Supersensitivity to levorin and amphotericin B in several Saccharomyces cerevisiae mutants resistant to nystatin]

104 mutants resistant to nystatin were isolated after UV-treatment of two haploid marked strains of Saccharomyces cerevisiae. The analysis of resistance to three polyene antibiotics allowed to determine 8 phenotype classes of mutants including those resistant to nystatin but in various combinations showing hypersensitivity to levorin and (or) amphotericin B. The analysis of UV absorption spectra of sterolic extracts prepared from cells of different mutants showed that similar quality changes in sterol composition could be associated both with polyresistant an supersensitive phenotype. New type of mutants resistant to nystatin and supersensitive to levorin and (or) amphotericin B seems to be promising for studies on the mechanisms of action of polyene antibiotics, the bases of resistance to them and also in consideration of the possibility to increase the efficiency of antimycotic antibiotic therapy.     

Toxicity of nystatin and its methyl ester toward parental and hybrid mammalian cells

Nystatin methyl ester (NME), the methyl ester derivative of the polyene macrolide antibiotic nystatin, is known to be effective against fungi and is now found to be relatively less toxic than the parent antibiotic nystatin (NYS) to animal cells in culture as measured by 51Cr release, cell survival at different posttreatment periods and cell growth. NYS and NME were tested on TK- mouse (B82) and hamster (B1) cells, HGPRT- mouse (RAG) cells, and on lysolecithin-fused cells selected in HAT medium and confirmed as B82-RAG and B1-RAG hybrids by chromosomal analysis plus polyacrylamide gel electrophoresis of lactate dehydrogenase. NME was less toxic and caused less immediate membrane damage than NYS when tested in all five cell systems. However, differences in innate polyene sensitivity were evident between the three parental cell types. B82 and B1 cells were more resistant than RAG cells to NYS and NME. B82-RAG hybrids reflected the higher level resistance of B82 parental cells, and B1-RAG hybrids reflected the higher level resistance of B1 cells. Where one parental cell type is relatively more polyene sensitive, the use of polyenes in the future may be applicable as selective agents in cell hybridization.     

Toxicity of nystatin and its methyl ester toward parental and hybrid mammalian cells

Summary Nystatin methyl ester (NME), the methyl ester derivative of the polyene macrolide antibiotic nystatin, is known to be effective against fungi and is now found to be relatively less toxic than the parent antibiotic nystatin (NYS) to animal cells in culture as measured by⁵¹Cr release, cell survival at different posttreatment periods and cell growth. NYS and NME were tested on TK⁻ mouse (B82) and hamster (B1) cells, HGPRT⁻ mouse (RAG) cells, and on lysolecithin-fused cells selected in HAT medium and confirmed as B82-RAG an B1-RAG hybrids by chromosomal analysis plus polyacrylamide gel electrophoresis of lactate dehydrogenase. NME was less toxic and caused less immediate membrane damage than NYS when tested in all five cell systems. However, differences in innate polyene sensitivity were evident between the three parental cell types. B82 and B1 cells were more resistant than RAG cells to NYS and NME. B82-RAG hybrids reflected the higher level resistance of B82 parental cells, and B1-RAG hybrids reflected the higher level resistance of B1 cells. Where one parental cell type is relatively more polyene sensitive, the use of polyenes in the future may be applicable as selective agents in cell hybridization. This investigation was supported by NIH Training Grant No. GM 507 from the National Institute of General Medical Sciences.     

Nystatin effects on vacuolar function in Saccharomyces cerevisiae.

The effects of nystatin, a polyene antibiotic, was studied in Saccharomyces cerevisiae by isolating and characterizing nystatin-sensitive mutants. We isolated a number of nystatin-sensitive mutants by ethylmethane sulfonate mutagenesis. One of these mutants, the nss1 mutant, was characterized in detail. The mutant was sensitive to stresses such as high temperature or high concentrations of monovalent and divalent cations. The nss1 mutants showed severe vacuolar protein sorting and vacuolar morphology defects. The nss1 mutant was demonstrated to have a mutational lesion in the known VPS16 gene, which is essential for vacuolar protein sorting in S. cerevisiae. All of the vacuolar deficient mutants (vps11, vps16, vps18, and vps33) were sensitive to nystatin. Nystatin was found to cause extensive enlargement of the vacuole in wild-type S. cerevisiae cells. These results are discussed with special reference to the vacuolar function of S. cerevisiae.     

How do the polyene macrolide antibiotics affect the cellular membrane properties?

In the 1970's great strides were made in understanding the mechanism of action of amphotericin B and nystatin: the formation of transmembrane pores was clearly demonstrated in planar lipid monolayers, in multilamellar phospholipid vesicles and in Acholeplasma laidlawii cells and the importance of the presence and of the nature of the membrane sterol was analyzed. For polyene antibiotics with shorter chains, a mechanism of membrane disruption was proposed. However, recently obtained data on unilamellar vesicles have complicated the situation. It has been shown that: membranes in the gel state (which is not common in cells), even if they do not contain sterols may be made permeable by polyene antibiotics, several mechanisms may operate, simultaneously or sequentially, depending on the antibiotic/lipid ratio, the time elapsed after mixing and the mode of addition of the antibiotic, there is a rapid exchange of the antibiotic molecules between the vesicles. Although pore formation is apparently involved in the toxicity of amphotericin B and nystatin, it is not the sole factor which contributes to cell death, since K+ leakage induced by these antibiotics is separate from their lethal action. The peroxidation of membrane lipids, which has been demonstrated for erythrocytes and Candida albicans cells in the presence of amphotericin B, may play a determining role in toxicity concurrently with colloid osmotic effect. On the other hand, it has been shown that the action of polyene antibiotics on cells is not always detrimental: at sub-lethal concentrations these drugs stimulate either the activity of some membrane enzymes or cellular metabolism. In particular, some cells of the immune system are stimulated. Furthermore, polyene antibiotics may act synergistically with other drugs, such as antitumor or antifungal compounds. This may occur either by an increased incorporation of the drug, under the influence of a polyene antibiotic-induced change of membrane potential, for example, or by a direct interaction of both drugs. That fungal membranes contain ergosterol while mammalian cell membranes contain cholesterol, has generally been considered the basis for the selective toxicity of amphotericin B and nystatin for fungi. Actually, in vitro studies have not always borne out this assumption, thereby casting doubt on the use of polyene antibiotics as antifungal agents in mammalian cell culture media.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane composition modulates the interaction between a new class of antineoplastic agents deriving from aromatic 2-chloroethylureas and lipid bilayers: a solid-state NMR study

This work presents the investigations of the interactions between nystatin, a polyene antibiotic, and phospholipids with various head groups (phosphatidylcholine and phosphatidylethanolamine) and acyl chains of different length and saturation degree. The experiments were performed with the Langmuir monolayer technique. Among phosphatidylethanolamines, DMPE, DPPE and DSPE were studied, while phosphatidylcholines were represented by DSPC and DOPC. The influence of the antibiotic on the molecular organization of the phospholipid monolayer was analysed with the compression modulus values, while the strength of nystatin/phospholipid interactions and the stability of the mixed monolayers were examined on the basis of the excess free energy of mixing values. The results obtained proved a high affinity of nystatin towards phospholipids. Nystatin was found to interact more strongly with phosphatidylcholines than with phosphatidylethanolamines. The most negative values of the excess free energy of mixing observed for the antibiotic and DOPC mixtures prove that nystatin favors the phospholipid with two unsaturated acyl chains. The results imply that nystatin/phospholipid interactions compete in the natural membrane with nystatin/sterol interactions, thereby affecting the antifungal activity of nystatin and its toxicity towards mammalian cells.     

Dissociation kinetics and equilibrium binding properties of polyene antibiotic complexes with phosphatidylcholine/sterol vesicles

The interactions of sonicated vesicles with the polyene antibiotics amphotericin B, candicidin, mediocidin , and a water-soluble, guanidine derivative of amphotericin B were examined by UV-visible spectroscopy at concentrations below which the polyenes become self-associated. The association constants, Kapp, and the numbers of binding sites per sterol or phospholipid molecule (n) were determined at 30 degrees C and pH 7.4. A single class of binding sites was found, with no evidence of cooperativity. For the binding of mediocidin , amphotericin B, and the guanidine derivative with phosphatidylcholine (PC), PC/cholesterol, and PC/ergosterol vesicles, Kapp was in the range of (1.0-3.0) X 10(6) M-1; Kapp was higher for candicidin-vesicle interaction, reaching 9.0 X 10(6) M-1 with PC/ergosterol vesicles. Binding of the guanidine derivative of amphotericin B to PC vesicles lacking sterol was extensive (n = 0.46); since the other polyenes, which have low aqueous solubilities, had n less than 0.05, positive charges in the mycosamine moiety appear to enhance the extent of polyene antibiotic interaction with the glycerophospholipid head group. Higher values of n (and, therefore, of nKapp ) were found with sterol-containing than with sterol-free vesicles, suggestive of penetration of the polyenes toward the interior of the bilayer when sterol is present. For binding to PC/sterol vesicles, nKapp followed the order of candicidin greater than guanidine derivative of amphotericin B greater than amphotericin B much greater than mediocidin . The values of n and nKapp were appreciably higher for amphotericin B-ergosterol than for amphotericin B-cholesterol interaction in vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of docosahexaenoic and arachidonic acids on the synthesis and distribution of aminophospholipids during neuronal differentiation of PC12 cells

We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.     

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific d-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.     

Is cholesterol the receptor for polyene antibiotic-induced B-lymphocyte activation

Stimulation induced by polyene antibiotics has previously been considered to be mediated via binding to cell membrane cholesterol. However, stimulation by polyenes such as nystatin was not affected differently than other polyclonal B-cell activators by the addition of cholesteral-blocking drugs such as digitonin, tomatin, or pimaricin, nor did cholesterol-containing serum diminish its mitogenic properties. Incubation of cells with lecithin-cholesterol vesicles, thereby increasing total cell cholesterol, greatly reduces cell reactivity toward polyenes to the same extent as toward other B- or T-cell activators. We therefore, tentatively suggest that polyene-induced mitogenesis is not mediated via binding to cell membrane cholesterol but via interaction with a distinct mitogen receptor on the cell surface.     

Inhibitory effect of sodium ursodeoxycholate on basal and stimulated short-circuit current across the isolated toad skin

The effect of sodium ursodeoxycholate (U) on short-circuit current (SCC), an index of basal and stimulated net ion transport across isolated skins of Bufo arenarum toads, was tested. U inhibited basal SCC when added to the epidermal side of the skins. The inhibitory effect was reversible after rinsing the preparation during 60 min. U also inhibited the natriferic response to oxytocin, db-cAMP and theophylline by 82%, 49% and 47%, respectively. Inhibition of SCC by exposure to U was reversed by the polyene antibiotic nystatin. In turn, SCC induced by nystatin in the amiloride-treated skin was insensitive to U and blocked by ouabain, a Na⁺, K⁺-ATPase inhibitor. These results strongly suggest that the effect of U is exerted at the apical membrane of sodium transporting cells, and rule out the existence of an additional site of inhibitory action of U.     

Enrichment of auxotrophic mutants inHansenula polymorpha

Summary An enrichment scheme was designed for the isolation of auxotrophic mutants of the thermotolerant and methanol-utilizing yeast,Hansenula polymorpha, by the use of the polyene antibiotic nystatin. This treatment resulted in auxotrophic isolates at a frequency of 75%. With or without nystatin enrichment, the vast majority of obtained auxotrophs required nucleic acid bases.     

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.     

Interaction of amphotericin B and its N-fructosyl derivative with murine thymocytes: a comparative study using fluorescent membrane probes

The polyene antibiotics amphotericin B (AmB) and N-(1-deoxy-D-fructos-1-yl)amphotericin (N-Fru-AmB) have different activity towards murine thymocytes (N-Fru-AmB is less toxic but is a potent immunomodulator). The interactions of the drugs with these cells have been studied by fluorescence methods. Fluorescence energy transfer from 1-[4-(trimethylammonio) phenyl]-6-phenylhexa-1,3,5-triene, p-toluenesulfonate (TMA-DPH) to polyenes was used to follow the binding of the two drugs to the plasma membrane. The results, confirmed by circular dichroism measurements, indicate that at saturation the ratio AmB bound/plasma membrane lipid is low (less than 1 molecule of polyene for 170 lipids). The slightly higher binding of AmB as compared to N-Fru-AmB demonstrates that affinity of the antibiotic for plasma membrane does not account for the activity of the polyenes towards lymphocytes. The effect of the two polyenes on membrane fluidity was studied by steady-state fluorescence anisotropy. The results suggest that AmB strongly perturbs the structure of the membrane whereas only a slight decrease of the anisotropy is observed with N-Fru-AmB in the range of concentration where the biological activity has been demonstrated. Polyene location was further investigated by comparing the energy transfer efficiency obtained with TMA-DPH and with the parental compound 1,6-diphenylhexa-1,3,5-triene, p-toluene sulfonate (DPH). While AmB binds to plasma membrane, as well as to intracellular structures, N-Fru-AmB seems to accumulate into the cell and bind to intracellular membrane structures.     

Interaction of the polyene antibiotics with lipid bilayer vesicles containing cholesterol.

The interaction of the polyene antibiotics, amphotericin B, nystatin and filipin with cholesterol-containing single bilayer lipid vesicles has been characterized using gel permeation chromatography and proton magnetic resonance. All three antibiotics bind to vesicles at low concentrations without causing a large amount of vesicle destruction. The strength of binding as determined by gel permeation studies is greater for filipin and amphotericin than for nystatin. Nystatin and amphotericin B at these low concentrations induce a rapid loss of internal vesicle contents consistents consistent with pore formation. Filipin induces no leakage beyond that expected from partial vesicle destruction or general detergent action. At antibiotic levels above 1:1 antibiotic: cholesterol ratios the NMR results show all three antibiotics to cause extensive vesicle destruction. The onset of this behavior, which appears to be independent of the total antibiotic concentraion, indicates a well defined antibiotic : cholesterol interaction stoichiometry. Despite the fact that cholesterol is required for antibiotic activity, the NMR spectra prior to vesicle destruction show no changes indicative of an antibiotic-induced reversal of cholesterol restriction of phosphatidylcholine mobility. The contrast with polyene antibiotic behavior in more extended bilayers is discussed.     

Interaction of the polyene antibiotics with lipid bilayer vesicles containing cholesterol

The interaction of the polyene antibiotics, amphotericin B, nystatin and filipin with cholesterol-containing single bilayer lipid vesicles has been characterized using gel permeation chromatography and proton magnetic resonance. All three antibiotics bind to vesicles at low concentrations without causing a large amount of vesicle destruction. The strength of binding as determined by gel permeation studies is greater for filipin and amphotericin than for nystatin. Nystatin and amphotericin B at these low concentrations induce a rapid loss of internal vesicle contents consistent with pore formation. Filipin induces no leakage beyond that expected from partial vesicle destruction or general detergent action.At antibiotic levels above 1 : 1 antibiotic : cholesterol ratios the NMR results show all three antibiotics to cause extensive vesicle destruction. The onset of this behavior, which appears to be independent of the total antibiotic concentration, indicates a well defined antibiotic : cholesterol interaction stoichiometry. Despite the fact that cholesterol is required for antibiotic activity, the NMR spectra prior to vesicle destruction show no changes indicative of an antibiotic-induced reversal of cholesterol restriction of phosphatidylcholine mobility. The contrast with polyene antibiotic behavior in more extended bilayers is discussed.     

Phospholipid uptake by Plasmodium knowlesi infected erythrocytes

The uptake of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in Plasmodium knowlesi infected erythrocytes has been studied. Whereas uptake of phospholipids, in the absence of phospholipid transfer proteins, is negligible in control cells, the infected cells can incorporate considerable amounts of added phospholipids. The uptake is enhanced by the presence of lipid transfer proteins. Doubly labeled [3H]oleate, [14C]choline) PC does not undergo any appreciable remodelling following uptake, which strongly suggests that plasma PC is used as such for the biogenesis of the parasite membranes. Transport of extracellularly offered PS and PE towards the intraerythrocytic parasite and utilization of these lipids by the parasite are confirmed by the observation that these lipids are converted into respectively PE and PC. The extent and rate of these conversions depend on the way the phospholipids are introduced into the infected cells.     

The modulation of ion channels by the inhalation general anaesthetics. A1H-NMR investigation using unilamellar phospholipid membranes

The modulation of a variety of mechanisms of channel-mediated transport across unilamellar phospholipid membranes by a range of halogenated inhalation general anaesthetics (chloroform, enflurane, halothane and methoxyflurane) was investigated using 1H-NMR spectroscopy. Transport of the probe ion Pr3+ across egg yolk phosphatidylcholine (PC) and dipalmitoyl phosphatidylcholine (DPPC) vesicular membranes in the presence of the channel forming polypeptides alamethicin 30 and melittin, and the polyene antibiotic nystatin, as well as the degree of vesicular lysis at the gel to liquid-crystal phase transition of DPPC vesicles was monitored. The observation that the inhalation general anaesthetics inhibit such membrane permeability independently of the channel system or type of lipid used, suggests that hydrogen-bonded water structure and/or hydrogen-bonding centres at dipolar lipid-polypeptide interfaces, can be likely sites of action of the general anaesthetics.     

Phospholipid asymmetry in cardiac sarcolemma. Analysis of intact cells and 'gas-dissected' membranes

The investigation focuses on the phospholipid composition of the sarcolemma of cultured neonatal rat heart cells and on the distribution of the phospholipid classes between the two monolayers of the sarcolemma. The plasma membranes are isolated by 'gas-dissection' technique and 38% of total cellular phospholipid is present in the sarcolemma with the composition: phosphatidylethanolamine (PE) 24.9%, phosphatidylcholine (PC) 52.0%, phosphatidylserine/phosphatidylinositol (PS/PI) 7.2%, sphingomyelin 13.5%. The cholesterol/phospholipid ratio of the sarcolemma is 0.5. The distribution of the phospholipids between inner and outer monolayer is defined with the use of two phospholipases A2, sphingomyelinase C or trinitrobenzene sulfonic acid as lipid membrane probes in whole cells. The probes have access to the entire sarcolemmal surface and do not produce detectable cell lysis. The phospholipid classes are asymmetrically distributed: (1) the negatively charged phospholipids, PS/PI are located exclusively in the inner or cytoplasmic leaflet; (2) 75% of PE is in the inner leaflet; (3) 93% of sphingomyelin is in the outer leaflet; (4) 43% of PC is in the outer leaflet. The predominance of PS/PI and PE at the cytoplasmic sarcolemmal surface is discussed with respect to phospholipid-ionic binding relations between phospholipids and exchange and transport of ions, and the response of the cardiac cell on ischemia-reperfusion.     

Evidence for nystatin micelles in L-cell membranes from fluorescence photobleaching measurements of diffusion

Diffusion of a nitrobenzoxadiazole derivative of the polyene antibiotic nystatin in the membranes of L cells is found to depend on the concentration of nystatin in the membrane. Its diffusion coefficient measured by fluorescence photobleaching decreases hyperbolically as the concentration of nystatin is increased. This behavior is reproduced when the concentration of the derivative is increased. In contrast, diffusion of a nitrobenzoxadiazole derivative of a phospholipid is insensitive to the nystatin concentration under these conditions. The nystatin-specific diffusion changes can be understood if nystatin exists in a monomer-micelle equilibrium within the membrane but cannot be accounted for by binding or phase partitioning.