Dimethyl sulfoxide enhances GLUT4 translocation through a reduction in GLUT4 endocytosis in insulin-stimulated 3T3-L1 adipocytes

Insulin increases muscle and fat cell glucose uptake by inducing the translocation of glucose transporter GLUT4 from intracellular compartments to the plasma membrane. Here, we have demonstrated that in 3T3-L1 adipocytes, DMSO at concentrations higher than 7.5% augmented cell surface GLUT4 levels in the absence and presence of insulin, but that at lower concentrations, DMSO only enhanced GLUT4 levels in insulin-stimulated cells. At a 5% concentration, DMSO also increased cell surface levels of the transferrin receptor and GLUT1. Glucose uptake experiments indicated that while DMSO enhanced cell surface glucose transporter levels, it also inhibited glucose transporter activity. Our studies further demonstrated that DMSO did not sensitize the adipocytes for insulin and that its effect on GLUT4 was readily reversible (t1/2∼12 min) and maintained in insulin-resistant adipocytes. An enhancement of insulin-induced GLUT4 translocation was not observed in 3T3-L1 preadipocytes and L6 myotubes, indicating cell specificity. DMSO did not enhance insulin signaling nor exocytosis of GLUT4 vesicles, but inhibited GLUT4 internalization. While other chemical chaperones (glycerol and 4-phenyl butyric acid) also acutely enhanced insulin-induced GLUT4 translocation, these effects were not mediated via changes in GLUT4 endocytosis. We conclude that DMSO is the first molecule to be described that instantaneously enhances insulin-induced increases in cell surface GLUT4 levels in adipocytes, at least in part through a reduction in GLUT4 endocytosis.     

Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.     

Ligand binding to truncated hemoglobin N from Mycobacterium tuberculosis is strongly modulated by the interplay between the distal heme pocket residues and internal water

The survival of Mycobacterium tuberculosis requires detoxification of host *NO. Oxygenated Mycobacterium tuberculosis truncated hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant (k'(NOD) approximately 745 x 10(6) m(-1) x s(-1)), which is approximately 15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-truncated hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O(2) binding is very rapid with rates approaching 1-2 x 10(9) m(-1) x s(-1). These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photoproduct of the *NO derivative of met-trHbN, where both the *NO and water can be directly followed, revealed that water rebinding is quite fast (approximately 1.49 x 10(8) s(-1)) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11).     

Risk Factors Associated with Ebola and Marburg Viruses Seroprevalence in Blood Donors in the Republic of Congo

BackgroundEbola and Marburg viruses (family Filoviridae, genera Ebolavirus and Marburgvirus) cause haemorrhagic fevers in humans, often associated with high mortality rates. The presence of antibodies to Ebola virus (EBOV) and Marburg virus (MARV) has been reported in some African countries in individuals without a history of haemorrhagic fever. In this study, we present a MARV and EBOV seroprevalence study conducted amongst blood donors in the Republic of Congo and the analysis of risk factors for contact with EBOV.Conclusions/SignificanceThis MARV and EBOV serological survey performed in the Republic of Congo identifies a probable role for environmental determinants of exposure to EBOV. It highlights the requirement for extending our understanding of the ecological and epidemiological risk of bats (previously identified as a potential ecological reservoir) and birds as vectors of EBOV to humans, and characterising the protection potentially afforded by EBOV-specific antibodies as detected in blood donors.     

RAL-1 controls multivesicular body biogenesis and exosome secretion

Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plasma membrane. Despite their importance in various processes, the molecular mechanisms controlling their formation and release remain unclear. Using nematodes and mammary tumor cells, we show that Ral GTPases are involved in exosome biogenesis. In Caenorhabditis elegans, RAL-1 localizes at the surface of secretory MVBs. A quantitative electron microscopy analysis of RAL-1–deficient animals revealed that RAL-1 is involved in both MVB formation and their fusion with the plasma membrane. These functions do not involve the exocyst complex, a common Ral guanosine triphosphatase (GTPase) effector. Furthermore, we show that the target membrane SNARE protein SYX-5 colocalizes with a constitutively active form of RAL-1 at the plasma membrane, and MVBs accumulate under the plasma membrane when SYX-5 is absent. In mammals, RalA and RalB are both required for the secretion of exosome-like vesicles in cultured cells. Therefore, Ral GTPases represent new regulators of MVB formation and exosome release.     

Aurora B prevents chromosome arm separation defects by promoting telomere dispersion and disjunction

The segregation of centromeres and telomeres at mitosis is coordinated at multiple levels to prevent the formation of aneuploid cells, a phenotype frequently observed in cancer. Mitotic instability arises from chromosome segregation defects, giving rise to chromatin bridges at anaphase. Most of these defects are corrected before anaphase onset by a mechanism involving Aurora B kinase, a key regulator of mitosis in a wide range of organisms. Here, we describe a new role for Aurora B in telomere dispersion and disjunction during fission yeast mitosis. Telomere dispersion initiates in metaphase, whereas disjunction takes place in anaphase. Dispersion is promoted by the dissociation of Swi6/HP1 and cohesin Rad21 from telomeres, whereas disjunction occurs at anaphase after the phosphorylation of condensin subunit Cnd2. Strikingly, we demonstrate that deletion of Ccq1, a telomeric shelterin component, rescued cell death after Aurora inhibition by promoting the loading of condensin on chromosome arms. Our findings reveal an essential role for telomeres in chromosome arm segregation.     

A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast Pichia pastoris

We produced bovine trypsinogen in the yeast Pichia pastoris. Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong aox1 promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells. We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin. This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors. Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation. Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism. This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation.     

Method for monitoring alginate released in biological fluids by high-performance anion-exchange chromatography with pulsed amperometric detection

The use of alginate-entrapped cells in cell therapy requires a method for monitoring possible released compound within biological fluids following either their implantation or inoculation in artificial organs. Oligomannuronic and oligoguluronic acids were prepared by enzymatic depolymerization with alginate lyase from Pseudomonas alginovora, characterized by high-performance anion-exchange chromatography with pulsed amperometric detection and quantitated in human, pig and rabbit blood, urine and tissue samples. The method was tested for linearity and detection limit, accuracy, intra- and inter-day precision. The limit of detection was 3 microgram/ml in both urine and plasma and 5 mg/g of tissues. The relative standard deviations (RSDs) of intra-day precision were 6.0-16.6% and 4.8-8.7% in plasma and urine, respectively; the RSDs of inter-day precision were 5.1-14.4% and 5.0-11.6% in plasma and urine, respectively. Thus, this method appears suitable for the measurement of released alginate from entrapped cells used in cell therapy.     

Virulence of an emerging pathogenic lineage of Vibrio nigripulchritudo is dependent on two plasmids

Vibrioses are the predominant bacterial infections in marine shrimp farms. Vibrio nigripulchritudo is an emerging pathogen of the cultured shrimp Litopenaeus stylirostris in New Caledonia and other regions in the Indo‐Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have revealed that recent pathogenic V. nigripulchritudo isolates from New Caledonia all cluster into a monophyletic clade and contain a small plasmid, pB1067. Here, we report that a large plasmid, pA1066 (247 kb), can also serve as a marker for virulent V. nigripulchritudo, and that an ancestral version of this plasmid was likely acquired prior to other virulence‐linked markers. Additionally, we demonstrate that pA1066 is critical for the full virulence of V. nigripulchritudo in several newly developed experimental models of infection. Plasmid pB1067 also contributes to virulence; only strains containing both plasmids induced the highest level of shrimp mortality. Thus, it appears that these plasmids, which are absent from non‐pathogenic isolates, may be driving forces, as well as markers, for the emergence of a pathogenic lineage of V. nigripulchritudo.