Effect of gamma-irradiation on some biological and physico-chemical properties of polyene antibiotics]

Deep effect of gamma-rays on polyenic antibiotics was studied. It was shown that gamma-radiation induced radiation-chemical oxidation of the substances. The chromatographic analysis showed that the levorin degradation products were identical to the polyenic products of the antibiotic oxidative destruction. As for mycoheptin and amphotericin B, destruction of their molecules to non-polyenic products was observed. It was found that toxicity of the levorin aromatic heptaen did not practically change after gamma-irradiation in high doses. The toxicity of mycoheptin and amphotericin B, non-aromatic heptaens increased after exposure to high doses of gamma-rays.     

Cation conductance and efflux induced by polyene antibiotics in the membrane of skeletal muscle fiber.

Cation conductance and efflux induced by polyene antibiotics amphotericin B (AMB), amphotericin B methyl ester (AME), nystatin, mycoheptin, and levorin on frog isolated skeletal muscle fibers and whole sartorius muscles were investigated. Conductance was measured under current-clamp conditions using a double sucrose-gap technique. Cation efflux was studied using flame emission photometry. Some new data were obtained concerning the effects of levorin and mycoheptin on biological membranes. The power dependence of polyene-induced cation transport on antibiotic concentration in muscle membrane was lower than that in bilayers. The decline in the equilibrium conductance caused by polyene removal (except for levorin) was very fast. There was reverse temperature dependence of AMB- and nystatin-induced conductances. Both induced conductance and efflux values demonstrated a correlation with the order of antifungal activities: levorin > AMB, mycoheptin > AME > nystatin, except for AME, which was more potent on yeastlike cells. These effects were interpreted in terms of possible differences in the kinetics of channel formation in biological and model membranes and in light of the role of nonconducting antibiotic forms in biological membranes.     

Lethal and mutagenic action of N-nitroso-N-methylbiuret on the producers of levorin, amphotericin and mycoheptin]

The lethal and mutagenic effect of N-nitrozo-N-methylbiuret (NMB) on the organisms producing levorin, amphotericin B and mycoheptin was studied. The mutagen effect depended on the dose, culture and physiological state of the spores. NMB had a low mutagenic effect on the levorin-producing organism characterized by high activity and genetic homogenicity with respect to the colony morphology and antibiotic production. As for the organisms producing amphotericin B and mycoheptin characterized by high genetic heterogenicity, significant variation of all the features studied was observed on their exposure to the mutagen. Inspite of diverse reaction of the organisms producing levorin, amphotericin B and mycoheptin to the effect of NMB mutants with increased antibiotic production were obtained from the three cultures. The lethal and mutagenic effect of NMB on the mycoheptin-producing organism depended on the process of the spore DNA replication. The spores during the DNA replication period were least sensitive to the lethal effect of the mutagen and most mutable with the respect to the colony morphology. For selection of highly active and stable strains exposure to NMB of the spores of the mycoheptin-producing organism during replication of DNA proved to be more effective than that of the spores during the lag-phase.     

Study of components of standard samples of polyene macrolide antibiotics by the methods of high performance liquid chromatography and SIEAP mass spectrometry]

The component composition of reference samples of polyenic macrolide antibiotics such as nystatin, mycoheptin, amphotericin B and levorin was studied by HPLC and chromatographic mass spectrometry in comparison to the WHO standards. It was shown that the samples were close by their component composition to the analogous samples of the WHO standards.     

Drug sensitivity of Candida in patients with kidney diseases receiving immunosuppressive therapy

It was shown that prednisolone and its combination with azathioprine++ increased contamination of the patients with yeast-like fungi and promoted development of candidiasis in them to a greater extent than cyclophosphamide. In the patients treated with the immunodepressants there was observed a carrier state in regard to various yeast-like fungi: 12 species belonging to 6 genera were isolated from the pathological materials. Determination of sensitivity to antifungal drugs in 200 Candida strains revealed that amphotericin B was the most active agent. Then followed mycoheptin, nystatin and nitroxolin. Levorin was the least active drug. The MICs of the drugs for the majority of the cultures were 0.5, 4-8, 8-16 and 32-64 micrograms/ml respectively. Candida resistant strains (mainly to levorin and mycoheptin) were isolated only from recipients of kidney transplants during the early postoperative period when the patients were subjected to intensive immunodepressive and prophylactic antifungal therapy. Among the fungi of the Candida genus C. guillermondii and C. parapsilosis proved to be the most resistant. Under the hospital conditions and in vitro studies it was found that cyclophosphamide and combinations of prednisolone with cytostatics increased resistance of Candida to the antifungal drugs. Rapid increasing of the fungi resistance to levorin and mycoheptin was observed. The increase in the resistance to amphotericin B was somewhat lower and that to nitroxolin and nystatin was extremely low. The study of the combined effect of the immunodepressants and antifungal drugs demonstrated that the immunodepressants increased the antifungal activity of amphotericin B, levorin and nitroxolin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative study of fungicidal and herbicidal activities of salts of polyenic macrolide antibiotics]

The fungicidal and herbicidal activities of water soluble salts of levorin, nystatin, mycoheptin and amphotericin B, polyenic macrolide antibiotics, were studied. The biological tests showed that the antibiotics had fungicidal and low herbicidal activities. They also had a growth regulating action.     

Nystatin-, mycoheptin- and levorin-induced conductance in the membrane of frog skeletal muscle fibres

The effects of the polyene antibiotics nystatin (2 × 10^–5–10^–4 mol/l), mycoheptin (1.3 × 10^–6–10^–5 mol/l) and levorin (10^–8–5 × 10^–5 mol/l)on isolated frog skeletal muscle fibres and whole sartorius muscles of the frog have been investigated. Cation conductance was measured under current clamp conditions using a double sucrosegap technique. Cation effluxes were studied by means of flame emission photometry. All three antibiotics increased the cation conductance and efflux rates; however, differences between the polyenes were found in the steady state values of induced cation transport at a given concentration. The values of both induced conductance g_A and efflux rate constants K_A formed the following sequence: levorin > mycoheptin > nystatin, demonstrating a correlation with the order of antifungal activities. The dose-response curves of lg polyene-induced cation transport against lg of antibiotic concentration in our experiments had slope values which were much lower than those in bilayers: 1.7 and 1.3 for nystatin and mycoheptin, respectively, whereas the aromatic heptaene levorin had an even smaller concentration dependence. The decline in the equilibrium conductance caused by nystatin- and mycoheptin removal was very fast (during the first minute = 0.74 and 2.39 min, respectively). In contrast, levorin-induced conductance was irreversible. It is proposed that the processes which limit the rate of channel formation are different in biological and model membranes. Correspondence to: N. E. Shvinka     

Possible use of a single test culture in determining the biological activity of polyene antibiotics

A possibility using a common test-culture in estimation of biological activity of levorin, amphotericin B, mycoheptin and nistatin was studied. It was found that C. guillier mondii, strain 40, may be used as a common test-culture. It provided satisfactory microbial growth, clear and rather large inhibition growth zones with respect to all the drugs tested.     

Ionization of the acid-base groups of polyene antibiotics in aqueous solutions]

The acid-base characteristics of polyenic antibiotics, such as nystatin, mycoheptin and levorin in aqueous solutions were studied. A special procedure provided the use of potentiometric titration for investigation of ionization of the groups of vater-insoluble substances. The ionization constants of the carboxylic and amine groups of the antibiotics at several temperatures were determined. It was found that ionization of the acid group did not practically depend on the temperature. At the same time the heat effect of the amine group ionization was significant and amounted to about 10 kcal/mole. Thermodynamic analysis of the ionization process of the polyenic antibiotics in aqueous solutions was performed. Integral components defining the process energetics were calculated.     

Stabilization of standard samples of levorin and nystatin with antioxidants]

To prolong the storage period of the reference samples of levorin and nystatin, polyenic macrolide antibiotics, their effect of 23 antioxidants was studied by using rapid inactivation. The antioxidants belonged to compounds of different classes. The inactivation was performed at 20, 37 and 50 degrees C in the presence of 1, 2 or 5 per cent of the antioxidants. An antioxidant of the class of partially hydrated oxyquinolines was shown to have the highest stabilizing action on levorin and nystatin. It may be recommended for stabilizing the levorin and nystatin reference samples.     

Action of polyene antibiotics on actinomycetes with a varying lipid makeup]

The effect of polyenic antibiotics, such as nystatin, levorin, amphotericin B and mycoheptin on 93 representatives of various species of the ray fungi was studied. It was shown that resistance of the actinomycetes to the polyens was connected with the absence or insufficient content of sterols (0.001--0.008 per cent in the dry mycelium). On addition of cholesterol to the nutrient media (100 microgram/ml) it was included into the membranes of some cultures and their sensitivity increased 2--60 times. Resistance of Actinomyces sp. LIA 0775 grown on the media with fats differing in their composition decreased 2--4 times. In these cases the culture lipids were characterized by lower content of phospholipids (35--45 per cent from the total lipids as compared to 70--80 per cent when grown on the control medium without fats) and significantly increased content of unsaturated fatty acids (3--4 times).     

Optical rotary dispersion of some heptaene antibiotics]

Experimental differences in the curves of the optic rotation dispersion (ORD) of cystrans-heptaenic antibiotics were found. The ORD curves of amphotericin B, mycoheptin, levorin components and isolevorin A2, components of criptomycin and candidin were registered. The curves of the ORD which were smooth had been prepared in dimethylsulphoxide in the spectral range at 450 to 600 nm. In the spectral range at 300 to 420 nm the ORD curves appeared to be anomal with a complex Kotton effect, they were prepared in methyl alcohol. The Kotton effect was probably due to asymmetry of the electron membranes of polyenic chromophore induced by the other part of the polyen molecule. This was evident from the fact that the curve of the Kotton effect was situated in the same spectral range as the absorption bond of the polyenic chromophore. The oscillating structure in the absorption spectrum and the curve of the complex Kotton effect were analogous.     

Dependence of Ion Channel Properties Formed by Polyene Antibiotics Molecules on the Lactone Ring Structure

The properties of ion channels formed in membranes by polyene antibiotics of various chemical structure of hydrophilic and hydrophobic chains are investigated. Small differences in a hydrophylic chain with a changed number of hydroxyl and carbonyl groups significantly influence the values of conductivity and selectivity of the polyene channel. The greater number of double bonds in a hydrophobic part of polyene molecules leads to the higher biological activity of antibiotics. Measurement of anion–cationic selectivity of the channels formed by polyenes showed that anionic selectivity, as well as conductivity of channels, decreases among antibiotics: amphotericin B, nystatin, candidin, mycoheptin, and levorin. The study of physical and chemical properties of the single and hybrid ion channels on the bilayer lipid membranes in the presence of polyene antibiotics makes possible to create a theoretically reasonable recommendation for the targeted synthesis of new antibiotics with the desired properties.     

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and protein peroxides generated by singlet oxygen attack.

Reaction of certain peptides and proteins with singlet oxygen (generated by visible light in the presence of rose bengal dye) yields long-lived peptide and protein peroxides. Incubation of these peroxides with glyceraldehyde-3-phosphate dehydrogenase, in the absence of added metal ions, results in loss of enzymatic activity. Comparative studies with a range of peroxides have shown that this inhibition is concentration, peroxide, and time dependent, with H2O2 less efficient than some peptide peroxides. Enzyme inhibition correlates with loss of both the peroxide and enzyme thiol residues, with a stoichiometry of two thiols lost per peroxide consumed. Blocking the thiol residues prevents reaction with the peroxide. This stoichiometry, the lack of metal-ion dependence, and the absence of electron paramagnetic resonance (EPR)-detectable species, is consistent with a molecular (nonradical) reaction between the active-site thiol of the enzyme and the peroxide. A number of low-molecular-mass compounds including thiols and ascorbate, but not Trolox C, can prevent inhibition by removing the initial peroxide, or species derived from it. In contrast, glutathione reductase and lactate dehydrogenase are poorly inhibited by these peroxides in the absence of added Fe2+-EDTA. The presence of this metal-ion complex enhanced the inhibition observed with these enzymes consistent with the occurrence of radical-mediated reactions. Overall, these studies demonstrate that singlet oxygen-mediated damage to an initial target protein can result in selective subsequent damage to other proteins, as evidenced by loss of enzymatic activity, via the formation and subsequent reactions of protein peroxides. These reactions may be important in the development of cellular dysfunction as a result of photo-oxidation.     

Identification of a novel class of endoperoxides from arachidonate autoxidation

Free radical-initiated lipid autoxidation in low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. Oxidation of the lipid components of LDL leads to a complex mixture of hydroperoxides, bicyclic endoperoxides, monocyclic peroxides, and serial cyclic peroxides. The oxidation compounds and/or their decomposition products can modify protein components, which may lead to various diseases. A novel class of peroxides (termed dioxolane-isoprostanes) having a bicyclic endoperoxide moiety characteristic of the isoprostanes and a dioxolane peroxide functionality in the same molecule was identified in the product mixture formed from in vitro autoxidation of cholesteryl arachidonate. The same products are also detected in in vitro oxidized LDL. Various mass spectrometric techniques have been applied to characterize these new peroxides. The structure of these compounds has also been confirmed by independent synthesis. We reason, based on the free radical mechanism of the transformation, that only the 12- and 8-peroxyl radicals (those leading to 12-HPETE and 8-HPETE) of arachidonate can form these new peroxides. We also suggest that the formation of these peroxides provides a rationale to explain the fact that 5- and 15-series isoprostanes are formed in preference to 8- and 12-series. Furthermore, series of other isoprostanes, such as dioxolane A(2), D(2), E(2), etc., can be derived from the dioxolane-isoprostane peroxides. These findings offer further insights into the oxidation products of arachidonate and the opportunity to study their potential biological relevance.     

Dependence of inhibition by levorin of amino acid incorporation into protoplasts and subcellular proteins of Candida albicans on the change of endocellular potassium concentration]

Levorin is found to decrease more efficiently potassium concentration in C. albicans protoplasts under their incubation in the presence of sodium than in the medium containing the equivalent amount of potassium. Minimal inhibitory concentration of levorin for resistant C. albicans cells incubated on potassium-depeleted medium was in 4 times lower than for cells incubated in potassium-enriched medium. The decrease of membrane permeability for 14C-amino acids and their incorporation into membrane, ribosomal and soluble proteins under the effect of levorin was more pronounced when protoplasts were cultivated in sodium-containing medium than in potassium-containing one. In both media the inhibition of 14C-amino acid incorporation by levorin into ribosomal and cytosol proteins was more efficient than into membrane proteins, but these differences were less pronounced in case of potassium-containing medium.     

Effect of the products of the vital activity of yeast-like fungi on levorin synthesis

The effect on levorin synthesis of the cells and fermentation broth filtrates of Candida tropicalis after their cultivation in the fermentation medium was studied. It was found that the yeast-like fungi belonging to Candida excreted during their development some products capable of stimulating the synthesis of levorin by 40--60 per cent. When the actinomycete producing levorin was grown on the medium containing 80 per cent of the filtrate the level of levorin synthesis was the same as that observed with mixed cultivation of the actinomycete and C. tropicalis. The study on the conditions providing accumulation of the stimulating substances showed the following: production of the stimulating substances started during the first hours of the yeast growth and reached its maximum by the 48th hour, these substances being consumed by the actinomycete during the fermentation process. Aeration is required for production of the stimulating substances but its high levels are not necessary.     

Chemical and biochemical aspects of superoxide radicals and related species of activated oxygen

The spectrum of biological processes in which oxygen is used by living systems is quite large, and the products include some damaging species of activated oxygen, particularly the superoxide radical (O-.2) and hydrogen peroxide (H2O2). Superoxide radicals and hydrogen peroxide, in turn, can lead to the formation of other damaging species: hydroxyl radicals (.OH) and singlet oxygen (1O2). Hydroxyl radicals react with organic compounds to give secondary free radicals that, in the presence of oxygen, yield peroxy radicals, peroxides, and hydroperoxides. Formation, interconversion, and reactivity of O-.2 and related activated oxygen species, methods available for their detection, and the basis of their biological toxicity are briefly reviewed.     

Use of paper discs in determining the biological activity of mycoheptin by the agar diffusion method]

Diffusion of mycoheptin from the discs into agar was studied. A procedure providing practical use of the discs for quantitative estimation of mycoheptin activity was developed. A possibility of using the discs in the assay of solutions containing significant amounts of an organic solvent was shown.     

The effectiveness of a lipid peroxide in oxidizing protein and non-protein thiols

1. Thiol oxidation by a lipid peroxide or hydrogen peroxide was as efficient in denatured non-haem proteins as in small thiols. Both peroxides were relatively ineffective in oxidizing haemoprotein thiols, especially at low pH. Increased amounts of haematin decreased greatly the efficiency of GSH oxidation by peroxides especially at low pH. 2. Other than the haematin ring, the thiol group was found to be probably the group in proteins most sensitive to modification by peroxides. 3. At low concentrations, the fatty acid moiety of a lipid peroxide appeared to impede thiol oxidation in proteins, probably by hydrophobic bonding to the protein, rather than to stimulate thiol oxidation by denaturing the protein and thereby increasing the exposure and reactivity of the thiol group. 4. The relative rates of thiol oxidation by peroxides in the different thiols were: haemoprotein thiols>small thiols>other protein thiols. In all cases, thiol oxidation was much more rapid by the lipid peroxide than by hydrogen peroxide.