Differential expression of mycothiol pathway genes: are they affected by antituberculosis drugs?

Mycothiol (MSH) is the major cellular thiol in Mycobacterium tuberculosis (M.tb). We hypothesize that the mycothiol-dependent detoxification pathway may serve an important role during oxygen stress management in M. tuberculosis, derived from normal aerobic metabolism, the macrophage environment and through the action of anti-tubercular antibiotics, such as Isoniazid (INH). Total mRNA and DNA were isolated from M. bovis BCG at different stages of growth in 7H9 mycobacterial medium. Three genes involved in mycothiol metabolism and encoding the enzymes mycothiol S-conjugate amidase (Mca, Rv1082), NADPH dependent mycothiol reductase (mtr, Rv2855), and N-Acetyl-1-D-myo-Inosityl-2-Amino-2-Deoxy-alpha-D-Glucopyranoside Deacetylase (GlcNAc-Ins deacetylase, Rv1170 or mshB) were investigated for genomic rearrangements and expression. The results show that the genomic domains of the genes remain conserved in evolutionary diverse and unrelated M. tuberculosis isolates. The genes encoding enzymes implicated in mycothiol reduction, mtr (Rv2855) and the mycothiol-dependant detoxification of electrophilic agents, Mca (Rv1082), are shown to be actively transcribed during logarithmic M. bovis BCG growth. The gene encoding GlcNAc-Ins deacetylase (the rate limiting mycothiol biosynthesis step) shows induction in the presence of INH. Antisense oligonucleotides to both GlcNAc-Ins deacetylase (Rv1170) and mtr (Rv2855) mRNA affect mycobacterial growth. In conclusion the results presented here suggest that these enzymes are sensitive to free radical generating antituberculosis drugs and may be useful targets for new drug development.     

Reactive oxygen species are important mediators of taurine release from skeletal muscle cells

The present study illustrates elements ofthe signal cascades involved in the activation of taurine effluxpathways in myotubes derived from skeletal muscle cells. Exposingprimary skeletal muscle cells, loaded with ¹⁴C-taurine, to1) hypotonic media, 2) the phospholipaseA₂ (PLA₂) activator melittin, 3)anoxia, or 4) lysophosphatidyl choline (LPC) causes anincrease in ¹⁴C-taurine release and a concomitantproduction of reactive oxygen species (ROS). The antioxidants butulatedhydroxy toluene and vitamin E inhibit the taurine efflux after cellswelling, anoxia, and addition of LPC. The muscle cells possess twoseparate taurine efflux pathways, i.e., a swelling- andmelittin-induced pathway that requires 5-lipoxygenase activity foractivation and a LPC-induced pathway. The two pathways aredistinguished by their opposing sensitivity toward the anion channelblocker DIDS and cholesterol. These data provide evidence forPLA₂ products and ROS as key mediators of the signalcascade leading to taurine efflux in muscle.

     

Environmental DNA metabarcoding of lake fish communities reflects long‐term data from established survey methods

Organisms continuously release DNA into their environments via shed cells, excreta, gametes and decaying material. Analysis of this ‘environmental DNA’ (eDNA) is revolutionizing biodiversity monitoring. eDNA outperforms many established survey methods for targeted detection of single species, but few studies have investigated how well eDNA reflects whole communities of organisms in natural environments. We investigated whether eDNA can recover accurate qualitative and quantitative information about fish communities in large lakes, by comparison to the most comprehensive long‐term gill‐net data set available in the UK. Seventy‐eight 2L water samples were collected along depth profile transects, gill‐net sites and from the shoreline in three large, deep lakes (Windermere, Bassenthwaite Lake and Derwent Water) in the English Lake District. Water samples were assayed by eDNA metabarcoding of the mitochondrial 12S and cytochrome b regions. Fourteen of the 16 species historically recorded in Windermere were detected using eDNA, compared to four species in the most recent gill‐net survey, demonstrating eDNA is extremely sensitive for detecting species. A key question for biodiversity monitoring is whether eDNA can accurately estimate abundance. To test this, we used the number of sequence reads per species and the proportion of sampling sites in which a species was detected with eDNA (i.e. site occupancy) as proxies for abundance. eDNA abundance data consistently correlated with rank abundance estimates from established surveys. These results demonstrate that eDNA metabarcoding can describe fish communities in large lakes, both qualitatively and quantitatively, and has great potential as a complementary tool to established monitoring methods.     

MPP: a microarray-to-phylogeny pipeline for analysis of gene and marker content datasets

MPP is a Java application, encompassing both new and established algorithms, for the analysis of gene and marker content datasets arising from high-throughput microarray techniques. MPP analyses flat file output from microarray experiments to determine the probability of the presence or absence of genes or markers within a genome. MPP can construct gene or marker content datasets for a number of genomes and can use the data to estimate an evolutionary tree or network. Results from gene content analyses may be validated by comparing them to known gene contents. MPP was initially developed to analyse data derived from comparative genome hybridization (CGH) microarray experiments in fungi and bacteria. It has recently been adapted to analyse retrotransposon-based insertion polymorphism (RBIP) marker scores derived from tagged microarray marker (TAM) experiments in pea. New analytical procedures may be added easily to MPP as plugins in order to increase the scope of the software. AVAILABILITY: MPP source code, executables and online help are available at http://cbr.jic.ac.uk/dicks/software/     

Emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants in a minority of HIV-1-infected patients following treatment with the CCR5 antagonist maraviroc is from a pretreatment CXCR4-using virus reservoir

Antagonists of the human immunodeficiency virus type 1 (HIV-1) coreceptor, CCR5, are being developed as the first anti-HIV agents acting on a host cell target. We monitored the coreceptor tropism of circulating virus, screened at baseline for coreceptor tropism, in 64 HIV-1-infected patients who received maraviroc (MVC, UK-427,857) as monotherapy for 10 days. Sixty-two patients harbored CCR5-tropic virus at baseline and had a posttreatment phenotype result. Circulating virus remained CCR5 tropic in 60/62 patients, 51 of whom experienced an HIV RNA reduction from baseline of >1 log(10) copies/ml, indicating that CXCR4-using variants were not rapidly selected despite CCR5-specific drug pressure. In two patients, viral load declined during treatment and CXCR4-using virus was detected at day 11. No pretreatment factor predicted the emergence of CXCR4-tropic virus during maraviroc therapy in these two patients. Phylogenetic analysis of envelope (Env) clones from pre- and posttreatment time points indicated that the CXCR4-using variants probably emerged by outgrowth of a pretreatment CXCR4-using reservoir, rather than via coreceptor switch of a CCR5-tropic clone under selection pressure from maraviroc. Phylogenetic analysis was also performed on Env clones from a third patient harboring CXCR4-using virus prior to treatment. This patient was enrolled due to a sample labeling error. Although this patient experienced no overall reduction in viral load in response to treatment, the CCR5-tropic components of the circulating virus did appear to be suppressed while receiving maraviroc as monotherapy. Importantly, in all three patients, circulating virus reverted to predominantly CCR5 tropic following cessation of maraviroc.     

Diversity of soluble methane monooxygenase-containing methanotrophs isolated from polluted environments

Methanotrophs were enriched and isolated from polluted environments in Canada and Germany. Enrichments in low copper media were designed to specifically encourage growth of soluble methane monooxygenase (sMMO) containing organisms. The 10 isolates were characterized physiologically and genetically with one type I and nine type II methanotrophs being identified. Three key genes: 16S rRNA; pmoA and mmoX, encoding for the particulate and soluble methane monooxygenases respectively, were cloned from the isolates and sequenced. Phylogenetic analysis of these sequences identified strains, which were closely related to Methylococcus capsulatus, Methylocystis sp., Methylosinus sporium and Methylosinus trichosporium. Diversity of sMMO-containing methanotrophs detected in this and previous studies was rather narrow, both genetically and physiologically, suggesting possible constraints on genetic diversity of sMMO due to essential conservation of enzyme function.     

Determinants of successful establishment and post‐translocation dispersal of a new population of the critically endangered St. Croix ground lizard (Ameiva polops)

Translocation to areas free of exotic predators, habitat degradation, or disease may be the most viable restoration option for many endangered species. We report on a successful translocation of the critically endangered St. Croix ground lizard, Ameiva polops, extirpated from St. Croix, U.S. Virgin Islands, Caribbean, by predation from introduced mongooses (Herpestes auropunctatus). We translocated 57 adult A. polops from Green Cay to Buck Island in May 2008. We placed 4 females and 3 males each in eight, 100 m², enclosures on Buck Island for 71 days, then the enclosures were opened. During the enclosure period, 20 individuals were identified and 32 others were seen. The average number sighted per survey was only 5.28 (range = 2–10). One hatchling was sighted in an enclosure, indicating a translocated female successfully nested. Body condition of the translocated individuals increased significantly by the end of the enclosure period. Population monitoring surveys at 61 sites across Buck Island showed that 5 years after the initial translocation in June 2013, the new population had grown to an estimated 1,473 individuals and occupied 58.9% of the island. We attribute eradication of mongoose, life history of the species, large propagule size, condition of habitat, soft‐release, use of adults, interagency collaboration, and systematic assessment as primary factors that facilitated this successful translocation. Our findings provide meaningful insights on factors that enhance the potential for successful translocations, and point to new strategies aimed at restoring populations of endangered reptiles in their native ranges.     

Evidence that IP3 and ryanodine-sensitive intra-cellular Ca2+ stores are not involved in acute hyposmotically-induced prolactin release in Tilapia.

Prolactin (PRL) cells from the euryhaline tilapia, Oreochromis mossambicus, behave like osmoreceptors by responding directly to reductions in medium osmolality with increased secretion of the osmoregulatory hormone PRL. Extracellular Ca(2+) is essential for the transduction of a hyposmotic stimulus into PRL release. In the current study, the presence and possible role of intracellular Ca(2+) stores during hyposmotic stimulation was investigated using pharmacological approaches. Changes in intracellular Ca(2+) concentration were measured with fura-2 in isolated PRL cells. Intracellular Ca(2+) stores were depleted in dispersed PRL cells with thapsigargin (1 microM) or cyclopiazonic acid (CPA, 10 microM). Pre-incubation with thapsigargin prevented the rise in [Ca(2+)](i) induced by lysophosphatidic acid (LPA, 1 microM), an activator of the IP(3) signalling cascade, but did not prevent the hyposmotically-induced rise in [Ca(2+)](i) in medium with normal [Ca(2+)] (2mM). Pre-treatment with CPA produced similar results. Prolactin release from dispersed cells followed a pattern that paralleled observed changes in [Ca(2+)](i). CPA inhibited LPA-induced prolactin release but not hyposmotically-induced release. Xestospongin C (1microM), an inhibitor of IP(3) receptors, had no effect on hyposmotically-induced PRL release. Pre-exposure to caffeine (10mM) or ryanodine (1microM) did not prevent a hyposmotically-induced rise in [Ca(2+)](i). Taken together these results indicate the presence of IP(3) and ryanodine-sensitive Ca(2+) stores in tilapia PRL cells. However, the rapid rise in intracellular [Ca(2+)] needed for acute PRL release in response to hyposmotic medium can occur independently of these intracellular Ca(2+) stores.     

Direct evidence for the presence of a rotenone-resistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria

Submitochondrial particles (SMP) were produced from Jerusalem artichoke (Helianthus tuberosus L.) mitochondria by sonication and differential centrifugation. The SMP were about 50% inside-out as measured by the access of reduced cytochrome c to cytochrome c oxidase. Uncoupled NADH oxidation (1 mM NADH) by the SMP was 120 nmol O₂ min^?1mg^?1, which was reduced to 98 nmol O₂ min^?1 (mg mitochondrial protein)^?1 in the presence of EGTA. In contrast, the oxidation of NADH by intact mitochondria was completely inhibited by EGTA (from 182 to 14 nmol O₂ min^?1mg^?1). The EGTA-resistant NADH oxidation by the SMP is ascribed to the NADH dehydrogenase(s) on the inside of the inner membrane and exposed to the medium in the inside-out SMP. In the presence of EGTA it could be shown that two NADH dehydrogenase activities were present in the SMP. One had an apparent K_m of 7 μM for NADH, a V_max of 80 nmol NADH min^?1mg^?1, and was rotenone-sensitive. This dehydrogenase is equivalent to the mammalian Complex I NADH dehydrogenase. The other dehydrogenase, which was rotenone-resistant, had a K_m of 80 μM and a V_max of 131 nmol NADH min^?1mg^?1; it is probably responsible for the rotenone-resistant oxidation of organic acids often observed in plant mitochondria. The redox poise of the pyridine nucleotides had only a small effect on the relative rates of the two internal dehydrogenases. Electron flow through these dehydrogenases appears, therefore, to be regulated mainly by the concentration of NADH in the matrix of the mitochondria.