[Changes in growth patterns and intracellular calcium concentrations in aspergillus awamori treated with amphotericin B].

Growth patterns and intracellular Ca2+ concentrations in the mutant strain Aspergillus awamori 66A, containing a recombinant aequorin gene were studied in the presence of a permeabilizing fungicidal agent amphotericin B. The cell response, i.e., changes in the growth and development of the fungus (initiation of spore germination, mycelial growth, and intensity ofsporulation) was dose-dependent. Low concentrations of amphotericin B (2.5 microM) stimulated spore germination: the number of germinating spores was 2-3 times higher than in the control (without the fungicide). At higher amphotericin concentrations (20 microM) spore germination was inhibited. Amphotericin B had a dose-dependent effect on mycelial growth and sporulation intensity on solid Vogel medium. Intracellular Ca2+ concentrations in the presence of amphotericin B were investigated using the luminescence of the photoprotein aequorin. High concentrations of amphotericin B (10 and 20 microM) were shown to cause an instantaneous increase in Ca2+ concentrations, compared to the control and lower amphotericin concentration (2.5 microM). Ca2+ concentrations remained elevated throughout the experiment and correlated with the inhibition of mycelial growth and development.

[Analysis of the Ca2+ response of mycelial fungi to external effects by the recombinant aequorin method]. / Analiz Ca2+-otveta mitselial'nykh gribov na vneshnie vozdeistviia s ispol'zovaniem rekombinantnogo ékvorina.

Using the mutant strain Aspergillus awamori 66A producing a recombinant Ca2+-dependent photosensitive protein aequorin, the dynamics of Ca2+ was studied for the first time in the cytosol of the micromycetes exposed to stressful factors, such as an increase in extracellular Ca2+ to 50 mM, hypoosmotic shock, and mechanical shock. Cell response to stress proved to involve an increase in the Ca2+ concentration in the cytosol, which was determined from the amplitude of aequorin luminescence and the time of the amplitude enhancement and relaxation. The level of Ca2+ response depended on the physiological stimulus. Inhibitory analysis with various agents that block Ca2+ channels and with agonists that specifically enhance the activity of the channels suggested that (1) the level of Ca2+ in the cytosol of micromycetes increases in response to stress because of the ion influx from both the growth medium and intracellular reservoirs and (2) the potential-dependent transport systems play the major role in the Ca2+ influx into the cytosol of the micromycete cells.

[Effect of a chemical analogue of autoinducers of microbial anabiosis on the Ca2+ response of mycelial fungi]. / Vliianie khimicheskogo analoga mikrobnykh autoinduktorov anabioza na Ca2+-otvet mitselial'nykh gribov.

The microbial alkylhydroxybenzenes (AHB), autoinducers of anabiosis, or d1 factors, participate in stress response of mycelial fungi, as determined from changes in intracellular Ca2+ concentration. By using the genetically modified strain Aspergillus awamori 66A, which produces a recombinant Ca2+-dependent protein aequorin, the dynamics of Ca2+ was studied in the cytosol of cells exposed to mechanical shock in the presence of the protective doses (0.001-0.01% w/vol) of a chemical AHB analogue, 4-n-hexylresorcinol. Like under stressful conditions, Ca2+ concentration increases in the cell cytosol in response to enhanced AHB level in a growing fungal culture; thus, AHB is perceived by cells as a stress signal. The level of cell response, which was determined from the amplitude of luminescence dependent on the Ca2+ concentration in cytosol was related to the physiological age of the cells and AHB concentration. Micromycete preincubation with AHB was found to protect cells from subsequent stress; this was reflected in the Ca2+ response. The protective AHB effect was manifested as (1) a significant decrease in the amplitude of luminescence and, thus, in Ca2+ accumulation in the cytosol during subsequent mechanical stress (as compared to the control--mechanical stress only); (2) development of the secondary Ca2+ response, which was not observed in the control; (3) a high level of Ca2+ retained in the cytosol for a long time in the presence of AHB (as compared to the control without preincubation with AHB). The mechanisms underlying the AHB effect on the Ca2+ transport systems are discussed.

[Effect of Bacillus intermedius ribonuclease on properties of the yeast Saccharomyces cerevisiae]. / Vliianie ribonukleazy Bacillus intermedius na svoistva drozhzhei Saccharomyces cerevisiae.

Effects of Bacillus intermedius ribonuclease on physiological, biochemical, and consumer properties of baker's yeast Saccharomyces cerevisiae were studied. This enzyme improved the yeast raising strength and increased the cell tolerance to various adverse factors. The antistress effect of RNase correlated with an earlier start of the stationary growth phase and increased trehalose pool.

[Dependence of the effect of RNAase from Bacillus intermedius on the growth of baker's yeast on the concentration of the exogenous enzyme]. / Zavisimost' vliianiia RNKazy Bacillus intermedius na rost pekarskikh drozhzhei ot kontsentratsii ékzogennogo fermenta.

Bacillus intermedius RNase added at a low concentration (0.001 microgram/ml) stimulated yeast growth, while a high RNase concentration (1500 micrograms/ml) was inhibitory to yeast growth. The inhibitory effect of RNase was transient and correlated with the increase in the trehalose pool of yeast cells. The number of unbudded cells in the yeast population tended to decrease under the action of low concentrations of bacillar RNase and to increase under the action of high concentrations of this enzyme.

[Stabilization of enzymes by anabiosis autoinducers as a possible mechanism of resistance of resting microbial forms]. / Stabilizatsiia fermentov autoinduktorami anabioza kak odin iz me khanizmov ustoichivosti pokoiashchikhsia form mikroorganizmov.

Alkyl-substituted hydroxybenzenes (AHBs), auto-inducers of microbial dormancy (or d1 factors), were found to stabilize the structure of protein macromolecules, making them metabolically less active and more resistant to stresses. In vitro experiments with the Bacillus intermedius ribonuclease and chymotrypsin showed that the degree of the physical and chemical stability of these enzymes treated with AHBs depends on their concentration and incubation time. Experiments with RNase, which is capable of refolding, i.e., renaturation after heat denaturation, revealed that AHBs efficiently interact with both intact and denatured proteins. The data obtained allow the inference to be made that d1 factors may play the role of natural chemical chaperons, blocking metabolism in dormant cells through the formation of catalytically inactive thermostable complexes with enzymes.

[Changes in the trophic organization of mitotic cycle of Candida utilis after exposure to RNAse from Bacillus intermedius]. / Izmenenie troficheskoi organizatsii mitoticheskogo tsikla drozhzhei Candida utilis pod deistviem RNKazy Bacillus intermedius.

The effect of the RNase from Bacillus intermedius on the growth and trophic cycle of Candida utilis was studied. The RNase at concentrations of 0.001-0.01 microgram/ml stimulated yeast growth by 30-40% as compared to the control, reduced the mitotic cycle of the yeast by shortening its G1 phase, and decreased the number of exotrophic cells in the G1 phase to a minimum. It was suggested that RNase is involved in the regulation of the transition of cells from the exo- to endotrophic state.

[Effect of the RNAase from Bacillus intermedius on growth and physiological characteristics of Escherichia coli]. / Issledavanie vliianiia RNKazy Bacillus intermedius na rost i fiziologicheskuiu kharakteristiku Esherichia coli.

The effect of the RNase from Bacillus intermedius on the growth of Escherichia coli was investigated. RNase added to growth medium enhanced the synthesis of DNA, RNA, and protein and stimulated cell division; the degree of stimulation depended on the enzyme concentration. A necessary condition for stimulation was the adsorption of the enzyme on the cell surface and its interaction with the cytoplasmic membrane, as demonstrated immunocytochemically. The adsorption of the enzyme was accompanied by a 43% decrease in the surface charge density. Other effects of RNase involved a 25% increase in the growth rate, a 38% biomass gain, and generation time shortening by 10 min. The stimulation of bacterial growth correlated with the stimulation of cellular respiration rate.

[The use of pancreatic RNAse in the production of baker's yeast]. / Primenenie pankreaticheskoi RNKazy v proizvodstve khlebopekarnykh drozhzhei.

Microdoses of a preparation of pancreatic RNase were shown to stimulate the growth of Saccharomyces cerevisiae yeast. The effect was only retained at a certain inoculum/enzyme preparation ratio. Industrial tests of the preparation showed that the addition of RNase to the first reservoirs for culture accumulation (an inoculator and a seeding device) increased the yield of bakers' yeast and improved their quality.

[Use of exogenous ribonuclease for improvement of Lactobacillus biological properties]. / Primenenie ékzogennoi ribonucleazy dlia uluchsheniia biologicheskikh svoistv laktobatsill.

The influence of exogenous RNAse on the dynamics of the acid formation by the industrial strain 8R-A3 of Lactobacillus plantarum, its antibiotic sensitivity and antagonistic activity was studied. In the presence of the RNAase growth stimulating dose both a decrease of the culture lag-phase and a more intensive accumulation of lactic acid in the incubation medium resulting in an increase of the Lactobacillus antagonistic activity were observed. It was shown that RNAase increased the Lactobacillus stability to tetracycline and erythromycin by 32 to 57 per cent as compared to the control.

[Effect of Bacillus intermedius RNAases with various catalytic activity on multiplication of Escherichia coli and Bacillus subtilis]. / Vliianie RNKazy Bacillus intermedius s raznoi kataliticheskoi aktivnost'iu na razmnozhenie Escherichia coli i Bacillus subtilis.

The effect of the Bacillus intermedius ribonuclease and its mutant forms derived by site-specific mutagenesis on the growth of gram-positive and gram-negative bacteria was studied. Both catalytically active and catalytically inactive (mutant) ribonucleases stimulated bacterial growth, the extent of stimulation correlating with the catalytic activity of the enzymes. It was suggested that the biological activity of exogenous ribonucleases is mainly due to their catalytic activity.

[The stimulating action of exogenous RNase on eubiotic strains of Lactobacillus, Bifidobacterium and Escherichia coli]. / Stimuliruiushchee deistvie ékzogennoi RNKazy na éubioticheskie shtammy laktobatsill, bifidobakterii i Escherichia coli.

The influence of Bacillus intermedius RNAase on the multiplication on B.B.bifidum, L.fermentum and E. coli was studied. The study revealed that the stimulating action depended on the dose of the enzyme, the microbial species and the growth phase of the inoculate. RNAase, added to the nutrient medium, was shown to induce the acceleration of the synthesis of DNA, RNA, protein, as well as mitosis. At the same time the stimulating action of RNAase was accompanied not only by the reduction of the duration of the lag phase, but in some cases even by an increase in the specific growth rate.

[Effect of Bacillus intermedius RNAase on growth of a Saccharomyces cerevisiae culture]. / Vliianie RNKazy Bacillus intermedius na rost kul'tury drozhzhei Saccharomyces cerevisiae.

The effects of RNase from Bacillus intermedius on proliferation of Saccharomyces cerevisiae were studied. The enzyme (0.01 microgram/ml) stimulated the yeast cell budding. This effect was dose-dependent and required an appropriate physiological stage of the growing culture cells. RNase produced maximal effects when added to exponentially growing cultures. Analysis of the age structure of the population showed that exogenous RNase stimulated the cell cycle at a stage preceding the initiation of DNA synthesis and budding of single yeast cells and cells occurring at the budding stage III. RNase did not decrease the buoyancy and osmotic sensitivity of baker's yeast.

[An immunocytochemical study of bacterial ribonuclease penetration into Candida utilis yeast cells and the effect of the enzyme on yeast multiplication]. / Immunotsitokhimicheskoe issledovanie proniknoveniia bakterial'noi ribonukleazy v kletki drozhzhei Candida utilis i vliianie fermenta na razmnozhenie drozhzhei.

Permeability of Candida utilis cells for exogenic Bacillus intermedius RNase was studied cytochemically. The enzyme was shown to penetrate through the outer membrane of C. utilis after a 20 min incubation with cells being in exponential phase, and to be connected with the yeast cytoplasmic membrane.

[Change in various biological properties of Lactobacillus as affected by ribonuclease]. / Izmenenie nekotorykh biologicheskikh svoistv laktobatsill pod deistviem ribonukleazy.

The influence of RNAse from Bacillus intermedius on the growth of the industrial strain Lactobacillus plantarum 8R-A3 was studied. It was shown that the stimulating effect of the enzyme depended on its dose and manifested itself in decreasing the growth lag phase. At the same time the growth stimulating dose of RNAse increased the Lactobacillus adhesion to the epithelial cells and promoted secretion of proteinases from Lactobacillus to the culture medium. The possible use of RNAse as a stimulant of the growth of industrial strains was demonstrated which is advantageous for colonization of the biological surfaces.

[Effect of membranotropic physiologically-active substances on the growth of Saccharomyces cerevisiae cultures]. / Deistvie membranotropnykh fiziologicheski aktivnykh veshestv na rost kul'tury Saccharomyces cerevisiae.

The effect of Bacillus intermedius RNase and yeast autoregulatory d2 factor on growth of Saccharomyces cerevisiae is reported. It is shown that 0.01 microgram/ml of RNase stimulates the growth of the yeast on malt wort and molasses, the peak of the effect being observed for the enzyme added in the middle exponential phase. Certain concentrations of the d2 factor can also potentiate yeast growth. The effect is more pronounced if the d2 factor and RNase are added in combination rather than singly. Although the cell membrane is a target for both d2 factor and RNase, the mechanisms of their action are presumably different.

[The effect of Bacillus intermedius RNAase on the growth and development of Bacillus mucilaginosus]. / Vliianie RNKazy Bacillus intermedius na rost i razvitie Bacillus mucilaginosus.

The effect of Bacillus intermedius RNAase on vegetative cells and spores of siliceons bacteria Bacillus mucilaginosus was investigated. It is shown that the enzyme stimulates the growth of vegetative cells of B. mucilaginosus at a concentration of 10 mkg/ml and promotes germination of spores at a 1000-fold lesser concentration (0.1 mkg/ml). The spores at stages of activation and initiation appear to be most susceptible to the enzyme action. The effect of RNAase on B. mucilaginosus multiplication correlates with intensification of bacterial leaching of bauxites.

[Penetration of pancreatic DNase into Candida tropicalis cells]. / Proniknovenie pankreaticheskoi DNKazy v kletki Candida tropicalis.

Permeability of Candida tropicalis cells for exogenic DNAse was studied by a cytochemical method. The enzyme were shown to penetrate yeast outer membrane and cell wall after a 20 minute incubation period when incubated together with cells at the beginning of the stationary phase.

[The effect of Bacillus intermedius RNAse on the multiplication of Candida tropicalis yeasts]. / Vliianie RNKazy Bacillus intermedius na razmnozhenie drozhzhei Candida tropicalis.

The effect of Bacillus intermedius RNAse on the reproduction of Candida tropicalis and synthesis of the main biopolymers in the yeast cells. It has been found that stimulating action of the enzyme appears at the concentration of 10(-5)-10(-6) mg/ml and does not depend on the physiological state of the sowing culture. The connection between the increase of the ionic penetration and stimulation of the RNA and proteins synthesis in the yeast cells subjected to the RNAse action is shown. The mechanism of chromatine-associated RNA-polymerase activation is suggested to include the alteration of the ionic penetration of cells under the RNAse action.

[The biochemical aspects of the stimulating action of exogenous RNAse]. / Biokhimicheskie aspekty stimuliruiushchego deistviia ékzogennykh RNKaz.

A sequence of biochemical reactions in yeast from moment of RNAse interaction with cell membrane to cell division has been studied. RNAse addition in growth medium causes the increase of Ca2+ entering rate in cells in 2.4 times. Under this condition the increase of membrane enzyme adenylate cyclase correlating with the growth of cAMP content in cell and rise of cAMP-dependent protein kinase activity have been observed. A hypothetic scheme of cellular response on the RNAse effect is suggested.