rhamnose production.

39-46

Authors: Yu. I. Sidorov

State of the art industrial lyophilic batch dryers of periodic action used for drying of heatsensitive products of the pharmaceutical and biotechnology industries, produced by wellknown leading companies such as Martin Christ, Niro, GEA Pharma Systems, Zirbus technology, Millrock Techno logy is considered. High technical level of the Asian manufacturers such as Tofflon, Luxun International Group, Changzhou Yibu Drying Equipment (China), Biotron (South Korea), Bioasset Technologies Private (India), which had been made by imitation of European goods brands or licensed production, was achieved. Despite a sufficient number of innovative solutions, lyophilizers of continuous action are not yet widely used because of lack of demand. On the market there is really the only company with a series of dryers Niro CONRAD for the manufacture of instant coffee.

57-65

Authors: L. M. Korshun, G. V. Kovtonjuk, L. M. Moysa, O. K. Kiselyova, L. O. Ganova, M. I. Vudmaska, M. J. Spivak

Specific interaction parameters such as affinity and avidity of serum IgG in the formation of immunocomplex with recombinant antigen of herpes simplex virus type 2 were determined. It was found that all the herpes simplex virus type 2 positive sera contained high-affinity antibodies (the exponent of the affinity constant was in the range 107–108 M–1). The highest value of avidity index (81,15±11,15)% was observed in serum antibody number 2 while for the samples number 1 and number 3 these values were lower: (62,50±15,50)% and (65,0±15,10%) respectively, although there was not significant diffe rence between the amples (P > 0,05). The results of the avidity estimation for these three sera were in good agreement with theoretical values of affinity constants which were measured by the indirect ELISA according to the method of Freguet and others using the equations of Scatchard, Klotz and the equation of Stevens. For one of the sera (№ 4) it was found the discrepancy between the affinity and avidity of antibody levels. Thus the affinity constant value calculated with the equation of Stevens was (2,08 ± 0,25)•108 M–1 and the avidity index (23,50 ± 6,50)% was significantly lower (P < 0,05) with respect to these three serums. Obtained results are a contribution to the improvement of existing methods of laboratory diagnosis of herpes simplex virus and the detection of virus-specific antibodies in serum.

73-77

Authors: Y. Ya. Kit, R. O. Bilyy, T. V. Skorokhoda, N. M. Boiko, N. S. Korniy, N. E. Mitina, A. S. Zaichenko, R. S. Stoika

With the aim to enhance therapeutic effectiveness, a drug doxorubicin was previously conjugated with surface-active water-soluble synthetic oligomer TF3 and water-soluble TF3-Dox conjugates were obtained possessing high cytotoxic activity towards tumor cells in vitro. As the therapeutic use of anticancer drugs is performed by intravenous administration, the purpose of the work was to determine the peculiarities of TF3 Docks interaction with proteins and human blood cells. Peripheral human blood was incubated with TF3-Dox or with pure doxorubicin, as control, afterwards blood was separated for plasma and cellular components by centrifugation. The affinity of doxorubicin and TF3-Dox to blood cells was evaluated by fluorescent microscopy. Plasma proteins were separated by electrophoresis in 1% agarose gel and detected via UV-light illumina tion. It was found that TF3-Dox unlike doxorubicin are able to be selectively accumulated in nucleated blood cells as well as to form stable complexes with serum albumin. Formation of such complexes can facilitate their endocytosis by nucleated blood cells. Obtained data suggest the perspectives of the use of TF3 carrier for targeted delivery of doxorubicin during onco-haematologic diseases.

85-94

Authors: К. G. Dreval, M. I. Boyko

Nutrient medium composition on sources of carbon and nitrogen for maximal activity of basidiomycetes cellulolytic enzymes has been optimized. It was found that among carbon sources the most pronounced effect on repressing the cellulases activity exhibits glucose. While being cultivated on nutrient mediums with disaccharides, basidiomycetes produced nonspecific cellobiase more than endoglucanase. To receive cultural liquids of basidiomycetes with highest total cellulolytic activity, strains should be cultivated on nutrient mediums containing filter paper as single carbon source in concentration 8 g/l. As a result of culture medium optimization for nitrogen source it was revealed that nitrites and urea display maximal inhibiting effect and maximuminduction occurs when nitrates are brought into the culture medium. The best nitrogen source for cellulases activity for К-1 Irpex lacteus strain is 1,5 g/l calcium nitrate; А-Дон-02 and Д-1 Irpex lacteus are 1,5 g/l ammonium nitrate; and AnSc-1 Daedaleopsis confragosa f. confragosa — 1,65 g/l ammonium sulfate. Optimal ratios of carbon to nitrogen for cellulases activity are 5,3:1 for К-1, А-Дон-02, Д-1 I. lacteus strains and 4,8:1 for AnSc-1 D. confragosa f. confragosa one.

101-106

Authors: L. V. Gorbunov, M. D. Bezugly

Technologies of the artificial fertilization and transplantation of сryopreservated sperm and embryos are the foundation of the breeding and selection of animals. The comparative analysis is carried out, theoretical generalization and the new solution of increase of reproducibility of results of experiment on the basis of the offered mathematical model of the estimation of a native condition of embryos of cows and efficiency of stages of their сryopreservation are given. Application of model repeatedly increases reproducibility of results and provides the condition to their comparability when using different ways of freezing of embryos of cows. Experimental test of the offered model showed that use of the generalized factors of viability and efficiency reduces the dispersion of results to size of an error of their detection. The design values of the generalized values were as follows: viability of embryos of cows, which depended on physiological condition of their donors — 97.1±1.0% and recipients — 95.1±3.9%; efficiency of сryopreservation — 96.0±4.2% and transplantation — 54.3±4.8%. The quantitative accounting of a condition of native bioobject, during determination of efficiency of stages of the different ways of the freeze, raised manifold the reproducibility of results of its сryopreservation, that allowed to reduce considerably the volume of the biological material and costs for carrying out the tests under condition of obtaining of reliable value.