Modulation of triglyceride and cholesterol ester synthesis impairs assembly of infectious hepatitis C virus

In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection

L6 skeletal muscle cells have functional V1-vasopressin receptors coupled to stimulated inositol phospholipid metabolism

AbstractThe effects of vasopressin and related peptides upon the rat skeletal muscle cell line, L6, have been examined. No effects upon cellular cyclic AMP levels were found indicating that L6 cells possess no functional V2-vasopressin receptors. Vasopressin and its analogues did, however, stimulate the rapid and dose-dependent accumulation of inositol phosphates. This effect and the rank order of potency of vasopressin analogues demonstrate the presence of functional V1-vasopressin receptors upon L6 cells. These results suggest that the L6 line may be a useful model for vasopressin effects upon skeletal muscle metabolism

Distinct phospholipase C-regulated signalling pathways in Swiss 3T3 fibroblasts induce the rapid generation of the same polyunsaturated diacylglycerols

AbstractProstaglandin F2α, platelet-derived growth factor (PDGF) and calcium ionophore A23187 stimulated the rapid (within 25 s) generation of polyunsaturated 1,2-diacylglycerol (DAG) species, in particular 18:0/20:3n-9, 18:0/20:4n-6 and 18:0/20:5n-3, in Swiss 3T3 fibroblasts. This was followed by a second sustained phase characterised by saturated, monounsaturated and diunsaturated DAG species derived, at least partially, from a phospholipase D/phosphatidate phosphohydrolase-linked pathway. This could be directly activated by phorbol ester. Assay of rat brain protein kinase C (PKC) in lipid vesicles showed that first phase, polyunsaturated-enriched DAG isolated from Swiss 3T3 cells was a more potent activator of kinase activity compared to that achieved with DAG from control or 5 min stimulated cells. Thus activation of distinct members of the phospholipase C family leads to the rapid and almost identical generation of polyunsaturated DAG species which are capable of preferentially activating protein kinase C (PKC)

The C(3P) + NH3 reaction in interstellar chemistry: II. Low temperature rate constants and modeling of NH, NH2 and NH3 abundances in dense interstellar clouds

A continuous supersonic flow reactor has been used to measure rate constants for the C + NH3 reaction over the temperature range 50 to 296 K. C atoms were created by the pulsed laser photolysis of CBr4. The kinetics of the title reaction were followed directly by vacuum ultra-violet laser induced fluorescence (VUV LIF) of C loss and through H formation. The experiments show unambiguously that the reaction is rapid at 296 K, becoming faster at lower temperatures, reaching a value of 1.8 10-10 cm3 molecule-1 s-1 at 50 K. As this reaction is not currently included in astrochemical networks, its influence on interstellar nitrogen hydride abundances is tested through a dense cloud model including gas-grain interactions. In particular, the effect of the ortho-to-para ratio of H2 which plays a crucial role in interstellar NH3 synthesis is examined

Evaluation of the accuracy of serum MMP-9 as a test for colorectal cancer in a primary care population

Background Bowel cancer is common and is a major cause of death. Meta-analysis of randomised controlled trials estimates that screening for colorectal cancer using faecal occult blood (FOB) test reduces mortality from colorectal cancer by 16%. However, FOB testing has a low positive predictive value, with associated unnecessary cost, risk and anxiety from subsequent investigation, and is unacceptable to a proportion of the target population. Increased levels of an enzyme called matrix metalloproteinase 9 (MMP-9) have been found to be associated with colorectal cancer, and this can be measured from a blood sample. Serum MMP-9 is potentially an accurate, low risk and cost-effective population screening tool. This study aims to evaluate the accuracy of serum MMP-9 as a test for colorectal cancer in a primary care population. Methods/Design People aged 50 to 69 years, who registered in participating general practices in the West Midlands Region, will be asked to complete a questionnaire that asks about symptoms. Respondents who describe any colorectal symptoms (except only abdominal bloating and/or anal symptoms) and are prepared to provide a blood sample for MMP9 estimation and undergo a colonoscopy (current gold standard investigation) will be recruited at GP based clinics by a research nurse. Those unfit for colonoscopy will be excluded. Colonoscopies will be undertaken in dedicated research clinics. The accuracy of MMP-9 will be assessed by comparing the MMP-9 level with the colonoscopy findings, and the combination of factors (e.g. symptoms and MMP-9 level) that best predict a diagnosis of malignancy (invasive disease or polyps) will be determined. Discussion Colorectal cancer is a major cause of morbidity and mortality. Most colorectal cancers arise from adenomas and there is a period for early detection by screening, but available tests have risks, are unacceptable to many, have high false positive rates or are expensive. This study will establish the potential of serum MMP-9 as a screening test for colorectal cancer. If it is confirmed as accurate and acceptable, this serum marker has the potential to assist with reducing the morbidity and mortality from colorectal cancer

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles

<p>Abstract</p> <p>Background</p> <p>Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear.</p> <p>Results</p> <p>Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion.</p> <p>Conclusion</p> <p>The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.</p

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Speed and sensitivity of phototransduction in Drosophila depend on degree of saturation of membrane phospholipids.

Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved we predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, we used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38 to ∼5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca(2+) influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ∼10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ∼2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade.This research was supported by the Biotechnology and Biological Sciences Research Council (BBSRC; to M.J.O.W. and Q.Z., BBSRC Grant BB/G006865/1 to R.C.H., BB/H013849/1 to M.J., and BBSRC doctoral awards to A.S.R. and S.A.D.), the State Key Laboratory of Cognitive Neuroscience and Learning Open Research Fund (to M.J.), Jane and Aatos Erkko Foundation Fellowship (to M.J.), the Leverhulme Trust Grant (RPG-2012-567 to M.J.), and the UK Medical Research Council (Career Development Award to K.F.).This is the final published version of the article, originally published in the Journal of Neuroscience, February 11, 2015, 35(6): 2731–2746, DOI: 10.1523/JNEUROSCI.1150-14.201