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Simple methods for determining free d-biotin were developed based on the latex agglutination reaction inhibition and competitive ELISA. Biotin-binding protein Str used as a biospecific marker was immobilized on the surface of colored polyacrolein latex particles or a nitrocellulose membrane. The sensitivity of both methods of the free d-biotin assay, which take 1.5 h, is 1.0-3.0 ng/ml.

The work was aimed at studying the effect of gramicidin S on the intact cells, spheroplasts and membrane specimens of Escherichia coli K12S with the natural resistance to this antibiotic. The resistance was shown to be caused by the barrier properties of the cell wall: the spheroplasts were highly sensitive to the lytic action of gramicidin S. The differences in the sensitivity to gramicidin S of substrate oxidation carried by the membranes of E. coli and Micrococcus luteus, a sensitive organism, were not of crucial significance for the manifestation of the resistance. The resistance was not associated with the decrease of gramicidin S adsorption: the cells were capable of binding large quantities of the antibiotic and remaining viable. Gramicidin S appeared to be attached to the cell walls (most likely, the outer membranes) rather than the cytoplasmic membranes.

The study on the possibility of eliminating gramicidin S from the bacterial cells which had adsorbed it showed that a part of the labeled antibiotic bound by the bacteria may be washed out with buffer or salines. When the cells which had adsorbed gramicidin S were treated with lecithin emulsion, a significant part of the bound antibiotic was transferred to the lecithin liposomes. This turned the gramicidin S effect to the cells: significant but not complete reduction of the membrane barrier properties and dehydrogenase reactivation. Elimination of gramicidin S also reduced the colony forming capacity in a part of the cells.

The effect of membrane active antibiotics, such as gramicidin S, its derivatives and carbonyl-conjugated pentaens on suspended bacterial protoplasts resulted in lysis of the protoplasts accompanied by a marked decrease in the optical density (OD) of the suspensions. However, when the drug concentrations were lower than those inducing the lysis, an increase in the suspension OD by 10-30 per cent as compared to the control values of the OD was often observed. The increase in the protoplast suspension OD was most pronounced with the use of the drugs with a relatively low lytic activity or under the conditions lowering the lytic activity of the antibiotics. Under such conditions no agglutination of the protoplasts was observed. The comparative estimation of the protoplast diameters by the method of Klenin et al. in the control suspensions of M. lysodeikticus protoplasts and in the suspensions with a stable increase in the OD showed that the OD increase was associated with swelling of the protoplasts: an increase in the suspension OD by approximately 30 per cent corresponded to an increase in the protoplast diameter by approximately 15 per cent. The observed increasing of the suspension OD must be due to the fact that the membrane active antibiotics induced a decrease in the osmotic stability of the protoplasts not sufficient for their lysis.

In concentrations of 0.05-0.10 gamma/ml geliomycin suppresses inclusion of C14-uracyl into the acid-soluble fractions of the staphylococcal cells. Inclusion of C14-leucine at the same concentrations of the antibiotics was suppressed to a lesser extent. The above concentrations of geliomycin had practically no effect on inclusion of C14-thimine. Suppression of C14-uracyl inclusion was observed in 5 minutes of the cell incubation with the antibiotic, a significant decrease in C14-leucine inclusion being observed only in 30 minutes of the incubation. The effect of geliomycin on the staphylococcal cells within the concentrations inhibiting the microbial growth and suppressing inclusion of labeled uracyl and leucine into the cells was of the bacteriostatic nature. It was supposed that the antibacterial effect of geliomycin was based on suppression of RNA synthesis.

The effect of dioxidine on the growth of freshly isolated clinical strains of microorganisms with various levels of resistance to antibiotics was studied. It was shown that strains of Ps.aeruginosa not resistant to carbenicillin, chloramphenicol and nalidixic acid were most sensitive to the drug. Similar correlation was found in Proteus strains with various levels of resistance to chloramphenicol. Sensitivity of these organisms to dioxidine did not depend on their resistance to gentamicin, ampicillin and neomycin. The antibacterial effect of the drug is based on inhibition of the synthesis of nucleic acids, the level of DNA synthesis inhibition being the highest. Dioxidine had practically no effect on protein synthesis.

The effect of cyclic decapeptide of gramicidin S on electrical conductivity of bilayer lipid membranes has been studied. The integral conductivity of bilayer has been shown to increase with the growth of antibiotic concentration. The integral conductivity increase occurs as series of conductivity discrete leaps, differing in amplitude from fluctuations of conductivity caused by linear gramicidins. In the series of selectivity of bilayer membranes for cations of alkaline metals the rubidium ion is before the cesium ion. This is the only difference between this series and the series of relative ionic mobility series of cations of alkaline metals in water solutions.

Heliomycin at a concentration of 100 mcg/ml had almost no effect on the growth of Escherichia coli, which was inhibited only upon the combined action of heliomycin and ethanol that facilitated the penetration of the antibiotic into the cell. Heliomycin inhibited in these conditions the synthesis of RNA and had no effect on the synthesis of DNA in the three studied strains of E. coli. The synthesis of protein either was not inhibited by heliomycin at all or was inhibited only to a small extent.

Variants of Micrococcus lysodeikticus resistant to 100 micrograms/ml of gramicidin S with preserved resistance in subcultures on media without the antibiotic were isolated as a result of prolonged adaptation on a solid medium with increasing concentrations of gramicidin. The sensitive and resistant cells did not differ by their ability to bind gramicidin. Under the antibiotic effect permeability of the cytoplasmic membranes of the intact cells in the sensitive bacteria appeared to be impaired to a greater extent than that of the membranes of the cells in the resistant variant. Comparison of the lytic activity of gramicidin and its derivatives with respect to the protoplasts prepared with the cells of the initial and resistant variants of M. lysodeikticus revealed much higher resistance of the resistant variant protoplasts to the membrane-disorganizing effect of the preparations. Malate dehydrogenase and NADH-oxidase in the membrane preparations of the resistant variant cells differed from analogous enzymes from the membranes of the initial strain by the levels of their activity and sensitivity to gramicidin. It is likely that during adaptation of M. lysodeikticus to gramicidin significant changes in the cell cytoplasmic membranes occurred.

The membrane activity of gramicidin S against intact cells of staphylococci and micrococci determined by the loss of intracellular low-molecular compounds with the adsorption maxima at 260 nm by the bacteria markedly increased in the presence of phosphates. Acetate and hydrochlorides had no effect on the membranotropic action of the antibiotic. Analogous results were obtained for gramicidin S derivatives by free ornithine amino groups possessing basic and antibiotic activity. The increased membrane action of the antibiotic on the cells in the presence of phosphates was probably due to the changes under these conditions in the aggregate state of the substance in solution. With the use of labeled gramicidin S it was shown that centrifugation of the antibiotic solution in a concentration of 50-1000 micrograms/ml at 18000-20000 rpm resulted in sedimentation of a significant part of gramicidin S dissolved in the phosphate buffer. Sedimentation of the drug in aqueous and NaCl solutions was insignificant. The presence of phosphates in the medium had no effect on the quantity of the antibiotic bound to the micrococcal membrane preparations. It is suggested that the increase in the level of cytoplasmic membrane disorganization in the presence of phosphates was due to binding of the antibiotic molecule associations to the membranes and/or additional intermolecular association of gramicidin S bound to the membranes.

When Streptomyces olivocinereus 11-98 MFU was grown in media containing L-arabinose or sucrose there was observed a converse relation between the culture growth and heliomycin biosynthesis. In media with two carbon sources: L-arabinose and glycerol or sucrose and glycerol at first L-arabinose or sucrose was consumed while the level of glycerol consumption remained low as compared to the control. After exhaustion of the first carbon source there was observed increased consumption of the second one i.e. glycerol. While the medium contained L-arabinose or sucrose the culture growth was mainly provided by these carbon sources and biosynthesis of heliomycin was inhibited. The culture started biosynthesis of heliomycin when L-arabinose or sucrose in the medium was exhausted. Probably control of heliomycin biosynthesis by L-arabinose or sucrose is achieved by catabolic type carbon regulation known as the general mechanism regulating biosynthesis of various antibiotics.

Heliomycin inhibited formation of the pool or purine and pyrimidine containing compounds in the cells of the growing cultures of Staphylococcus aureus and Escherichia coli both in the lag phase at the maximum rate of formation of the pool of compounds absorbing at 260 nm and in the exponential phase. A decrease in the concentration of purine and pyrimidine containing compounds in the acid-soluble fraction of the cells by the antibiotic was not caused by the disturbed permeability of the membranes. Apparently, heliomycin interfered with the regulation of RNA synthesis, either directly or through its action on the energy metabolism of the cells.

Gramicidin S and its derivatives by the free amino groups of the ornithine moieties were studied with respect to their effect on interface tension of mercury and the polarographic background for comparative estimation of the surface active properties of these compounds. The same way as the compounds preserving their main properties, the neutral and acid derivatives lowered the polarographic maximum. No correlation between the level of the antimicrobial and membrane activity and the surface active properties of the tested compounds was observed. It is likely that the direct cause of the induced biological effect is not connected with the surface active properties responsible for possible interaction of the tested substances with the cell (protoplast).

Heliomycin inhibited synthesis of RNA in Staph. aureua which was clearly shown in the study of the antibiotic effect on RNA synthesis in the lag phase of the culture development: heliomycin markedly lowered the maximum RNA level in the biomass observed in the culture at the beginning of the exponential growth. On further growth of the culture heliomycin induced a significant retardness of the process of the natural decrease in the RNA biomass level resulting in increased content of RNA in the cells growing in the presence of heliomycin as compared to the control culture. Retarded natural decrease in the RNA biomass level in the presence of heliomycin was observed also on the antibiotic addition just at the beginning of the exponential growth, during the period of maximum RNA accumulation in the cells. Heliomycin had no effect on synthesis and biomass levels of DNA. Heliomycin inhibited the protein synthesis and was close to chloramphenicol by the level of inhibition of the summation protein synthesis in the biomass. However, comparison of the effect of the above antibiotics on synthesis of beta-galactosidase, an individual enzyme protein showed that heliomycin was much less active as an inhibitor of protein synthesis in comparison to chloramphenicol.

The work was concerned with studying the effect of gramicidin S derivatives with modified free amino groups of ornithine residues on bacterial cells and protoplasts. The substitution of the amino groups with neutral or carboxyl-containing groups eliminated or sharply decreased the antibacterial activity of gramicidin S, its binding to the cells, and the ability to change the permeability of the cytoplasmic membranes of the intact cells. However, the neutral derivatives and the derivative with acidic properties showed a considerable lytic activity when they were incubated with the protoplasts of Micrococcus lysodeikticus, Bacillus megaterium and Bacillus subtilis. Hence, these compounds preserved a certain membranotropic level. Those gramicidin S derivatives with modified ornithine amino groups which possessed basic properties were similar to gramicidin S in the antibiotic activity, the modified permeability of the membranes, the ability to bind with the cells, and the lytic action on the protoplasts.

The rate of incorporation of labeled precursors for RNA ([14C]uracil) and protein ([14C]DL-leucine) into the cells of the synchronous culture of Candida utilis VKMY-1668 (the optimum temperature of growth, 31--32 degrees C) was studied as a function of different temperatures (28, 31, 32, 34, 36, 38, and 41 decrees C). The yeast was grown on a simple mineral medium containing glycerol. RNA synthesis was found to be more susceptible to elevated temperature than protein synthesis: the maximum rate of incorporation was registered at 32--34 degrees C for [14C]DL-leucine and only at 32 degrees C for [14C]uracil (the rate of its incorporation at 34 degrees C decreased by 50% as compared to that at 32 degrees C). The rate of incorporation of [14C]uracil at 34 degrees C reached 100% (the rate at 32 degrees C) when yeast autolysate was added to the medium, and 75 and 70%, respectively, upon the addition of DL-methionine or Mg2+ (as compared to 50% without them).

Influence of gramicidin S on electric parameters of nitrocellulose ultrafilter as a biomembrane model was studied, the ultrafilters being impregnated with fatty acids or their ethers. It was shown that addition of the antibiotic to the solution over one side of the model membrane resulted in generation of electric potential. With increasing of the drug concentration by one order there was observed more than a 10-fold drop in the membrane resistance while the electric capacitance actually remained unchanged. It was suggested that gramicidin S was localized in thin water layers covering the surface of the ultrafilter pores and separating the polymer matrix and impregnating liquid filling the pores. Such incorporation led to changes in the state of water and water channel surfaces which defined the increase in the model membrane electric conductivity.

The effect of copper ions and unfavourable pH values of the medium on the incorporation of labelled precursors of protein and RNA was studied in Candida utilis. Specific inhibition of protein synthesis by copper ions and alkaline conditions was found. No specific inhibition of protein or RNA was detected at low pH values of the medium.

The effect of some antibiotics on in vitro blast transformation of lymphocytes of healthy donors stimulated with phytohemagglutinin was studied. The stimulation level was estimated by incorporation of 3H-thymidine into lymphocytes. It was shown that minocycline had a markedly pronounced inhibitory activity. Chloramphenicol and tetracycline also proved to be active inhibitors. Penicillin, cephalotin and carbenicillin had no effect on lymphocyte mitogenesis. Gentamicin proved to be a rather active stimulator of mitogenesis, especially with the use of a suboptimal concentration of phytohemagglutinin. The method is rather sensitive and informative. It may be used for preliminary determination of the immunotropic properties of drugs and chemical compounds with respect to immunocompetent cells of man.

The rates of incorporation of labelled precursors of protein (14C-DL-leucine) and RNA (14C-uracil) into the cells of synchronous yeast cultures of Pichia membranaefaciens, Hansenula anomala, Saccharomyces cerevisiae and Saccharomyces (fabospora) fragilis were studied at different temperatures. Synthesis of RNA and then of protein was inhibited in H. anomala if the temperature was increased above the optimal one. This is manifested even more distinctly in Sacch. cerevisiae and P. membranaefaciens. In the thermotolerant yeast Sacch. fragilis, the incorporation of 14C-uracil was inhibited at temperatures above 40 degrees C while the rate of protein synthesis did not decrease.

Heliomycin inhibited in vitro the RNA-polymerase reaction catalyzed by the preparation of DNA-dependent RNA-polymerase from E. coli. The blocking effect increased with a rise in the antibiotic concentration. The inhibitory effect of heliomycin decreased, when the amount of RNA-polymerase in the system increased. Yet, it did not depend on the content of DNA and the nature of the DNA preparation. Preincubation of RNA-polymerase with DNA resulting in formation of the enzyme-matrix complex did not prevent blocking RNA synthesis by heliomycin. Suppression of the RNA-polymerase reaction did not depend on the time of the antibiotic addition to the polymerizing system. Heliomycin had a significant activity not only with respect to the bacterial RNA-polymerase, but also in the system containing the enzyme isolated from the cells of Crithidia oncopelti.