<Emphasis Type="Italic">Poseidonocella pacifica</Emphasis> gen. nov., sp. nov. and <Emphasis Type="Italic">Poseidonocella sedimentorum</Emphasis> sp. nov., novel alphaproteobacteria from the shallow sandy sediments of the Sea of Japan

The taxonomic study of two Gram-negative, aerobic, non-pigmented bacteria KMM 9010^T and KMM 9023^T isolated from a sandy sediment sample collected from the Sea of Japan seashore was performed. On the basis of the nearly complete 16S rRNA gene sequences, strains KMM 9010^T and KMM 9023^T clustered with the Roseobacter lineage (class Alphaproteobacteria) forming a distinct phylogenetic line adjacent to the genus Donghicola. Novel strains shared the highest sequence similarity of 96.4% to each other and lower than 96.1% similarities to other validly named genera of the class Alphaproteobacteria. In both strains, ubiquinone Q-10 was found to be the major respiratory quinone; phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidic acid, and an unknown aminolipid were the major polar lipids and C_18:1ω7c and 11-methyl C_18:1ω7c were predominant fatty acids. The DNA G+C content was 60.5 mol% (KMM 9010^T) and 65.4 mol% (KMM 9023^T). Based on phenotypic properties and phylogenetic evidence, strains KMM 9010^T and KMM 9023^T should be classified as two novel species in a new genus, Poseidonocella gen. nov., with Poseidonocella pacifica sp. nov., the type species with the type strain KMM 9010^T (= NRIC 0794^T = JCM 17310^T), and Poseidonocella sedimentorum sp. nov. as the second species with the type strain KMM 9023^T (= NRIC 0796^T = JCM 17311^T).     

Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR

Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q_D) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q_D site (MSQ_D) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with ¹H₂O/²H₂O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ_D binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in ¹H₂O and ²H₂O samples and by selective detection of the exchanged deuterons using Q-band ²H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b_D. Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q_D site are discussed, in light of the unusually high thermodynamic stability of MSQ_D.     

Hazardous alcohol consumption is a major factor in male premature mortality in a typical Russian city: prospective cohort study 2003-2009

Introduction

Russia has experienced massive fluctuations in mortality at working ages over the past three decades. Routine data analyses suggest that these are largely driven by fluctuations in heavy alcohol drinking. However, individual-level evidence supporting alcohol having a major role in Russian mortality comes from only two case-control studies, which could be subject to serious biases due to their design.

Methods and Findings

A prospective study of mortality (2003–9) of 2000 men aged 25–54 years at recruitment was conducted in the city of Izhevsk, Russia. This cohort was free from key limitations inherent in the design of the two earlier case-control studies. Cox proportional hazards regression was used to estimate hazard ratios of all-cause mortality by alcohol drinking type as reported by a proxy informant. Hazardous drinkers were defined as those who either drank non-beverage alcohols or were reported to regularly have hangovers or other behaviours related to heavy drinking episodes.Over the follow-up period 113 men died. Compared to non-hazardous drinkers and abstainers, men who drank hazardously had appreciably higher mortality (HR = 3.4, 95% CI 2.2, 5.1) adjusted for age, smoking and education. The population attributable risk percent (PAR%) for hazardous drinking was 26% (95% CI 14,37). However, larger effects were seen in the first two years of follow-up, with a HR of 4.6 (2.5, 8.2) and a corresponding PAR% of 37% (17, 51).

Interpretation

This prospective cohort study strengthens the evidence that hazardous alcohol consumption has been a major determinant of mortality among working age men in a typical Russian city. As such the similar findings of the previous case-control studies cannot be explained as artefacts of limitations of their design. As Russia struggles to raise life expectancy, which even in 2009 was only 62 years among men, control of hazardous drinking must remain a top public health priority.     

Analysis of the role of the coat protein N-terminal segment in Potato virus X virion stability and functional activity

Previously, we have reported that intact Potato virus X (PVX) virions cannot be translated in cell-free systems, but acquire this capacity by the binding of PVX-specific triple gene block protein 1 (TGBp1) or after phosphorylation of the exposed N-terminal segment of intravirus coat protein (CP) by protein kinases. With the help of in vitro mutagenesis, a nonphosphorylatable PVX mutant (denoted ST PVX) was prepared in which all 12 S and T residues in the 20-residue-long N-terminal CP segment were substituted by A or G. Contrary to expectations, ST PVX was infectious, produced normal progeny and was translated in vitro in the absence of any additional factors. We suggest that the N-terminal PVX CP segment somehow participates in virion assembly in vivo and that CP subunits in ST virions may differ in structure from those in the wild-type (UK3 strain). In the present work, to test this suggestion, we performed a comparative tritium planigraphy study of CP structure in UK3 and ST virions. It was found that the profile of tritium incorporation into ST mutant virions in some CP segments differed from that of normal UK3 virions and from UK3 complexed with the PVX movement protein TGBp1. It is proposed that amino acid substitutions in ST CP and the TGBp1-driven remodelling of UK3 virions induce structural alterations in intravirus CPs. These alterations affect the predicted RNA recognition motif of PVX CP, but in different ways: for ST PVX, labelling is increased in α-helices 6 and 7, whereas, in remodelled UK3, labelling is increased in the β-sheet strands β3, β4 and β5.     

A Comparison of Brain Gene Expression Levels in Domesticated and Wild Animals

Domestication has led to similar changes in morphology and behavior in several animal species, raising the question whether similarities between different domestication events also exist at the molecular level. We used mRNA sequencing to analyze genome-wide gene expression patterns in brain frontal cortex in three pairs of domesticated and wild species (dogs and wolves, pigs and wild boars, and domesticated and wild rabbits). We compared the expression differences with those between domesticated guinea pigs and a distant wild relative (Cavia aperea) as well as between two lines of rats selected for tameness or aggression towards humans. There were few gene expression differences between domesticated and wild dogs, pigs, and rabbits (30–75 genes (less than 1%) of expressed genes were differentially expressed), while guinea pigs and C. aperea differed more strongly. Almost no overlap was found between the genes with differential expression in the different domestication events. In addition, joint analyses of all domesticated and wild samples provided only suggestive evidence for the existence of a small group of genes that changed their expression in a similar fashion in different domesticated species. The most extreme of these shared expression changes include up-regulation in domesticates of SOX6 and PROM1, two modulators of brain development. There was almost no overlap between gene expression in domesticated animals and the tame and aggressive rats. However, two of the genes with the strongest expression differences between the rats (DLL3 and DHDH) were located in a genomic region associated with tameness and aggression, suggesting a role in influencing tameness. In summary, the majority of brain gene expression changes in domesticated animals are specific to the given domestication event, suggesting that the causative variants of behavioral domestication traits may likewise be different.     

Reversible derivatization to enhance enzymatic synthesis: chemoenzymatic synthesis of doxorubicin-14-O-esters

An efficient three-step, chemoenzymatic synthesis of unprotected doxorubicin-14-O-esters from doxorubicin hydrochloride salt is described. The key step is a lipase-catalyzed regioselective transesterification/esterification using commercially available acyl donors and doxorubicin reversibly derivatized with N-alloc to improve substrate loadings. The overall yield is ca. 60% and chromatographic purification is not required, thereby making the process more amenable to scale-up.     

Reversibility and "pH-T phase diagrams" of Rapana venosa hemocyanin and its structural subunits

We have studied the stability and reassociation behaviour of native molecules of Rapana venosa hemocyanin and its two subunits, termed RvH1 and RvH2. In the presence of different concentrations of Ca(2+) and Mg(2+) ions and pH values, the subunits differ not only in their reassociation behaviour, but also in their formation of helical tubules and multidecamers. RvH1 revealed a greater stability at higher pH values compared to RvH2. Overall, the stability of reassociated RvH and its structural subunits was found to be pH-dependent. The increasing stability of native Hc and its subunits, shown by pH-induced CD transitions (acid and alkaline denaturation), can be explained with the formation of quaternary structure. The absence of a Cotton effect at temperatures 20-40 degrees C in the pH-transition curves of RvH2 indicates that this subunit is stabilized by additional "factors", e.g.: non-ionic/hydrophobic stabilization and interactions of carbohydrate moieties. A similar behaviour was observed for the T-transition curves in a wide pH interval for RvH and its structural subunits. At higher temperatures, many of the secondary structural elements are preserved especially at neutral pH, even at extreme high temperatures above 90 degrees C the protein structures resemble a "globule state".     

N-Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface.

The primary structures of N-terminal 19-mer peptides, released by limited trypsin treatment of coat protein (CP) subunits in intact virions of three potato virus X (PVX) isolates, were analyzed. Two wild-type PVX strains, Russian (Ru) and British (UK3), were used and also the ST mutant of UK3 in which all 12 serine and threonine residues in the CP N-terminal segment were replaced by glycine or alanine. With the help of direct carbohydrate analysis and MS, it was found that the acetylated N-terminal peptides of both wild-type strains are glycosylated by a single monosaccharide residue (galactose or fucose) at NAcSer in the first position of the CP sequence, whereas the acetylated N-terminal segment of the ST mutant CP is unglycosylated. Fourier transform infrared spectra in the 1000-4000 cm(-1) region were measured for films of the intact and in situ trypsin-degraded PVX preparations at low and high humidity. These spectra revealed the presence of a broad-band in the region of valent vibrations of OH bonds (3100-3700 cm(-1)), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups. On calculating difference ('wet' minus 'dry') spectra, it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle. This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX. It is proposed that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation of a columnar-type shell from bound water molecules around the virions, which probably play a major role in maintaining the virion surface structure.     

Water permeability ofElodeacells and cytoplasm motion

The reduction of the rotational chloroplast motion velocity (CMV) during the exposure of Elodea cells to hypertonic solutions of PEG-4000 is satisfactorily described by an exponent. The dynamics of reduction correlates with the dynamic equilibration time (T_eq) between osmotic potentials of cell cytoplasm and external solution. The acceleration of the chloroplast motion, for example by gadolinium ions, results in the reduction of T_eq, and this is supposed to be due to the increase of the con tribution of the near-membrane Nernst layers to transmembrane transfer resistance resulting from the «blowing-off» of a part of the layer by moving cytoplasm.