Meta-Analysis of Quantification Methods Shows that Archaea and Bacteria Have Similar Abundances in the Subseafloor

There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined “yield” to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly.     

Glutamine Supplementation Alleviates Vasculopathy and Corrects Metabolic Profile in an In Vivo Model of Endothelial Cell Dysfunction

Endothelial Cell Dysfunction (ECD) is a recognized harbinger of a host of chronic cardiovascular diseases. Using a mouse model of ECD triggered by treatment with L-Nω-methylarginine (L-NMMA), we previously demonstrated that renal microvasculature displays a perturbed protein profile, including diminished expression of two key enzymes of the Krebs cycle associated with a Warburg-type suppression of mitochondrial metabolism. We hypothesized that supplementation with L-glutamine (GLN), that can enter the Krebs cycle downstream this enzymatic bottleneck, would normalize vascular function and alleviate mitochondrial dysfunction. To test this hypothesis, mice with chronic L-NMMA-induced ECD were co-treated with GLN at different concentrations for 2 months. Results confirmed that L-NMMA led to a defect in acetylcholine-induced relaxation of aortic rings that was dose-dependently prevented by GLN. In caveolin-1 transgenic mice characterized by eNOS inactivation, L-NMMA further impaired vasorelaxation which was partially rescued by GLN co-treatment. Pro-inflammatory profile induced by L-NMMA was blunted in mice co-treated with GLN. Using an LC/MS platform for metabolite profiling, we sought to identify metabolic perturbations associated with ECD and offset by GLN supplementation. 3453 plasma molecules could be detected with 100% frequency in mice from at least one treatment group. Among these, 37 were found to be differentially expressed in a 4-way comparison of control vs. LNMMA both with and without GLN. One of such molecules, hippuric acid, an “uremic toxin” was found to be elevated in our non-uremic mice receiving L-NMMA, but normalized by treatment with GLN. Ex vivo analysis of hippuric acid effects on vasomotion demonstrated that it significantly reduced acetylcholine-induced vasorelaxation of vascular rings. In conclusion, functional and metabolic profiling of animals with early ECD revealed macrovasculopathy and that supplementation GLN is capable of improving vascular function. Metabolomic analyses reveal elevation of hippuric acid, which may further exacerbate vasculopathy even before the development of uremia.     

An essential role for the proximal but not the distal cytoplasmic tail of glycoprotein M in murid herpesvirus 4 infection

Murid herpesvirus-4 (MuHV-4) provides a tractable model with which to define common, conserved features of gamma-herpesvirus biology. The multi-membrane spanning glycoprotein M (gM) is one of only 4 glycoproteins that are essential for MuHV-4 lytic replication. gM binds to gN and is thought to function mainly secondary envelopment and virion egress, for which several predicted trafficking motifs in its C-terminal cytoplasmic tail could be important. We tested the contribution of the gM cytoplasmic tail to MuHV-4 lytic replication by making recombinant viruses with varying C-terminal deletions. Removing an acidic cluster and a distal YXXPhi motif altered the capsid distribution somewhat in infected cells but had little effect on virus replication, either in vitro or in vivo. In contrast, removing a proximal YXXPhi motif as well completely prevented productive replication. gM was still expressed, but unlike its longer forms showed only limited colocalization with co-transfected gN, and in the context of whole virus appeared to support gN expression less well. We conclude that some elements of the gM cytoplasmic tail are dispensible for MuHV-4 replication, but the tail as a whole is not.     

Mycoplasma insons sp. nov., a twisted mycoplasma from green iguanas (Iguana iguana)

Mycoplasma insons sp. nov., first cultured from the choanae and tracheae of healthy green iguanas (Iguana iguana) from El Salvador, was readily distinguished from all previously described mollicutes and assigned to the Mycoplasma fastidiosum phylogenetic cluster by 16S rRNA gene sequence comparisons. Growth inhibition assays distinguished the isolates serologically from the other two members of that cluster. Many M. insons cells exhibit a remarkable twisted rod morphology despite lacking a cell wall. The organism is nonmotile, produces acid from glucose, but does not hydrolyze arginine, esculin, or urea. Mycoplasma insons 16S rRNA gene was also detected by PCR in packed blood cells from culture-negative iguanas. The type strain I17P1(T) has been deposited with the Mollicutes Collection at Purdue University and with the American Type Culture Collection (ATCC BAA-1435) in the USA. A limited number of cultures generated by the authors have also been deposited with the Culture Collection, University of G?teborg, in Sweden (CCUG 53461).     

Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

Structural Brain Changes in Chronic Pain Reflect Probably Neither Damage Nor Atrophy

Chronic pain appears to be associated with brain gray matter reduction in areas ascribable to the transmission of pain. The morphological processes underlying these structural changes, probably following functional reorganisation and central plasticity in the brain, remain unclear. The pain in hip osteoarthritis is one of the few chronic pain syndromes which are principally curable. We investigated 20 patients with chronic pain due to unilateral coxarthrosis (mean age 63.25±9.46 (SD) years, 10 female) before hip joint endoprosthetic surgery (pain state) and monitored brain structural changes up to 1 year after surgery: 6–8 weeks, 12–18 weeks and 10–14 month when completely pain free. Patients with chronic pain due to unilateral coxarthrosis had significantly less gray matter compared to controls in the anterior cingulate cortex (ACC), insular cortex and operculum, dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex. These regions function as multi-integrative structures during the experience and the anticipation of pain. When the patients were pain free after recovery from endoprosthetic surgery, a gray matter increase in nearly the same areas was found. We also found a progressive increase of brain gray matter in the premotor cortex and the supplementary motor area (SMA). We conclude that gray matter abnormalities in chronic pain are not the cause, but secondary to the disease and are at least in part due to changes in motor function and bodily integration.     

Erratum to: Synthesis and Anticancer Activity of Functionalized Thieno[2,3-d]pyrimidine Compounds and Their Triazinyl and Tetrazinyl Derivatives

Russian Journal of Bioorganic Chemistry - erratum     

Iso-coenzyme A

Iso-coenzyme A is an isomer of coenzyme A in which the monophosphate is attached to the 2'-carbon of the ribose ring. Although iso-CoA was first reported in 1959 (Moffatt, J. G., and Khorana, H. G. (1959) J. Am. Chem. Soc. 81, 1265-1265) to be a by-product of the chemical synthesis of CoA, relatively little attention has been focused on iso-CoA or on acyl-iso-CoA compounds in the literature. We now report structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and beta-hydroxybutyryl-iso-CoA using mass spectrometry (MS), tandem MS, and homonuclear and heteronuclear NMR analyses. Although the 2'-phosphate isomer of malonyl-CoA was recently identified in commercial samples, previous characterizations of iso-CoA itself have been based on chromatographic analyses, which ultimately rest on comparisons with the degradation products of CoA and NADPH or have been based on assumptions regarding enzyme specificity. We describe a high performance liquid chromatography methodology for separating the isomers of several CoA-containing compounds. We also report here the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl transfer reactions. Finally, we describe a simple synthesis of iso-CoA from CoA, which utilizes beta-cyclodextrin to produce iso-CoA with high regioselectivity, and we demonstrate a plausible mechanism that accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. We anticipate that these results will provide methodology and impetus for investigating iso-CoA compounds as potential pseudo-substrates or inhibitors of the >350 known CoA-utilizing enzymes.     

Low density lipoprotein receptor-related protein 1 (LRP1) controls endocytosis and c-CBL-mediated ubiquitination of the platelet-derived growth factor receptor beta (PDGFR beta)

The low density lipoprotein receptor-related protein 1 (LRP1) has been implicated in intracellular signaling functions as well as in lipid metabolism. Recent in vivo and in vitro studies suggest that LRP1 is a physiological modulator of the platelet-derived growth factor (PDGF) signaling pathway. Here we show that in mouse fibroblasts LRP1 modulates PDGF-BB signaling by controlling endocytosis and ligand-induced down-regulation of the PDGF receptor beta (PDGFRbeta). In LRP1-deficient fibroblasts, basal PDGFRbeta tyrosine kinase activity was derepressed, and PDGF-BB-induced endocytosis and degradation of PDGFRbeta were accelerated as compared with control cells. This was accompanied by rapid uptake of receptor-bound PDGF-BB into the cells and by attenuated ERK activation in response to PDGF-BB stimulation. Pulse-chase analysis indicated that the steady-state turnover rate of PDGFRbeta was also accelerated in LRP-deficient fibroblasts. The rapid degradation of PDGFRbeta in the LRP1-deficient fibroblasts was prevented by MG132 and chloroquine. Furthermore, the association of PDGFRbeta with c-Cbl, a ubiquitin E3-ligase, as well as the ligand-induced ubiquitination of PDGFRbeta were increased in LRP1-deficient fibroblasts. We show that LRP1 can directly interact with c-Cbl, suggesting a Sprouty-like role for LRP1 in regulating the access of the PDGFRbeta to the ubiquitination machinery. Thus, LRP1 modulates PDGF signaling by controlling ubiquitination and endocytosis of the PDGFRbeta.