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For the first time, data were obtained on the effect of lectins of two strains of azospirillum—Azospirillum brasilense Sp7 (epiphyte) and Azospirillum brasilense Sp245 (endophyte)—on germination and growth characteristics of the host plant under abiotic stresses; the range of optimal concentrations is set and the possible protective and growth-stimulating effect of these glycoproteins was evaluated. As a result of the studies, it was shown that lectins A. brasilense Sp7 and Sp245 with different efficiency neutralized the negative effects of simulated abiotic stresses—heavy metals (СuSO4, CoSO4, ZnSO4, Pb(CH3COO)2), hypo- and hyperthermic stress, salinization and drought—causing a decrease in seed germination. For both strains, the most pronounced effect on germination was observed in the case of exposure to heavy metals. A positive effect of lectins on the morphometric parameters of the roots of wheat seedlings was found. The results demonstrated a stimulating effect of lectins on the length and number of roots. The lectin of the endophytic strain showed a higher efficiency, expressed in a higher level of effect and in lower concentrations compared to the lectin of the epiphytic strain.

It was shown that the lectins Azospirillum brasilense Sp7 (epiphyte) and Sp245 (endophyte) with different efficacy changed the content of ascorbate and glutathione in the initial period of exposure to CuSO4, CoSO4, ZnSO4, Pb(CH3COO)2 on the wheat seedling roots.

The accumulation of nanoparticles of colloidal silver with spherical shape in culture liquid of Azospirillum brasilense has been shown by transmission electron microscopy. Bacterial extracellular Mn-peroxidases were found to participate in silver reduction from silver nitrate with the formation of nanoparticles. A mechanism of extracellular biosynthesis of silver nanoparticles by A. brasilense bacteria was proposed

Effect of stressors (unfavorable pH and temperature or carbon and nitrogen limitation) on the synthesis of the components of the NO synthase signaling system was studied in submerged cultures of xylotrophic basidiomycetes Lentinula edodes and Grifola frondosa. Marker compounds of the NO synthase signaling system were found in both cultures. A simultaneous increase of the concentrations of NO and citrulline in the culture liquid of the basidiomycetes grown at superoptimal pH and in nitrogen-limited medium indicates the activation of the NO synthase signaling system under such stress conditions.

The lectins of associative nitrogen-fixing strains Azospirillum brasilense Sp7 and Sp245 were shown to exerte a multidirectional effect on the activity of acidic (pH 3.5), neutral (6.8), and alkaline (pH 7.8) proteinases. The lectin of the epiphytic A. brasilense Sp7 decreased proteolytic activity at all pH values, whereas the lectin of the endophytic A. brasilense Sp245 activated neutral and alkaline proteinases, while not affecting the alkaline ones. Experiments with protease inhibitors made it possible to conclude that the lectins of the studied A. brasilense strains alter the ratio between the activities of different protease types in germinating seeds. The activity of trypsin inhibitors in wheat seedling roots was found to increase in the presence of the lectins. Our results indicate a broader spectrum of effects of azospirilla lectins on the host plant organism.

Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability of lectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect of lectin in under 10 min in a concentration of 10 μg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.

Activation of expression of the lcc4 and tir genes encoding laccase and tyrosinase was observed during transition of a xylotrophic basidiomycete Lentinus edodes from the vegetative to the generative growth stages. This was especially pronounced in the brown mycelial mat (the stage preceding formation of the fruiting bodies). Development of this structure was shown to be associated with a sharp increase of laccase and tyrosinase activities, as well as with rearrangements in the phenol oxidase complex. Formation of the tissues with thickened cell walls was associated with enhanced expression of the chi and exg1 genes encoding chitinase and glucanase, respectively. Exogenous treatment of the vegetative mycelium with laccase preparation from the brown mycelial mat promoted formation of this morphological structure. Activation of the lcc4, tir, chi, and exg1 genes may be used as a marker of readiness to fruition in xylotrophic fungi.

The lectins of associative nitrogen-fixing bacteria Azospirillum brasilense Sp7 and its mutant A. brasilense Sp7.2.3 were shown to have different effects on the components of the wheat seedling root signal system, namely to regulate the levels of cAMP, nitric oxide, diacylglycerol, and salicylic acid, as well as to induce the activities of superoxide dismutase and lipoxygenase. Our results make it possible to consider azospirilla lectins as inducers of the signal systems in wheat seedling roots, since they cause development of several flows of primary signals. These data are of general biological importance, since lectins are present in all living organisms and most ot the functions of lectins remain insufficiently understood.

A gas sensor was developed on the basis of extracts of mycelium mushroom Pleurotus ostreatus for determining phenol in air. The procedure of sensor preparation was optimized using a complete factorial experiment. The quantitative and kinetic parameters were found for the adsorption of some organic compounds (alcohols, ketones, alkyl acetates, phenols, aromatic amines) on films with a biological modifier.

We studied the effect of lectins of Azospirillum brasilense Sp 7 on germination capacity of tomato, cucumber, pea, wheat, and onion seeds. The lectins proved to inhibit seed germination. We studied the effect of different concentrations of lectins from four azospirilla strains on the rate of wheat seed germination. Both inhibiting and stimulating effects were demonstrated. Lectin-induced changes in the mitotic state of plant cells were demonstrated with histological sections of the root growing point in onion.

It is shown that lectins of nitrogen-fixing associative bacteria of the genus Azospirillum are able to activate nitric oxide production in wheat seedling roots through the NO signal system and thereby can be inducers of plant adaptation processes.

The time course of changes in the endogenous content of salicylic acid, the ratio between the acid's free and bound forms, and changes in the activities of phenylalanine ammonia-lyase and catalase in wheat seedling roots under the effect of lectins of two strains of the associative nitrogen-fixing bacterium Azospirillum (A. brasilense Sp7 and its mutant defective in lectin activity, A. brasilense Sp7.2.3) is investigated. Differences in plant response to the action of the lectins from these two strains are established. On the basis of the obtained data, a model is proposed for lectin-assisted induction of resistance, according to which the lectin effect on the roots of seedlings results in the accumulation of free salicylic acid, which inhibits catalase activity, ultimately leading to accumulation of hydrogen peroxide and formation of induced resistance.

Plant-associated nitrogen-fixing soil bacteria Azospirillum brasilense were shown to reduce the gold of chloroauric acid to elemental gold, resulting in formation of gold nanoparicles. Extracellular phenoloxidizing enzymes (laccases and Mn peroxidases) were shown to participate in reduction of Au+3 (HAuCl4) to Au(0). Transmission electron microscopy revealed accumulation of colloidal gold nanoparticles of diverse shape in the culture liquid of A. brasilense strains Sp245 and Sp7. The size of the electron-dense nanospheres was 5 to 50 nm, and the size of nanoprisms varied from 5 to 300 nm. The tentative mechanism responsible for formation of gold nanoparticles is discussed.

This is the first study to demonstrate that the medicinal basidiomycete Lentinula edodes can reduce gold (III) ions from hydrogen tetrachloaurate (chloroauric acid) H[AuCl4] to the elementary state with the formation of spherical nanoparticles (nanospheres). When a culture was grown under submerged conditions in the presence of chloroauric acid, the appearance of an intense purple-red color of L. edodes filamentous hyphae was recorded, which indicates that gold ions were reduced to gold nanoparticles. Using transmission electron microscopy and X-ray fluorescence, we observed accumulation of colloidal gold by the fungal mycelium in the form of electron-dense nanospheres of 5 to 50 nm in diameter on the surface and inside fungal cells.

The auxin formation in a submerged culture of the xylotrophic basidiomycete Lentinus edodes (Berk.) Sing (Lentinula edodes (Berk.) Pegler) (shiitake) is studied. Biologically active substances of an indole nature are identified, "the effect of small doses" of which lies in not only the stimulation of growth of the mycelium (indole-3-acetic acid, 2 x 10(-7)-2 x 10(-4) g/l), but also in the induction of tryptophan-independent paths of auxin biosynthesis. The above-mentioned path is realized in the presence of exogenous indole (1 x 10(-3)-1 x 10(-4) g/l), as well as while inducing the biosynthesis of indole-3-acetic acid by its microadditives (1 x 10(-5)-1 x 10(-8) g/l), and is accompanied by the formation of anthranilic acid (up to 1.5 mg/l). Induction of the generative development stage ofshiitake by indole derivatives is revealed. It was found that among the studied compounds only indoleacetamide at a concentration of an order of x 10(-4) g/l in the culture fluid of L. edodes had a pronounced stimulatory effect on the formation of shiitake's brown mycelial film.

Homogenous Mn-peroxidase of a 26-fold purity grade was isolated from a culture of Azospirillum brasilense Sp245 cultivated on a medium containing 0.1 mM pyrocatechol. The molecular weight of the enzyme is 43 kD as revealed by electrophoresis in SDS-PAAG. It was shown that the use of pyrocatechol and 2,2'-azino-bis(3-ethylbenzotiazoline-6-sulfonate) at concentrations of 0.1 and I mM as inductors increased the Mn-peroxidase activity by a factor of 3.

Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability oflectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect oflectin in under 10 min in a concentration of 10 microg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.

Submerged mycelium of a xylotrophic basidiomycete Lentinus edodes produces an extracellular glycolipid, S3, associated with a lectin. Galactose glycan residue, as well as the lipid pool composition, which includes nonhydroxylated short-chain fatty acids, is uncommon for basidiomycetes. The glycolipid consists of D-galactopyranose (15% of S3 contains galactose sulfate) acylated by octadecanoic and nonadecanoic fatty acid residues (28 and 72%, respectively). The glycolipid structure and composition are confirmed by physicochemical analysis. The glycolipid is assumed to be a regulator of lectin activity.

A number of lectins varying in polypeptide composition and carbohydrate specificity were isolated from Lentinus edodes at different stages of its morphogenesis: nonpigmented mycelium, brown mycelium film, and fruiting body. Three lectins were identified at the nonpigmented mycelium stage, two of them being dimers consisting of 16 and 45 kDa and 16 and 42 kDa subunits; the third is a tetramer of 16, 39, 42, and 45 kDa subunits. The fractions with lectin activity obtained at the brown mycelium film stage contained polypeptides of 24, 30, and 38 kDa, characteristic of this morphological structure. The fruiting body was shown to contain two lectins of 43 and 55 kDa. All of the isolated lectins expressed the highest affinity towards L,D-melibiose, D-lactose, and D-galactose.

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes.

We studied changes of the hemagglutinating activity of intracellular lectins of the basidiomycete Lentinus edodes (shiitake) at various stages of its morphogenetic development depending on erythrocyte type, growth medium, and lectin purification degree. Under certain experimental conditions, the specific lectin activity at the brown mycelium film stage exceeded the corresponding value for nonpigmented mycelium. The sensitivity of the lectins towards trypsin-treated rabbit erythrocytes was no less than a hundredfold higher than towards any other erythrocyte type studied. The general regularities of specific activity change did not depend on nutrient medium composition. With purification of intracellular shiitake lectins, their sensitivity to human erythrocytes decreased seventyfold or more, whereas their sensitivity to rabbit erythrocytes increased by the same factor.

The possibility of application of the Pleurotus ostreatus D1-soil microflora to bioremediation of oil-polluted soils was studied. The fungus degraded mainly the aromatic fraction, whereas soil microflora intensely degraded paraffin and naphthene oil fractions. Introduction of the fungus Pleurotus ostreatus D to soil induces degradation of a wider range of oil hydrocarbons. It is reasonable to further investigate the discovered phenomenon in order to improve procedures of remediation of oil-polluted soils.

Lectins from the surface of Azospirillum brasilense Sp7 and Azospirillumn brasilense Sp7.2.3 (a mutant with impaired lectin activity) were shown to induce a stabilizing effect on the activity of almond beta-glucosidase under conditions of thermoinactivation and proteolytic enzyme treatment. Differences were revealed in the influence of lectins with various antigenic properties. Our results indicate that the effects of lectins on the catalytic activity of the enzyme are mainly associated with conformational changes in lectin molecules during mutagenesis, but not with carbohydrate specificity (general property). These data should be taken into account in evaluating the role of lectins in the formation of nitrogen-fixing associations.

For the first time, from the surface of the dikaryotic mycelium of the xylotrophic basidiomycete Grifola frondosa 0917 a lectin has been isolated with a molecular mass of 68 +/- 1 kDa, consisting of two subunits of 33-34 kDa each. The lectin is a hydrophilic glycoprotein with the protein : glycan ratio of 3 : 1. It exhibits high affinity to native rabbit erythrocytes and to human erythrocytes of the 0 blood group, but not to trypsin-treated ones. The hemagglutination (HA) caused by lectin was not blocked by any of the 25 tested mono-, di-, and amino sugars; it was also not blocked by some of glyco derivatives. Only 13.9 microg/ml of the homogeneous preparation of a polysaccharide, a linear D-rhamnan with the structure of the repetitive component --2)-beta-D-Rhap-(1 --3)-alpha-D-Rhap-(1 --3)-alpha-D-Rhap-(1 --2)-alpha-D-Rhap-(1 --2)-alpha-D-Rhap-(1 --blocked hemagglutination completely. The analysis of the amino acid composition of the lectin showed the greatest percentage of amino acids with positively charged R groups, arginine, lysine, and histidine, as well as the complete absence of sulfur-containing amino acids, cysteine, and methionine. D-glucose and D-glucosamine were detected in the carbohydrate part.

The effect of temperature and lectin from bacteria of the genues Azospirillum with blocked activity on human adipose tissue cells has been studied. The temperature used was 43.5 +/- 0.5 degrees C. Comparative results are given for the effect of lectin with the blocked and active carbohydrate-binding centers on adipocytes during heating, and the time course of the structural changes of adipocytes is described. When lectin with the active carbohydrate-binding centers was used for treatment, the heat-treated cells of a healthy obesity-prone subject died on the average in 55 +/- 5 min, whereas cells treated with lectin with the L-fucose-blocked carbohydrate-binding centers died in 80 +/- 5 min. The heat-treated cells of a diabetic obesity-prone patient died in 150 +/- 10 min on average when exposed to both active and inactive lectin. Consequently, when the lectin center is blocked with L-fucose, the effectiveness of lectin action on adipose cells of healthy obesity-prone persons decreases.

Impact of Azospirillum brasilense Sp7 lectin with carbohydrate specificity to L-fucose on T- and B-lymphocytes production, activity of IL-1alpha, and TNF-alpha was studied in vitro and compared with influence of red kidney bean phytohemagglutinin (PHA) on the same parameters. Increase of growth of T- and B-cells subpopulations has been shown that point to stimulatory effect of the bacterial lectin. Bacterial lectin of A. brasilense also increased synthesis of IL-1alpha and TNF-alpha. It has been shown that the bacterial lectin has more pronounced stimulatory effect on functional activity of lymphocytes compared with PHA.

It was found that Azospirillum brasilensis strain Sp7 is able to produce extracellular proteolytic enzymes. The enzymes were active within a broad range of pH values, with two peaks of activity being located in the acid and alkaline pH areas; required calcium ions; and exhibited substrate specificity with respect to azogelatin. Zymography allowed at least four proteolytic enzymes with molecular weights of 32, 45, 52, and 174 kDa to be detected in A. brasilense Sp7 culture liquid. It was shown that the lectin from A. brasilense Sp7 can inhibit proteolytic enzymes.

The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under the conditions of submerged culture was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and pH of culture medium. The activity of lectin in culture medium was maximal when the fungus was grown in a medium containing L-arabinose as a source of carbon and L-asparagine as a source of nitrogen (C : N ratio, (9.5-12): 1)) on the day 15-18 of culturing at pH 8-9.

The activity of the extracellular lectins of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] and the formation of a pigmented mycelial film by this fungus upon submerged cultivation in a synthetic medium were found to depend on the presence of some amino acids (particularly, asparagine) and Ca2+ and Mn2+ ions in the medium. Quantum chemical calculations suggest that the different character of the interaction of amino acids with the aforementioned ions is due to differences in the hydrophobicity of the amino acids rather than to differences in the electron structure of the amino acid zwitterions.

The complex study of the influence of A. brasilense Sp 7 lectin with carbohydrate specificity to L-fucose and D-galactose on the dynamics of cell populations in different structural functional zones of the white mice mesentery, as well as on the capacity of immunocompetent cells of mesenterial lymph nodes for synthesizing cytokines (interleukin-1, interleukin-6 and tumor necrosis factor), was carried out. A single oral administration of bacterial lectin in a dose of 4.5 micrograms produced a pronounced immunostimulating effect.

The activities of alpha-glucosidase, beta-glucosidase, and beta-galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilense Sp7 and Azospirillum lipoferum 59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol-acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied among the azospirilla studied. The cofunction of the A. brasilense Sp7 lectin and beta-galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum 59b lectin and glucosidases was revealed. The lectin from A. lipoferum 59b may possess saccharolytic activity.

The mutant strain Azospirillum brasilense Sp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilense Sp7(S) both in the exponential and stationary growth phases. The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla. The specific binding of the A. brasilense Sp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters. The interaction of the lectins of A. brasilense 75, A. brasilense Sp7, and A. lipoferum 59b with the polysaccharide-containing complexes that were isolated from these strains was not specific. No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found. A coflocculation of A. brasilense Sp7 cells with Bacillus polymyxa 1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.

The hemagglutinating (HA) activity of the submerged mycelium and the culture liquid (CL) of four strains of Lentinus edodes was studied. The HA activity of CLs proved to be much higher than that of mycelia. The carbohydrate specificity of fungal agglutinating factors was determined. HA activity was investigated as a function of the inoculum size, cultivation temperature, and culture age. The agglutinating activity of different morphogenetic structures of L. edodes F-249, including mycelium, brown mycelial mat (MM), primordia, and fruiting bodies, was studied, MM was found to possess the maximum HA activity, which can be explained by the possible involvement of agglutinins in the formation of MM, which is composed of glued hyphae.

Previous work from this Lab has shown that the proteins, lectins detected on the surfaces of the associative nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and Azospirillum lipoferum 59b come into play in interaction with plants at the early stages of establishment of an associative system (Nikitina V.E. et al., 1996). The present work demonstrates the influence of the lectins from A. brasilense Sp7 and A. lipoferum 59b on the activity of β-glucosidase of sweet almonds. Bacteria were grown on synthetic medium (Sadasivan L. et al., 1985) at 37 °C for 18 hours. Agglutinating activity of the cells was estimated with the help of the reaction of hemagglutination, with the use of rabbit erythrocytes treated with trypsin (Lis H. et al., 1972). Isolation of lectins from the cell surface was carried out in accordance with Eshdat (Eshdat Y. et al., 1978). The activity of β-glucosidase was evaluated spectrophotometrically at 425 nm by the amount of the formed nitrophenol (Buglova T.T. et al., 1991). It has been demonstrated that the addition of the increasing concentrations of lectins from A. brasilense Sp7 and A. lipoferum 59b caused the decrease of enzyme activity, hence exerts the inhibiting effect. Maximum inhibiting effect for the lectin from A. brasilense Sp7 was observed at the concentration of 70 mkg/ml (in this case enzyme activity decreased to 85%). The inhibiting effect of the lectin from A. lipoferum 59b was better pronounced (the enzyme activity decreased to 11%) and exhibited at reasonably lower concentrations, namely, 0.32; 0.64; 1.28 mkg/ml.

One particle distributions ofπ − produced in the collisions of deuterons, alphas, and carbon nuclei with Ta target at 4.2 GeV/c per nucleon momentum, registered in the propane bubble chamber, are presented. Comparison of the data with multiple scattering model and additive quark model is attempted.

Physico-chemical properties and chemical composition of the cell wall of pigmentless variants B-3 and B-6 differ from those of the parent strain of Staphylococcus aureus 209-P. As to morphology, the wall is more thick and loose. The mutants differ also by the phagotype, the ability for sorption of acridine orange, and the rate of sedimentation of cell suspensions. The properties of teichoic acids and the content of peptides in peptidoglycan change in the cell wall of the mutants.