Peroxide modification of skeletal muscle sarcoplasmic reticulum in antioxidant deficiency and under the action of ionol. I. Calcium transport into sarcoplasmic reticulum membranes]

It is shown that in case of antioxidant insufficiency (AOI) activation of NADPH- and ascorbate-dependent lipid peroxidation (LPO) in sarcoplasmic reticulum (SR) of skeletal muscles proceeds 1.7 and 4.1 times faster, respectively. Activation of lipid peroxidation in AOI leads to damage of Ca2+ transport processes in SR of skeletal muscles. Under these conditions ATP-dependent accumulation of 45Ca (by 88%) and Ca(2+)-ATPase (by 14%) activity in SR of skeletal muscles falls. In case of AOI a significant disturbance of passive Ca2+ transport in SR of skeletal muscles takes place, being characterized by an increased passive 45Ca output from vesicles due to breakage of the biomembrane permeability as a result of lipid peroxidation of membranes. Treatment of animals with ionol, a synthetic antioxidant, causes a decrease of activated NADPH- and ascorbate-dependent LPO in SR of skeletal muscles and stabilization of Ca2+ transport processes.     

Evaluation of the effect of late blight-resistant potato plants on the structure of bacterial associations in soil

We studied the compositions of microbial associations isolated from soils where nontransgenic and transgenic late blight-resistant lines of potato varieties Lugovskoi, Charodei, and Golubizna had been grown. The analysis was based on denaturing gradient gel electrophoresis of total amplificates of 16S rRNA gene fragments and analysis of clone libraries of nifH gene fragments. Neither method revealed significant differences in the structure of the microbial associations isolated from soils with control or transgenic plants. The minor differences detected in the microflora ranges were no greater than those in the rhizospheres of different nontransgenic potato varieties.     

Analysis of the diversity of diazotrophic bacteria in peat soil by cloning of the <Emphasis Type="Italic">nifH</Emphasis> gene

The diversity of nitrogen-fixing microorganisms in the soil of an oligotrophic Sphagnum peat bog was studied by molecular cloning of fragments of the nifH gene encoding one of the main components of the nitrogenase complex. The fragments were amplified from the DNA isolated from the peat samples collected at the same site in January (library I) and November (library II), 2005. Analysis of the nifH sequence libraries revealed high diversity of diazotrophic bacteria in peat soil: the first library consisted of 237 clones and 55 unique sequence types, the second one included 171 clones and 52 sequence types. Comparison of the two clone libraries showed that the composition and population structure of the nitrogen-fixing community depended greatly on the sampling time; they shared only 11 phylotypes. The sequences of representatives of the class Alphaproteobacteria prevailed in both libraries (27% and 57% of clones in libraries I and II, respectively). Representatives of the classes Deltaproteobacteria and Chlorobea were minor components of library I (6% and 7% of clones, respectively), whereas they prevailed in library II (18% and 24% of clones, respectively). Members of the class Chloroflexi were present only in library I, while members of the classes Bacilli, Clostridia, and Methanomicrobia were present only in library II. Our studies demonstrated that, for complete evaluation of the diversity of natural nitrogen-fixing communities, nifH libraries should consist of at least 200–300 clones.     

The effect of alpha-tocopherol and ionol on the physical structure of the membranes of rat liver microsomes under conditions of antioxidant insufficiency

Physiochemical conformity of the alpha-tocopherol interaction with hepatic microsomal membranes has been studied by means of fluorescent probes (pyrene and 1-anilinonaphthalene-8-sulphonate). The microsomal membrane microviscosity is shown to sharply decrease under conditions of the antioxidant deficiency with vitamin E expelled into animals normalizes microviscosity, but feebly influences the microsomal surface charge. Microcalorimetry has been used to establish that penetration of tocopherol into microsomal membranes was accompanied by the exothermic effect.     

Peroxide modification of membranes and isomorphic composition of cytochrome P-450 of rat liver microsomes during antioxidant deficiency]

Lipid peroxidation (LPO), physico-chemical properties of the membranes and isoformic composition of microsomal cytochrome P-450 from the rat liver were studied under conditions of antioxidant insufficiency (AOI) which was modelled by exclusion of alpha-tocopherol from the animals' ration. An insignificant accumulation of microsomal diene conjugates and schiff bases against a sharp increase of the ability to the prooxidant stimulated LPO in vitro took place. A significant decrease of membrane lipid microviscosity and a change in surface properties of microsomal membranes of rats with AOI was determined. Absence of alpha-tocopherol in the ration was accompanied by a significant change in the content of separate isoforms of cytochrome P-450 exhibited in growth of a polypeptide with m. w. 54 kDa and the lowering of proteins with m. w. 48 and 50 kDa. Less intensive quenching of tryptophan fluorescence by acrylamide was also revealed, which testified to a lower accessibility of the quencher to membrane proteins or their fluorophore sites. Modification of lipid composition and of physicochemical properties of the rat liver membrane microsomes which was observed at AOI was significantly correlated by pretreatment with the antioxidant 4-methyl-2,6-ditretbutylphenol (ionol).     

The role of peroxidation processes and antioxidant protection in nitrite-induced hypoxia and its correction with vitamins]

Different doses of sodium nitrite were studied for their action in acute and chronic experiments on rats. Nitrite (NaNO2) hypoxia in rats was simulated to show how the methemoglobin (MtHb) level in blood depends on NaNO2 doses and the method of introduction. Lethal and sublethal doses of NaNO2 (50% of MtHb and more) promoted a decrease of lipid peroxidation (LP) in the liver microsomes, while the average and easy level of hypoxia activated it. Introduction of NaNO2 has led to dose-dependent activation of superoxide dismutase (SOD) in the liver, blood and heart tissues as well as to disturbances in the DNA structure. An average level (40 mg NaNO2 per kg of rat weight daily during one month) of chronic nitrite hypoxia has led to the same changes of metabolism as acute one. Vitamin E normalized LP, but not the MtHb level.     

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.     

Natronobacillus azotifigens gen. nov., sp. nov., an anaerobic diazotrophic haloalkaliphile from soda-rich habitats

Gram-positive bacteria capable of nitrogen fixation were obtained in microoxic enrichments from soda soils in south-western Siberia, north-eastern Mongolia, and the Lybian desert (Egypt). The same organisms were obtained in anoxic enrichments with glucose from soda lake sediments in the Kulunda Steppe (Altai, Russia) using nitrogen-free alkaline medium of pH 10. The isolates were represented by thin motile rods forming terminal round endospores. They are strictly fermentative saccharolytic anaerobes but tolerate high oxygen concentrations, probably due to a high catalase activity. All of the strains are obligately alkaliphilic and highly salt-tolerant natronophiles (chloride-independent sodaphiles). Growth was possible within a pH range from 7.5 to 10.6, with an optimum at 9.5–10, and within a salt range from 0.2 to 4 M Na⁺, with an optimum at 0.5–1.5 M for the different strains. The nitrogenase activity in the whole cells also had an alkaline pH optimum but was much more sensitive to high salt concentrations compared to the growing cells. The isolates formed a compact genetic group with a high level of DNA similarity. Phylogenetic analysis based on 16S-rRNA gene sequences placed the isolates into Bacillus rRNA group 1 as a separate lineage with Amphibacillus tropicus as the nearest relative. In all isolates the key functional nitrogenase gene nifH was detected. A new genus and species, Natronobacillus azotifigens gen. nov., sp. nov., is proposed to accommodate the novel diazotrophic haloalkaliphiles. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The GenBank accession numbers for the 16S rRNA gene of the novel strains are EU143681-EU143690 and EU850814-EU850816; for the nifH gene the accession numbers are EU542601, EU563380-EU563386 and EU850817-EU850819.