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The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that - at a subsequent stage of transfection into yeast cells - it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein's secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 µg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

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The analysis of clusters of iodine-scarce states formation among the children's population of the Saratov Region is submitted. The hygienic assessment of iodine content in raw food materials and foodstuffs grown up and made on the territory of the Saratov Region is given. It has been revealed that various food groups were characterized by a low iodine content, which was (mcg/100 g) in the bread--4.5 +/- 1.0; 3,6 +/- 0.8 in wheat grain; 2.7 +/- 0.6 in buckwheat; 2.5 +/- 1.1 in beans; 1.95 +/- 0.9 in peas. The actual iodine content in local vegetables and fruits was 1.3-1.6 fold below than the standard chemical composition according to the national tables, in river fish--twice. The quality analysis of the iodated salt sold in the trading network has been carried out. It has been found out that the use of the iodated salt with low iodine content has decreased the additional direct population risk from 12.34% in 2000 to 0.48% in 2010. For the last few years the use of the iodated salt tends to increase in the Saratov Region. In 2011 the iodated salt was used by 37.2% of urban population and 29.0% of villagers. In 2011 88.6% of school students were provided with the iodated salt in educational institutions of the area. It has been shown that the formation of mass iodine-scarce states among the population in the Saratov Region has lasted for a long time. The screening researches of urine excretion in the organized groups of surveyed children revealed an average level of iodine deficiency. In 2011 the iodine deficiency of moderate degree was registered in 18 areas of the Saratov Region, in 6--of easy degree, whereas the moderately severe deficiency areas were not registered.

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In the article there is presented an analysis of clusters of the formation of iodine deficiency in schoolchildren of the Saratov region. There is given a hygienic assessment of iodine content in food raw materials and food, grown and produced in the territory of the region. For various groups of food the low content of trace element iodine was shown to be typical. The analysis of the quality and diversity of iodized salt sold in the trading network of the region has been performed. The formation of the mass iodine deficiency states in the Saratov region was shown to bear a longtime character According to the results of urine screening studies of examined children of organized groups an average level of iodine deficiency has been revealed.

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Thymus involution is one of the most pronounced manifestations of aging immune system, associated with increase susceptibility to infections, autoimmune diseases and cancer. Its nature in normal aging is actively under investigation. Much less attention is paid to the study of mechanisms of accelerated thymic involution. Previously, we showed the connection of accelerated senescence in OXYS rats with accelerated thymic involution, the mechanisms of which remain unclear. The aim of the present work was to study multifunctional condition of thymic epithelial cells in aging OXYS rats. Immunohistochemical analysis showed the reduction of thymic epithelial cell net in OXYS rats, significant decrease in volume and surface area of epithelial cells in cortical substance as compared to control Wistar rats. Electron microscopic study revealed the marked changes of epithelial cell ultrastructure, namely the reduction of cytoplasm volume, sharp decrease in size and quantity of secretory vacuoles, the presence of multiple autophagosomes and phagolysomes. The results indicate that one of the possible mechanisms of epithelial cell net reduction in thymus of senescence-accelerated OXYS rats can be the aggravation of autophagy, probably associated with mitochondrial dysfunction typical for OXYS rats. Despite the known fact of slowing autophagy with aging in some tissues, the example of OXYS rats allows to suggest that chronic deviation of intensity of this process from physiological level, either to decrease or activation, can lead to degenerative changes in organs and finally form the progeric phenotype of the whole organism.

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Oxidative damage of biomolecules increases with age and is postulated to be a major causal factor of various neurodegenerative disorders. Consequently, the concept of neuroprotection by antioxidants has been developed. Recently we have shown that the behaviour of young senescent-accelerated OXYS rats is similar to the behaviour of old Wistar animals. To determine the role of oxidative stress in this phenomenon we investigated age-related changes in protein carbonyls (PrC), lipid peroxides (LP), reduced glutathione (GSH), alpha-tocopherol (TP) and SOD activity in the brain of OXYS and Wistar rats. We also studied the effect of long-term supplementation with bilberry extract (2g/kg of diet) and Vitamin E (140 mg/kg of diet) on oxidative stress markers and on learning in passive avoidance test. In both rat strains LP, PrC and TP increased with age and at 24 months PrC was significantly higher (p<0.0001) in OXYS rats. At 3 months GSH was higher and SOD activity was lower in OXYS rats than in Wistar rats. SOD activity decreased with age in OXYS whereas increased in Wistar rats. Cognitive impairments in OXYS rats were manifested earlier than significant differences in the level of brain oxidative stress markers between two strains. By contrast, differences in antioxidant systems of Wistar and OXYS rats were registered at 3 months. Antioxidants attenuated cognitive deficits in OXYS rats, providing evidence for therapeutic role of antioxidants. Nevertheless, the exact mechanisms of neuroprotective effects of antioxidants in vivo and the real impact of oxidative stress on the development of cognitive impairments in OXYS rats still needs to be further investigated.

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After stress (immobilization in cages) plasma corticosterone level in prematurely aging OXYS rats was higher, while the content of NO metabolites was much lower than in Wistar rats. Stress increased blood pressure in OXYS and Wistar rats, the maximum values were observed in control and stressed OXYS rats. The concentration of reduced glutathione in the brain of OXYS rats was lower than in Wistar rats. After immobilization the concentration of reduced glutathione decreased in animals of both strains. The concentration of oxidized protein increased by 1.5 times only in OXYS rats. SOD activity remained unchanged, but in OXYS rats this parameter was higher than in Wistar rats. It can be hypothesized that high blood pressure, low NO content, high corticosterone concentration, and stress-induced deficiency of the antioxidant system (or combined effects of these factors) contribute to the development of neurodegenerative changes in the brain of OXYS rats.

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The authors studied contemporary peculiarities of work conditions of individuals engaged into agriculture, cause-effect relationships in occupational morbidity and priority directions in occupational diseases prophylaxis.

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The authors studied influence of work conditions and temperature mode on functional state of stove female workers. The results include leading factor of work operations, that determinates work hardiness and jeopardy class for work conditions. Physiologic research demonstrated changes in functional state of some body systems, especially cardiovascular and neuromuscular ones.

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Seleno-organic compounds are known as efficient "scavengers" of peroxynitrite (PN). Here we studied the protective effect of selenolipoic acid (SeLA), the seleno-containing analogue of lipoic acid, on peroxynitrite-dependent inactivation of NADPH-cytochrome P450 reductase. 3-Morpholinosydnonimine hydrochloride (SIN-1) was used as a source of peroxynitrite. The reductase was irreversibly inactivated by PN generated from SIN-1. The inactivation occurred with the rate constant of about 3 x 10(4) M-1 s-1. The presence of SeLA at low concentration (0.5 microM) led to synergistic increase of the reductase inactivation by PN. Our results suggest the formation of a reactive derivative of SeLA in the reaction of SeLA with PN, probably selenolseleninate, that mediates the aggravation of reductase inactivation. In the presence of SeLA, the inactivation was reversible under the action of thiols, allowing us to conclude that the observed action of SeLA may be considered as protective.

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The formation of reactive oxygen species (ROS) in hemolymph and hemocytes of Galleria mellonella and Dendrolimus superans sibiricus larvae was studied by ESR spectroscopy using spin-trap 1-hydroxy-3-carboxy-pyrrolidine (CP-H). The background level of ROS formation was detected in the intact hemolymph. The addition of dihydroxyphenylalanine (DOPA) into free cells of the hemolymph increased CP-H oxidation about two times for G. mellonella and about four times for D. superans sibiricus. This increase was completely inhibited by a specific inhibitor of phenoloxidase, phenylthiourea. The presence of exogenous superoxide dismutase (SOD) did not change CP-H oxidation in the hemolymph. The data obtained in hemocytes showed only a DOPA-induced increase in CP-H oxidation. Phagocytosis activators did not affect ROS formation in hemocytes of both insect species. SOD decreased DOPA-induced CP-H oxidation 20-30% in suspension of hemocytes of D. superans sibiricus only. Our results are in agreement with the contribution of superoxide radical and DOPA-derived quinones/semiquinones in the immune response of insects.

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NADPH-cytochrome-P450 reductase both purified from rat hepatic microsomes and involved in microsomal fraction was inactivated by treatment with alpha-lipoic acid. Since alpha-lipoic acid contains disulfide bond, it reacts with SH-groups of the reductase via the reaction of thiol-disulfide exchange resulting in the loss of the enzyme reducing activity. NADP+ completely protected reductase from the inactivation. The modification of reductase was reversible: the modified enzyme was partially reactivated with dithiothreitol and dihydrolipoic acid in the case when cytochrome c was used as a substrate of reductase. In the case when inorganic substrate, K3Fe(CN)6, was used for assay the activity of modified reductase no reactivation was observed. It was found that the order of the reaction of inactivation of membrane-bound microsomal reductase is equal to 1.2 +/- 0.2, which is in an agreement with pseudo-first order kinetics, and the second-order-rate constant of 26 M-1min-1. The results have shown that well known therapeutic agent alpha-lipoic acid is an efficient inhibitor of both purified and microsomal reductase.

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