Effects of clary sage oil and its main components, linalool and linalyl acetate, on the plasma membrane of Candida albicans: an in vivo EPR study.

The effects of clary sage (Salvia sclarea L.) oil (CS-oil), and its two main components, linalool (Lol) and linalyl acetate (LA), on cells of the eukaryotic human pathogen yeast Candida albicans were studied. Dynamic and thermodynamic properties of the plasma membrane were investigated by electron paramagnetic resonance (EPR) spectroscopy, with 5-doxylstearic acid (5-SASL) and 16-SASL as spin labels. The monitoring of the head group regions with 5-SASL revealed break-point frequency decrease in a temperature dependent manner of the plasma membrane between 9.55 and 13.15 °C in untreated, in CS-oil-, Lol- and LA-treated membranes. The results suggest a significant increase in fluidity of the treated plasma membranes close to the head groups. Comparison of the results observed with the two spin labels demonstrated that CS-oil and LA induced an increased level of fluidization at both depths of the plasma membrane. Whereas Lol treatment induced a less (1 %) ordered bilayer organization in the superficial regions and an increased (10 %) order of the membrane leaflet in deeper layers. Acute toxicity tests and EPR results indicated that both the apoptotic and the effects exerted on the plasma membrane fluidity depended on the composition and chemical structure of the examined materials. In comparison with the control, treatment with CS-oil, Lol or LA induced 13.0, 12.3 and 26.4 % loss respectively, of the metabolites absorbing at 260 nm, as a biological consequence of the plasma membrane fluidizing effects. Our results confirmed that clary sage oil causes plasma membrane perturbations which leads to cell apoptosis process.

Linalool-induced oxidative stress processes in the human pathogen Candida albicans.

The present study investigated the linalool (Lol)-induced effects in acute toxicity tests in the human pathogen Candida albicans (C. albicans). Lol treatments induced reduced germ tube formation of the pathogen, which plays a crucial role in the virulence. In comparison with the untreated control, the exposure of 107 cells ml-1 to 0.7 mM or 1.4 mM Lol for one hour induced 20% and 30% decrements, respectively, in the colony-forming ability. At the same time, these treatments caused dose-dependent decrease in the levels of superoxide anion radical and total reactive oxygen species, while there was 1.5 and 1.8-fold increases in the concentrations of peroxides and lipid peroxides, respectively, indicating oxidative stress induction in the presence of Lol. Lol treatments resulted in different adaptive modifications of the antioxidant system. In 0.7 mM-treated cells, decreased specific activities of superoxide dismutase and catalase were detected, while exposure to 1.4 mM Lol resulted in the up-regulation of catalase, glutathione reductase and glutathione peroxidases.

Regulation of unbalanced redox homeostasis induced by the expression of wild-type HIV-1 viral protein R (NL4-3Vpr) in fission yeast.

The wild-type viral protein R (Vpr) of human immunodeficiency virus type 1 exerts multiple effects on cellular activities during infection, including the induction of cell cycle G2 arrest and the death of human cells and cells of the fission yeast Schizosaccharomyces pombe. In this study, wild-type Vpr (NL4-3Vpr) integrated as a single copy gene in S. pombe chromosome was used to investigate the molecular impact of Vpr on cellular oxidative stress. NL4-3Vpr triggered an atypical response in early (14-h), and a wellregulated oxidative stress response in late (35-h) log-phase cultures. Specifically, NL4-3Vpr expression induced oxidative stress in the 14-h cultures leading, to decreased levels of superoxide anion (O(2)(·-)), hydroxyl radical (·OH) and glutathione (GSH), and significantly decreased activities of catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione S-transferase. In the 35-h cultures, elevated levels of O(2)(·-) and peroxides were accompanied by increased activities of most antioxidant enzymes, suggesting that the Vpr-induced unbalanced redox state of the cells might contribute to the adverse effects in HIV-infected patients.

Susceptibility of clinically important dermatophytes against statins and different statin-antifungal combinations.

The investigation of the antifungal activities of drugs whose primary activities are not related to their antimicrobial potential is in the current forefront of research. Statin compounds, which are routinely used as cholesterol-lowering drugs, may also exert direct antimicrobial effects. In this study, the in vitro antifungal activities of various statins (lovastatin, simvastatin, fluvastatin, atorvastatin, rosuvastatin and pravastatin) were examined against one isolate each of four dermatophyte species (Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Microsporum gypseum). Basically, statins were effective in inhibiting all dermatophyte studied, but were particularly active against M. canis and T. mentagrophytes. Fluvastatin and simvastatin were active against all of the tested fungi causing a complete inhibition of their growth at very low concentrations (6.25-12.5 µg/ml). Lovastatin and rosuvastatin had inhibitory effects at higher concentrations (25-128 µg/ml), while atorvastatin and pravastatin proved the less effective. The in vitro interactions between statins and different antifungals (ketoconazole, itraconazole, fluconazole, amphotericin B, nystatin, griseofulvin, terbinafine and primycin) were also investigated using a standard chequerboard broth microdilution method. Synergetic interactions were observed in several cases, most of them were noticed when statins were combined with terbinafine and the different azoles. Some combinations were particularly active (ketoconazole-simvastatin or terbinafine-simvastatin), as they were found to exert synergistic effect against all of the investigated isolates. The other antifungals showed synergistic interactions with statins in only certain cases. These results suggest that statins exert substantial antifungal effects against dermatophyte fungi and they should be promising components in a combination therapy as they can act synergistically with a number of clinically used antifungal agents.

Adaptation to tert-butyl hydroperoxide at a plasma membrane level in the fission yeast Schizosaccharomyces pombe parental strain and its t-BuOOH-resistant mutant.

The one-gene mutant hyd1-190 of the fission yeast Schizosaccharomyces pombe displayed four-fold resistance to tert-butyl hydroperoxide (t-BuOOH) in comparison with its parental strain hyd(+). The cells of hyd1-190 exhibited a quantitative alteration in the sterol content and hence in the fatty acid composition of the plasma membrane, reflected in a two-fold amphotericin B sensitivity, increased rigidity of the plasma membrane, revealed by an elevated (Δ7.9 °C) phase-transition temperature, measured by means of electron paramagnetic resonance spectroscopy, and a significantly decreased uptake of glycerol. Treatment of the strains with a subinhibitory concentration (0.2 mM) of t-BuOOH induced adaptation via modification of the sterol and fatty acid compositions, resulting in increased (Δ3.95 °C) and decreased (Δ6.83 °C) phase-transition temperatures of the hyd(+) and hyd1-190 strains, respectively, in order to defend the cells against the consequences of t-BuOOH-induced external oxidative stress. However, in contrast with hyd(+), hyd1-190 lacks the ability to adapt to t-BuOOH at a cell level.

tert-Butyl hydroperoxide-induced differing plasma membrane and oxidative stress processes in yeast strains BY4741 and erg5Δ.

The molecular mechanism of tert-butyl hydroperoxide (t-BuOOH) elicited cytotoxicity and the background of t-BuOOH sensitivity were studied in the Saccharomyces cerevisiae ergosterol-less gene deletion mutant erg5Δ and its parental strain BY4741. In comparison to BY4741, untreated erg5Δ cells exhibited alterations in sterol and fatty acid compositions of the plasma membrane, as reflected by the inherent amphotericin B resistance, an elevated level (31%) of plasma membrane rigidity and a decreased uptake of glycerol. Surprisingly, the untreated erg5Δ cells exhibited an unbalanced intracellular redox state, accompanied by the continuous upregulation of the antioxidant enzymes Mn superoxide dismutase, catalase, and glutathione S-transferase, which resulted in decreased specific concentrations of superoxide and peroxides and elevated levels of the hydroxyl radical and thiols. The 2.5-fold sensitivity of erg5Δ to t-BuOOH suggested that the oxidative stress adaptation processes of the mutant could not restore the redox homeostasis of the cells and there is an overlap between sterol and redox homeostases. t-BuOOH treatment of both strains induced adaptive modification of the sterol and fatty acid compositions, increased the plasma membrane fluidity and elevated the specific activities of most antioxidant enzymes through specific regulation processes in a strain-dependent manner.

Direct in vivo interaction of the antibiotic primycin with the plasma membrane of Candida albicans: an EPR study.

The direct interaction of the antibiotic primycin with the plasma membrane was investigated by employing the well-characterized ergosterol-producing, amphotericin B-sensitive parental Candida albicans strain 33erg(+) and its ergosterol-less amphotericin B-resistant plasma membrane mutant erg-2. The growth inhibition concentration in shaken liquid medium was 64 µgml(-1) for 33erg(+) and 128 µgml(-1) for erg-2, suggesting that the plasma membrane composition influences the mode of action of primycin. To determine the primycin-induced changes in the plasma membrane dynamic, electron paramagnetic resonance (EPR) spectroscopy methods were used, the spin-labeled fatty acid 5-(4,4-dimethyloxazolidine-N-oxyl)stearic acid) being applied for the in vivo measurements. The phase transition temperatures of untreated strain 33erg(+) and its mutant erg-2 were 12.5°C and 11°C, respectively. After 128 µgml(-1) primycin treatment, these values increased to 17.5°C and 16°C, revealing a significant reduction in the phospholipid flexibility. Saturation transfer EPR measurements demonstrated that, the rotational correlation times of the spin label molecule for the control samples of 33erg(+) and erg-2 were 60 ns and 100 ns. These correlation times gradually decreased on the addition of increasing primycin concentrations, reaching 8 µs and 1 µs. The results indicate the plasma membrane "rigidizing" effect of primycin, a feature that may stem from its ability to undergo complex formation with membrane constituent fatty acid molecules, causing alterations in the structures of phospholipids in the hydrophobic surface near the fatty acid chain region.

Overexpression of the YAP1, PDE2, and STB3 genes enhances the tolerance of yeast to oxidative stress induced by 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine.

7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT) is an antifungal agent that induces oxidative stress and enhances the activity of other antifungals with different modes of action. A genome-wide screening of Saccharomyces cerevisiae genomic library in the high-copy-number plasmid revealed three genes, YAP1, PDE2, and STB3, which increased the CTBT tolerance of the parental strain. The YAP1 gene is known to activate many genes in response to oxidants. The PDE2 and STB3 genes encode the high-affinity cAMP phosphodiesterase and the transcription factor recognizing the ribosomal RNA processing element in promoter sequences, respectively. The protective effects of their overexpression against CTBT toxicity was observed in the absence of certain proteins involved in stress responses, cell wall integrity signaling, and chromatin remodeling. The enhanced CTBT tolerance of the YAP1, PDE2, and STB3 transformants was a consequence of their high antioxidant enzyme activities at the beginning of CTBT treatment in comparison with that of the parental strain, for that they inactivated the CTBT-induced reactive oxygen species. These results point to the complex interplay among the oxidant sensing, cAMP-protein kinase A signaling, and transcription reprogramming of yeast cells, leading to their better adaptation to the stress imposed by CTBT.

Effect of the fungal mycotoxin patulin on the chromatin structure of fission yeast Schizosaccharomyces pombe.

The fungal mycotoxin patulin is produced by several molds, especially by Aspergillus and Penicillium. The aim of this study was to clarify whether patulin causes alterations in plasma membrane permeability of Schizosaccharomyces pombe lead to cellular shrinkage charateristic to apoptosis or increases cell size indicating necrosis in cells. Transmission and scanning electronmicroscopy revealed that lower concentrations of patulin induced cellular shrinkage and blebbing, higher concentration caused expansion without cellular disruption. Large-scale morphological changes of individual cells were followed by time lapse video microscopy. Patulin caused the elongation and stickiness of cells or rounded up their shapes. To visualize chromatin structures of S. pombe nuclei upon patulin treatment, protoplasts were isolated from S. pombe and subjected to fluorescent microscopy. Chromatin changes in the presence of 50 µM patulin concentration were characterized by elongated nuclei containing sticky fibrillary chromatin and enlarged round shaped nuclei trapped at the fibrillary stage of chromatin condensation. Short (60 min) incubation of S. pombe cells in the presence of high (500 µM) patulin concentration generated patches of condensed chromatin bodies inside the nucleus and caused nuclear expansion, with the rest of chromatin remaining in fibrillary form. Longer (90 min, 500 µM) incubation resulted in fewer highly condensed chromatin patches and in nuclear fragmentation. Although, high patulin concentration increased the size of S. pombe size, it did not lead to necrotic explosion of cells, neither did the fragmented nuclei resemble apoptotic bodies that would have indicated programmed cell death. All these morphological changes and the high rate of cell survival point to rapid adaptation and mixed type of fungistatic effects.

Oxidative stress induced by HIV-1 F34IVpr in Schizosaccharomyces pombe is one of its multiple functions.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during infection, including induction of cell cycle G(2) arrest and cell death in both human and the fission yeast Schizosaccharomyces pombe cells. In this study, a mutant derivative of Vpr (F34IVpr), which causes transient G2 arrest with little or no effect of cell killing, was used to study the molecular impact of Vpr on cellular oxidative stress responses in S. pombe. We demonstrated here that F34IVpr triggers low level of complex and atypical oxidative stress responses in comparison with its parental strain SP223 in early (14-h) and late (35-h) log phase cultures. Specifically, F34IVpr production in S. pombe causes significantly elevated levels of reactive oxygen species such as superoxide and peroxides; meanwhile, it also induces decreased levels of glutathione, hydroxyl radical concentrations and specific enzyme activities such as those of antioxidant enzymes including superoxide dismutases, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione transferase. These observations may provide functional insights into the significance of Vpr-induced oxidative stress as part of the multifaceted functions of Vpr, and contribute to the development of future new strategies aimed to reduce the adverse Vpr-mediated effects in HIV-infected patients.

Interference of chromium with biological systems in yeasts and fungi: a review.

This paper deals with the interactions of chromium (Cr) with biological systems, focusing in particular on yeasts and fungi. These interactions are analysed with primarily regard to biochemical functions, but higher levels of organization are also considered. Thus, the morphological and cytological characteristics of selected microorganisms in response to exposure to chromium ions are evaluated. The different oxidation states of chromium and reactive oxygen species (ROS) generated in redox reactions with chromium ions are presented and characterized. The interactions of the most exposed subcellular structures, including the cell wall, plasma membrane and nuclei, have been deeply investigated in recent years, for two major reasons. The first is the toxicity of chromium ions and their strong impact on the metabolism of many species, ranging from microbes to humans. The second is the still disputed usefulness of chromium ions, and in particular trivalent chromium, in the glucose and fat metabolisms. Chromium pollution is still an important issue in many regions of the world, and various solutions have been proposed for the bioremediation of soil and water with selected microbial species. Yeasts and especially moulds have been most widely investigated from this aspect, and the biosorption and bioaccumulation of chromium for bioremediation purposes have been demonstrated. Accordingly, the mechanisms of chromium tolerance or resistance of selected microbes are of particular importance in both bioremediation and waste water treatment technologies. The mechanisms of chromium toxicity and detoxification have been studied extensively in yeasts and fungi, and some promising results have emerged in this area.

Occurrence and genetic variability of Candida parapsilosis sensu lato in Hungary.

The occurrence and genetic variability of Candida parapsilosis isolates in two Hungarian hospitals, located in Debrecen and Pécs, were examined. Among the 209 Candida isolates examined, 20 were found to belong to C. parapsilosis sensu lato, based on morphological, physiological and molecular data. The frequency of occurrence of C. parapsilosis isolates (9.6%) was lower than that observed in Europe but higher than that observed previously in Hungary. The genetic variability of C. parapsilosis sensu lato isolates was also examined using random amplified polymorphic DNA (RAPD) analysis and sequence analysis of the intergenic transcribed spacer (ITS) region of the rRNA gene cluster. The genetic variability of the isolates was relatively high, as revealed by RAPD analysis. Two isolates were found to belong to the recently described Candida metapsilosis species (C. parapsilosis group III), based on ITS sequence data, RAPD analysis and phenotypic data. These two isolates could also be distinguished from C. parapsilosis sensu stricto isolates using a primer pair developed for the detection of C. parapsilosis group I isolates. To the best of the authors' knowledge, this is the first report on the identification of C. metapsilosis from bloodstream infection.

Pro-oxidative versus antioxidative reactions between Trolox and Cr(VI): The role of H(2)O(2).

The effect of the Vitamin E model compound Trolox in reactions with Cr(VI) in the presence or absence of hydrogen peroxide was investigated. The aim of this study was to establish and discuss potential Trolox-mediated pro-oxidative reactions. The importance of the Trolox:Cr(VI) ratio in the Cr(VI) reduction process was determined from the EPR spectra and DNA cleavage reactions. In the absence of hydrogen peroxide, reduction of Cr(VI) occurred with concomitant oxidation of Trolox to the phenoxyl radical. In the presence of hydrogen peroxide, Cr(V), produced by the reduction of Cr(VI), reduced hydrogen peroxide to the hydroxyl radical. The latter was detected by spin-trapping the methyl radical following reaction with N-methyl sulfoxide. During Cr(VI) reduction with Trolox, DNA single- or double-strand breaks due to Trolox radical formation were not observed. Relaxed DNA appeared only when H(2)O(2) was added to Trolox/Cr(VI) mixtures most probably due to hydroxyl radical formation during the redox cycling of Cr(V/IV)-species. Fenton-like reactions do not play a significant role in the Trolox/Cr(VI) system in the absence of added H(2)O(2).

Regulation of the antioxidant system in cells of the fission yeast Schizosaccharomyces pombe after combined treatment with patulin and citrinin.

The effects of combined treatment with patulin (PAT) and citrinin (CTN) on Schizosaccharomyces pombe cells were investigated in acute toxicity tests. In comparison with the controls the exposure of fission yeast cells (10(7) cells ml(-1)) to PAT + CTN (250 µM each) for 1 h at a survival rate of 66.6% significantly elevated the concentration of total reactive oxygen species (ROS) via increased levels of peroxides without affecting the concentrations of superoxides or the hydroxyl radical. This treatment induced a 3.08-fold increase in the specific concentration of glutathione and elevated specific activities of catalase and glutathione S-transferase, while at the same time the activity of glutathione reductase decreased. The pattern of the ROS was the same as that induced by CTN (Máté et al., 2014), while the presence of PAT in the PAT + CTN combination treatment modified the activities of the antioxidant system (Papp et al., 2012) in comparison with the individual PAT or CTN treatment, suggesting toxin-specific regulation of glutathione and the enzymes of the antioxidant system and the possibility that the transcription factor (pap1 and atf1) -regulated processes might be influenced directly by ROS.

Effects of hexavalent chromium on the plasma membranes of sensitive and tolerant mutants of Schizosaccharomyces pombe. An EPR study.

The interactions of chromium(VI) with the plasma membranes of chromium-sensitive (chr-51S) and chromium-tolerant (chr1-66T) mutants and their parental strain (6chr(+)) of a Schizosaccharomyces pombe strain were studied by electron paramagnetic resonance (EPR) spectroscopy. 5-doxylstearic acid (5-SASL) and 3-doxylbutyric acid (HO-185) spin probes were used to label the membranes. The order parameter S from the EPR spectra was calculated at different temperatures (0-25 degrees C) in order to characterize the internal dynamics of the membranes. In control experiments, both mutants exhibited differences in structural transitions in the both 5-SASL- and the HO-185-labeled membranes in comparison with their parental strain, suggesting differences in the membrane composition and/or rotational dynamics of these mutants. Addition of K(2)Cr(2)O(7) (225 microM) induced small decreases in the phase transition temperatures of the 5-SASL-labeled membranes of the parental and chromium-sensitive strains. More pronounced effects of the chromium compound on the HO-185-labeled membranes were detected as evidence that the membrane perturbations are mostly localized in the environment of the lipid-water interface.

Regulation of oxidative stress-induced cytotoxic processes of citrinin in the fission yeast Schizosaccharomyces pombe.

In this study, the citrinin (CTN)-induced accumulation of reactive oxygen species (ROS) and the regulation of the activities of antioxidant enzymes were investigated in acute toxicity tests in Schizosaccharomyces pombe. 30% of the CTN was accumulated by the cells in 1000 µM CTN solution. In comparison with the control, exposure of 10(7) cells ml(-1) to 1000 µM CTN for 60 min at pH = 4.5 induced significantly (p < 1%) elevated levels of peroxides and total ROS, but not of superoxide or hydroxyl radicals, while there was a 3-fold increase in the concentration of glutathione. ROS-induced adaptation processes at cell and molecular levels via activation of the redox-sensitive transcription factors Pap1 and (in part) Atf1 resulted in significantly increased specific activities of glutathione peroxidases, glucose-6-phosphate dehydrogenase and glutathione S-transferase and in decreased levels of catalase and glutathione reductase, but no changes were detected in the activities of superoxide dismutases. This treatment caused a G2/M cell cycle arrest and elevated the number of fragmented nuclei, which is one of the markers of apoptosis. Comparison of these results with those for the positive control, 200 µM H2O2, suggested that CTN induced a medium level of oxidative stress.

Antifungal activity of the primycin complex and its main components A1, A2 and C1 on a Candida albicans clinical isolate, and their effects on the dynamic plasma membrane changes.

The in vitro antifungal activities of the macrolide lactone antibiotic complex primycin (PC) and its main components, A1 (50%), A2 (7.3%) and C1 (13%), against the opportunistic pathogenic fungus Candida albicans 33erg(+)were determined by microdilution testing. The MIC(100) (the minimal concentration required for 100% growth inhibition) values found, A2 (2 µg ml(-1)), PC (32 µg ml(-1)), A1 (32 µg ml(-1)) and C1 (64 µg ml(-1)), suggested that the biological activity of PC is highly dependent on the proportions of its constituents. In vivo measurements of the biophysical properties of plasma membranes were carried out by electron paramagnetic resonance (EPR) spectroscopic methods, using the spin probe 5-(4,4-dimethyloxazolidine-N-oxyl)stearic acid. Conventional EPR measurements demonstrated altered phase transition temperatures (Tm) of the plasma membrane of strain 33erg(+) as a consequence of treatment with PC or its constituents: for cells treated with 128 µg ml(-1) PC, A1, A2 or C1 for 15 min, Tm was 17, 21, 14.5 and 15 °C, respectively; that is significantly higher than the Tm of untreated cells, 12 °C. The molecular motions of the near-surface hydrophobic region of the plasma membrane, estimated by saturation transfer EPR spectroscopy, reflected changes in the membrane phases after the treatment. Two physiological membrane phases were detected in control samples: liquid-ordered and liquid-disordered, characterized by molecular movements ∼10(-6)-10(-8) s and ≥10(-9) s. The cells treated with the investigated compounds showed the strong presence of a non-physiological gel phase additional to the above phases, characterized by movements ≤10(-5) s.

Regulation of cytotoxic, non-estrogenic, oxidative stress-induced processes of zearalenone in the fission yeast Schizosaccharomyces pombe.

This study investigates the non-estrogenic mode of zearalenone (ZEA) toxicity in a novel aspect via accumulation of reactive oxygen species (ROS) and the regulation of the activities of antioxidant enzymes in the Schizosaccharomyces pombe in acute toxicity tests. In comparison with the control, 500 µM ZEA treatment caused 66% decrease in the concentration of glutathione (GSH), which was a consequence, in the absence of ZEA-GSH interaction, of the GSH-consuming processes of the antioxidant system; this depletion of GSH initiated a 1.8- and 2.0-fold accumulation of the superoxide anion and hydrogen peroxide, but did not increase the concentration of the hydroxyl radical; ROS-induced adaptation processes via activation of the Pap1 transcription factor resulted in significantly increased activities of superoxide dismutases, catalase, glutathione reductase and glutathione S-transferase, and decreased activities of glutathione peroxidase and glucose-6-phosphate dehydrogenase. This treatment altered the sterol composition of the cells by inducing decreased concentrations of ergosterol, squalene and 24-methylene-24,25-hydrolanosterol, and also elevated the number of fragmented nuclei. Cells strived to correct the unbalanced redox state by regulation of the antioxidant system, but this was not enough to defend the cells from the disturbed sterol composition, the cell cycle arrest, and the fragmentation of nuclei.

Citrinin-induced fluidization of the plasma membrane of the fission yeast Schizosaccharomyces pombe.

Citrinin (CTN) is a toxic fungal metabolite that is a hazardous contaminant of foods and feeds. In the present study, its acute toxicity and effects on the plasma membrane of Schizosaccharomyces pombe were investigated. The minimum inhibitory concentration of CTN against the yeast cells proved to be 500 µM. Treatment with 0, 250, 500 or 1000 µM CTN for 60 min resulted in a 0%, 2%, 21% or 100% decrease, respectively, in the survival rate of the cell population. Treatment of cells with 0, 100, 500 or 1000 µM CTN for 20 min induced decrease in the phase-transition temperature of the 5-doxylstearic acid-labeled plasma membrane to 16.51, 16.04, 14.18 or 13.98°C, respectively as measured by electron paramagnetic resonance spectroscopy. This perturbation was accompanied by the efflux of essential K⁺ from the cells. The existence of an interaction between CTN and glutathione was detected for the first time by spectrofluorometry. Our observations may suggest a direct interaction of CTN with the free sulfhydryl groups of the integral proteins of the plasma membrane, leading to dose-dependent membrane fluidization. The change in fluidity disturbed the ionic homeostasis, contributing to the death of the cells, which is a novel aspect of CTN cytotoxicity.

Complex formation between primycin and ergosterol: entropy-driven initiation of modification of the fungal plasma membrane structure.

The interaction of the antibiotic primycin with the main fungal sterol, ergosterol, was investigated in vitro in order to monitor the effect of primycin on the fungal plasma membrane at the molecular level. The thermodynamic parameters of complex formation were determined by measuring Rayleigh scattering as a signal sensitive to particle size. The Benesi-Hildebrand method validated the 1 : 1 stoichiometry of the primycin-ergosterol complexes. A very low enthalpy change (ΔH=-1.14 kJ mol(-1)) was measured during the complex formation, which itself cannot be responsible for the molecular association. However, the entropy production (ΔS=29.78 J mol K(-1)) observed during the complex formation can describe the molecular interaction. This effect is probably due to the partial destruction of the solvation shell of the interacting species before the interlinking of the molecules. The results highlight the importance of ergosterol as concerns the mode of effect of primycin in the treatment of fungal infections. As the entropy has a determinant role in the ergosterol-primycin interaction, this interaction exhibits a very high temperature dependence, with the important consequence that the effect exerted by primycin on the cell membranes increases with rising temperature, and the effect is therefore pronounced in fevered bodies.