A. G. Medentsev

Biosynthesis of Naphthoquinone Pigments by Fungi of the Genus Fusarium

We studied the biosynthesis of colored naphthoquinone metabolites by Fusarium decemcellulare, F. graminearum, and F. bulbigenum fungi. Depending on the conditions of cultivation, F. bulbigenum and F. graminearum synthesized bikaverin and aurofusarin, respectively, whereas F. decemcellulare synthesized soluble extracellular naphthoquinones of the naphthazarin structure (javanicin, anhydrojavanicin, fusarubin, anhydrofusarubin, bostricoidin, and novarubin) or extracellular dimeric naphthoquinone aurofusarin. The biosynthesis of naphthoquinone pigments was shown to be the main response of the fungi to stress, observed under conditions of growth inhibition or arrest.

Tolerance of the yeast Yarrowia lipolytica to oxidative stress

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H2O2, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H2O2 (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H2O2, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.

Adaptation of the Phytopathogenic Fungus Fusarium decemcellulare to Oxidative Stress

The adaptive response of the phytopathogenic fungus Fusarium decemcellulare to the oxidative stress induced by hydrogen peroxide and juglone (5-hydroxy-1,4-naphthoquinone) was studied. At concentrations higher than 1 mM, H2O2 and juglone completely inhibited the growth of the fungus. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H2O2 (0.25 mM) and juglone (0.1 mM) led to the development of a resistance to high concentrations of these oxidants. The stationary-phase cells were found to be more resistant to the oxidants than the logarithmic-phase cells. The adaptation of fungal cells to H2O2 and juglone was associated with an increase in the activity of cellular catalase and superoxide dismutase, the main enzymes involved in the defense against oxidative stress.

Kinetic characteristics of 1-en-dehydrogenation of 6α-methylhydrocortisone by cells of Arthrobacter globiformis 193

Abstract Biochemical characteristics of 6α-methylhydrocortisone 1-en-dehydrogenation by bacterial cells of Arthrobacter globiformis 193 have been studied. The reaction follows the kinetics of substrate inhibition; the inhibition reveals itself at the level of the respiratory chain. A mathematical model describing multiple substrate inhibition during the process of 6α-methylhydrocortisone 1-en-dehydrogenation with whole cells was proposed. The solution of the constructed model agrees well with experimental data. There is little or no inhibitory effect at low substrate concentrations, and the reaction rate is determined by the enzyme-substrate interaction rather than the respiratory chain activity.

Adaptation of the yeast Yarrowia lipolytica to heat shock

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45°C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. A 60-min preincubation of cells at 37°C or pretreatment with low concentrations of H2O2 (0.5 mM) or menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).

Gossypol Inhibits Electron Transport and Stimulates ROS Generation in Yarrowia lipolytica Mitochondria

This work studied the effect of gossypol on the mitochondrial respiratory chain of Yarrowia lipolytica. The compound was shown to inhibit mitochondrial electron transfer and stimulate generation of reactive oxygen species. The inhibition kinetics in oxidation of various substrates (NADH, succinate, α-glycerophosphate and pyruvate + malate) by isolated mitochondria was investigated. Analysis of the kinetic parameters showed gossypol to inhibit two fragments of the mitochondrial electron transfer chain: a) between coenzyme Q and cytochrome b with KIIIi of 118.3 μM (inhibition by the noncompetitive type), and b) at the level of exogenous NADH dehydrogenase with of KIi 17.2 μM (inhibition by the mixed type).

Adaptation of the yeast Yarrowia lipolytica to ethanol

Resistance of the yeast Yarrowia lipolytica to ethanol stress was studied under different ethanol concentrations and treatment duration. Cell viability was shown to increase in the variants including preliminary treatment with small doses of ethanol, oxidants, or soft thermal exposure. The study of the respiratory activity under ethanol stress revealed the involvement of an alternative cyanide-resistant oxidase in the adaptive response of the cells. The level of intracellular cAMP decreased in response to the action of ethanol, which correlated with increased activity of the antioxidant systems (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, glutathione reductase) and NAD+-dependent alcohol dehydrogenase.

Respiratory activity of yeast Yarrowia lipolytica under oxidative stress and heat shock

Heat shock (45°C) and the effect of oxidants (H2O2) resulted in a decrease of the respiratory activity of yeast cells and their survival rate. Increased resistance to stress effects after mild heat treatment (37°C) or treatment with a nonlethal dose of oxidants (0.5 mM H2O2) for 60 min) was accompanied by appearance of an alternative (cyanide-resistant) oxidative pathway in the mitochondria, which promotes survival due to retention of the capacity for ATP synthesis in the first coupling point at the level of endogenous NADH dehydrogenase. The alternative oxidative pathway is more resistant to the effect of stressors that disrupt electron transfer in the cytochrome site of the respiratory chain.

Ultrastructural changes in Yarrowia lipolytica cells under stress conditions

Ultrastructural organization of the aerobic yeast Yarrowia lipolytica was studied under conditions of oxidative, heat, and ethanol stresses. It was shown that the following uniform changes in cell ultrastructure did not depend on the type of stress: enlargement of mitochondria, enhanced number and enlargement of peroxisomes, and formation of lipid granules. Similar ultrastructural changes also occurred during the transition of cells to the stationary growth phase. It was shown for the first time that accumulation of polyphosphate granules occurred as a stress response in yeasts. Moreover, numerous globular structures of unknown nature appeared on the cell wall surface under oxidative or heat stress. Under ethanol stress, the cells developed clearly marked deep invaginations of the cytoplasmic membrane. (The same changes in the cytoplasmic membrane were observed in the cells grown on ethanol.) Variations of the cell envelope structure along with the formation of polyphosphate granules were not observed in the stationary growth phase. Ultrastructural changes in the cells under stress conditions are in agreement with the previous data on survival, respiratory activity, and variations of the antioxidant systems.

Activation of the alternative oxidase of Yarrowia lipolytica by adenosine monophosphate

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and the submitochondrial particles of Yarrowia lipolytica showed that adenosine-5′-monophosphate (AMP) did not stimulate the respiration of the intact mitochondria. The incubation of the mitochondria at room temperature (25°С) for 3–5 h or their treatment with ultrasound, phospholipase A, and the detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated by AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability, in the following descending order: AMP = GMP > GDP > GTP > XMP > IMP. The apparent reaction rate constant Km for AMP upon the reactivation of the alternative oxidase of mitochondria treated with Triton X-100 or incubated at 25°C was 12.5 and 20 μM, respectively. The Km for AMP upon the reactivation of the alternative oxidase of submitochondrial particles was 15 μM. During the incubation of yeast cells under conditions promoting the development of alternative oxidase, the content of adenine nucleotides (AMP, ADP, and ATP) in the cells and their respiration tended to decrease. The subsequent addition of cyanide to the cells activated their respiration, diminished the intracellular content of ATP by three times, and augmented the content of AMP by five times. These data suggest that the stimulation of cell respiration by cyanide may be due to the activation of alternative oxidase by AMP.