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J Bolard

Publications and source records attributed to J Bolard.

At least 37 records · Page 2Linked to original sources
PubMed1994

Na+, K+ and Cl- selectivity of the permeability pathways induced through sterol-containing membrane vesicles by amphotericin B and other polyene antibiotics.

Membrane diffusion potentials induced by amphotericin B (AmB), amphotericin B methyl ester (AmE), N-fructosyl AmB (N FruAmB) and vacidin, an aromatic polyene antibiotic, in ergosterol- or cholesterol-containing egg yolk phosphatidylcholine large unilamellar vesicles (LUV), were measured in various media, in order to determine the relative selectivity of Na+, K+, Cl- and other ions in these environments. Changes in the membrane potential were followed by fluorescence changes of 3,3'-dipropylthiadicarbocyanine (diS-C3-(5)). Subtle changes in intercationic selectivity were monitored by measuring biionic potentials, using the fluorescent pH sensitive probe pyranine. In all the cases studied, the intercationic selectivity of the permeability pathways induced by the four antibiotics was weak compared to that of specific biological channels, though distinct differences were noted. With AmB the selectivity appeared to be concentration dependent. Above 5 x 10(-7) M, the sequence determined for sterol-free small unilamellar vesicles (SUV) and cholesterol-containing SUV and LUV, Na+ > K+ > Rb+ > or = Cs+ > Li+ (sulfate salts), corresponded closely to Eisenman selectivity sequence number VII. At 5 x 10(-7) M and below the selectivity switched from Na+ > K+ to K+ > Na+. In contrast, Li+ was the most permeant ion for AmB channels in the presence of ergosterol. The selectivity between Na+ or K+ vs. Cl- varied with the antibiotic. It was very strong with vacidin at concentrations below 5 x 10(-7) M, smaller with AmB, nil with AmE and N FruAmB. The selectivities observed were antibiotic, concentration and time dependent, which confirms the existence of different types of channels.

Amphotericin B↗
PubMed1994

The endocytic process in CHO cells, a toxic pathway of the polyene antibiotic amphotericin B.

We describe the fate of the polyene antibiotic amphotericin B (AmB) after its interaction with Chinese hamster ovary (CHO) cells. The global uptake of AmB by these cells was measured at 37 degrees C after a 1-h incubation in the presence of 5% fetal bovine serum. It increased with the total concentration of drug and reached a plateau of approximately 1 nmol/mg of cell protein for an external concentration of 25 microM. The same experiment performed at 5 degrees C revealed a drastic decrease in uptake. The distribution of the drug among plasma membranes, endosomes, and lysosomes was then investigated after the separation of the postnuclear fractions by a Percoll gradient. After a 10-min incubation, AmB was found only in the plasma membrane fraction, regardless of the drug concentrations used (5 to 100 microM). After 60 min, at low drug concentrations (5 and 10 microM) AmB was found to be incorporated mainly in plasma and lysosomal fractions. At high concentrations (50 microM) AmB accumulated in endosomal fractions and plasma membranes. At intermediate concentrations (25 microM) AmB was distributed among the three fractions. When the same experiment was carried out at 5 degrees C, AmB was associated only with the plasma membrane even after 60 min, which was consistent with the absence of endocytotic process at low temperature. The effect of AmB on the endocytic process resulted in the increased uptake of sulforhodamine B, a fluid-phase marker of endocytosis, as well as by the accumulation of sulforhodamine in spots scattered in the cytoplasms of AmB-treated cells, in contrast to the accumulation around the nuclei observed in the control cells. These results are interpreted as indicating that AmB is internalized by the cells through endocytosis and that high concentrations of the drug block the fusion between endosomes and/or the fusion between endosomes and lysosomes.

Amphotericin B↗
PubMed1994

Amphotericin B incorporated into egg lecithin-bile salt mixed micelles: molecular and cellular aspects relevant to therapeutic efficacy in experimental mycoses.

The cellular activities of amphotericin B (AmB) used as Fungizone were compared with those of AmB complexed to either egg lecithin and glycocholate (Egam) or egg lecithin and deoxycholate (Edam). Under conditions in which leakage of K+ from erythrocytes and cultured L cells treated with Fungizone was almost complete, Egam and Edam containing concentrations of AmB severalfold greater than the concentration of AmB in Fungizone had no effect but retained the ability to decrease the level of retention of K+ in fungal cells. Analysis by absorption and circular dichroism spectroscopy demonstrated that when these formulations containing AmB at concentrations of less than 10(-5) M were added to buffer, the AmB dissociated slowly as monomers from Egam or Edam and dissociated rapidly as a mixture of monomers and self-associated species from Fungizone. We propose that in Egam and Edam, the absence of free AmB in the self-associated form reduces the toxicity of AmB to mammalian cells, whereas the presence of monomeric AmB results in the retention of the antifungal activities of these complexes.

Amphotericin B↗
PubMed1994

Effect of ketoconazole on lethal action of amphotericin B on Leishmania mexicana promastigotes.

The effect of ergosterol depletion by ketoconazole on the leishmanicidal activity of the pore-forming antibiotic amphotericin B (AmB) was investigated. Leishmania mexicana promastigotes were lysed within minutes by the addition of micromolar concentrations of AmB (0.5 microM) but became insensitive to AmB after growth in the presence of ketoconazole (0.25 microM, 90 h). Lipid chromatographic analysis indicated that under such conditions, ketoconazole depleted the major Leishmania sterols, dehydroepisterol and ergosterol. Plasma membrane vesicles prepared from ketoconazole-treated promastigotes exhibited a much reduced enhancement of their salt permeability after the addition of AmB at concentrations as high as 5 microM. This finding clearly indicates that upon ketoconazole treatment, the capacity of pore formation by the antibiotic is substantially impaired. The reduction of desmethyl sterols by ketoconazole was accompanied by a significant increase of 14-alpha-methyl sterols, but exogenous cholesterol remained unchanged. This ability of Leishmania promastigotes to incorporate cholesterol from the external medium may explain why ketoconazole-treated cells exhibited a much decreased but significative response to AmB when they were exposed to high AmB concentrations (2.5 or 5.0 microM). Parallel measurements by using a fluorescence energy transfer method indicated that binding of AmB to ketoconazole-treated Leishmania promastigotes and heat-transformed leishmanias was also decreased but to different extents, a finding that may be related to the differences in their sterol content. The results obtained clearly indicate that the specific interaction of AmB with desmethyl sterols, such as dehydroepisterol, ergosterol, and even exogenous cholesterol, is an absolute requirement for the lethal action exerted by this polyene antibiotic on L. mexicana promastigotes.

Amphotericin B↗
PubMed1994

Influence of diet on experimental toxicity of amphotericin B deoxycholate.

The effects of pre- and postprandial levels of lipids in serum on the experimental in vivo and in vitro toxicities of amphotericin B deoxycholate (AmB-d) were studied. Normal OF1 mice were tested at baseline, after normal feeding, after 3 h of fasting, or after a sequence of feeding and fasting and vice versa. The 50% lethal dose (LD50) of AmB-d was significantly higher in fed mice than in mice which fasted or at baseline (2.38 +/- 0.12 versus 1.53 +/- 0.2 and 1.50 +/- 0.1 mg/kg of body weight, respectively; P < 0.05). When different nutritional regimens were alternated over a short period, the level of in vivo AmB-d toxicity was dictated by the last feeding regimen. Serum triglycerides, but not cholesterol in very-low-density and low-density lipoproteins, correlated significantly (P < 0.01) with the LD50 of AmB. In vitro experiments showed that the addition of human serum reduced AmB-d-induced toxicity against human erythrocytes, but serum drawn after fasting was less protective than postprandial serum. However, neither serum decreased the in vitro activity of AmB-d against Candida albicans. Circular dichroism, a method that enables the amount of free AmB to be measured, showed that both mouse and human total serum lipoproteins bound more AmB-d when serum was isolated postprandially than when it was obtained after fasting. Our results show that AmB-d toxicity is reduced by feeding-induced modifications in serum lipids. The influence of food intake on the clinical toxicity of the drug merits being investigated.

Amphotericin B↗
PubMed1993

Liposomal amphotericin B bases of therapeutic index increase.

Liposomes represent for amphotericin B (AmB) a promising delivery system that may reduce the important toxicity of the drug. Liposomal AmB has a higher therapeutic index than AmB alone and allows to use larger doses. The mechanism of reduced toxicity may be related to a better targeting ability, a protective effect, and a selective transfer of liposomes. Anyway the liposomes intercalated AmB tends to be uptaken by organs that belong to the reticuloendothelial system, where fungi, the target cells, are localized.

Amphotericin B↗
PubMed1992

Polyene macrolide antibiotics: indirect stimulation of the Na+/H+ exchanger of BALB/c B lymphoid cell line, A20.

The fluorescent pH probe, 2'-7'-bis (carboxyethyl) 5-carboxyfluorescein, was used to follow changes in internal pH (pHi) induced by aromatic polyene antibiotics in the BALB/c lymphoid cell line A20. The antibiotics studied were vacidin, which contains a free carboxylic group in the position C18 of the macrolide ring, and vacidin glycyl methyl ester and perimycin, which are without free carboxylic groups. Although all of them induced transmembrane Na+ and K+ movements, only vacidin had protonophoric activity, as previously demonstrated for red blood cells [Cybulska B et al., Biochem Pharmacol 38: 1755-1762, 1989]. However, with all three antibiotics, pHi changes were observed in A20 cells. It was demonstrated that the transmembrane H+ movements resulted to different degrees, principally in the case of perimycin and vacidin glycyl methyl ester, or partially in the case of vacidin, from the stimulation of the Na+/H+ exchanger by the induced Na+ permeability. The non-aromatic polyene antibiotic amphotericin B had a low ability to increase proton permeability.

Amiloride↗
PubMed1992

Emerging role of lipids of Candida albicans, a pathogenic dimorphic yeast.

It is clear that C. albicans lipids have gained tremendous importance in recent years. In addition to being a barrier for entrance of various metabolites, it also provides the site of action for the synthesis of enzyme(s) involved in cell wall morphogenesis and antifungal action. While alterations in lipid composition during a yeast to mycelia transition have been observed, in most of the studies, lipid fluctuations reported could have been due to various environmental factors involved in the induction of morphogenesis [4,5]. A clear understanding of lipid biosynthesis and metabolic blocks due to antifungal action is likely to shed further light on selective interactions of antifungals. Despite the multifacet role of lipids in various functions of this pathogenic yeast, their exact involvement is poorly understood. The situation is little better with regard to ergosterol and its metabolism. Ergosterol is, indeed, important for anti-candidal activity and appears to be involved in the morphogenesis of C. albicans. The fluctuation in phospholipid composition have led to altered properties of plasma membrane namely, membrane fluidity, transport activities and drug sensitivity, which suggest that-a critical level of individual phospholipid is important for proper functioning of the plasma membrane. What the exact role is of individual phospholipid is far from clear. Many unanswered questions relating to the role of PI and sphingomyelin in signal transduction, involvement of phospholipases in the maintenance of phospholipid composition, and role of lipid transfer proteins in assembly and asymmetry of lipids are some aspects which merit further work.

Candida albicans↗
PubMed1992

Inhibiting cholesterol synthesis reduces the binding and toxicity of amphotericin B against rabbit renal tubular cells in primary culture.

Renal tubular cells are a target of amphotericin B (AmB) toxicity, but the mechanisms involved in the tubular cell-AmB interactions are unknown. Ketoconazole was selected to lower the cholesterol content of rabbit renal tubular cells in primary culture. The consequences of cholesterol depletion on AmB nephrotoxicity was investigated in vitro as the inhibition of Na(+)-dependent phosphate uptake. After 1 h of exposure, AmB decreased phosphate uptake (49%, 77%, and 82% inhibition with 5, 10, and 20 microM of AmB, respectively). Pretreatment of cells with ketoconazole (10 microM for 24 h) reduced by 50% (P less than .01) the phosphate uptake inhibition induced by AmB, decreased cellular cholesterol synthesis (greater than 80% inhibition), and decreased AmB binding to cell membrane by 50%, as measured by the fluorescence extinction of a probe bound to tubular cell membrane. Incubation with exogenous exchangeable cholesterol again increased AmB binding to plasma membrane and restored AmB toxicity. These results demonstrate that the first step of AmB renal tubular toxicity is mediated by cellular cholesterol content and is parallel to the binding of AmB to cell membrane.

Amphotericin B↗
PubMed1992

Effects of aggregation and solvent on the toxicity of amphotericin B to human erythrocytes.

In aqueous suspensions of amphotericin B (AmB), a polyene antibiotic and antifungal agent, three forms of AmB coexist: monomers, water-soluble oligomers, and non-water-soluble aggregates. The toxicity of the water-soluble self-associated form of AmB compared with that of the non-water-soluble self-associated form was tested by measuring induction of K+ leakage from human erythrocytes, using different suspensions containing the antibiotic and phosphate-buffered saline. These suspensions were obtained from various stock solutions of the antibiotic in dimethyl formamide or dimethyl sulfoxide. Their circular dichroism spectra around 340 nm, indicative of the degree of AmB self-association, were strongly dependent on the concentration of organic solvent in the suspensions. The nonsoluble self-associated form was separated from the water-soluble form by centrifugation. The nonsoluble form was favored by a high concentration of AmB of the stock solution. The kinetics of AmB-induced K+ leakage from human erythrocytes also appeared to be strongly dependent on the AmB concentration of the stock solution being much weaker with concentrated stock solutions. It was concluded that the only form of AmB toxic to human erythrocytes is the water-soluble self-associated form (in contrast with fungal cells on which the monomeric form is also active). This result may be important in the design of new less toxic AmB derivatives and in the understanding of the mechanism of action of liposomal AmB.

Amphotericin B↗
PubMed1992

Influence of phospholipid/amphotericin B ratio and phospholipid type on in vitro renal cell toxicities and fungicidal activities of lipid-associated amphotericin B formulations.

We studied the influence of the lipid/amphotericin B (AMB) ratio and the phospholipid type on the in vitro renal cell toxicity and antifungal efficacy of lipid-associated AMB (L-AMB). L-AMB was prepared at one of two different lipid/AMB ratios (1 and 40) by incubating AMB with empty small unilamellar vesicles, made from one of three different phospholipids: dipalmitoyl-, dimirystoyl-, and distearoylphosphatidylcholine (DPPC, DMPC, and DSPC, respectively). Renal cell toxicity, investigated through an assessment of the Na-dependent uptake of phosphate by proximal tubular cells, and fungicidal effect against Candida albicans were studied after 1 h of treatment at 37 degrees C. The amount of unbound AMB present in each L-AMB formulation was studied by use of circular dichroism. At a lipid/AMB ratio of 40, the three lipidic formulations were not toxic for renal cells but were less effective against C. albicans than AMB; however, DSPC-AMB, which contained 50% unbound AMB, was more effective against C. albicans than DPCC-AMB or DMPC-AMB, containing 0 and 13% unbound AMB, respectively. At a lipid/AMB ratio of 1, the antifungal effects of L-AMB and AMB were similar, whatever the phospholipid used, but only DMPC-AMB remained highly protective against AMB renal cell toxicity, despite the presence of the same amount of unbound AMB (50%) in DMPC-AMB and DPPC-AMB. We conclude that the in vitro activities and renal cell toxicities of different L-AMB formulations are influenced by the phospholipid type and the lipid/AMB ratio. The optimal ratio depends on the phospholipid itself. At a lipid/AMB ratio of 40, the antifungal activity depends mainly on the amount of unbound AMB in the formulation. At a lipid/AMB ratio of 1, the renal cell toxicity also depends on the fluidity of the phospholipid.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

One-sided action of amphotericin B on cholesterol-containing membranes is determined by its self-association in the medium.

The inducement of K+ permeability through membranes by the polyene antibiotic amphotericin B (AmB) has been analyzed as a measure of the antibiotic activity. Dose-response curves have been obtained with cholesterol- and ergosterol-containing egg yolk phosphatidylcholine large unilamellar vesicles (LUVs), human erythrocytes, and Saccharomyces cerevisiae cells. Conductance changes induced by AmB in sterol-containing planar bilayer membranes have also been studied. AmB self-association in aqueous buffer was determined by circular dichroism (CD) as a function of the antibiotic concentration. Electronic absorption and CD spectra of AmB were recorded in the presence of LUVs. For given AmB concentrations, the extent of permeability inducement is dependent on the lipid concentration. On the other hand, for cholesterol-containing LUVs or erythrocytes, a critical AmB concentration had to be reached before any permeability is observed. Independent of lipid concentration, this concentration was directly related to antibiotic self-association in the aqueous buffer. The same observation was made for erythrocytes and nystatin. The AmB absorption and CD spectra were totally different for ergosterol- and cholesterol-containing LUVs. Formation of single channels by one-sided addition of AmB could be observed only in ergosterol-containing membranes. These data lead us to propose that the permeability pathways induced by amphotericin B or nystatin, in ergosterol- and in cholesterol-containing membranes, are of different natures. In the latter case the antibiotics are only active, by single-sided addition, in the self-associated form. These findings offer important clues for the design of less toxic derivatives of AmB: they should have a low degree of self-association in water.

Absorption↗
PubMed1991

SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry: a critical assessment.

The use of SNARF-1-AM (seminaphtorhodafluor-1-acetoxymethylester) to measure the internal pH of a single living cell by laser microspectrofluorometry has been analyzed with a lymphocyte murine B cell line A20. After incubation of the cells at 37 degrees C in the presence of 10 microM SNARF-1-AM, the internal concentration of SNARF-1 was approximately 200 microM. The enhancement of fluorescent intensity of the probe is concomitant with its leakage out of the cells. During the measurement period, this induces a continuous increase of the contribution of the external probe to the total fluorescence intensity. This prevented classical spectrofluorometry measurements, but did not preclude microspectrofluorometry measurements of internal pH. The ratio R was calculated from fluorescence intensities at 635 and 590 nm and used as an indicator of the intracellular pH. Calibration curves of the intracellular pH were obtained in the presence of nigericin and valinomycin. It appeared that both the fluorescence intensity and the ratio R were lower inside the cell than those values obtained in aqueous solutions. Possible interactions with the main biological macromolecules (i.e., DNA, proteins, membranes) were investigated as well as a possible compartmentation of the probe in cellular organelles. The modifications of probe characteristics inside the cells were attributed to the binding of the probe to cellular proteins. The intracellular pH of A20 cells, measured by SNARF-1 on 84 cells, was found to be 7.18 +/- 0.10 (with an external pH of 7.40 +/- 0.05), which corresponded with values obtained by conventional fluorometric methods.

Animals↗
PubMed1991

The amphiphilic alpha-helix concept. Consequences on the structure of staphylococcal delta-toxin in solution and bound to lipids.

Staphylococcal delta-toxin, a synthetic analogue and a fragment were studied in order to determine their structure in solution and bound in lipids. In solution, a self-association process is observed. Analytical ultracentrifuge and quasi-elastic light-scattering experiments suggest an isodesmic aggregation in the high concentration domain above 2 microM up to very large asymmetrical species. Decreasing concentrations below 2 microM of delta-toxin and the analogue allows dissociation, probably into monomers. The self-associated species are essentially alpha-helical (70%) with buried and highly immobilized Trp either at position 15 for natural delta-toxin or 16 for the analogue. At the lowest concentration studied, the alpha-helix content severely decreases down to 35% while Trp fluorescence shows that these residues are exposed to buffer. The fragment 11-26 is always monomeric and structureless. From all the data, a structural model of aggregated species is proposed with stacked antiparallel amphipathic rods. When bound to lipids, whatever their initial structure in solution, 26-residue long peptides mainly adopt an alpha-helix conformation (80%) while fragment 11-26 exhibits about 50% alpha-helix. The lipid-peptide interactions were quantitatively analysed. For fragment 11-26, a single-step mechanism fits the spectroscopic changes and defines a single monomeric bound structure. On the other hand, for the 26-residue-long analogue, multiple-step processes must occur. The data were analysed with a partition of tetramers into lipids followed by a partial dissociation. Finally, the affinity of fragment 11-26 severely decreases from micelles to fluid and gel-state bilayers. The partition coefficient of the delta-toxin analogue is higher than those of other more apolar peptides, such as melittin and alamethicin, correlating with Eisenberg's hydrophobic moments. It is therefore proposed that delta-toxin probably lies parallel to the surface, only penetrating weakly in lipids, depending on their packing.

Amino Acid Sequence↗
PubMed1991

Dark interaction of 5-methoxypsoralen and 8-methoxypsoralen with erythrocyte ghosts: a fluorescence and circular dichroism study.

The dark interaction of 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) with plasma membranes was studied using human erythrocyte ghosts as a model. In the presence of ghosts, modifications of the fluorescence characteristics of 5-MOP were observed, together with a quenching of the fluorescence of the tryptophan (Trp) residues of membrane proteins (up to 25%). Moreover, the appearance of an induced circular dichroism indicates that 5-MOP is located in a chiral environment. In contrast, only slight effects were observed in the case of 8-MOP. It is concluded that 5-MOP molecules are located partly within chiral protein sites of the membrane in such a way that a Förster energy transfer can occur from the Trp residues to the psoralen molecules.

5-Methoxypsoralen↗
PubMed1990

Early membrane potential and cytoplasmic calcium changes during mitogenic stimulation of WEHI 231 cell line by polyene antibiotics, lipopolysaccharide and anti-immunoglobulin.

Changes in free cytosolic calcium concentration and in membrane voltage are thought to be important initiating events in lymphocyte activation. The antifungal agent amphotericin B (AmB) holds interesting immunomodulating properties and its N-thiopropionyl derivative (AmBSH) is a potent polyclonal B-cell activator. These molecules may then exert their stimulating activity through the production of early ionic signals similar to those delivered by the classical activators lipopolysaccharide (LPS) and anti-immunoglobulin (anti-Ig). We addressed this question in a B-cell line (WEHI 231) which has previously been shown to exhibit characteristic response to LPS and anti-Ig. AmBSH protected these cells against anti-Ig-induced cell growth inhibition, providing a LPS-like response. In contrast, the parental compound AmB did not. The two polyene antibiotics did not modify the resting Ca2+i level of the cells, neither did LPS, whereas anti-Ig induced a rapid increase in the cytosolic calcium concentration. On the other hand, polyene antibiotics and LPS promoted membrane depolarization, whereas membrane voltage remained unchanged after anti-Ig treatment. Polyene antibiotics-induced depolarization originated from the increase of membrane permeability to Na+ ions and occurred independently of Ca2+i changes. The relationship between membrane potential and Ca2+i changes in lymphocyte activation are discussed on the basis of these results. Our conclusion was that constitutive Ca2+(-)dependent K+ channels are absent in the WEHI 231 cell line.

Amphotericin B↗
PubMed1990

Affinity of amphotericin B for phosphatidylcholine vesicles as a determinant of the in vitro cellular toxicity of liposomal preparations.

Candida albicans and human erythrocytes were treated with liposomal amphotericin B (AmB) obtained by incubation of free AmB with small unilamellar vesicles (SUV) composed of unsaturated fatty acyl chains phosphatidylcholine (egg-yolk PC). Cellular effects were determined by changes in the K+ internal content of cells and in the number of colonies formed by fungal cells or as hemolysis, measured as a decrease in haemoglobin retention by erythrocytes. Dose-response curves were obtained by two procedures: either the ratio of AmB to phospholipids was kept constant over the AmB concentration range used (R = 10(-2] or the phospholipid concentration was kept constant (C = 0.2 mM) and the concentration of AmB varied. The liposomal preparations of AmB were as active against fungi as AmB in dimethylsulfoxide but less active (internal K+ changes) or inactive (hemolysis) against erythrocytes. On the other hand the binding of AmB to the SUV, as a function of the AmB concentration, was monitored by circular dichroism, fluorescence and UV absorption, in the two conditions used for the cellular studies. The amount of AmB bound when the total concentration of antibiotic was 2.10(-7) M was very low but increased with concentration and reached 90% at 10(-5) M. In all the assays we used, the anticellular effects could be attributed to the levels of AmB remaining free (unbound to the lipids). The variations of these levels with total AmB concentration could therefore explain the increased selectivity of liposomal AmB in toxicity against fungi and erythrocytes as compared to that of AmB added as a solution in dimethylsulfoxide. Indeed fungal cells are sensitive to low concentrations of AmB in dimethylsulfoxide; at these concentrations, in liposomal preparations, AmB is not bound to phospholipids and therefore as active as if added in dimethylsulfoxide. By contrast erythrocytes are only sensitive to much higher concentrations of AmB in dimethylsulfoxide; at these concentrations AmB is almost totally bound to phospholipids and therefore much less active.

Amphotericin B↗