[FEATURES OF FORMATION OF BACTERIAL BIOFILMS IN CONDITIONS OF SPACE FLIGHT].

AIM: Study the effect of microgravitation on the formation of Lactobacillusplantarum 8PA-3 bacterial biofilms in the conditions of space flight. MATERIALS AND METHODS: Information on the ef- fect of microgravitation on the development of biofilms was obtained during study of L. plantarum 8PA-3 probiotic lactobacilli in special equipment in the process of execution of space experiments in the Russian segment of the International Space Station. Comparative analysis of growth of plankton and biofilm forms of cells developing in the conditions of space flight and surface conditions was carried out by microbiologic and electron-microscopy methods using scanning and transmission electron microscopy. RESULTS: Accelerated dynamics of formation of L. plantarum 8PA-3 lactobacilli biofim on the surface polymer substrate was shown for the first time during the space experiment. Microbiological anfalysis of the bacterial culture has also confirmed the accelerated growth of L.plantarum 8PA-3 under microgravitation compared with surface conditions. Ultrastructure of plankton form of L. plantarum 8PA-3 taking part in formation of biofilms inconditions ofmicrogravitationwas detected for the first time inthe space experiment. CONCLUSION: Data on comparative electron-microscopic analysis obtained in space experiments are important for scientific justification of the effect of icrogravitation on bacterial communities developig as biofilms - the most natural form of existence of microorganisms. The results obtained could be taken into consideration during creation of novel antibacterial means and disinfectants as well as methods of treatment of surfaces of modules of piloted space complexes that could allow to clarify methods of effective prophylaxis of biofilm spread which pose a risk of health of the crew and normal functioning of equipment in the International Space Station.

[Sorption properties of various polysaccharide matrixes to Lactobacillus plantarum 8RA-3 bacteria].

AIM: Study of sorption properties of various spherical polysaccharide matrixes designated as Spherocell to probiotic Lactobacillus plantarum 8RA-3 bacteria. MATERIALS AND METHODS: Industrial strain of L. plantarum 8PA-3 was used. The process of immobilization of lactobacilli on 3 variants of spherical sorbents was studied. The first sorbent - neutral, composed of nonpolar cellulose matrix with ("0") charge, the second--DEAE obtained by modification of cellulose by diethylaminoethyl groups with positive ("+") charge and the third--CM (carboxymethyl) with negative ("-") charge. Cellulose matrixes were designated by us by the term Spherocell. Immobilization of bacterial cells on Spherocell was performed by addition of suspension containing 1.0 x 10(9) CFU/ml. The effect of bacterial immobilization was evaluated by CFU/ ml titration and by electron microscopy. RESULTS: The dependence on matrix charge of adsorption immobilization on sorbent granules of lactobacilli cells was shown. At certain equal parameters (granule size, surface characteristics, charge value) the positively charged matrix sorbed 3-10 times more cells than neutral and 20-25 times more than negatively charged matrix. Each 100-180 microm Spherocell DEAE particle could sorb more than 1000 viable bacterial cells. CONCLUSION: Positively charged polysaccharide matrix Spherocell DEAE obtained by modification of cellulose by diethylaminoethyl groups is promising for creation of immobilized probiotic preparations.

[The cultivation of cells on the porous titanium implants with the different structure].

The data on human dermal fibroblasts and rabbit mesenchymal stromal cells cultivation on porous titanium implants are presented in given paper. Two types of implants were used: type 1--with irregular pores formed by pressed titanium particles and type 2--with regular pores formed by coalescence of one-size titanium particles into implant. The goal of this study was to choose the type of titanium implant porosity which ensures the tightest interaction of titanium implant with surrounding tissue cells after implantation in the body. Cells were cultivated on implants for 7 days and in both cases they formed confluent monolayer on the implants surfaces. That indicated adhesion, migration and proliferation of cells on such implants. Condition of cells cultured on titanium implants was controlled by scanning electron microscopy. The character of fibroblasts interaction with given implants was different depending on porosity type of implants. On implants with irregular pores, the cells were more spread and overlapped the pores spreading over neighbored particles. On implants with regular pores that formed by one-size particles into implant, the fibroblasts covered these particles not overlapping the pores and seldom interacted with neighbored particles by small outgrowths. There was no tight interaction of particles into implant. In implants formed by pressed particles, the cells grew not only on the surface but also in the depth of implant. Thereby, we suppose that more tight interaction of cells with titanium implant and, supposedly, tissues with implant in an organism will take place in the case of implant structure formed by pressed titanium particles.

Cell Cultivation on Porous Titanium Implants with Various Structures.

The paper presents data on the cultivation of human dermal fibroblasts and rabbit mesenchymal stromal cells on two types of porous titanium implants, i.e., those with irregular pores formed by pressed titanium particles and those with regular pores formed by the cohesion of one-size titanium particles inside the implant. The goal of this study was to determine what type of titanium implant porosity ensured its strongest interaction with cells. Cells were cultivated on implants for 7 days. During this period, they formed a confluent monolayer on the implant surface. Cells grown on titanium implants were monitored by scanning electron microscopy. Fibroblasts interaction with implants depended on the implant porosity structure. On implants with irregular pores cells were more spread. On implants with regular pores fibroblasts enveloped particles and were only occasionally bound with neighboring particles by small outgrowths. There was no tight interaction of particles inside the implant. In implants formed by pressed particles, cells grow not only on surface, but also in the depth of the implant. Thus, we suppose that a tighter interaction of cells with the titanium implant and, supposedly, tissues with the implant in the organism will take place in the variant when the implant structure is formed by pressed titanium particles, i.e., cellular interaction was observed inside the implant. In implants with irregular pores, cells grew both on the surface and in the depth. Thus, cells exhibited more adequate interactions with irregular pore titanium implants in vitro and hopefully the same interaction will be true in tissues after the implantation of the prosthesis into the organism.

[Using of Spherocelle sorbents for construction of immobilized probiotics].

AIM: To assess sorption properties of Spherocelle beads consisting of particles of macroporous celiulose with various charges in relation to bacterial cells of manufacturing probiotic strains from different taxonomic groups. MATERIALS AND METHODS: The following manufacturing strains: Bifidobacterium bifidum 1, Lactobacillus plantarum 8PA-3 and Escherichia coli M-17, as well as 3 variants of Spherocelles' matrix: neutral, with positive and negative charges, were used. RESULTS: Spherocelle globules DEAE with a positive charge of the matrix were successively used for designing of immobilized probiotic preparations. Efficacy of sorbent is determined by sorption of > or =1000 viable cells as well as bacterial metabolites interacting in conditions of sorbent-regulated pH on each globule with diameter 100-180 microm. It provides, on the one hand, prolonged viability of probiotic bacteria in culture fluid within 6 months and, on the other hand, optimal pharmacokinetics of preparation due to gradual desorption of metabolites from sorbent globules. CONCLUSION: Sorbent Spherocell DEAE is biocompatible with cells of manufacturing strains of lactobacilli, bifidobacteria and E. coli and recommended for designing of immobilized probiotics.

Effect of melanins from black yeast fungi on proliferation and differentiation of cultivated human keratinocytes and fibroblasts.

The effects of melanin preparations from black yeast fungi (BYF) on the proliferation and differentiation of normal cultivated human skin keratinocytes and embryonic pulmonary fibroblasts have been investigated. Melanin preparations in the range of 5-0.1 microg/ml were optimally active, with a more pronounced effect on keratinocyte than on fibroblast proliferation. Of 17 dihydroxynaphthalene (DHN) natural melanin preparations and two commercial dihydroxyphenylalanine (DOPA) melanin preparations, only one preparation--DOPA melanin (of animal origin) significantly stimulated proliferation of keratinocytes at 5 microg/ml; four preparations (DHN melanin from BYF) significantly inhibited proliferation of these cells at 5 or 1 microg/ml. The remaining preparations had no significant effect. Similarly, of the 17 preparations of DHN melanin from BYF, one preparation significantly stimulated fibroblast proliferation, and four significantly inhibited proliferation at 5 microg/ml, one at all the concentrations, and three from 1 down to 0.1 microg/ml. These melanin preparations were also shown to affect the in vitro differentiation of keratinocytes.

[The "air pouch" model in mice and a study of the wound fluid proteolytic activity]. / Model' "vozdushnogo puzyria" u myshei i izuchenie proteoliticheskoi aktivnosti ranevogo ékssudata.

The experimental model of a cutaneous wound in mice has been offered, aimed to study the proteolytic activity of the wound fluid produced at early stages of wound healing, and to examine the continuous inflammatory state. The presence of metalloproteinases MMP-2 and MMP-9 in the wound fluid matrix was found to correlate with the existence of neutrophils and macrophages in the tissue.

Photoprotective activity of melanin preparations from black yeast-like fungus during UV irradiation of human skin: dependence on the concentration.

The effect of melanin solutions on the skin exposed to UV irradiation (1,050 kJ/m(2)) depended on its dose and varied from photoprotection (0.005 mg/ml) to photosensitization and phototoxicity (burn, 0.1 mg/ml). These results suggest that doses of melanin preparations should be empirically selected to achieve optimum photoprotective effect.

Photoprotective activity of melanin preparations in human skin exposed to UV irradiation: dependence on previous photoexposure.

Photoprotective effects of three melanin preparations (from black yeast fungi and Sepia sp.) were studied. These preparations in aqueous solutions (5 g/ml, dark exposure for 7 days) demonstrated high photomodification capacity upon exposure to visible light in doses of up to 1.8 kJ/m2. Preliminary exposure of these solutions to visible light in a dose of 360 kJ/m2 notably decreased the photoprotective effect of melanins during UV exposure of the skin treated with these solutions (at UV dose of 3.4 kJ/m2). This necessitates empirical selection of the dose and storage condition of melanin preparations for attaining the optimal photoprotective effect.

[Impact of high-energy laser irradiation on pulmonic fibroblast in human embryo]. / Vliianie vysokoénergeticheskikh lazernykh izluchenii na legochnye fibroblasty émbriona cheloveka.

This impact was attained by medicinal laser application to alexandrite, dye, erbium and gallium arsenid in various conditions on cultures of pulmonic fibroblasts of human embryo. Obtained results were estimated by transmission and scanning microscopy. The highest cell destruction was observed in dye and alexandrite, being less expressed in gallium arsenid under laser irradiation. Impulsive action of erbium laser did not cause any substantional cell destruction. So, laser application to dye alexandrite and gallium arsenide for healing scar and contracture damages is completely justified for skin pathogeny.

[Effect of melanins from black yeast fungi on cultured human cells. II. Differentiation of keratinocytes in vitro]. / Deistvie melaninov iz chernykh drozhzhevykh gribov na kul'tiviruemye kletki cheloveka. II. Differentsirovka keratinotsitov in vitro.

Data on the influence of the black yeast melanin (3 samples) on the in vitro differentiation of human keratinocytes are presented. The effect of melanins was estimated by the morphological state of keratinocytes using electron microscopy. The obtained differences in the state of the formed multilayer keratinocyte sheets depended on the melanin sample.

[The cultural characteristics of keratinocytes from newborn rats]. / Osobennosti kul'tury keratinotsitov novorozhdennykh krys.

Results of cultivation of newborn rat keratinocytes are presented, differences between growing rat and human keratinocytes in culture being revealed in addition to possibilities of experimental use of these results. It has been shown that a rat keratinocyte layer forms faster than a human one: the former it is thinner and contains twice less layers. The main peculiarity of rat keratinocytes in culture is their ability to grow successfully without feeder cells, while the presence of feeder cells is crucial for human keratinocyte growth in culture.

[Experiments with Bacillus thuringiensis protoplasts. II. Study of the potential interspecies fusion of bacterial protoplasts: Bacillus thuringiensis and Bacillus megaterium]. / Eksperimenty s protoplastami Bacillus thuringiensis. Soobshchenie II. Issledovanie vozmozhnosti mezhvidovogo sliianiia bakterial'nykh protoplastov: Bacillus thuringiensis i Bacillus megaterium.

In the present study the possibility of obtaining interspecific bacterial hybrids by polyethylene glycol-assisted fusion of protoplasts from Bacillus thuringiensis and Bac. megaterium has been examined. Electron microscopic and genetic data allow to confirm with great probability that cytological fusion takes place. However, genetic analysis revealed that neither of methods applied for protoplast fusion gave stable recombinants. Apparently, it is due to the lack of recombination or the death of recombinants that follows the functioning of the cell correction system. The mechanism of protoplast fusion and its most important steps are also studied in the present work.

[Experiments with Bacillus thuringiensis protoplasts. I. Isolation of protoplasts and their reversion to bacillary form]. / Eksperimenty s protoplastami Bacillus thuringiensis. Soobshchenie I. Poluchenie protoplastov i ikh reversiia v batsilliarnuiu formu.

A method for protoplastization of crystal- and spore-forming Bacillus thuringiensis bacterian and consequent cell wall regeneration on a solid hypertonic medium is presented. Up to 50% of the protoplasts prepared were viable and formed colonies under special conditions; at the same time, less than 0,01% of the cells treated with lysozyme were resistant to the osmotic shock; bacterial autolytic system takes part in protoplasts formation. Electron microscopic studies of protoplasts and cells confirm the fact of cell wall removal and support the proposed mechanism of protoplast formation.