Changes in the membrane ATPase activity of erythrocytes and Candida guilliermondii under the action of roseofungin

Changes in the Mg-ATPase and Na, K-ATPase activity of the rat erythrocyte and Candida guilliermondii membranes under the effect of roseofungin were studied. The antibiotic was totally bound to the isolated plasmatic membranes of Candida guilliermondii, up to 3 micrograms of the antibiotic per 1 microgram of the yeast protein. The Mg-APTase activity of these membranes was slightly inhibited by the antibiotic. The activity of Na, K-ATPase was almost completely inhibited even at 0.04 mg of roseofungin per 1 mg of protein. Much higher concentrations of the antibiotic inhibited the Mg-ATPase and Na, K-ATPase activity of the erythrocyte membranes to a less extent.     

Growth inhibitory effect of antibiotic tetaine on yeast and mycelial forms of Candida albicans

The mycelial (M) form of Candida albicans is more sensitive to the action of the antibiotic tetaine than the yeast (Y) form. Tetaine, at low concentrations about 1 microgram/ml also inhibits Y-M transition. It causes severe deformation of cells, agglutination and inhibits septum formation in the yeast forms. Tetaine action is reversed by dipeptides in both forms and by tripeptides in M form. N-acetyl glucosamine is a powerful antagonist of tetaine action on both morphological forms. Tetaine action on mycelial forms is slightly antagonised by N-acetyl mannosamine and very powerfully by glutamine.     

Comparative study of different variants of Actinomyces roseoflavus producing the polyene antibiotic roseofungin

The respiration chain in the membranes of whole Actinomyces roseoflavus (var. roseofungini) cells from the parent and secondary cultures is sensitive to KCN, non-sensitive to Triton X-100 treatment removing the antibiotic roseofungin from the cells, and has a very high for the bacteria respiration control. When the cells are in contact with atomic tritium at the temperature of liquid nitrogen, roseofungin is tritiated and binds to A. roseoflavus isolated membranes and whole cells, mostly to those of the parent culture as compared to the secondary culture. A fraction of membranes which lost NADH dehydrogenase in the course++ of purification was isolated from the cells disintegrated in the frozen state.     

Comparison of anti-Vibrio activities of potassium sorbate, sodium benzoate, and glycerol and sucrose esters of fatty acids.

The effects of fatty acids and their glycerol and sucrose esters, potassium sorbate, and sodium benzoate on growth of Vibrio parahaemolyticus in laboratory media at pH 6.7 were evaluated. The minimum concentrations at which inhibition by esters of glycerol could be detected were lowest for monolaurin (5 microgram/ml) and monocaprin (40 microgram/ml); these concentrations were lower than those observed for inhibition by lauric and capric acids, respectively. Inhibitory action of sucrose caprylate was detected at 40 microgram/ml, whereas sucrose caprate was effective at 100 microgram/ml; sucrose esters of lauric, myristic, and palmitic acids were ineffective at 100 microgram/ml. Potassium sorbate and sodium benzoate inhibited growth at concentrations as low as 30 and 300 microgram/ml, respectively, and enhanced the rate of thermal inactivation of V. parahaemolyticus at slightly higher concentrations. Fatty acid esters of glycerol and sucrose offer potential as perservatives for slightly acid or alkaline low-fat foods which do not lend themselves to the full antimicrobial action of traditional food preservatives such as potassium sorbate and sodium benzoate.     

Enzymatic degradation of nitriles by a Candida guilliermondii UFMG-Y65

Candida guilliermondii UFMG-Y65, isolated from a gold mine, was able to utilize different nitriles and the corresponding amides as sole source of nitrogen, at concentrations up to 2 M. Resting cells cultivated on YCB-acetonitrile medium showed nitrile hydrolyzing enzyme activities against acrylonitrile and benzonitrile. These enzymes were inducible and intracellular; the optimum pH was 7.0-8.0, and the optimum temperature 25 degrees C-30 degrees C. Liquid chromatographic analysis indicated that C. guilliermondii UFMG-Y65 metabolized 12 mM benzonitrile to 11 mM benzoic acid and 10 mM acrylonitrile to 7.9 mM acrylic acid. The results suggest that C. guilliermondii UFMG-Y65 may be useful for the bioproduction of amides and acids, and for the bioremediation of environments contaminated with nitriles.     

Effects of rifampicin and doxycycline on the production of hydrogen peroxide by macrophages]

The influence of rifampicin and doxycycline on oxidative metabolism of macrophages was estimated in vitro by production of hydrogen peroxide. It was shown that low concentrations of rifampicin and doxycycline stimulated production of hydrogen peroxide by macrophages of guinea pigs. In concentrations of 1 to 10 micrograms/ml corresponding to the mean therapeutic ones doxycycline increased both the spontaneous and zymosan-induced production of hydrogen peroxide by the macrophages. The potentiating activity of doxycycline on the cells activated by opsonized zymosan was higher. The maximum increase in the induced production of hydrogen peroxide (by 40 per cent) was observed when the antibiotic concentration was 1 microgram/ml. Rifampicin in concentrations of 0.1 to 1 microgram/ml corresponding to the mean therapeutic ones stimulated the zymosan-induced production of hydrogen peroxide by the macrophages. The maximum increase in the production of hydrogen peroxide (by 22 per cent) was noted at the rifampicin concentration of 1 microgram/ml.     

Virus-inhibiting properties of the carbonyl-conjugated pentaene roseofungin

The antiinfluenza activity of roseofungin, a polyenic macrolide antibiotic was studied in vitro on surviving fragments of the chick embryo chorionallantoic membranes and in ovo on growing chick embryos. It was shown that the antibiotic activity against influenza A and B viruses was sufficiently high. The activity of roseofungin against influenza A virus did not differ from that of remantadin, the most active inhibitor of influenza virus reproduction. However, the activity of roseofungin against influenza B virus was an advantage of this antibiotic over remantadin, which had practically no effect on this virus type. A statistically significant protective effect of roseofungin (p less than 0.05) was shown on the animals with experimental influenza. The study on the antiviral activity of roseofungin against the DNA-containing variolovaccine virus revealed that it markedly inhibited the plague reduction. Roseofungin had a pronounced inhibitory effect on cell neoplastic transformation induced by the RNA-containing oncogenic virus of Rous sarcoma.     

Enhanced antibiotic production by Streptomyces sindenensis using artificial neural networks coupled with genetic algorithm and Nelder-Mead downhill simplex

Antibiotic production with Streptomyces sindenensis MTCC 8122 was optimized under submerged fermentation conditions by artificial neural network (ANN) coupled with genetic algorithm (GA) and Nelder-Mead downhill simplex (NMDS). Feed forward back-propagation ANN was trained to establish the mathematical relationship among the medium components and length of incubation period for achieving maximum antibiotic yield. The optimization strategy involved growing the culture with varying concentrations of various medium components for different incubation periods. Under non-optimized condition, antibiotic production was found to be 95 microgram/ml, which nearly doubled (176 microgram/ml) with the ANN-GA optimization. ANN-NMDS optimization was found to be more efficacious, and maximum antibiotic production (197 microgram/ml) was obtained by cultivating the cells with (g/l) fructose 2.7602, MgSO4 1.2369, (NH4)2PO4 0.2742, DL-threonine 3.069%, and soyabean meal 1.952%, for 9.8531 days of incubation, which was roughly 12% higher than the yield obtained by ANN coupled with GA under the same conditions.     

Mode of Action of Lactococcin B, a Thiol-Activated Bacteriocin from Lactococcus lactis

Lactococcin B (LcnB) is a small, hydrophobic, positively charged bacteriocin produced by Lactococcus lactis subsp. cremoris 9B4. Purified LcnB has a bactericidal effect on sensitive L. lactis cells by dissipating the proton motive force and causing leakage of intracellular substrates. The activity of LcnB depends on the reduced state of the Cys-24 residue. Uptake and efflux studies of different solutes suggest that LcnB forms pores in the cytoplasmic membrane of sensitive L. lactis cells in the absence of a proton motive force. At low concentrations of LcnB, efflux of those ions and amino acids which are taken up by proton motive force-driven systems was observed. However, a 150-fold higher LcnB concentration was required for efflux of glutamate, previously taken up via a unidirectional ATP-driven transport system. Strains carrying the genetic information for the immunity protein against LcnB were not affected by LcnB. The proton motive force of immune cells was not dissipated, and no leakage of intracellular substrates could be detected.     

In vitro antibacterial activity and antifungal mode of action of flocculosin, a membrane-active cellobiose lipid

Aims:  To investigate the in vitro antibacterial activity and antifungal mode of action of flocculosin, a cellobiose lipid produced by Pseudozyma flocculosa .
Methods and Results:  When tested against clinical bacterial isolates, the compound was particularly active against Gram-positive bacteria and its effect was not mitigated against isolates known as resistant to other antibiotics. The antifungal activity of flocculosin was found to be rapid and concentration-dependent. At lethal concentrations against Candida albicans , flocculosin caused a rapid leakage of intracellular potassium and inhibited acidification of the medium by plasma membrane ATPases suggesting a physical rather than a biochemical effect. TEM observations of cells exposed 6 h to flocculosin revealed disrupted membranes and disorganized mitochondria.
Conclusions:  Data obtained in this study confirm that flocculosin acts by disrupting the membrane surface of sensitive micro-organisms.
Significance and Impact of the Study:  The elucidation of an antifungal mode of action of flocculosin can be exploited in furthering its antimicrobial potential against fungi and bacteria whose cell membranes are particularly sensitive to the action of the molecule.     

Differential Effects of Monovalent and Divalent Ions upon the Mode of Action of the Polyene Antibiotic Candicidin

The polyene antibiotic candicidin is a potent membrane active agent, the action of which can be inhibited by the presence of certain ions. The destruction of the selective permeability of yeast membranes by candicidin allows small molecules to leak into the environment. Loss of intracellular potassium ions inhibits yeast glycolysis. This inhibition may be reversed by extracellular concentrations of potassium or ammonium ions. Monovalent ions did not prevent antibiotic absorption or protect yeast growth from the action of the antibiotic. Divalent ions did not protect yeast glycolysis from the action of candicidin, but were able to reduce antibiotic-induced membrane damage and allowed yeast growth in the presence of antibiotic. It is suggested that divalent ions may interact with membrane sterols creating steric hindrance to subsequent candicidin absorption.     

Effects of cloacin DF13 on the functioning of the cytoplasmic membrane

Adsorption of cloacin DF13 onto sensitive cells induces a leakage of potassium ions after a lag of 15–20 min. The rate of the potassium efflux is proportional to the bacteriocin concentration used. The extent of the potassium loss depends on the concentrations of bacteriocin and of extracellular potassium. Leakage of potassium ions is accompanied by an uptake of sodium ions. Cloacin DF13 does not affect the transport of magnesium ions, phosphate ions or amino acids. About 10 min after addition of the bacteriocin the ATP content of the cells begins to decrease gradually to about 25% of the original level. Anaerobiosis, lack of energy source and inhibition of oxidative phosphorylation may protect the cells if applied within 10 min after addition of the bacteriocin. The results suggest that the action of cloacin DF13 proceeds through at least two distinct phases. Phase I is a period after bacteriocin adsorption which is reversible and in some way depends on oxidative phosphorylation. This phase does not cause any cellular damage. Phase II is irreversible and results in a disturbance of several cellular functions.     

Changes in the trophic organization of mitotic cycle of Candida utilis after exposure to RNAse from 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.     

Sodium and potassium content and membrane transport properties in red blood cells from newborn puppies

The intracellular sodium and potassium concentrations and membrane transport properties for these ions were investigated in red blood cells from newborn puppies and adult dogs. At birth the intracellular concentrations of sodium and potassium are much higher than those found in adult dog red cells. During the first few weeks of life the intracellular concentrations of these ions gradually decrease until the adult level is reached. Changes in the membrane transport properties develop concurrently. The rate of active potassium influx, as measured by ouabain-sensitivity, and the pump to leak ratio are greater in red cells from newborn puppies than in those from adult animals. No ouabain-sensitive sodium efflux could be demonstrated in red cells from older puppies or adult dogs. When either puppy or adult dog red cells are depleted of ATP (by incubation at 37°C with no substrate), potassium permeability increases, and the permeability of the membrane to sodium decreases. The addition of adenosine reverses the effect of depletion.     

Ultrastructural study of concanavalin-A binding to the surface of preimplantation mouse embryos

Receptors for Con-A were labelled (using the peroxidase-diaminobenzidine technique) on the plasma membrane of unfertilized and fertilized mouse eggs, cleavage stage embryos, trophoblast and inner cell mass (ICM) of the blastocyst. Embryos were exposed to Con-A concentrations of 10 microgram/ml, 50 microgram/ml, or 1,000 microgram/ml and the lowest concentration was observed to be the most suitable for discerning differences between stages of embryonic development. On the surface of unfertilized and fertilized eggs and 2-cell embryos, reaction product appeared as a thin, discontinuous layer. The surface of 4- and 16-cell stage embryos had a thicker, continuous, although non-uniform, layer of the reaction product. On the surface of the cells of the late morula, and on the trophoblastic cells of the blastocyst, clustering of reaction product was observed. Cells of ICM of intact blastocyst were free of the reaction product, showing that either Con-A and/or peroxidase cannot penetrate tight junctions between trophoblastic cells. Reaction product in the form of a thin, uniform layer covered the free surface of the cells of the ICM after they had been isolated (using immunosurgery) and exposed to 50 microgram/ml of Con-A. The amount and distribution of Con-A receptors is discussed, along with their redistribution and mobility in relation to the agglutinability of preimplantation mouse embryos.     

Role of gramicidin S in the sporulation of the R+ and R- variants of Bacillus brevis var. G.-B.]

The effect of gramicidin S added to the cultivation medium on sporulation of the gramicidin S-producing P+ variant and gramicidin S-nonproducing P- variant of Bacillus brevis var. G.-B. was studied. Gramicidin S added to the synthetic medium with glucose in an amount of 30 and 100 microgram/ml 4 and 7 hours after inoculation with the vegetative cells of R- variant had no effect on the growth of the culture but retarded its sporulation. When gramicidin S was added in an amount of 100 microgram/ml 4 hours after inoculation, the sporulation rate of R- variant strongly decreased, rohile sporulation was not suppressed as it was noted before with respect to R+ variant. Active stimulation of Bacillus brevis var. G.-B. sporulation was observed after addition of gramicidin S 13 hours after development of R+ and R- variants without the antibiotic biosynthesis. Synthesis of gramicidin S by R+ strain was suppressed by the specific inhibitor beta-phenyl-beta-alanine. The amount of gramicidin S added to the medium during the sporulation process of R+ and R- variants decreased. On addition of 30 microgram/ml of the antibiotic it was practically not detectable when the culture showed the greatest number of the spores. Therefore, gramicidin S added to the medium is probably adsorbed by the cells of Bac. brevis var. G.-B. and affects sporulation of R- and R+ variants thus accelerating or retarding this process depending on the cultivation conditions.     

Uptake and nature of the intracellular binding of cyclosporin A in a murine thymoma cell line, BW5147

In a survey of malignant cell lines including a variety of leukemias and lymphomas, BW5147, a T lymphoma from the spontaneous virus-associated thymoma in AKR mice, was found to be the most sensitive to growth inhibition by cyclosporin A (Cs A). Inhibition of growth was cell cycle phase-independent and inhibition of macromolecular precursor uptake was relatively nonspecific. Uptake of radiolabeled Cs A by these cells was characterized by two components: one that appeared saturable at low drug concentrations (0.03 to 1.0 microgram/ml), and another that was nonsaturable at higher drug concentrations (1.0 microgram/ml or higher). Most of the drug concentrated by cells (70 to 80%) was located in the cytosol (100,000 X G supernatant of lysed cells). The apparent m.w. of the drug-macromolecule complex was 15,000 to 20,000 as determined by m.w. exclusion columns. This complex could also be formed by adding drug to cytosol prepared from unexposed cells. The low m.w. complex migrated on a preparative isoelectric focusing column to form two peaks with isoelectric points of 6.8 and 8.5. A method was developed to assay for the binding component, and a sequence of m.w. exclusion columns and isoelectric focusing was used to effect partial purification of the Cs A binding component.     

Adsorption of 14C-labeled gramicidin S on cell of bacteria

Gramicidin S (GS) containing 14C-labeled proline was synthesized by a solid-phase method, and the labeled GS dihydrochloride was obtained as crystals. The labeled GS exhibited same antibacterial activity as natural GS. Strains sensitive to GS (B. subtilis and S. aureus) and an insensitive strain (E. coli) were treated with the labeled GS, and the amount of the labeled GS adsorbed on the cells was measured. GS was adsorbed rapidly on the cells of the sensitive strains; the amount adsorbed increased linearly with GS concentration up to 1-1.5 microgram/ml, and at a lower rate at above 1.5 microgram/ml. GS molecules covered most of the cell surface at the minimum inhibitory concentration of 1.5 microgram/ml; the number of molecules adsorbed per cell was 1.3-1.4 x 10(6). No GS was adsorbed by the insensitive strain.     

Osmoregulation in the Extremely Euryhaline Marine Micro-Alga Chlorella autotrophica

Chlorella autotrophica (Clone 580) grows over the external salinity range of 1 to 400% artificial sea water (ASW), can photosynthesize over the range from 1 to 600% ASW, and survives the complete evaporation of seawater. The alga grown at high salinities shows an increase in cell volume and a small decrease in cell water content. Measurements of ion content were made by neutron activation analysis on cells washed in isoosmotic sorbitol solutions which contained a few millimolar of major ions to prevent ion leakage. Cells grown at various ASW concentrations contain large quantities of sodium, potassium, and chloride ions. Measurements of cations associated with cell wall and intracellular macromolecules were made to determine intracellular concentration of free ions. The proline content of cells increases in response to increases in external salinity. Cells in 300% ASW contain 1500 to 1600 millimolar proline.     

Bactericidal activity of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv. glycines, on phytopathogenic Xanthomonas campestris pv. vesicatoria cells

The ability of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv. glycines 8ra, to kill closely related bacteria has been demonstrated previously by our group. In the present study, we aimed at determining the glycinecin A-induced cause of death. Treatment with glycinecin A caused slow dissipation of membrane potential and rapid depletion of the pH gradient. Glycinecin A treatment also induced leakage of potassium ions from X. campestris pv. vesicatoria YK93-4 cells and killed sensitive bacterial cells in a dose-dependent manner. Sensitive cells were killed within 2 h of incubation, most likely due to the potassium ion efflux caused by glycinecin A. These results suggest that the bactericidal mechanism of action of glycinecin A is correlated with the permeability of membranes to hydroxyl and potassium ions, leading to the lethal activity of the bacteriocin on the target bacteria.