Genome of Methylobacillus flagellatus, molecular basis for obligate methylotrophy, and polyphyletic origin of methylotrophy

Along with methane, methanol and methylated amines represent important biogenic atmospheric constituents; thus, not only methanotrophs but also nonmethanotrophic methylotrophs play a significant role in global carbon cycling. The complete genome of a model obligate methanol and methylamine utilizer, Methylobacillus flagellatus (strain KT) was sequenced. The genome is represented by a single circular chromosome of approximately 3 Mbp, potentially encoding a total of 2,766 proteins. Based on genome analysis as well as the results from previous genetic and mutational analyses, methylotrophy is enabled by methanol and methylamine dehydrogenases and their specific electron transport chain components, the tetrahydromethanopterin-linked formaldehyde oxidation pathway and the assimilatory and dissimilatory ribulose monophosphate cycles, and by a formate dehydrogenase. Some of the methylotrophy genes are present in more than one (identical or nonidentical) copy. The obligate dependence on single-carbon compounds appears to be due to the incomplete tricarboxylic acid cycle, as no genes potentially encoding alpha-ketoglutarate, malate, or succinate dehydrogenases are identifiable. The genome of M. flagellatus was compared in terms of methylotrophy functions to the previously sequenced genomes of three methylotrophs, Methylobacterium extorquens (an alphaproteobacterium, 7 Mbp), Methylibium petroleiphilum (a betaproteobacterium, 4 Mbp), and Methylococcus capsulatus (a gammaproteobacterium, 3.3 Mbp). Strikingly, metabolically and/or phylogenetically, the methylotrophy functions in M. flagellatus were more similar to those in M. capsulatus and M. extorquens than to the ones in the more closely related M. petroleiphilum species, providing the first genomic evidence for the polyphyletic origin of methylotrophy in Betaproteobacteria.     

Multisubstrate kinetics of PAH mixture biodegradation: analysis in the double-logarithmic plot

The proposed method of kinetic analysis of aqueous-phase biodegradation of polycyclic aromatic hydrocarbons (PAH) mixture presupposes representation of kinetic curves for each pair of mixture components, S _x and S _y , in double-logarithmic coordinates (ln S _x ; ln S _y ). If PAH mixture conversion corresponds to the multisubstrate model with a common active site, then the graphs in double-logarithmic coordinates are straight lines with the angular coefficients equal to the ratio of respective first-order rate constants k_x^y = fracV_y K_x K_y V_x k_{x}^{y} = {frac{{V_{y} K_{x} }}{{K_{y} V_{x} }}} , where K _x and K _y are half-saturation constants, V _x and V _y are the maximum conversion rates for substrates S _x and S _y ; the graph slope does not depend on any concentrations and remains constant during the change of reaction rates as a result of inhibition, induction/inactivation of enzymes or biomass growth. The formulated method has been used to analyze PAH mixture conversion by the culture of Sphingomonas sp. VKM B-2434. It has been shown that this process does not satisfy the multisubstrate model with a single active site. The results suggest that the strain VKM B-2434 contains at least two dioxygenases of different substrate specificity: one enzyme converts phenanthrene and fluoranthene and the other converts acenaphthene and acenaphthylene. The ratios of first-order rate constants have been obtained for these pairs of substrates.     

Downregulation of microsomal glutathione-S-transferase 1 modulates protective mechanisms in differentiated PC12 cells

Microsomal glutathione-S-transferase 1 (Mgst1) plays a specific role in protection of cells against oxidative stress. In this study, we assayed the effect of Mgst1 downregulation on cells behavior using differentiated PC12 line, a widely accepted neuronal model system. We have developed stable transfected cells with downregulated Mgst1 (PC12_M), which were differentiated with 1 mM dibutyryl-cAMP (db-cAMP). Mgst1 reduction induced necrosis, decreased ATP amount, and increased thiobarbituric acid reacting substances (TBARS) content. However, in PC12_M cell population, we detected more intensive neuritogenesis than that in mock-transfected cells. Interestingly, total glutathione as well as GSH level were significantly higher than those in control PC12 line. Real-time PCR and Western blot analyses showed elevated expression of enzymes involved in glutathione metabolism—a rate-limiting γ-glutamylcysteine ligase and glutathione reductase. The present study shows for the first time that under stress conditions induced by Mgst1 downregulation, a rescue pathway can be activated and thereby enables differentiated PC12 cells to survive. Since Mgst1expression was reported to decline with age, our results could represent a putative adaptive process during aging. It could also be an early mechanism protecting neuronal cells against some neurodegenerative insults.     

Some trends in the somatic development of children and adolescents under iodine-deficiency: materials from the Saratov region

2,106 girls and 2,169 boys from 7 to 17 were investigated in 2002-2004 in three urban settlements of the Saratov region (Povolzhje area): the town of Khvalynsk, population 15,000, with a low level of industrialization; the city of Balakovo, population 220,000, highly industrialized and with a nuclear power station; and the city of Saratov, population around 1,000,000 a regional capital, and also highly industrialized. The whole area, particularly the location of Khvalynsk, is also characterized by iodine deficiency (iodine concentration is 0.78 mkg/kg v. normal values of 5-7 mkg/kg).The program included about 30 anthropometric measurements, evaluation of developmental stages of secondary sexual characteristics, and information on menarcheal age by the status quo method. Information on parental occupation and education, as well as number of children per family was collected by questionnaire. For the analysis all the data were standardized, and further comparisons were made irrespective of age groups. The significance of differences was assessed by one-way ANOVA. For height, weight and chest circumference there are significant differences among the three populations (p < 0.001), with Khvalynsk children being the smallest in body height and weight. However, in chest circumference they are close to or even bigger (girls) than Balakovo children. The children from Khvalynsk are characterized by the lowest values for subcutaneous fat layer, both on the trunk and extremities. For the age of menarche, Khvalynsk girls have the highest values: 13 years 5 months (13.42). In Balakovo and Saratov, the corresponding figures are identical: 13 years 2 months (13.17). Secular changes in Khvalynsk and Saratov children are discussed as compared to the literature.     

Prevention of cardiolipin oxidation and fatty acid cycling as two antioxidant mechanisms of cationic derivatives of plastoquinone (SkQs)

The present state of the art in studies on the mechanisms of antioxidant activities of mitochondria-targeted cationic plastoquinone derivatives (SkQs) is reviewed. Our experiments showed that these compounds can operate as antioxidants in two quite different ways, i.e. (i) by preventing peroxidation of cardiolipin [Antonenko et al., Biochemistry (Moscow) 73 (2008) 1273–1287] and (ii) by fatty acid cycling resulting in mild uncoupling that inhibits the formation of reactive oxygen species (ROS) in mitochondrial State 4 [Severin et al. Proc. Natl. Acad. Sci. USA 107 (2009), 663–668]. The quinol and cationic moieties of SkQ are involved in cases (i) and (ii), respectively. In case (i) SkQH₂ interrupts propagation of chain reactions involved in peroxidation of unsaturated fatty acid residues in cardiolipin, the formed SkQ^? being reduced back to SkQH₂ by heme b_H of complex III in an antimycin-sensitive way. Molecular dynamics simulation showed that there are two stable conformations of SkQ1 with the quinol residue localized near peroxyl radicals at C₉ or C₁₃ of the linoleate residue in cardiolipin. In mechanism (ii), fatty acid cycling mediated by the cationic SkQ moiety is involved. It consists of (a) transmembrane movement of the fatty acid anion/SkQ cation pair and (b) back flows of free SkQ cation and protonated fatty acid. The cycling results in a protonophorous effect that was demonstrated in planar phospholipid membranes and liposomes. In mitochondria, the cycling gives rise to mild uncoupling, thereby decreasing membrane potential and ROS generation coupled to reverse electron transport in the respiratory chain. In yeast cells, dodecyltriphenylphosphonium (С₁₂TPP), the cationic part of SkQ1, induces uncoupling that is mitochondria-targeted since С₁₂TPP is specifically accumulated in mitochondria and increases the H⁺ conductance of their inner membrane. The conductance of the outer cell membrane is not affected by С₁₂TPP.     

The western and eastern roots of the Saami--the story of genetic "outliers" told by mitochondrial DNA and Y chromosomes

The Saami are regarded as extreme genetic outliers among European populations. In this study, a high-resolution phylogenetic analysis of Saami genetic heritage was undertaken in a comprehensive context, through use of maternally inherited mitochondrial DNA (mtDNA) and paternally inherited Y-chromosomal variation. DNA variants present in the Saami were compared with those found in Europe and Siberia, through use of both new and previously published data from 445 Saami and 17,096 western Eurasian and Siberian mtDNA samples, as well as 127 Saami and 2,840 western Eurasian and Siberian Y-chromosome samples. It was shown that the “Saami motif” variant of mtDNA haplogroup U5b is present in a large area outside Scandinavia. A detailed phylogeographic analysis of one of the predominant Saami mtDNA haplogroups, U5b1b, which also includes the lineages of the “Saami motif,” was undertaken in 31 populations. The results indicate that the origin of U5b1b, as for the other predominant Saami haplogroup, V, is most likely in western, rather than eastern, Europe. Furthermore, an additional haplogroup (H1) spread among the Saami was virtually absent in 781 Samoyed and Ob-Ugric Siberians but was present in western and central European populations. The Y-chromosomal variety in the Saami is also consistent with their European ancestry. It suggests that the large genetic separation of the Saami from other Europeans is best explained by assuming that the Saami are descendants of a narrow, distinctive subset of Europeans. In particular, no evidence of a significant directional gene flow from extant aboriginal Siberian populations into the haploid gene pools of the Saami was found.     

Hyperinduction of nitrilases in filamentous fungi

2-Cyanopyridine proved to act as a powerful nitrilase inducer in Aspergillus niger K10, Fusarium solani O1, Fusarium oxysporum CCF 1414, Fusarium oxysporum CCF 483 and Penicillium multicolor CCF 2244. Valeronitrile also enhanced the nitrilase activity in most of the strains. The highest nitrilase activities were produced by fungi cultivated in a Czapek-Dox medium with both 2-cyanopyridine and valeronitrile. The specific nitrilase activities of these cultures were two to three orders of magnitude higher than those of cultures grown on other nitriles such as 3-cyanopyridine or 4-cyanopyridine.     

Pallister-Killian syndrome: rapid decrease of isochromosome 12p frequency during amniocyte subculturing. Conclusion for strategy of prenatal cytogenetic diagnostics.

Pallister-Killian syndrome (PKS) is characterized cytogenetically by mosaic tetrasomy of chromosome 12p. Routine prenatal diagnosis of PKS is still complicated because of the difficulties of discriminating between the supernumerary isochromosome 12p and the duplication 21q and because of the variable level of mosaicism. The frequency of cells with an extra metacentric chromosome i(12)(p10) is usually determined by tissue-limited or tissue-specific mosaicism. We demonstrated a decrease of the abnormal clone with extra i(12p) in the amniotic fluid cells of the PKS fetus during amniocyte subculturing. The rapid loss of the i(12p) in the course of amniocyte subculturing should be the focus of attention during prenatal karyotyping. This is especially necessary for cultures with slow growth, which require further interpretation of the result during cytogenetic diagnosis of PKS.