Probing Reactivity and Substrate Specificity of Both Subunits of the Dimeric \u3ci\u3eMycobacterium tuberculosis\u3c/i\u3e FabH Using alkyl-CoA Disulfide Inhibitors and acyl-CoA Substrates

The dimeric Mycobacterium tuberculosis FabH (mtFabH) catalyses a Claisen-type condensation between an acyl-CoA and malonyl-acyl carrier protein (ACP) to initiate the Type II fatty acid synthase cycle. To analyze the initial covalent acylation of mtFabH with acyl-CoA, we challenged it with mixture of C6-C20 acyl-CoAs and the ESI-MS analysis showed reaction at both subunits and a strict specificity for C12 acyl CoA. Crystallographic and ESI-MS studies of mtFabH with a decyl-CoA disulfide inhibitor revealed a decyl chain bound in acyl-binding channels of both subunits through disulfide linkage to the active site cysteine. These data provide the first unequivocal evidence that both subunits of mtFabH can react with substrates or inhibitor. The discrepancy between the observed C12 acyl-CoA substrate specificity in the initial acylation step and the higher catalytic efficiency of mtFabH for C18-C20 acyl-CoA substrates in the overall mtFabH catalyzed reaction suggests a role for M. tuberculosis ACP as a specificity determinant in this reaction

Separate Entrance and Exit Portals for Ligand Traffic in Mycobacterium tuberculosis FabH

SummaryMycobacterium tuberculosis FabH initiates type II fatty acid synthase-catalyzed formation of the long chain (C16–C22) acyl-coenzyme A (CoA) precursors of mycolic acids, which are major constituents of the bacterial cell envelope. Crystal structures of M. tuberculosis FabH (mtFabH) show the substrate binding site to be a buried, extended L-shaped channel with only a single solvent access portal. Entrance of an acyl-CoA substrate through the solvent portal would require energetically unfavorable reptational threading of the substrate to its reactive position. Using a class of FabH inhibitors, we have tested an alternative hypothesis that FabH exists in an “open” form during substrate binding and product release, and a “closed” form in which catalysis and intermediate steps occur. This hypothesis is supported by mass spectrometric analysis of the product profile and crystal structures of complexes of mtFabH with these inhibitors

Study on stress-strain state and deformations occurring in existing roller tables

The design of a new diverting roller table is presented, containing continuous series of sections with diverting rollers. Using the program product of finite element analysis of Autodesk Inventor, the stress-strain state rollers of the new outrigger roller table is calculated. It is proved that the maximum concentrations of stresses and deformations are observed in barrels and necks of rollers a new outrigger roller table. At the same time, the value of these indices is much smaller in comparison with the values of stresses and deformations occurring in existing roller tables

Plastid DNA sequence diversity in wild grapevine samples (Vitis vinifera subsp. sylvestris) from the Caucasus region

DNA sequence diversity was investigated in three plastid regions (the trnH-psbA intergenic spacer, accDpsaI intergenic spacer and the rpl16 intron) in a group of 40 wild grape (Vitis vinifera subsp. sylvestris) samples from the South Caucasus. This group included 22 samples from Georgia, 9 samples from Azerbaijan, 2 samples from Armenia and 7 samples from Turkey. The South Caucasus region is widely believed to be the area in which grape domestication began, and the study of genetic diversity in this region is viewed as key to understanding grape domestication in general. Four plastid haplotypes are evident in the 40 samples, and are designated by their character states at each of the 4 polymorphic positions: AAAT – 22 samples, ATTT – 6 samples, GTAC – 1 sample, and ATAT – 11 samples. The AAAT haplotype is restricted to Georgia and Azerbaijan, the ATAT haplotype is distributed across the entire study area, the ATTT haplotype is distributed in the southern part of the study area from the Black Sea to the Caspian Sea. The single GTAC haplotype was only found in southwestern Georgia. The AAAT haplotype is restricted to both wild (V. vinifera subsp. sylvestris) and cultivated (V. vinifera subsp. vinifera) grape samples from the Caucasus. This observation and the presence of all other plastid haplotypes observed in a previous study of worldwide grape cultivars highlight both unique and high levels of genetic variation in wild grape (V. vinifera subsp. sylvestris) from the greater Caucasus region.

ЭМБРИОТРОПНЫЕ СВОЙСТВА НОВОЙ КОМПОЗИЦИИ ФЕНБЕНДАЗОЛА И АЛБЕНДАЗОЛА (ПАНАВЕРМ ПЛЮС)

The drug Panaverm plus administered at the doses of 10,0; 15,0; 25,0; 35,0 and 45,0 mg/kg body weight (by 4.5 times increased dose) showed no embryotropic activity. Panaverm plus used at the dose of 55,0 mg / kg body weight did not cause changes in the development  of embryos, except  one case of hydrocephaly and reduction of embryo weight. Objective of research: studies of the embryotropic properties of preparation Panaverm plus. Materials and methods: The  studies of embryotropic properties of  preparation  Panaverm plus were conducted on 40 outbreed white female rats with weight 200-250 g. in accordance with «Methodical recommendations  for estimation  of drug effects on reproductive function of animals»  approved by Ministry of Healthcare of the Russian Federation (1997). Results and conclusion: Panaverm plus administered at the doses of 10,0; 15,0; 25,0; 35,0 and 45,0 mg/kg body weight (by 4.5 times increased dose) showed no embryotropic activity. Panaverm plus used at the dose of 55,0 mg / kg body weight did not cause changes in the development of embryos, except one case of hydrocephaly and reduction of embryo weight. Цель исследования  – изучение эмбриотропных свойств препарата панаверм плюс на основе фенбендазола и альбендазола. Материалы и методы. Исследования  по изучению эмбриотропных свойств панаверма плюс проводили на 40 беспородных белых самках крыс массой  200–250 г согласно нормативно-методического документа «Методические  рекомендации по  оценке  влияния  препаратов  на генеративную функцию животных», одобренного Минздравом РФ (1997). Результаты и обсуждение. Панаверм плюс после назначения в дозах 10, 15, 25, 35 и  45 мг/кг массы тела (в 4,5 раза увеличенной дозе) не проявил эмбриотропную активность. Доза панаверма плюс 55 мг/кг массы тела не вызывала изменений в развитии эмбрионов, за исключением одного случая гидроцефалии и снижения массы эмбриона. 

Drosophila selenophosphate synthetase 1 regulates vitamin B6 metabolism: prediction and confirmation

<p>Abstract</p> <p>Background</p> <p>There are two selenophosphate synthetases (SPSs) in higher eukaryotes, SPS1 and SPS2. Of these two isotypes, only SPS2 catalyzes selenophosphate synthesis. Although SPS1 does not contain selenophosphate synthesis activity, it was found to be essential for cell growth and embryogenesis in <it>Drosophila</it>. The function of SPS1, however, has not been elucidated.</p> <p>Results</p> <p>Differentially expressed genes in <it>Drosophila </it>SL2 cells were identified using two-way analysis of variance methods and clustered according to their temporal expression pattern. Gene ontology analysis was performed against differentially expressed genes and gene ontology terms related to vitamin B6 biosynthesis were found to be significantly affected at the early stage at which megamitochondria were not formed (day 3) after <it>SPS1 </it>knockdown. Interestingly, genes related to defense and amino acid metabolism were affected at a later stage (day 5) following knockdown. Levels of pyridoxal phosphate, an active form of vitamin B6, were decreased by <it>SPS1 </it>knockdown. Treatment of SL2 cells with an inhibitor of pyridoxal phosphate synthesis resulted in both a similar pattern of expression as that found by <it>SPS1 </it>knockdown and the formation of megamitochondria, the major phenotypic change observed by <it>SPS1 </it>knockdown.</p> <p>Conclusions</p> <p>These results indicate that SPS1 regulates vitamin B6 synthesis, which in turn impacts various cellular systems such as amino acid metabolism, defense and other important metabolic activities.</p

Structured headache services as the solution to the ill-health burden of headache: 1. Rationale and description

In countries where headache services exist at all, their focus is usually on specialist (tertiary) care. This is clinically and economically inappropriate: most headache disorders can effectively and more efficiently (and at lower cost) be treated in educationally supported primary care. At the same time, compartmentalizing divisions between primary, secondary and tertiary care in many health-care systems create multiple inefficiencies, confronting patients attempting to navigate these levels (the “patient journey”) with perplexing obstacles. High demand for headache care, estimated here in a needs-assessment exercise, is the biggest of the challenges to reform. It is also the principal reason why reform is necessary. The structured headache services model presented here by experts from all world regions on behalf of the Global Campaign against Headache is the suggested health-care solution to headache. It develops and refines previous proposals, responding to the challenge of high demand by basing headache services in primary care, with two supporting arguments. First, only primary care can deliver headache services equitably to the large numbers of people needing it. Second, with educational supports, they can do so effectively to most of these people. The model calls for vertical integration between care levels (primary, secondary and tertiary), and protection of the more advanced levels for the minority of patients who need them. At the same time, it is amenable to horizontal integration with other care services. It is adaptable according to the broader national or regional health services in which headache services should be embedded. It is, according to evidence and argument presented, an efficient and cost-effective model, but these are claims to be tested in formal economic analyses

Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus

Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.This work was supported by the Medical Research Council (MRC) grant MR/N022890/1 to EH and grant 1001764 to RML; National Institutes of Health (NIH) grant RO1-GM09285 to CRE; Spanish Ministry of Economy and competitiveness (MINECO) grant BIO2013-46414-P to ML and AFM is supported by a Doc.Mobility fellowship from the Swiss National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript