Miklós Pesti

Anti yeast activities of some essential oils in growth medium, fruit juices and milk.

The anti-yeast activities of four essential oils (EOs) from clary sage, juniper, lemon and marjoram against wild-type isolates of the food-related yeasts Geotrichum candidum, Pichia anomala, Saccharomyces cerevisiae and Schizosaccharomyces pombe in malt extract (ME) medium, apple juice and milk were investigated. Minimal inhibitory concentrations (MICs) for the EOs and their main components were determined and the checkerboard method was used to calculate fractional inhibitory concentration (FIC) indices for the combinations of EOs or components. The most sensitive yeast was S. pombe (MICs of 0.0625-0.125 μl/ml) while G. candidum proved to be the most insensitive (MICs of 0.5-2 μl/ml). In general, the lag phases were lengthened by increasing EO concentrations, while significant reduction of growth rates was obtained only at the highest EO concentrations. The anti-yeast effects of the EOs were good in the acidic pH range optimal for yeasts growth. Combinations of juniper and clary sage EOs resulted in additive effects in the case of S. cerevisiae and G. candidum, but all other combinations showed no interaction. The combination of α-pinene and limonene led to synergism, while the combination of α-pinene with linalool resulted in an additive effect. Cloudy apple juice protected the yeasts against the effect of lemon EO: the lag phases were shorter and the growth rates higher than in clear apple juice. Lemon EO decreased the growth rate of G. candidum in skimmed milk in a dose-independent manner. Our results show that by adding lemon EO to clear apple juice a new, harmonic taste can be achieved and open storage time could be prolonged.

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. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Effect of hexavalent chromium on eukaryotic plasma membrane studied by EPR spectroscopy

The effect of Cr(VI) anion on an ergosterol-producing strain of eukaryotic yeast Candida albicans and its mutant with ergosterol-less membrane was studied with EPR spectroscopy. 5- and 14-doxyl stearic acid spin probes were used to label the protoplast membrane after removal of the cell wall. In control experiments, the mutant strain exhibited larger rigidity in the membrane than its parental strain. Addition of Cr(VI), at a minimum inhibitory concentration of 0.6 mM, increased the rotational mobility of the spin labels significantly and decreased the temperature of the structural changes in both strains, in the temperature range between 0 and 30 degrees C. The ergosterol-less mutant, having a membrane composition with increased polyunsaturated fatty acid content, exhibited higher Cr(VI) sensitivity. Treatment of the membrane with Cr(VI) for 10 min already resulted in an increase in membrane fluidity. An EPR signal of Cr(V) was detected which reached maximum amplitude after 120 min of treatment with Cr(VI). Further chemical reduction of Cr(V) in the absence of extracellular Cr(VI) led to a lack of detectable paramagnetic chromium intermediates within 200 min.

Prevention of intracellular oxidation in yeast: the role of vitamin E analogue, Trolox (6-hydroxy-2,5,7,8-tetramethylkroman-2-carboxyl acid)

Reactive oxygen species (ROS) are not only generated in conditions of cellular stress but are also constitutively produced in most cell types by specific metabolic processes. This research focused on a potential antioxidant Trolox (model compound for α‐tocopherol), with the aim to establish exact mechanisms of Trolox intracellular oxidation prevention on model organism Saccharomyces cerevisiae. Measuring intracellular oxidation of Trolox‐treated yeast cells revealed that Trolox decreased intracellular oxidation during normal metabolism. Trolox treatment decreased cyto‐ and geno‐toxicity of treated yeast cells in MES buffer, lowered intracellular oxidation, decreased intracellular peroxides formation, and increased H2O2 degradation and superoxide quenching yeast extract ability.

In vitro synergistic interactions of the effects of various statins and azoles against some clinically important fungi

The treatment of opportunistic fungal infections is often difficult as the number of available antifungal agents is limited. Nowadays, there is increasing interest in the investigation of the antifungal activity of nonantifungal drugs, and in the development of efficient antifungal combination therapy. In this study, the in vitro interactions of the effects of various statins (lovastatin, simvastatin, fluvastatin, atorvastatin (ATO), rosuvastatin (ROS) and pravastatin) and various azole antifungals [miconazole, ketoconazole, itraconazole and fluconazole (FLU)] against different opportunistic pathogenic fungi were investigated using a standard chequerboard broth microdilution method. When the investigated strains were sensitive to both compounds of the combination, additive interactions were frequently noticed. Synergistic interactions were observed in many cases when a strain was sensitive only to the azole compound (as in certain combinations with ATO or ROS) or the statin compound (as in certain combinations with FLU). In many combinations with an additive effect, the concentrations of drugs needed for total growth inhibition could be decreased by several dilution steps. Similar interactions were observed when the variability of the within-species sensitivities to some selected drug combinations was investigated.

Chromate sensitivity in fission yeast is caused by increased glutathione reductase activity and peroxide overproduction

The Cr(VI)‐sensitive mutant chr‐51S of the Schizosaccharomyces pombe accumulated chromate (CrO42–) and reduced Cr(V) to much greater extent, than did its parental strain 6 chr+. Sublethal doses of K2Cr2O7 did not induce any adaptive stress response, while H2O2 or menadione pretreatment proved protective against the cell injuries caused by Cr(VI). The intracellular GSH concentration in chr‐51S cells was approximately half of that for the 6 chr+. Moreover, the glutathione disulfide reducing capacity of chr‐51S was characterized by significantly increased glutathione reductase (GR) and glucose‐6‐phosphate dehydrogenase activities. These data strongly suggested that, instead of GSH, the NADPH/GR system was the major one‐electron Cr(VI) reductant in vivo. The increased Cr(V) reduction in chr‐51S mutant was accompanied by high intracellular superoxide and peroxide concentrations, required for formation of the hydroxyl radical (•OH). The decreased intracellular GSH levels and the Cr(VI)‐sensitive phenotype of the chr‐51S cells indicates that GSH might act effectively against chromate by scavenging •OH.

In vitro interactions between primycin and different statins in their effects against some clinically important fungi.

The in vitro antifungal activities of primycin (PN) and various statins against some opportunistic pathogenic fungi were investigated. PN completely inhibited the growth of Candida albicans (MIC 64 microg ml(-1)) and Candida glabrata (MIC 32 microg ml(-1)), and was very effective against Paecilomyces variotii (MIC 2 microg ml(-1)), but had little effect on Aspergillus fumigatus, Aspergillus flavus or Rhizopus oryzae (MICs >64 microg ml(-1)). The fungi exhibited different degrees of sensitivity to the statins; fluvastatin (FLV) and simvastatin (SIM) exerted potent antifungal activities against a wide variety of clinically important fungal pathogens. Atorvastatin, rosuvastatin and lovastatin (LOV) had a slight effect against all fungal isolates tested, whereas pravastatin was completely ineffective. The in vitro interactions between PN and the different statins were investigated using a standard chequerboard titration method. When PN was combined with FLV, LOV or SIM, both synergistic and additive effects were observed. The extent of inhibition was higher when these compounds were applied together, and the concentrations of PN and the given statin needed to block fungal growth completely could be decreased by several dilution steps. Similar interactions were observed when the variability of the within-species sensitivities was investigated.

In vivo direct patulin-induced fluidization of the plasma membrane of fission yeast Schizosaccharomyces pombe.

Patulin is a toxic metabolite produced by various species of Penicillium, Aspergillus and Byssochlamys. In the present study, its effects on the plasma membrane of fission yeast Schizosaccharomyces pombe were investigated. The phase-transition temperature (G) of untreated cells, measured by electron paramagnetic resonance spectrometry proved to be 14.1 degrees C. Treatment of cells for 20 min with 50, 500, or 1000 microM patulin resulted in a decrease of the G value of the plasma membrane to 13.9, 10.1 or 8.7 degrees C, respectively. This change in the transition temperature was accompanied by the loss of compounds absorbing light at 260 nm. Treatment of cells with 50, 500 or 1000 microM patulin for 20 min induced the efflux of 25%, 30.5% or 34%, respectively, of these compounds. Besides its cytotoxic effects an adaptation process was observed. This is the first study to describe the direct interaction of patulin with the plasma membrane, a process which could definitely contribute to the adverse toxic effects induced by patulin.

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

Patulin (PAT), is one of the most widely disseminated mycotoxins found in agricultural products. In this study the PAT-induced accumulation of reactive oxygen species (ROS) and the regulation of the specific activities of antioxidant enzymes were investigated in the single cell eukaryotic organism Schizosaccharomyces pombe. In comparison with the untreated cells, 500 μM PAT treatment caused a 43% decrease in the concentration of the main intracellular antioxidant, glutathione (GSH); this depletion of GSH initiated a 2.44- and a 2.6-fold accumulation of superoxide anion and hydrogen peroxide, respectively, but did not increase the concentration of hydroxyl radicals; the reduction of ROS-induced adaptation processes via the activation of Pap1 transcription factor resulted in significantly increased specific activities of Cu/Zn superoxide dismutase, catalase and glutathione S-transferase to protect the cells against the ROS-induced unbalanced redox state. However, no change was measured in the activities of glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase. It seems reasonable to assume that the temporary PAT-induced ROS accumulation plays a crucial role in adaptation processes. The adverse effects of PAT may be exerted mainly through the destruction of cellular membranes and protein/enzyme functions.

Effects of decreased specific glutathione reductase activity in a chromate-tolerant mutant of Schizosaccharomyces pombe

A chromate-tolerant mutant chr1-663T bearing a stable one-gene mutation and its parental strain 6chr+ were used to investigate the background of CrVI tolerance in the fission yeast Schizosaccharomyces pombe. The mutant chr1-663T displayed a significantly decreased specific glutathione reductase (GR) activity coded by the pgr1+ gene compared with its parental strain. Transformants of the mutant chr1-663T with a nonintegrative pUR18N vector expressing the pgr1+ gene exhibited the same CrVI sensitivity and specific GR activity as their parental strain, demonstrating the importance of the GR-NADPH system in CrVI tolerance. Transformants, nevertheless, exhibited an increased intracellular peroxide concentration, a decreased CrVI-reducing and HO•-producing ability, which suggested an unbalanced oxidoreduction state of cells and partial complementation of the GR function. No mutation was found in the sequences of the pgr1+ and the pap1+ (transcriptional regulatory gene of GR) genes of the CrVI-tolerant mutant by sequence analysis.