Healthy brain aging: Interplay between reactive species, inflammation and energy supply.

Brains' high energy expenditure with preferable utilization of glucose and ketone bodies, defines the specific features of its energy homeostasis. The extensive oxidative metabolism is accompanied by a concomitant generation of high amounts of reactive oxygen, nitrogen, and carbonyl species, which will be here collectively referred to as RONCS. Such metabolism in combination with high content of polyunsaturated fatty acids creates specific problems in maintaining brains' redox homeostasis. While the levels of products of interaction between RONCS and cellular components increase slowly during the first two trimesters of individuals' life, their increase is substantially accelerated towards the end of life. Here we review the main mechanisms controlling the redox homeostasis of the mammalian brain, their age-dependencies as well as their adaptive potential, which might turn out to be much higher than initially assumed. According to recent data, the organism seems to respond to the enhancement of aging-related toxicity by forming a new homeostatic set point. Therefore, further research will focus on understanding the properties of the new set point(s), the general nature of this phenomenon and will explore the limits of brains' adaptivity.

FREE RADICALS, REACTIVE OXYGEN SPECIES, OXIDATIVE STRESSES AND THEIR CLASSIFICATIONS.

The phrases "free radicals" and "reactive oxygen species" (ROS) are frequently used interchangeably although this is not always correct. This article gives a brief description of two mentioned oxygen forms. During the first two-three decades after ROS discovery in biological systems (1950-1970 years) they were considered only as damaging agents, but later their involvement in organism protection and regulation of the expression of certain genes was found. The physiological state of increased steady-state ROS level along with certain physiological effects has been called oxidative stress. This paper describes ROS homeostasis and provides several classifications of oxidative stresses. The latter are based on time-course and intensity principles. Therefore distinguishing between acute and chronic stresses on the basis of the dynamics, and the basal oxidative stress, low intensity oxidative stress, strong oxidative stress, and finally a very strong oxidative stress based on the intensity of the action of the inductor of the stress are described. Potential areas of research include the development of this field with complex classification of oxidative stresses, an accurate identification of cellular targets of ROS action, determination of intracellular spatial and temporal distribution of ROS and their effects, deciphering the molecular mechanisms responsible for cell response to ROS attacks, and their participation in the normal cellular functions, i.e. cellular homeostasis and its regulation.

[Carbohydrate restriction in the larval diet causes oxidative stress in adult insects of Drosophila melanogaster].

The influence of 20 and 1% glucose and fructose, which were components of larval diet, on the level of oxidized proteins and lipids, low molecular mass antioxidant content as well as activities of antioxidant and associated enzymes in adult fruit fly Drosophila melanogaster were investigated. The restriction of carbohydrates in larval diet leads to oxidative stress in adult insects. It is supported by 40-50% increased content of protein carbonyl groups and by 60-70% decreased level of protein thiol groups as well as by a 4-fold increase of lipid peroxide content in 2-day-old flies of both sexes, developed on the diet with 1% carbohydrates. Oxidative stress, induced by carbohydrate restriction of the larval diet, caused the activation of antioxidant defence, differently exhibited in male and female fruit flies. Caloric restriction increased activity of superoxide dismutase and thioredoxin reductase associating only in males with 2-fold higher activity of NADPH-producing enzymes--glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase. Carbohydrate restriction in the larval diet caused the increase of uric acid content, but the decrease in catalase activity in males. In females the values of these parameters were changed in opposite direction compared with males. The obtained results let us conclude the different involvement of low molecular mass antioxidants, glutathione and uric acid, and antioxidant enzyme catalase in the protection of male and female fruit fly macromolecules against oxidative damages, caused by calorie restriction of larval diet.

Effect of short-term salt stress on oxidative stress markers and antioxidant enzymes activity in tocopherol-deficient Arabidopsis thaliana plants.

Changes of carotenoids and anthocyanins content, lipid peroxidation, and activity of antioxidant enzymes were studied in wild type and tocopherol-deficient lines vte1 and vte4 of Arabidopsis thaliana subjected to 200 mM NaCI during 24 h. The salt stress enhanced the intensity of lipid peroxidation to different extent in all three plant lines. Salt stress resulted in an increase of carotenoid content and activity of catalase, ascorbate peroxidase, guaiacol peroxidase and glutathione reductase in wild type and tocopherol-deficient vte1 mutant. However, the increase in anthocyanins concentration was observed in vte1 mutants only. In vte4 mutant, which contain gamma-tocopherol instead of alpha-tocopherol, the response to salt stress occurred via coordinative action of superoxide dismutase and enzymes of ascorbate-glutathione cycle, in particular, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and glutathione-S-transferase. It can be concluded, that salt stress was accompanied by oxidative stress in three studied lines, however different mechanisms involved in adaptation of wild type and tocopherol-deficient lines to salt stress.

Effect of sodium chloride and nitroprusside on protein carbonyl groups content and antioxidant enzyme activity in leaves of corn seedlings Zea mays L.

The effect of sodium nitroprusside (SNP) and sodium chloride (NaCl) on protein carbonyl group content and activity of antioxidant enzymes was investigated in leaves of maize seedlings. Incubation with NaCl and SNP+NaCl increased the content of carbonyl proteins after 24 h. Treatment with SNP+NaCl during 48 h showed lower and after 72 h higher carbonyl protein content than that in the control. Catalase activity was higher in the leaves of SNP+NaCl-treated than in the leaves of SNP-treated seedlings after 24 h. Ascorbate peroxidase activity increased after incubation with 0.2 mM SNP for 24 h. Significant increment of guaiacol peroxidase activity was obtained in all treated groups in comparison with the control after 72 h. Glutathione-S-transferase activity increased after 48 h seedling treatment with NaCl or SNP and 72 h seedling incubation with NaCl. Under experimental conditions used, glutathione reductase activity was virtually not affected. It is proposed that SNP can be used to prevent salt-induced oxidative stress in maize.

[Mild oxidative stress in fruit fly Drosophila melanogaster caused by products of sucrose hydrolysis].

The influence of 6% sucrose and equimolar mixture of glucose and fructose in larva diet on the level of oxidized proteins and lipids as well as the activity of antioxidant and associated enzymes in adult fruit fly Drosophila melanogaster was investigated. Larva growing on the diet with sucrose led to the mild oxidative stress development in adult insects, which was differently expressed in both sexes. In males mainly molecules of proteins were subjected to oxidative damages, whereas in females--lipid molecules. This is evidenced by 77% increased content of protein carbonyl groups and decreased (by 40%) level of protein SH-groups in males fed on sucrose. In females fed on sucrose the content of lipid peroxides was by 44% higher, than in individuals, hold on the diet with equimolar mixture of glucose and fructose. The oxidative stress in females was accompanied with increased activity of catalase, superoxide dismutase and thiredoxin reductase by 30, 15 and 34%, respectively. The obtained results suggest that uptake mode of glucose and fructose affects free radical processes in fruit flies.

[Effect of high sodium chloride concentrations on the pigment content and free-radical processes in corn seedlings leaves].

The effect of sodium chloride on general morphometrical parameters of seedlings, and biochemical parameters in the leaves of corn seedlings was studied. Exposure to 100 and 200 mM NaCl slowed down the growth of stem and roots, whereas 100 and 200 mM NaCl during 24 h enhanced the concentration of chlorophylls, carotenoids, anthocyans, and thiobarbituric acid reactive substances. The decrease in protein carbonyl groups was found at 24-hour exposure to 200 mM salt. The treatment during 24, 48 and 72 h to 200 mM salt increased the level of total and high molecular mass thiols, whereas low molecular mass thiol content was by 20-25% higher at 48 h exposure to all used salt concentrations. The activity of guaiacol peroxidase was higher only at 24 h exposure to 100 and 200 mM salt, and catalase--at 50 mM during 48 h. At 72-hour exposure, catalase activity was by 27 and 41% higher in seedlings, exposed to 50 and 200 mM NaCl, respectively. Therefore, it is concluded the plant exposure to 50-200 mM salt initially developed oxidative stress, inducing adaptive response--an increase in antioxidant potential and efficiency of systems of energy production. That results in plant adaptation to unfavourable conditions.

Effect of sodium nitroprusside and S-nitrosoglutathione on pigment content and antioxidant system of tocopherol-deficient plants of Arabidopsis thaliana.

Sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) were used as a source of exogenous nitric oxide (NO) to investigate their effects on biochemical parameters and antioxidant enzyme response in leaves of wild type Columbia and tocopherol-deficient vte4 and vte1 mutant lines of Arabidopsis thaliana plants and possible tocopherol involvement in regulation of antioxidant response under NO-induced stress. SNP enhanced the activity of the enzymes, that scavenge hydrogen peroxide in leaves of all studied lines, and increased glutathione reductase and glutathione-S-transferase activity there. In addition, it decreased the intensity of lipid peroxidation in vte1 mutant line leaves. At the same time, GSNO increased the levels of protein carbonyls and inactivated enzymes ascorbate peroxidase, guaiacol peroxidase and dehydroascorbate reductase in almost all investigated plant lines. In contrast to wild type, GSNO increased superoxide dismutase activity and decreased catalase activity and chlorophyll a/b ratio in the leaves of two mutant lines. It can be assumed that tocopherols in some way are responsible for plant protection against NO-induced stress. However the mechanisms of this protection remain unknown.

[Comparative characteristic of lactate dehydrogenase properties from the white muscles and liver of goldfish (Carassius auratus L.)].

Lactate dehydrogenase (LDH) from the liver and white muscles of the gold fish Carassius auratus was partially purified by differential precipitation with ammonium sulfate. The enzyme from the liver had relatively low Vmax--1.85 +/- 0.31 U/mg protein, the muscle enzyme--3.74 +/- 0.27 U/mg protein. LDH from the liver was less sensitive to substrate inhibition (I50 - 9.92 +/- 1.09 mM) compared to white muscles isozyme (I50 - 5.87 +/- 1.39 mM). The studied isozymes had pH-dependencies with pH optima at 7.0-8.0 and 7.25-8.75 for LDH from the white muscle and liver, respectively. In this work the inactivation of partially purified LDH in the system free radicals oxidations Fe2+/H2O2 has been conducted. During incubation for 5 min of both isozymes with H2O2 (10-50 mM) and FeSO4 (20-500 microM), approximately 50% of their initial activities were lost. The level of enzyme inactivation increased with the increase of iron ion and hydrogen peroxide concentrations. During incubation in the presence of 20 microM FeSO4 and 10 mM H2O2 for 60 min white muscles isozyme lost 44% while liver isozyme--26%, independent of buffer which was used.

Regulatory protein Yap1 is involved in response of yeast Saccharomyces cerevisiae to nitrosative stress.

The goal of this work was to investigate the possible involvement of protein transcription factor Yap1 in regulation of activity of antioxidant enzymes superoxide dismutase and catalase during yeast response to nitrosative stress. It was found that the inactivation of the YAP1 gene, encoding Yap1p, cancelled the activation of superoxide dismutase and catalase by NO-donors. Then, using chimeric protein Yap1-GFP, we found the accumulation of Yap1p in the nucleus in response to nitrosative stress. Therefore, we conclude that these results in combination with previous data clearly demonstrate the involvement of Yap1p in upregulation of superoxide dismutase and catalase in yeast cells in response to nitrosative stress.

Oxidative stress in yeast.

The mechanisms of production and elimination of reactive oxygen species in the cells of the budding yeast Saccharomyces cerevisiae are analyzed. Coordinative role of special regulatory proteins including Yap1p, Msn2/4p, and Skn7p (Pos9p) in regulation of defense mechanisms in S. cerevisiae is described. A special section is devoted to two other well-studied species from the point of view of oxidative stress -- Schizosaccharomyces pombe and Candida albicans. Some examples demonstrating the use of yeast for investigation of apoptosis, aging, and some human diseases are given in the conclusion part.

[Chemotaxis of Rhizobium leguminosarum bv. viciae to organic substances].

The chemotaxis of nodule bacteria cultures of pea Rhizobium leguminosarum bv. vicia RRL1, RRL3, RRL6 i RRL7 isolated from the plants of Ivano-Frankivsk district to different organic substances was investigated. Saccharose, pyruvate and tartaric acid were the most active attractants among carbohydrates and organic acids studied for all used nodule bacteria strains. Serine, alanine, glycine, tyrosine and cysteine were the most active attractants of amino acids. Dependence of pea nodule bacteria chemotaxic activity on time of growth and attractant concentrations was revealed. The isolate specificity towards organic substances for different strains of Rhizobium leguminosarum bv. vicia was found.

Possible pathways involved in activation of catalase and superoxide dismutase with sodium nitroprusside in yeast Saccharomyces cerevisiae.

The effect of nitric oxide (*NO) on biological systems depends very much on many circumstances. Nitric oxide can activate redox sensitive pathways that in many cases results in an increase of antioxidant potential of the cell. However, the direct effects of nitric oxide on the activity of principal antioxidant enzymes such as catalase and superoxide dismutase (SOD) have not been studied. In the present work we exploited the yeast model to elucidate a possibility of regulation of the mentioned activity by NO-donor sodium nitroprusside (SNP). We demonstrated that nitric oxide spontaneously generated at SNP decomposition increased the activity of catalase and SOD 1.3 times. Using inhibitors of mRNA (actinomycin D) and protein (cycloheximide) synthesis, the strain deficient in Yap1p, a master regulator coordinating yeast adaptive response to oxidative stress, we have found that these enzymes are up-regulated via synthesis of new molecules at transcription and translation levels. This response is mediated by Yap1p. Despite the increase of SOD activity in yeast cells possibly includes the activation of the present apoprotein by Ccs1p, the ways of nitric oxide regulation of Ccs1p activity are still unclear.

[Activation of enzymes of soxRS-regulon by hydrogen peroxide in Escherichia coli].

A mutant strain (delta soxR), a derivative of strain of Escherichia coli AB 57, was obtained by general transduction. The authors have investigated the effect of oxidative stress on activity of enzymes of regulons oxyR (catalase, peroxidase, glutathione reductase) and soxRS(superoxide dismutase (SOD) and glucose-6-phosphate dehydrogenase (G6PDH) in the donor strain DJ901, recipient strain AB1157 and transductant. Oxidative stress induced by phenasine metasulfate had no effect on activity of the studied enzymes of regulon oxyR in either used strain, and on enzymes of regulon soxRS in DJ901 transductant, but caused an 1.6- and 1.3-fold increase in activity of SOD and G6PDH in AB 1157, respectively. The stress induced by 20 microM hydrogen peroxide in the last strain increased the enzyme activity of both regulons, and SOD activity increased by 28 % and that of G6PDH--by 42%. Under the same kind of stress only SOD activity increased by 20% in the transductant. Possible ways of regulation of activity in enzymes being the components of regulon soxRS are discussed.

Inactivation of genes encoding superoxide dismutase modifies yeast response to S-nitrosoglutathione-induced stress.

Antioxidant enzymes can modify cell response to nitrosative stress induced, for example, by nitric oxide or compounds decomposing with its formation. Therefore, we investigated the effects of S-nitrosoglutathione (GSNO) on cell survival, activity of antioxidant enzymes, and concentrations of reduced and oxidized glutathione in parental and isogenic strains defective in Cu,Zn- or Mn-superoxide dismutases (Cu,Zn-SOD and Mn-SOD, respectively), or in both of them. Stress was induced by incubation of the yeast with 1-20 mM GSNO. The strains used demonstrated different sensitivity to GSNO. A Cu,Zn-SOD-defective strain survived the stress better than the parental strain, while the double mutant was the most sensitive to GSNO. The (*)NO-donor at low concentrations (1-5 mM) increased SOD activity, but its high concentrations (10 and 20 mM) decreased it. The activity of catalase in all strains was enhanced by GSNO. Inhibition of protein synthesis by cycloheximide did not prevent the activation of SOD, but it prevented the activation of catalase. These facts suggest that SOD was activated at a posttranslational level and catalase activity was enhanced via de novo synthesis. A GSNO-induced increase in oxidized glutathione level in the studied yeast strains might account for cell killing by GSNO due to the development of oxidative/nitrosative stress.

Effect of VTE1 AND VTE4 gene inactivation on salt stress response in Arabidopsis thaliana.

The effect of inactivation of VTE1 and VTE4 genes, encoding enzymes involved in tocopherol biosynthesis, on concentrations of chlorophylls, carotenoids, anthocyanins and activity of catalase and guaiacol peroxidase in Arabidopsis thaliana under salt stress conditions were studied. It was shown, that the inactivation of the VTE4 gene in A. thaliana caused the decrease in concentrations of chlorophylls and carotenoids, and at the same time, inactivation of VTE1 gene resulted in 3.6-fold increase of catalase activity in comparison with the wild type. Under salt stress, the activities of guaiacol peroxidase increased in all investigated plant groups, while the concentrations of carotenoids increased only in the wild type and vte4 mutant line of A. thaliana. Salt stress did not change the concentrations of protein carbonyl groups and activities of catalase.

[Fluorescein transport and antioxidant systems in the yeast Saccharomyces cerevisiae under acid stress].

The influence of acetic acid induced stress on the activity of fluorescein extrusion system and cell survival in the yeast Saccharomyces cerevisiae has been studied. It was shown that acetic acid caused the inhibition of fluorescein efflux from the cells of both parental strain and its derivative defective in the transcriptional factor War1 which regulates the system of acetate efflux from the cell. The stress induced by 200 mM CH3COOH decreased almost 10 times the survival of strains deficient in the regulatory proteins War1 and Yap1 as compared with respective wild strains. However, pretreatment of the yeast by sublethal concentrations of hydrogen peroxide resulted in the increased resistance to acid stress. Thus it may be supposed that several systems exist which are responsible for acetate extrusion from the yeast cells. Regulatory proteins War1 and Yap1 are involved in the yeast adaptation to the stress induced by acetic acid.

[Redox-sensors of microorganisms].

This review summarizes available literature data on the existence and operation of redox sensors of microorganisms. It is partially focused on the activation by hyrdrogen peroxide OxyR protein and by superoxide anion SoxR protein in bacteria Escherichia coli and the activation by hyrdrogen peroxide and superoxide anion of Orp1-Yap1 protein system in yeast Saccharomyces cerevisiae. The similarities and peculiarities of redox signal sensing in pro- and eukaryotes have been discussed.

[Comparative characteristics of lactate dehydrogenase from the liver and white muscles of common carp (Cyprinus carpio)].

Lactate dehydrogenase from the liver and white muscles of common carp (Cyprinus carpio) was purified by the differential precipitation of supernatants with ammonium sulfate. The results of native electrophoresis of partially purified lactate degydrogenase (LDH) and LDH from some tissues exhibited the different isozyme composition there. However, the kinetic properties of these isozymes from two tissues were not different, except for the value of K(m) for pyruvate, which was 72.5 +/- 11 and 217 +/- 32 microM for the liver and muscle, respectively. LDH from the liver and white muscle had similar pH-dependences with pH-optima for the both LDH activities between 6.75 and 8.00. But studied isozymes demonstrated different thermostability. The partially purified LDH from the liver showed 450% activity after 2 h at 50 degrees C. In contrast, the partially purified LDH from the muscle exhibited about 300% activity after 3 h at 65 degrees C. The heating of partially purified LDH from the muscle caused complete purification of this isozyme.

[Acid stress in yeast Saccharomyces cerevisiae].

The data concerning the influence of weak organic acid on microorganisms, particularly yeast Saccharomyces cerevisiae are summarized in the work. The main pathways of weak organic acid arrival into the cell and their metabolism are presented. Responsible mechanisms of microorganism's resistance to the action of weak acids are described. The information regarding the role of H+-ATPase, ABC-transporters and antioxidant enzymes in yeast during acid stress adaptation is given. Special attention is paid to the role of a transport protein Pdr12 and its transcription factor War1 in the response of S. cerevisiae to the stress induced by high concentration of weak organic acid anions in the cell.