Role of the General Base Glu-268 in Nitroglycerin Bioactivation and Superoxide Formation by Aldehyde Dehydrogenase-2

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) plays an essential role in nitroglycerin (GTN) bioactivation, resulting in formation of NO or a related activator of soluble guanylate cyclase. ALDH2 denitrates GTN to 1,2-glyceryl dinitrate and nitrite but also catalyzes reduction of GTN to NO. To elucidate the relationship between ALDH2-catalyzed GTN bioconversion and established ALDH2 activities (dehydrogenase, esterase), we compared the function of the wild type (WT) enzyme with mutants lacking either the reactive Cys-302 (C302S) or the general base Glu-268 (E268Q). Although the C302S mutation led to >90% loss of all enzyme activities, the E268Q mutant exhibited virtually unaffected rates of GTN denitration despite low dehydrogenase and esterase activities. The nucleotide co-factor NAD caused a pronounced increase in the rates of 1,2-glyceryl dinitrate formation by WT-ALDH2 but inhibited the reaction catalyzed by the E268Q mutant. GTN bioactivation measured as activation of purified soluble guanylate cyclase or release of NO in the presence of WT- or E268Q-ALDH2 was markedly potentiated by superoxide dismutase, suggesting that bioavailability of GTN-derived NO is limited by co-generation of superoxide. Formation of superoxide was confirmed by determination of hydroethidine oxidation that was inhibited by superoxide dismutase and the ALDH2 inhibitor chloral hydrate. E268Q-ALDH2 exhibited ∼50% lower rates of superoxide formation than the WT enzyme. Our results suggest that Glu-268 is involved in the structural organization of the NAD-binding pocket but is not required for GTN denitration. ALDH2-catalyzed superoxide formation may essentially contribute to oxidative stress in GTN-exposed blood vessels.Aldehyde dehydrogenases (ALDH; EC 1.2.1.3)² catalyze the oxidation of aliphatic and aromatic aldehyde substrates to the corresponding carboxylic acids with NAD(P) serving as electron accepting co-factor (1). The mitochondrial isoform (ALDH2), a homotetrameric protein with subunits of ∼54 kDa, appears to be essential for detoxification of ethanol-derived acetaldehyde, as indicated by significantly lowered alcohol tolerance of individuals expressing a low activity mutant of the protein (2, 3). Aldehyde oxidation by ALDH2 is thought to involve nucleophilic reaction of the substrate with a critical cysteine residue in the active site (Cys-302 in the human protein), resulting in formation of a thiohemiacetal intermediate, followed by hydride transfer to NAD, yielding a thioester intermediate that is hydrolyzed to the carboxylic acid product in a reaction that involves activation of H₂O by an adjacent glutamate residue (Glu-268). In addition to aldehyde oxidation, ALDH2 catalyzes ester hydrolysis (4). The esterase activity is stimulated by NAD, but the co-factor is not essential for the reaction, which is initiated by nucleophilic attack of the substrate by Cys-302, resulting in formation of a thioester and release of the corresponding alcohol by hydrolysis of the intermediate through activation of water by Glu-268 (4).The beneficial therapeutic effects of the antianginal drug GTN are thought to involve bioactivation of the organic nitrate in vascular smooth muscle to yield NO or a related species that activates sGC, resulting in cGMP-mediated vasorelaxation (5). In a seminal paper published in 2002, Stamler and co-workers (6) discovered that ALDH2 essentially contributes to vascular GTN bioactivation, and this has been confirmed in numerous later studies (for review see Ref. 7). Stamler and co-workers (6) proposed that GTN denitration involves the established esterase activity of ALDH2, i.e. nucleophilic attack of a nitro group of GTN by Cys-302, resulting in formation of a thionitrate intermediate and release of the corresponding alcohol, preferentially 1,2-glyceryl dinitrate (1,2-GDN). The thionitrate intermediate would then release nitrite either through nucleophilic attack of one of the adjacent cysteine residues (Cys-301 or Cys-303), resulting in formation of a disulfide in the active site, or through Glu-268-aided hydrolysis yielding a sulfenic acid derivative of Cys-302, which could undergo S-thiolation (8) to form a cysteinyl disulfide with one of the adjacent cysteine residues. This mechanism would be compatible both with the effect of NAD, which is not essential but increases reaction rates, and with GTN-triggered enzyme inactivation that is partially prevented by reduced thiols with two SH groups like DTT or dihydrolipoic acid. According to a brief statement in a paper on the structure of the East Asian (E487K) variant, mutation of Cys-302 and Glu-268 resulted in an almost complete loss of GTN reductase activity of ALDH2 (3), but so far the proposed role of these residues in GTN metabolism has not been thoroughly studied, and the mechanism underlying bioactivation of the nitrate is still unknown.     

Dyselectrolytemia in Chronic Obstructive Pulmonary Diseases with acute exacerbation

Chronic Obstructive Pulmonary Disease (COPD) is one of the leading causes of mortality and morbidity world wide. Due to lack of awareness about the precipitating factors and predictors of prognosis, cases of acute exacerbation of COPD often suffer the fatal outcomes. In our study we assessed the levels of serum sodium and potassium in subjects with acute episodes of COPD and their healthy controls. We found a significantly low level of serum sodium (133± 6.86 meq/lit) and potassium (3.39 ± 0.96 meq/L)) in subjects with acute exacerbation of COPD than their healthy counterparts [sodium-142 ± 2.28 meq/L and potassium- 4.52 ± 0.02 meq/L (p <0.05)]. Therefore, our study findings suggest that, serum sodium and potassium levels may get deranged in subjects with acute exacerbations of COPD which should be routinely checked for to avoid fatal outcomes.     

Combination HIV Prevention among MSM in South Africa: Results from Agent-based Modeling

HIV prevention trials have demonstrated the effectiveness of a number of behavioral and biomedical interventions. HIV prevention packages are combinations of interventions and offer potential to significantly increase the effectiveness of any single intervention. Estimates of the effectiveness of prevention packages are important for guiding the development of prevention strategies and for characterizing effect sizes before embarking on large scale trials. Unfortunately, most research to date has focused on testing single interventions rather than HIV prevention packages. Here we report the results from agent-based modeling of the effectiveness of HIV prevention packages for men who have sex with men (MSM) in South Africa. We consider packages consisting of four components: antiretroviral therapy for HIV infected persons with CD4 count <350; PrEP for high risk uninfected persons; behavioral interventions to reduce rates of unprotected anal intercourse (UAI); and campaigns to increase HIV testing. We considered 163 HIV prevention packages corresponding to different intensity levels of the four components. We performed 2252 simulation runs of our agent-based model to evaluate those packages. We found that a four component package consisting of a 15% reduction in the rate of UAI, 50% PrEP coverage of high risk uninfected persons, 50% reduction in persons who never test for HIV, and 50% ART coverage over and above persons already receiving ART at baseline, could prevent 33.9% of infections over 5 years (95% confidence interval, 31.5, 36.3). The package components with the largest incremental prevention effects were UAI reduction and PrEP coverage. The impact of increased HIV testing was magnified in the presence of PrEP. We find that HIV prevention packages that include both behavioral and biomedical components can in combination prevent significant numbers of infections with levels of coverage, acceptance and adherence that are potentially achievable among MSM in South Africa.     

Four new species of Orbiliaceae from Yunnan, China

During the fungal survey in YongPing County, Dali area in southwest China, a total of 50 specimens of Orbiliaceae were collected. Among them, were four apparently uncommon taxa growing on bark and wood of decayed branches of undetermined broad-leaved trees lying on the moist ground. They are described here as new species: Orbilia acicularis sp. nov., O. arcospora sp. nov., O. limoniformis sp. nov., and Hyalorbilia nodulosa sp. nov. Orbilia acicularis is characterized by needle-shaped straight ascospores, O. arcospora by spores being curved like an arch, O. limoniformis according to its lemon-shaped spores, and Hyalorbilia nodulosa by the knob-like glassy processes on the marginal excipular cells, the three Orbilia species also by filiform to subulate spore bodies (SBs) in the living spores.     

Risk Factors for HIV and Unprotected Anal Intercourse among Men Who Have Sex with Men (MSM) in Almaty, Kazakhstan

Introduction

Men who have sex with men (MSM) are at high risk for HIV infection. MSM in Central Asia, however, are not adequately studied to assess their risk of HIV transmission. Methods: This study used respondent driven sampling methods to recruit 400 MSM in Almaty, the largest city in Kazakhstan, into a cross-sectional study. Participation involved a one-time interviewer-administered questionnaire and rapid HIV screening test. Prevalence data were adjusted for respondent network size and recruitment patterns. Multivariate logistic regression was used to investigate the association between HIV and selected risk factors, and unprotected anal intercourse (UAI) and selected risk factors.

Results

After respondent driven sampling (RDS) weighted analysis, 20.2% of MSM were HIV-positive, and 69.0% had unprotected sex with at least one male partner in the last 12 months. Regression analysis showed that HIV infection was associated with unprotected receptive anal sex (AOR: 2.00; 95% CI: 1.04–3.84). Having unprotected anal intercourse with male partners, a measure of HIV risk behaviors, was associated with being single (AOR: 0.38; 95% CI: 0.23–0.64); very difficult access to lubricants (AOR: 11.08; 95% CI: 4.93–24.91); STI symptoms (AOR: 3.45; 95% CI: 1.42–8.40); transactional sex (AOR: 3.21; 95% CI: 1.66–6.22); and non-injection drug use (AOR: 3.10; 95% CI: 1.51–6.36).

Conclusions

This study found a high HIV prevalence among MSM in Almaty, and a population of MSM engaging in multiple high-risk behavior in Almaty. Greater access to HIV education and prevention interventions is needed to limit the HIV epidemic among MSM in Almaty.     

Colipila, a new genus in the Helotiales

Colipila, a new member of the Helotiales, is erected for two previously undescribed lignicolous species resembling Dasyscyphella and Lachnum by macroscopy. Species of Colipila are characterized by their long, entirely smooth, hyaline, thin-walled, multiseptate, subulate to basally fusoid hairs that tend to be curved on the stipe and lower flanks, and dimorphic, partly strongly protruding paraphyses which closely resemble the hairs. The type species, C. masduguana, is recorded repeatedly in southern France on rotten decorticated branches and trunks of Castanea sativa on the moist forest floor in sub-Mediterranean regions with siliceous soils, but also once on Quercus robur in a temperate forest with calcareous soil. The second species, C. pilatensis, was found on wood of an unidentified member of Rosaceae in a calcareous region of the Northern Alps and is known only from the holotype. The phylogenetic position of C. masduguana within the Helotiales was not resolved based on the analysis of nuclear LSU ribosomal DNA sequences. A key to the species of Colipila is provided.     

Wolf Lethal Control and Livestock Depredations: Counter-Evidence from Respecified Models

We replicated the study conducted by Wielgus and Peebles (2014) on the effect of wolf mortality on livestock depredations in Montana, Wyoming and Idaho states in the US. Their best models were found to be misspecified due to the omission of the time index and incorrect functional form. When we respecified the models, this replication failed to confirm the magnitude, direction and often the very existence of the original results. Wielgus and Peebles (2014) reported that the increase in the number of wolves culled the previous year would increase the expected number of livestock killed this year by 4 to 6%. But our results showed that the culling of one wolf the previous year would decrease the expected number of cattle killed this year by 1.9%, and the expected number of sheep killed by 3.4%. However, for every wolf killed there is a corresponding 2.2% increase in the expected number of sheep killed in the same year. The increase in sheep depredation appears to be a short term phenomenon.     

A Comparison of Complex Electron Donors for Anaerobic Dechlorination of PCE

The potential of sugar, flour, corn steep liquor, molasses, non-fat milk, and whey to serve as electron donors for anaerobic dechlorination of tetrachloroethene (PCE) was examined. The electron donors were compared based on acclimation time, the extent of PCE dechlorination achieved, the minimum electron donor dose necessary to achieve PCE removal, and unit cost. The time required to achieve routine dechlorination of PCE (to any daughter product) for each donor was (in days): corn steep liquor (10), milk (10), whey (10), methanol (12), molasses (14), sugar (26), flour (30). Ethene production was achieved by milk-, whey-, and methanol-fed cultures, whereas the other donors did not facilitate ethene production over a 135-day period. Corn steep liquor-, whey-, molasses-, and sugar-fed cultures needed five times the stoichiometric amount (e.g., donor per eq PCE to ethene) to facilitate PCE conversion to dichloroethene (DCE). Cultures fed milk and flour needed 20 times the stoichiometric amount, and methanol-fed cultures required 50 times the stoichiometric amount, perhaps due to competition from methanogenic organisms. Minimum laboratory-scale electron donor costs to achieve stoichiometric conversion of PCE to DCE are ($ per pound [lb] PCE) whey (0.04), molasses (0.07), milk (0.14), corn steep liquor (0.19), sugar (0.38), methanol (0.58), and flour (1.30).