Regulation of the unbalanced redox state in a Schizosaccharomyces pombe tert-butyl hydroperoxide-resistant mutant.

The one-gene mutation in the tert-butyl hydroperoxide-resistant mutant hyd1-190 of the fission yeast Schizosaccharomyces pombe led to a 4-fold increase in resistance to t-BuOOH and decreased specific concentrations of superoxide and total thiols in comparison with the parental strain hyd+. It suggested an unbalanced redox state of the cells, which induced continuously increased specific activities of glutathione peroxidase, glutathione reductase and glutathione S-transferase and decreased activities of the antioxidant enzymes superoxide dismutases and glucose-6-phosphate dehydrogenase to regulate the redox balance of the mutation-induced permanent, low-level but tolerable internal stress. These results may contribute to the understanding of internal, oxidative stress-related human diseases.

In vivo direct interaction of the antibiotic primycin on a Candida albicans clinical isolate and its ergosterol-less mutant.

Interaction of primycin antibiotic with plasma membrane, and its indirect biological effects were investigated in this study. The antifungal activity of primycin against 13 human pathogenic Candida ATCC and CBS reference species and 74 other Candida albicans clinical isolates was investigated with a microdilution technique. No primycin-resistant strain was detected. Direct interaction of primycin with the plasma membrane was demonstrated for the first time by using an ergosterol-producing strain 33erg+ and its ergosterol-less mutant erg-2. In growth inhibition tests, the 33erg+ strain proved to be more sensitive to primycin than its erg-2 mutant, indicating the importance of the plasma membrane composition in primycin-induced processes. The 64 µg ml-1 (56.8 nM) primycin treatment induced an enhanced membrane fluidity and altered plasma membrane dynamics, as measured by steady-state fluorescence anisotropy applying a trimethylammonium-diphenylhexatriene (TMA-DPH) fluorescence polarization probe. The following consequences were detected. The plasma membrane of the cells lost its barrier function, and the efflux of 260-nm-absorbing materials from treated cells of both strains was 1.5-1.8 times more than that for the control. Depending on the primycin concentration, the cells exhibited unipolar budding, pseudohyphae formation, and a rough cell surface visualized by scanning electron microscopy.

Characterization of stress processes of Phaffia rhodozyma stress-resistant mutant.

A carotenoid-less Phaffia rhodozyma mutant (MCP 325) exhibited significantly higher resistance to oxidative stressors such as menadione, H2O2 and K2Cr2O7 than its astaxanthin-producing parental strain (MCP 324). The absence of carotenoids in the mutant did not explain this phenomenon. The cause of the decreased superoxide, hydroxyl radical and glutathione contents, the increased peroxide concentration and the elevated specific activity of catalase under uninduced conditions may be a second mutation. Peroxide treatment induced specific catalase activity in the mutant but not in the parental strain. Regulation of these processes led to the result that, in spite of the mutations, the two strains exhibited the same multiplication rate and generation time.

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 Cr(VI) 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 Cr(VI) sensitivity and specific GR activity as their parental strain, demonstrating the importance of the GR-NADPH system in Cr(VI) tolerance. Transformants, nevertheless, exhibited an increased intracellular peroxide concentration, a decreased Cr(VI)-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 Cr(VI)-tolerant mutant by sequence analysis.

Pro-oxidative vs antioxidative properties of ascorbic acid in chromium(VI)-induced damage: an in vivo and in vitro approach.

The effect of antioxidant ascorbic acid (vitamin C) pretreatment on chromium(VI)-induced damage was investigated using the yeast Saccharomyces cerevisiae as a model organism. The objective of this study was to pretreat yeast cells with the antioxidant ascorbic acid in an effort to increase cell tolerance against reactive chromium intermediates and reactive oxygen species formed during chromium(VI) reduction. Intracellular oxidation was estimated using the fluorescence indicators dihidro-2,7-dichlorofluorescein, dihydroethidium and dihydrorhodamine 123. The role of ascorbic acid pretreatment on chromium(VI) toxicity was determined by measuring mitotic gene conversion, reverse mutations, 8-OHdG, hydroxyl radical, superoxide anion and chromium(V) formation. The chromium content in the biomass was determined by flame atomic absorption spectrometry. In the absence of chromium, ascorbic acid effectively protected the cells against endogenous reactive oxygen species formed during normal cellular metabolism. In vitro measurements employing EPR and the results of supercoiled DNA cleavage revealed that the pro-oxidative action of ascorbic acid during Cr(VI) reduction was concentration-dependent and that harmful hydroxyl radical and Cr(V) had formed following Cr(VI) reduction. However, the in vivo results highlighted the important role of increased cytosol reduction capacity related to modification of Cr(V) formation, increased chromium accumulation, better scavenging ability of superoxide anions and hydrogen peroxide, and consequently decreased cytotoxicity and genotoxicity in ascorbic acid pretreated cells. Ascorbic acid influenced Cr(VI) toxicity both as a reducing agent, by decreasing Cr(V) persistence, and as an antioxidant, by decreasing intracellular superoxide anion and hydrogen peroxide formation and by quenching free radicals formed during Cr(VI) to Cr(III) reduction. Increased 8-OHdG and decreased reduced glutathione in ascorbic acid-treated cells might induce an endogenous antioxidant defense system and thus increase cell tolerance against subsequent Cr-induced stress.

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 alpha-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 H(2)O(2) degradation and superoxide quenching yeast extract ability. This study suggests that Trolox treatment provides prevention against intracellular ROS formation. Trolox application as therapeutic agent against intracellular ROS formation would be worth considering. Additionally, results indicate that yeasts are good model organisms for studying intracellular oxidation and oxidative stress. The obtained results on yeast cells might be useful to direct further human-related search for the Trolox evaluation as a human supplement used for protecting cells against intracellular free radical formation.

In vivo interaction of trivalent chromium with yeast plasma membrane, as revealed by EPR spectroscopy.

The in vivo effects of CrCl(3) on an ergosterol-producing 33 erg(+) strain of the eukaryotic yeast Candida albicans, and on its ergosterol-deficient erg(-)2 mutant, were studied by using electron paramagnetic resonance spectroscopy. A 5-doxylstearic acid spin probe was applied to label the membranes. The absence of ergosterol, an increased accumulation of Delta(8) sterols, a decreased fatty acid chainlength and a lower proportion of unsaturated fatty acids of the erg(-)2 mutant resulted in a higher membrane rigidity and an increased sensitivity to Cr(III) than those of the parental 33 erg(+) strain. Cr(III) significantly increased the fluidity of the spin labelled membranes, this being more pronounced for the erg(-)2 mutant. The break in the slopes measured for the erg(-)2 mutant was decreased (DeltaAT approximately 4 degrees C) from 17 to 13 degrees C. Cr(III) treatment for 10 h caused a loss of metabolites adsorbing at 260 nm: this loss was 40% for 33 erg(+) and 60% for erg(-)2. This decriptification process might be the main cause of growth inhibition and cell killing by the impermeable Cr(III) ions.