Interactive Effects of Viral and Bacterial Production on Marine Bacterial Diversity

A general model of species diversity predicts that the latter is maximized when productivity and disturbance are balanced. Based on this model, we hypothesized that the response of bacterial diversity to the ratio of viral to bacterial production (VP/BP) would be dome-shaped. In order to test this hypothesis, we obtained data on changes in bacterial communities (determined by terminal restriction fragment length polymorphism of 16S rRNA gene) along a wide VP/BP gradient (more than two orders of magnitude), using seawater incubations from NW Mediterranean surface waters, i.e., control and treatments with additions of phosphate, viruses, or both. In December, one dominant Operational Taxonomic Unit accounted for the major fraction of total amplified DNA in the phosphate addition treatment (75±20%, ± S.D.), but its contribution was low in the phosphate and virus addition treatment (23±19%), indicating that viruses prevented the prevalence of taxa that were competitively superior in phosphate-replete conditions. In contrast, in February, the single taxon predominance in the community was held in the phosphate addition treatment even with addition of viruses. We observed statistically robust dome-shaped response patterns of bacterial diversity to VP/BP, with significantly high bacterial diversity at intermediate VP/BP. This was consistent with our model-based hypothesis, indicating that bacterial production and viral-induced mortality interactively affect bacterial diversity in seawater.     

Synthesis and antimicrobial activities of N6-hydroxyagelasine analogs and revision of the structure of ageloximes

(+)-N⁶-Hydroxyagelasine D, the enantiomer of the proposed structure of (?)-ageloxime D, as well as N⁶-hydroxyagelasine analogs were synthesized by selective N-7 alkylation of N⁶-[tert-butyl(dimethyl)silyloxy]-9-methyl-9H-purin-6-amine in order to install the terpenoid side chain, followed by fluoride mediated removal of the TBDMS-protecting group. N⁶-Hydroxyagelasine D and the analog carrying a geranylgeranyl side chain displayed profound antimicrobial activities against several pathogenic bacteria and protozoa and inhibited bacterial biofilm formation. However these compounds were also toxic towards mammalian fibroblast cells (MRC-5). The spectral data of N⁶-hydroxyagelasine D did not match those reported for ageloxime D before. Hence, a revised structure of ageloxime D was proposed. Basic hydrolysis of agelasine D gave (+)-N-[4-amino-6-(methylamino)pyrimidin-5-yl]-N-copalylformamide, a compound with spectral data in full agreement with those reported for (?)-ageloxime D.     

AMP-activated protein kinase phosphorylates and inactivates liver glycogen synthase

Recombinant muscle GYS1 (glycogen synthase 1) and recombinant liver GYS2 were phosphorylated by recombinant AMPK (AMP-activated protein kinase) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favourable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (UDP-glucose) [assayed in the absence of Glc-6-P (glucose-6-phosphate)] and Glc-6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with the pharmacological AMPK activators AICA riboside (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by glucagon treatment was transient. In hepatocytes from mice harbouring a liver-specific deletion of the AMPK catalytic α1/α2 subunits, GYS2 inactivation by AICA riboside and A769662 was blunted, whereas inactivation by glucagon was unaffected. The results suggest that GYS inactivation by AMPK activators in hepatocytes is due to GYS2 Ser7 phosphorylation.     

Compartmentation of newly synthesized phosphatidylethanolamine in rat brain microsomes

Summary The compartmentation of the phosphatidylethanolamine newly synthesized in brain microsomesin vitro either by base exchange or net synthesis has been studied, using difluorodinitrobenzene as a chemical probe. The experimental results demonstrate that in rat brain microsomes the phosphatidylethanolamine molecules synthesized by base exchange and the bulk membrane lipid belong to different pools. Ca²⁺ bound to microsomes seems to be involved in the maintenance of the compartmentation of phosphatidylethanolamine. In the presence of Ca²⁺ the newly synthesized phosphatidylethanolamine molecules react with difluorodinitrobenzene as though they are organized in clusters. After biosynthesisin vivo orin vitro through the cytidine pathway, the compartmentation of the newly formed phosphatidylethanolamine appears less marked than after the synthesis through base exchange.     

Increase in stretch-induced rhythmic motor activity in the diabetic rat colon is associated with loss of ICC of the submuscular plexus

Diabetes affects many aspects of gastrointestinal motility, in part due to changes in interstitial cells of Cajal (ICC). The effect of diabetes on the colon, however, is not well characterized, and the aim of the present study was to investigate possible relationships between altered colonic motility as a consequence of streptozotocin-induced diabetes and injury to ICC. Physiological, immunohistochemical, and ultrastructural techniques were employed. The motor pattern of the rat colon was dominated by rhythmic high-amplitude, low-frequency contractions that were primarily myogenic in origin. These rhythmic contractions were induced by stretch associated with increased tension; the amplitude of the superimposed rhythmic contractions increased with increasing applied tension. In diabetic rats, the stretch-induced rhythmic contractile activity remained robust and of similar frequency but was significantly higher in amplitude compared with that in control rats. At 700 mg of applied tension, the force of contraction in circular colonic muscle strips of the diabetic rats was 370% of control values. This robust presence of low-frequency contractions is consistent with the unaffected pacemaker, the ICC associated with Auerbach's plexus, and the increased amplitude correlates with loss of and injury to ICC of the submuscular plexus and intramuscular ICC. Loss of inhibitory nitrergic nerves does not appear to be a factor based on unaltered nNOS immunoreactivity.     

Synthesis,binding and bioactivity of γ-methylene γ-lactam ecdysone receptor ligands: Advantages of QSAR models for flexible receptors

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or ‘attractors’. We describe the synthesis, in vitro binding and selected in vivo toxicity data for γ-methylene γ-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.     

Dissociation between cardiomyocyte function and remodeling with beta-adrenergic receptor blockade in isolated canine mitral regurgitation

The low-pressure volume overload of isolated mitral regurgitation (MR) is associated with increased adrenergic drive, left ventricular (LV) dilatation, and loss of interstitial collagen. We tested the hypothesis that beta1-adrenergic receptor blockade (beta1-RB) would attenuate LV remodeling after 4 mo of MR in the dog. beta1-RB did not attenuate collagen loss or the increase in LV mass in MR dogs. Using MRI and three-dimensional (3-D) analysis, there was a 70% increase in the LV end-diastolic (LVED) volume-to-LV mass ratio, a 23% decrease in LVED midwall circumferential curvature, and a >50% increase in LVED 3-D radius/wall thickness in MR dogs that was not attenuated by beta1-RB. However, beta1-RB caused a significant increase in LVED length from the base to apex compared with untreated MR dogs. This was associated with an increase in isolated cardiomyocyte length (171+/-5 microm, P<0.05) compared with normal (156+/-3 microm) and MR (165+/-4 microm) dogs. Isolated cardiomyocyte fractional shortening was significantly depressed in MR dogs compared with normal dogs (3.73+/-0.31 vs. 5.02+/-0.26%, P<0.05) and normalized with beta1-RB (4.73+/-0.48%). In addition, stimulation with the beta-adrenergic receptor agonist isoproterenol (25 nM) increased cardiomyocyte fractional shortening by 215% (P<0.05) in beta1-RB dogs compared with normal (56%) and MR (50%) dogs. In summary, beta1-RB improved LV cardiomyocyte function and beta-adrenergic receptor responsiveness despite further cell elongation. The failure to attenuate LV remodeling associated with MR could be due to a failure to improve ultrastructural changes in extracellular matrix organization.     

Frozen tissue can provide reproducible proteomic results of subcellular fractionation

Differential detergent fractionation (DDF) is frequently used to partition fresh cells and tissues into distinct compartments. We have tested whether DDF can reproducibly extract and fractionate cellular protein components from frozen tissues. Frozen kidneys were sequentially extracted with three different buffer systems. Analysis of the three fractions with liquid chromatography–tandem mass spectrometry (LC–MS/MS) identified 1693 proteins, some of which were common to all fractions and others of which were unique to specific fractions. Normalized spectral index (SI_N) values obtained from these data were compared to evaluate both the reproducibility of the method and the efficiency of enrichment. SI_N values between replicate fractions demonstrated a high correlation, confirming the reproducibility of the method. Correlation coefficients across the three fractions were significantly lower than those for the replicates, supporting the capability of DDF to differentially fractionate proteins into separate compartments. Subcellular annotation of the proteins identified in each fraction demonstrated a significant enrichment of cytoplasmic, cell membrane, and nuclear proteins in the three respective buffer system fractions. We conclude that DDF can be applied to frozen tissue to generate reproducible proteome coverage discriminating subcellular compartments. This demonstrates the feasibility of analyzing cellular compartment-specific proteins in archived tissue samples with the simple DDF method.     

High N,dry: Experimental nitrogen deposition exacerbates native shrub loss and nonnative plant invasion during extreme drought

Hotter, longer, and more frequent global change‐type drought events may profoundly impact terrestrial ecosystems by triggering widespread vegetation mortality. However, severe drought is only one component of global change, and ecological effects of drought may be compounded by other drivers, such as anthropogenic nitrogen (N) deposition and nonnative plant invasion. Elevated N deposition, for example, may reduce drought tolerance through increased plant productivity, thereby contributing to drought‐induced mortality. High N availability also often favors invasive, nonnative plant species, and the loss of woody vegetation due to drought may create a window of opportunity for these invaders. We investigated the effects of multiple levels of simulated N deposition on a Mediterranean‐type shrubland plant community in southern California from 2011 to 2016, a period coinciding with an extreme, multiyear drought in the region. We hypothesized that N addition would increase native shrub productivity, but that this would increase susceptibility to drought and result in increased shrub loss over time. We also predicted that N addition would favor nonnatives, especially annual grasses, leading to higher biomass and cover of these species. Consistent with these hypotheses, we found that high N availability increased native shrub canopy loss and mortality, likely due to the higher productivity and leaf area and reduced water‐use efficiency we observed in shrubs subject to N addition. As native shrub cover declined, we also observed a concomitant increase in cover and biomass of nonnative annuals, particularly under high levels of experimental N deposition. Together, these results suggest that the impacts of extended drought on shrubland ecosystems may be more severe under elevated N deposition, potentially contributing to the widespread loss of native woody species and vegetation‐type conversion.     

6-Nitro-2,3-dihydroimidazo[2,1-b][1,3]thiazoles: Facile synthesis and comparative appraisal against tuberculosis and neglected tropical diseases

As part of a quest for backups to the antitubercular drug pretomanid (PA-824), we investigated the unexplored 6-nitro-2,3-dihydroimidazo[2,1-b][1,3]-thiazoles and related -oxazoles. The nitroimidazothiazoles were prepared in high yield from 2-bromo-4-nitroimidazole via heating with substituted thiiranes and diisopropylethylamine. Equivalent examples of these two structural classes provided broadly comparable MICs, with 2-methyl substitution and extended aryloxymethyl side chains preferred; albeit, S-oxidised thiazoles were ineffective for tuberculosis. Favourable microsomal stability data for a biaryl thiazole (45) led to its assessment in an acute Mycobacterium tuberculosis mouse model, alongside the corresponding oxazole (48), but the latter proved to be more efficacious. In vitro screening against kinetoplastid diseases revealed that nitroimidazothiazoles were inactive versus leishmaniasis but showed interesting activity, superior to that of the nitroimidazooxazoles, against Chagas disease. Overall, “thio-delamanid” (49) is regarded as the best lead.