[Ultrastructural study of the action of cyclosporine on cells of the fungus Aspergillus niger]. / Ul'trastrukturnoe izuchenie deistviia tsiklosporina na kletki griba Aspergillus niger.

Electron microscopy of the ultrastructure of the Aspergillus niger cells exposed to cyclosporine in acute experiments revealed changes in the mycelium morphology, hyphae growth and cell structure in the organism sensitive to cyclosporine. The analysis of the changes showed that the primary target of the cyclosporine action was the cell cytoplasmic membrane. The changes in the matrix of the cytoplasmic membrane, mitochondria and cell wall were likely mediated or secondary after the impairment of the cytoplasmic membrane.

[Effect of cyclosporin on various metabolic processes in fungi]. / Vliianie tsiklosporina na nekotorye metabolicheskie protsessy gribov.

Sensitivity of the representatives of Aspergillus and Piricularia to various concentrations of cyclosporine (in the submerged culture) was studied for choosing the test object for the subcellular investigation of the mechanism of the cyclosporine antifungal action. Changes in the main physiological and biochemical parameters of the fungal cells under the action of cyclosporine in concentrations of 10 to 80 micrograms per 1 ml of the medium were characterized. Low concentrations of cyclosporine (10 to 20 micrograms/ml) did not inhibit the growth of Aspergillus niger but induced stimulation of the growth processes. Only at a concentration of 80 micrograms/ml the inhibitory effect was observed. At the same time there was detected a dramatic stimulation of the culture respiration via the basic respiration chain. In the culture of Piricularia oryzae only inhibition of the culture growth even at the minimum concentration of cyclosporine (10 micrograms/ml) was recorded. Both the cyclosporine sensitive test objects i.e. A. niger and P. oryzae had an alternative cyanide resistant system of the electron transport. In the experiments with A. niger the cyclosporine effect on the synthesis of protein and RNA was revealed. The study provided data on the changes in the morphogenesis of the A. niger cells under the effect of cyclosporine.

[Effect of H-ATPase stimulators on cyclosporin biosynthesis]. / Vliianie stimuliatorov H-ATFazy na biosintez tsiklosporina.

An analogy was observed between the mechanisms of action of phytohormones on plant cells and cells of the fungus producing cyclosporine. Fusicoccin and cytokinin were shown to have a high stimulating action on the biosynthesis of cyclosporine. The stimulating concentrations of the phytohormones and the time of their maximum effect were determined. The electron microscopic studies demonstrated that an increase in the level of the cyclosporine synthesis correlated with a significant increase in the number of the cells in the state of the coagulation necrosis.

[Dynamics of the content of glycolysis enzymes in Streptomyces rimosus as it relates to the problem of regulation of oxytetracycline biosynthesis]. / Dinamika soderzhaniia fermentov glikoliza u Streptomyces rimosus v sviazi s problemoi reguliatsii biosinteza oksitetratsiklina.

The activities of the key glycolytic enzymes i.e. hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase were detected and investigated in 3 strains of Streptomyces rimosus differing in the level of the oxytetracycline production. The samples were assayed at different fermentation stages and under different aeration conditions. Correlation between the level of the pyruvate kinase activity within the first days of the fermentation process and the strain capacity for the antibiotic accumulation at the final stage of the fermentation process was observed.

[Isolation of protoplasts of Xanthomonas rubrilineans and their use in the study of localization of aminopeptidases]. / Poluchenie protoplastov Xanthomonas rubrilineans i ikh ispol'zovanie dlia izucheniia lokalizatsii aminopeptidaz.

The culture of Xanthomonas rubrilineans was able to synthesize a number of intracellular aminopeptidases. To study localization of the enzymes in the cells, a protoplasting procedure was developed providing the yield of 99.7 per cent. The following subcellular fractions were isolated: periplasmic, cytoplasmic and membranous. It was shown that alanine aminopeptidase was a cytoplasmic enzyme and glutamate peptidase was a membrane-bound enzyme.

[Activity of glycolysis enzymes in cyclosporine-producing Tolypocladium sp]. / Aktivnost' fermentov glikoliza u Tolycocladium sp.-produtsenta tsiklosporina.

Enzymes of various glycolysis stages, i.e. hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase, were detected in cyclosporine-producing organisms belonging to Tolypocladium. The initial activity of the enzymes in the highly active strain was much higher than that in the starting low active strain. During the fermentation the activity of the glycolysis enzymes per 1 mg of mycelium protein in the both strains increased. This was accompanied by a decrease in respiration activity. Therefore, there was a direct correlation between cyclosporine biosynthesis and glycolytic activity of the mycelium in Tolypocladium sp.

[Cloning in Escherichia coli of a mitochondrial DNA fragment from Acremonium chrysogenum containing a region responsible for DNA replication]. / Klonirovanie v Escherichia coli fragmenta mitokhondrial'noi DNK Acremonium chrysogenum, soderzhashchego uchastok, otvetstvennyi za replikatsiiu.

A bireplicone plasmid pSU901,4.6 kb in length, was constructed on the basis of plasmid pUC19 and the pstIB fragment, 1.9 kb in length, from mitochondrial DNA of A. chrysogenum. Based on the hybrid plasmid pSU901 and kanamycin resistance determinant, an autonomically replicating vector for A. chrysogenum, a culture producing cephalosporin C, is being constructed.

[High-molecular polyphosphates and pyrophosphate in cyclosporin- producing Tolypocladium sp. and their role in the processes of growth and antibiotic synthesis]. / Vysokomolekuliarnye polifosfaty i pirofosfat u produtsenta tsiklo- sporina Tolypocladium sp. i ikh rol' v protsessakh rosta i antibiotikoobrazovaniia.

The contents of high-energy phosphorous compounds, i.e. three fractions of polyphosphates, pyrophosphate, and ATP were determined in isogenic strains of Tolypocladium sp. differing in cyclosporine production levels. The content of polyphosphates was 1 to 2 orders of magnitude greater than that of pyrophosphate or ATP and did not depend on the strain productivity. During the period of the mycelial intensive growth and at the beginning of antibiotic synthesis, the level of polyphosphates lowered 2-3-fold and the content of pyrophosphate markedly decreased as well. The activities of polyphosphatase and pyrophosphatase during the culture growth and cyclosporine biosynthesis was higher in the highly productive strain.

[Cyclosporin biosynthesis and dynamics of bioenergetic processes in cyclosporin-producing strain]. / Biosintez tsiklosporina i dinamika bioénergeticheskikh protsessov u produtsenta tsiklosporina.

An attempt was made to show a correlation between definite bioenergetic parameters of the cells of the cyclosporine-producing culture and biosynthesis of cyclosporine. It was found that the three strains producing cyclosporine used in the study had an alternative cyanide-resistant pathway along with the classical cytochrome chain. In the strain forming only traces of the cyclopeptide during fermentation of the cyanide-resistant respiration constituted 60 to 80%. In the isogenic highly productive strains the cyanide-resistant respiration appeared to be markedly decreased beginning from the 1st day of fermentation and during the maximum biosynthesis of cyclosporine (on day 4 or 5 of fermentation) it reached zero. The ATP content in the cells of the highly productive strain, despite its decrease by the antibiotic biosynthesis peak, remained at a much higher level than that in the strain producing only traces of cyclosporine. A procedure for isolating functionally active mitochondria from the protoplasts was developed and a bioenergetic characterization of the mitochondria isolated from the strains with different antibiotic productions is presented.

[Isolation and characteristics of mitochondrial DNA from Acremonium chrysogenum]. / Vydelenie i kharakteristika mitokhondrial'noí DNK iz Acremonium chrysogenum.

Circular mDNAs 26.85 and 26.94 kb in length were isolated from two isogenic strains of A. chrysogenum producing cephalosporin C. The strains differed in antibiotic production capacity. Restriction analysis of the mDNAs was performed with using 6 endonucleases. Comparison of the restriction data revealed identity of mDNAs. A restriction map of the mDNAs was constructed. It is useful as a basis for further studies with molecular cloning.

[Pathways of the synthesis of glutamic acid by cephalosporin C-producing Acremonium chrysogenum]. / Puti obrazovaniia glutaminovoi kisloty u Acremonium chrysogenum, produtsenta tsefalosporina C.

There were observed two pathways of glutamic acid formation in two strains of Acremonium chrysogenum differing in the production levels of cephalosporin C. The pathway involving glutamate dehydrogenase is known. The other pathway involved amination catalyzed by glutamine synthetase. Activity of both the enzymes during intensive synthesis of the antibiotic was higher in the highly productive strain. Under conditions of limited nitrogen content in the medium production of glutamate during the antibiotic biosynthesis depended on glutamine synthetase. When there was an excess of nitrogen in the medium the main role in production of glutamic acid at the phase of cephalosporin synthesis was played by the other enzyme i. e. glutamate dehydrogenase. By the dynamics the curve of the glutamate dehydrogenase activity correlated with that of the antibiotic production.

[The protoplasts of Acremonium chrysogenum. Biochemical and morphological studies]. / Protoplasty Acremonium chrysogenum. Biokhimicheskie i morfologicheskie issledovaniia.

A procedure for preparing stable A. chrysogenum protoplasts capable of 60 per cent regeneration was developed. Two morphogenetic types of the regeneration were detected. The variants isolated after the protoplast regeneration were characterized by wide ranges of morphological variation. Capacity for the antibiotic production varied from 60 to 160 per cent of the activity of the starting strain. A procedure for isolating functionally active mitochondria from protoplasts of A. chrysogenum was also developed. Their main bioenergetic parameters were studied. In the respiratory chain of the A. chrysogenum mitochondria there were detected three conjugation sites of oxidative phosphorylation.

[Cytological study of early stages of regeneration of protoplasts of fungi and Streptomyces]. / Tsitologicheskoe izuchenie rannikh étapov regeneratsii protoplastov gribov i streptomitsetov.

Early stages of Penicillium chrysogenum 51 and Streptomyces lividans 66 protoplast regeneration on solid media were studied microscopically under conditions of microcompartments. It was shown that at the early regeneration stages there were both rapid reversion into the mycelial form and a retarded one. In P. chrysogenum retarded regeneration resulted in formation of hypha-like structures or protoplast breaking into fragments of various sizes. Some of the fragments restored the cell walls and mycelial organization whereas the others lysed. As a result of the breaking and compartmentalization of the viable areas one protoplasts formed several centers of P. chrysogenum colony reversion. Retarded regeneration of protoplasts in S. lividans 66 resulted in their growth and multiplication in the protoplast-like L-form. On media with penicillin, glycine and horse serum there were isolated colonies of S. lividans L-forms subject to passages or reversion depending on the medium composition.

[Valinomycin biosynthesis and the dynamics of the content of macroergic phosphorus compounds in Streptomyces cyaneofuscatus]. / Biosintez valinomitsina i dinamika soderzhaniia makroérgicheskikh fosfornykh soedinenii u Streptomyces cyaneofuscatus.

The pattern of accumulation and consumption of macroergic phosphoric compounds such as polyphosphates, pyrophosphate and ATP in the mycelium of the valinomycin-producing organism was studied. The content of high polymeric polyphosphates in the high productive strain A of S. cyaneofuscatus was much lower than that in the isogenic low productive strain B, which was indicative of their participation in providing bioenergetics of antibiotic production. Cultivation of the low productive strain B in the presence or absence of the A-factor showed that the mutant reduced its sporulation and provided a 40-fold increase in biosynthesis of valinomycin. However, no difference in consumption of diverse polyphosphate fractions was observed.

[Metabolic characteristics of polyphosphates and other macroergic phosphorus compounds in relation to the degree of penicillin production and growth conditions of Penicillium chrysogenum]. / Osobennosti obmena polifosfatov i drugikh makroérgicheskikh fosfornykh soedinenii v zavisomosti ot intensivnosti obrazovaniia penitsillina i uslovii rosta Penicillium chrysogenum.

Metabolism of macroergic phosphorus compounds was studied in high- and low-productive isogenic strains of Penicillium chrysogenum. It was shown that the levels of the high-polymer polyphosphates (fractions PP1, PP2 and PP3) in the strain intensively producing penicillin were 2-3 times higher than those in the low-productive strain by the 2nd day of the fermentation process (the period of penicillin production). The levels of pyrophosphate and ATP in the mycelium during the fermentation process did not significantly differ in the strains. The study on the relation between metabolism of the high-molecular polyphosphates and conditions of the culture growth and antibiotic production revealed that their accumulation was connected with biosynthetic processes giving rise to the growth of P. chrysogenum, while their consumption with penicillin production. The dynamics of the pyrophosphatase and polyphosphatase activity in the mycelium of the strains was studied.

[Possible role of the respiratory system of Fusidium coccineum in regulating fusidic acid biosynthesis]. / Vozmozhnaia rol' dykhatel'noi sistemy Fusidium cocineum v reguliatsii biosinteza fuzidievoi kisloty.

The respiration system was studied in three strains of the fungus Fusidium coccineum differing in their capability to synthesize fusidic acid. In all of the three strains, the system of oxidative phoshorylation predominated in supplying the cells with energy. In the strains with low and zero activities, the terminal oxidation of reduced equivalents occurred mainly via the respiration chain with cytochrome oxidase as a terminal component. In the strain with a high activity, there was an alternative cyanide resistant pathway, along with the classical cytochrome chain, and the complete switching to the alternative pathway coincided with the period of the antibiotic maximal accumulation. The induction of the alternative pathway in the strain with a high activity did not involve inhibition of the cytochrome region of the respiration chain. It was shown for the first time that the antibiotic synthesis and the character of cell differentiation can be changed by modifying the pathways of oxidation with specific inhibitors such as chloramphenicol and salicyl hydroxamate. Apparently, there is some general mechanism involved in regulating the production of the antibiotic, cell differentiation, and switching to the alternative oxidative pathway.

[Energy metabolism study of Fusidium coccineum strains with varying levels of antibiotic formation]. / Izuchenie énergeticheskogo obmena shtammov Fusidium coccineum s razlichnym urovnem antibiotikoobrazovaniia.

Energy parameters of the two strains of the inperfect fungus Fusidium coccineum with different levels of antibiotic production were studied comparatively. It was shown that the respiration activity of the both strains was almost completely suppressed by KCN+ salycyl hydroxomate. It provided a supposition that the respiration activity was almost selectively connected with the mitochondria. The analysis of the energy parameters and especially the efficiency of the substrate consumption indicated that the system of oxidative phospholiration was dominant in the energy supply in both the strains. The differences between the strains were observed in the growth rate and the level of KCN-stable respiration. A change in the respiration of the active strain to the alternative KCN-stable path was observed by the end of the 6-8 day fermentation process when significant amounts of fusidin accumulated in the fermentation broth. Correlation between the level of KCN-stability and the antibiotic production was found.