Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis

Biological sulfide oxidation is a reaction occurring in all three domains of life. One enzyme responsible for this reaction in many bacteria has been identified as sulfide:quinone oxidoreductase (SQR). The enzyme from Rhodobacter capsulatus is a peripherally membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer. In this work, SQR from Rb. capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli. Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis. The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine. A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes. Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone. Two conserved histidine residues have been mutated individually to alanine. Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction. Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods. On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.     

CD200 is a ligand for all members of the CD200R family of immunoregulatory molecules

CD200Fc, a chimeric molecule including the extracellular domain of CD200 and a murine IgG2a Fc region, regulates immune responses following engagement of a cell surface receptor, CD200R, expressed on cells of the myeloid and T cell lineage. A recent report focused attention on a family of CD200Rs, but concluded that only one member used CD200 as its ligand. We have also cloned and sequenced a family of CD200Rs, but identify an amino terminus to two of the three isoforms not recognized by previous researchers. We show by FACS, using FITC-labeled CD200Fc, that COS7 cells transfected with all CD200R isoforms bind CD200 as ligand, although the functional consequences of this binding likely differs between the different isoforms. mAbs directed against the CD200 R1/R4 isoforms altered IL-2/IL-4 cytokine production and suppressed CTL responses in a fashion comparable to CD200Fc, with a significantly lesser effect seen following addition of anti-CD200 R2/R3.     

Transcriptional Heterogeneity of Beta Cells in the Intact Pancreas

1. Download high-res image (385KB)
2. Download full-size image

     

R-2-hydroxyglutarate attenuates aerobic glycolysis in leukemia by targeting the FTO/m6A/PFKP/LDHB axis

1. Download : Download high-res image (167KB)
2. Download : Download full-size image

     

Naturally occurring monoterpenoids in needles of Picea abies (L.) Karst

Summary An analysis was made of the effects of different sampling and extraction techniques on the amounts and pattern of monoterpenoids isolated from needles of Norway spruce. The following isolation and analysis procedure was finally adopted: liquid nitrogen-cooled needles were pulverized by a microdismembrator, extracted with pentane overnight at 2°–3°C and concentrated to a volume not less than 3 ml/g fresh weight on a Vigreux column. The crude extract was injected splitless (with solvent split) onto a cold programmed temperature vaporized (PTV) precolumn of a gas chromatograph and the vaporizable compounds heated to a capillary column. This method was tested for production of artefacts and quantitative extraction and applied to needles of eleven 80-year-old spruce trees.     

Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product.

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5')tetraphospho(5')adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.     

Construct Validity and Factor Structure of the Pittsburgh Sleep Quality Index and Epworth Sleepiness Scale in a Multi-National Study of African,South East Asian and South American College Students

Background

The Pittsburgh Sleep Quality Index (PSQI) and the Epworth Sleepiness Scale (ESS) are questionnaires used to assess sleep quality and excessive daytime sleepiness in clinical and population-based studies. The present study aimed to evaluate the construct validity and factor structure of the PSQI and ESS questionnaires among young adults in four countries (Chile, Ethiopia, Peru and Thailand).

Methods

A cross-sectional study was conducted among 8,481 undergraduate students. Students were invited to complete a self-administered questionnaire that collected information about lifestyle, demographic, and sleep characteristics. In each country, the construct validity and factorial structures of PSQI and ESS questionnaires were tested through exploratory and confirmatory factor analyses (EFA and CFA).

Results

The largest component-total correlation coefficient for sleep quality as assessed using PSQI was noted in Chile (r = 0.71) while the smallest component-total correlation coefficient was noted for sleep medication use in Peru (r = 0.28). The largest component-total correlation coefficient for excessive daytime sleepiness as assessed using ESS was found for item 1 (sitting/reading) in Chile (r = 0.65) while the lowest item-total correlation was observed for item 6 (sitting and talking to someone) in Thailand (r = 0.35). Using both EFA and CFA a two-factor model was found for PSQI questionnaire in Chile, Ethiopia and Thailand while a three-factor model was found for Peru. For the ESS questionnaire, we noted two factors for all four countries

Conclusion

Overall, we documented cross-cultural comparability of sleep quality and excessive daytime sleepiness measures using the PSQI and ESS questionnaires among Asian, South American and African young adults. Although both the PSQI and ESS were originally developed as single-factor questionnaires, the results of our EFA and CFA revealed the multi- dimensionality of the scales suggesting limited usefulness of the global PSQI and ESS scores to assess sleep quality and excessive daytime sleepiness.     

Synthesis,docking, and biological studies of phenanthrene β-diketo acids as novel HIV-1 integrase inhibitors

In the present study we report the synthesis of halogen-substituted phenanthrene β-diketo acids as new HIV-1 integrase inhibitors. The target phenanthrenes were obtained using both standard thermal- and microwave-assisted synthesis. 4-(6-Chlorophenanthren-2-yl)-2,4-dioxobutanoic acid (18) was the most active compound of the series, inhibiting both 3′-end processing (3′-P) and strand transfer (ST) with IC₅₀ values of 5 and 1.3 μM, respectively. Docking studies revealed two predominant binding modes that were distinct from the binding modes of raltegravir and elvitegravir, and suggest a novel binding region in the IN active site. Moreover, these compounds are predicted not to interact significantly with some of the key amino acids (Q148 and N155) implicated in viral resistance. Therefore, this series of compounds can further be investigated for a possible chemotype to circumvent resistance to clinical HIV-1 IN inhibitors.     

Control of pollen-mediated gene flow in transgenic trees

Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees.     

Connectivity of Caribbean coral populations: complementary insights from empirical and modelled gene flow

Understanding patterns of connectivity among populations of marine organisms is essential for the development of realistic, spatially explicit models of population dynamics. Two approaches, empirical genetic patterns and oceanographic dispersal modelling, have been used to estimate levels of evolutionary connectivity among marine populations but rarely have their potentially complementary insights been combined. Here, a spatially realistic Lagrangian model of larval dispersal and a theoretical genetic model are integrated with the most extensive study of gene flow in a Caribbean marine organism. The 871 genets collected from 26 sites spread over the wider Caribbean subsampled 45.8% of the 1900 potential unique genets in the model. At a coarse scale, significant consensus between modelled estimates of genetic structure and empirical genetic data for populations of the reef-building coral Montastraea annularis is observed. However, modelled and empirical data differ in their estimates of connectivity among northern Mesoamerican reefs indicating that processes other than dispersal may dominate here. Further, the geographic location and porosity of the previously described east-west barrier to gene flow in the Caribbean is refined. A multi-prong approach, integrating genetic data and spatially realistic models of larval dispersal and genetic projection, provides complementary insights into the processes underpinning population connectivity in marine invertebrates on evolutionary timescales.