Inhibition of virus-induced plaque formation by atoxic derivatives of purified cobra neutrotoxins

Venom from Naja naja siamensis was resolved into 16 toxic and nontoxic fractions by chromatography on SP-Sephadex, C-25. The principal neurotoxin preparations were chromatographically and electrophoretically homogeneous.Of the purified constituents, only the principal neurotoxin and minor neurotoxins were precursors of inhibitors of plaque formation among baby hamster kidney fibroblasts infected with Semliki Forest Virus.     

Sodium-coupled solute transport in charophyte algae: A general mechanism for transport energization in plant cells?

Ion-gradient-coupled transport systems in plants are normally electrophoretic and carry inward current. Rapid inward electrical currents elicited by K⁺, by urea and by lysine in the freshwater acidophilic alga Nitella translucens Agh. are all very strongly dependent on the presence of Na⁺ or (except in the case of K⁺) Li⁺. These results indicate that Na⁺-coupled solute transport in plants, which had previously been demonstrated only in an alkalophilic species (Chara australis), did not evolve recently as an alternative to H⁺-coupled transport in high-pH environments, and might therefore be more widely distributed than has hitherto been recognised.We are very grateful to Professor E.A.C. MacRobbie and Mr J. Banfield (Botany School, University of Cambridge) for supplies of Nitella translucens. Financial support for this work was obtained from the Australian Department of Industry, Technology and Commerce and the Joint Research Council's Biotechnology Collaboration Scheme between Britain and Australia (to N.A.W. and D.S.), and from an Agricultural and Food Research Council Grant (PG 87/501 to D.S.). D.S. was a Nuffield Foundation Science Research Fellow.     

AGPAT6 is a novel microsomal glycerol-3-phosphate acyltransferase

AGPAT6 is a member of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family that appears to be important in triglyceride biosynthesis in several tissues, but the precise biochemical function of the enzyme is unknown. In the current study, we show that AGPAT6 is a microsomal glycerol-3-phosphate acyltransferase (GPAT). Membranes from HEK293 cells overexpressing human AGPAT6 had higher levels of GPAT activity. Substrate specificity studies suggested that AGPAT6 was active against both saturated and unsaturated long-chain fatty acyl-CoAs. Both glycerol 3-phosphate and fatty acyl-CoA increased the GPAT activity, and the activity was sensitive to N-ethylmaleimide, a sulfhydryl-modifying reagent. Purified AGPAT6 protein possessed GPAT activity but not AGPAT activity. Using [(13)C(7)]oleic acid labeling and mass spectrometry, we found that overexpression of AGPAT6 increased both lysophosphatidic acid and phosphatidic acid levels in cells. In these studies, total triglyceride and phosphatidylcholine levels were not significantly altered, although there were significant changes in the abundance of specific phosphatidylcholine species. Human AGPAT6 is localized to endoplasmic reticulum and is broadly distributed in tissues. Membranes of mammary epithelial cells from Agpat6-deficient mice exhibited markedly reduced GPAT activity compared with membranes from wild-type mice. Reducing AGPAT6 expression in HEK293 cells through small interfering RNA knockdown suggested that AGPAT6 significantly contributed to HEK293 cellular GPAT activity. Our data indicate that AGPAT6 is a microsomal GPAT, and we propose renaming this enzyme GPAT4.     

Genetic upregulation of matriptase-2 reduces the aggressiveness of prostate cancer cells in vitro and in vivo and affects FAK and paxillin localisation

The cellular function and the role of matriptase-2 in cancer progression are poorly understood. This study assesses the importance of this protease in prostate cancer cell lines. Two prostate cancer cell lines, PC-3 and DU-145, previously displaying minimal expression of matriptase-2, were forced to over-express matriptase-2 using a human mammalian expression construct. Over-expression of matriptase-2 significantly reduced the invasive capacity and significantly slowed the migration rates of PC-3 and DU-145 cells in vitro. Similarly, PC-3 cells containing the matriptase-2 expression plasmid were dramatically less able to survive, grow and develop into noticeable tumours, compared to control PC-3 cells containing an empty plasmid alone, following subcutaneous inoculation into CD1 nude mice. This trend was observed throughout the experiment, becoming apparent after the initial reading on day 7 (P = 0.0002) and continuing to the experimental end point at day 27 (P = 0.0002). Enhanced matriptase-2 levels were also seen to correlate with increased fluorescent staining of the paxillin and FAK adhesion molecules, where a greater extent of these molecules were localised to the focal adhesion complexes. This data suggests a suppressive role for matriptase-2 in the invasion and migration of prostate cancer cells in vitro and also in their development and growth in vivo, highlighting the potential of this molecule to interfere with key stages of metastasis. Furthermore, the data presented implies a possible connection between matriptase-2 and the paxillin and FAK adhesion molecules which may ultimately contribute to the reduced migration rates seen in this study.     

Improving the corn-ethanol industry: studying protein separation techniques to obtain higher value-added product options for distillers grains

Currently in America the biofuel ethanol is primarily being produced by the dry grind technique to obtain the starch contained in the corn grains and subsequently subjected to fermentation. This so-called 1st generation technology has two setbacks; first the lingering debate whether its life cycle contributes to a reduction of fossil fuels and the animal feed sectors future supply/demand imbalance caused by the co-product dry distillers grains (DDGS). Additional utilization of the cellulosic components and separation of the proteins for use as chemical precursors have the potential to alleviate both setbacks. Several different corn feedstock layouts were treated with 2nd generation ammonia fiber expansion (AFEX) pre-treatment technology and tested for protein separation options (protease solubilization). The resulting system has the potential to greatly improve ethanol yields with lower bioprocessing energy costs and satisfy a significant portion of the organic chemical industry.     

Gross deletions involving IGHM, BTK, or Artemis: a model for genomic lesions mediated by transposable elements

Most genetic disruptions underlying human disease are microlesions, whereas gross lesions are rare with gross deletions being most frequently found (6%). Similar observations have been made in primary immunodeficiency genes, such as BTK, but for unknown reasons the IGHM and DCLRE1C (Artemis) gene defects frequently represent gross deletions ( approximately 60%). We characterized the gross deletion breakpoints in IGHM-, BTK-, and Artemis-deficient patients. The IGHM deletion breakpoints did not show involvement of recombination signal sequences or immunoglobulin switch regions. Instead, five IGHM, eight BTK, and five unique Artemis breakpoints were located in or near sequences derived from transposable elements (TE). The breakpoints of four out of five disrupted Artemis alleles were located in highly homologous regions, similar to Ig subclass deficiencies and Vh deletion polymorphisms. Nevertheless, these observations suggest a role for TEs in mediating gross deletions. The identified gross deletion breakpoints were mostly located in TE subclasses that were specifically overrepresented in the involved gene as compared to the average in the human genome. This concerned both long (LINE1) and short (Alu, MIR) interspersed elements, as well as LTR retrotransposons (ERV). Furthermore, a high total TE content (>40%) was associated with an increased frequency of gross deletions. Both findings were further investigated and confirmed in a total set of 20 genes disrupted in human disease. Thus, to our knowledge for the first time, we provide evidence that a high TE content, irrespective of the type of element, results in the increased incidence of gross deletions as gene disruption underlying human disease.