Nitric oxide scavenging by the cobalamin precursor cobinamide

Nitric oxide (NO) is an important signaling molecule, and a number of NO synthesis inhibitors and scavengers have been developed to allow study of NO functions and to reduce excess NO levels in disease states. We showed previously that cobinamide, a cobalamin (vitamin B12) precursor, binds NO with high affinity, and we now evaluated the potential of cobinamide as a NO scavenger in biologic systems. We found that cobinamide reversed NO-stimulated fluid secretion in Drosophila Malpighian tubules, both when applied in the form of a NO donor and when produced intracellularly by nitricoxide synthase. Moreover, feeding flies cobinamide markedly attenuated subsequent NO-induced increases in tubular fluid secretion. Cobinamide was taken up efficiently by cultured rodent cells and prevented NO-induced phosphorylation of the vasodilator-stimulated phosphoprotein VASP both when NO was provided to the cells and when NO was generated intracellularly. Cobinamide appeared to act via scavenging NO because it reduced nitrite and nitrate concentrations in both the fly and mammalian cell systems, and it did not interfere with cGMP-induced phosphorylation of VASP. In rodent and human cells, cobinamide exhibited toxicity at concentrations > or =50 microM with toxicity completely prevented by providing equimolar amounts of cobalamin. Combining cobalamin with cobinamide had no effect on the ability of cobinamide to scavenge NO. Cobinamide did not inhibit the in vitro activity of either of the two mammalian cobalamin-dependent enzymes, methionine synthase or methylmalonyl-coenzyme A mutase; however, it did inhibit the in vivo activities of the enzymes in the absence, but not presence, of cobalamin, suggesting that cobinamide toxicity was secondary to interference with cobalamin metabolism. As part of these studies, we developed a facile method for producing and purifying cobinamide. We conclude that cobinamide is an effective intra- and extracellular NO scavenger whose modest toxicity can be eliminated by cobalamin.     

Construction of the model for the Genetic Analysis Workshop 14 simulated data: genotype-phenotype relationships,gene interaction,linkage, association,disequilibrium, and ascertainment effects for a complex phenotype

The Genetic Analysis Workshop 14 simulated dataset was designed 1) To test the ability to find genes related to a complex disease (such as alcoholism). Such a disease may be given a variety of definitions by different investigators, have associated endophenotypes that are common in the general population, and is likely to be not one disease but a heterogeneous collection of clinically similar, but genetically distinct, entities. 2) To observe the effect on genetic analysis and gene discovery of a complex set of gene x gene interactions. 3) To allow comparison of microsatellite vs. large-scale single-nucleotide polymorphism (SNP) data. 4) To allow testing of association to identify the disease gene and the effect of moderate marker x marker linkage disequilibrium. 5) To observe the effect of different ascertainment/disease definition schemes on the analysis. Data was distributed in two forms. Data distributed to participants contained about 1,000 SNPs and 400 microsatellite markers. Internet-obtainable data consisted of a finer 10,000 SNP map, which also contained data on controls. While disease characteristics and parameters were constant, four "studies" used varying ascertainment schemes based on differing beliefs about disease characteristics. One of the studies contained multiplex two- and three-generation pedigrees with at least four affected members. The simulated disease was a psychiatric condition with many associated behaviors (endophenotypes), almost all of which were genetic in origin. The underlying disease model contained four major genes and two modifier genes. The four major genes interacted with each other to produce three different phenotypes, which were themselves heterogeneous. The population parameters were calibrated so that the major genes could be discovered by linkage analysis in most datasets. The association evidence was more difficult to calibrate but was designed to find statistically significant association in 50% of datasets. We also simulated some marker x marker linkage disequilibrium around some of the genes and also in areas without disease genes. We tried two different methods to simulate the linkage disequilibrium.     

Anthelmintic efficacy of genistein, the active principle of Flemingia vestita (Fabaceae): alterations in the free amino acid pool and ammonia levels in the fluke, Fasciolopsis buski

The crude root-peel extract of Flemingia vestita, its active principle genistein and the reference flukicide oxyclozanide were tested against Fasciolopsis buski, the giant intestinal trematode. The amino acid composition of F. buski was demonstrated using HPLC and it was observed that the free amino acid (FAA) pool of the control worm consisted of aspartate, threonine, serine, glutamic acid, glutamine, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, lysine, histidine, arginine, phosphoserine, taurine, citrulline, ornithine, β-alanine, and γ-amino butyric acid (GABA). Of the amino acids detected valine was found to be the maximum in quantitative analysis. In qualitative analysis the FAA pool of the parasites under various treatments remained same as that of the control; however, quantitatively the level of various FAAs in the parasite was significantly affected. The treated parasites showed a marked decrease in the levels of arginine, ornithine, tyrosine, leucine, isoleucine, valine, alanine, glycine, proline, serine, threonine, and taurine following treatment with 20 mg/ml of crude peel extract, 0.5 mg/ml of genistein and 20 mg/ml of the reference drug, though an increase in the levels of glutamic acid, glutamine, phosphoserine, citrulline and GABA was noticeable. Enhanced levels of GABA and citrulline under the influence of genistein may be implicated in alterations of nitric oxide release and consequent neurological change (e.g. paralysis) in the parasite. Ammonia in the tissue homogenate as well as in the incubation medium showed a quantitative increase compared to the controls after treatment with the various test materials. The ammonia level increased by 40.7%, 66.4% and 18.16% in treatments with F. vestita, genistein and oxyclozanide, respectively, at the mentioned dosages. The changes in the levels of the amino acids and nitrogen components post treatment suggest that the amino acid metabolism in the parasite may have been altered under the influence of the test materials.     

Prophylactic Efficacy of <Emphasis Type="Italic">Coriandrum sativum</Emphasis> (Coriander) on Testis of Lead-Exposed Mice

Lead poisoning is a worldwide health problem, and its treatment is under investigation. The aim of this study was to access the efficacy of Coriandrum sativum (coriander) in reducing lead-induced changes in mice testis. Animal exposed to lead nitrate showed significant decrease in testicular SOD, CAT, GSH, total protein, and tissue lead level. This was accompanied by simultaneous increase in the activities of LPO, AST, ALT, ACP, ALP, and cholesterol level. Serum testosterone level and sperm density were suppressed in lead-treated group compared with the control. These influences of lead were prevented by concurrent daily administration of C. sativum extracts to some extent. Treating albino mice with lead-induced various histological changes in the testis and treatment with coriander led to an improvement in the histological testis picture. The results thus led us to conclude that administration of C. sativum significantly protects against lead-induced oxidative stress. Further work need to be done to isolate and purify the active principle involved in the antioxidant activity of this plant.     

Isolation and functional characterization of DNA damage repair protein (DRT) from Lepidium latifolium L.

We have isolated and in silico characterized a cold regulated plastocyanin encoding gene from Lepidium latifolium L designated as LlaDRT. Its cDNA sequence (JN214346) consists of a 504 bp ORF, 48 and 205 bp of 5′ and 3′ UTR regions, respectively encoding a protein of 17.07 KDa and pI 4.95. In silico and phylogenetic analysis of LlaDRT suggested that the protein has features of a typical plastocyanin family member and of a nearest relative of the predominant isoform of Arabidopsis (PETE2) plastocyanin. Validation of stress response of LlaDRT by qPCR under different abiotic stress regulators viz salicylic acid, jasmonic acid, calcium chloride, ethylene and abscisic acid revealed its possible regulation and crosstalk amongst different pathways.     

Costs and Impacts of Scaling up Voluntary Medical Male Circumcision in Tanzania

Background

Given the proven effectiveness of voluntary medical male circumcision (VMMC) in preventing the spread of HIV, Tanzania is scaling up VMMC as an HIV prevention strategy. This study will inform policymakers about the potential costs and benefits of scaling up VMMC services in Tanzania.

Methodology

The analysis first assessed the unit costs of delivering VMMC at the facility level in three regions—Iringa, Kagera, and Mbeya—via three currently used VMMC service delivery models (routine, campaign, and mobile/island outreach). Subsequently, using these unit cost data estimates, the study used the Decision Makers'' Program Planning Tool (DMPPT) to estimate the costs and impact of a scaled-up VMMC program.

Results

Increasing VMMC could substantially reduce HIV infection. Scaling up adult VMMC to reach 87.9% coverage by 2015 would avert nearly 23,000 new adult HIV infections through 2015 and an additional 167,500 from 2016 through 2025—at an additional cost of US$253.7 million through 2015 and US$302.3 million from 2016 through 2025. Average cost per HIV infection averted would be US$11,300 during 2010–2015 and US$3,200 during 2010–2025. Scaling up VMMC in Tanzania will yield significant net benefits (benefits of treatment costs averted minus the cost of performing circumcisions) in the long run—around US$4,200 in net benefits for each infection averted.

Conclusion

VMMC could have an immediate impact on HIV transmission, but the full impact on prevalence and deaths will only be apparent in the longer term because VMMC averts infections some years into the future among people who have been circumcised. Given the health and economic benefits of investing in VMMC, the scale-up of services should continue to be a central component of the national HIV prevention strategy in Tanzania.     

The peculiar biology of mouse mesenchymal stromal cells—oxygen is the key

Because of the ability to manipulate their genome, mice are the experimental tool of choice for many areas of scientific investigation. Moreover, established experimental mouse models of human disease are widely available and offer a valuable resource to obtain proof-of-concept for many cell-based therapies. Nevertheless, efforts to establish reliable methods to isolate mesenchymal stromal cells (MSCs) from mouse bone marrow have been elusive. Indeed, a variety of physical and genetic approaches have been described to fractionate MSCs from other cell lineages in bone marrow, but few have achieved high yields or purity while maintaining the genomic integrity of the cells. We provide a historic overview of published procedures dedicated to the isolation of mouse MSCs from bone marrow and compact bone. We also review current findings indicating that growth-restrictive conditions imposed by atmospheric oxygen promotes immortalization of mouse MSCs and how expansion in a low-oxygen environment enhances cell yields and maintains genomic stability. Finally, we provide basic recommendations for isolating primary mouse MSCs and discuss potential pitfalls associated with these isolation methods.     

Design,synthesis and preliminary screening of novel 3-(2-N,N-dimethylaminoethylthio) indole derivatives as potential 5-HT6 receptor ligands

The synthesis and potential 5-hydroxytryptamine₆ receptor (5-HT₆R) antagonist activity of a novel series of N-arylsulfonyl-3-(2-N,N-dimethylaminoethylthio) indoles has been reported. The molecular modeling, synthesis and in-vitro radioligand binding data of this series are discussed. The present article describes 37 derivatives of the title series. It was observed that the increased side-chain length with the insertion of a sulfur atom did not lead to the loss of binding affinity of these compounds, although the affinities were reduced. The compounds exhibited moderate affinity and selectivity to human 5-HT₆ receptors.