The polar polysaccharide capsule of Hyphomonas adhaerens MHS-3 has a strong affinity for gold.

Select groups of bacteria, including prothescate species, have an unusual capacity to sequester gold and bioconcentrate it to very high levels. Hyphomonas adhaerens MHS-3 (MHS-3) is one such species, as demonstrated by Energy Dispersive Spectroscopy. Transmission electron microscopy revealed that the binding site was specific on the polar polysaccharide capsule. A capsuleless mutant and periodate-treated wild type did not sequester gold. The gold may interact with the same sites in the capsule that naturally adhere MHS-3 to surfaces in the marine environment.

G(alpha)11 signaling through ARF6 regulates F-actin mobilization and GLUT4 glucose transporter translocation to the plasma membrane.

The action of insulin to recruit the intracellular GLUT4 glucose transporter to the plasma membrane of 3T3-L1 adipocytes is mimicked by endothelin 1, which signals through trimeric G(alpha)q or G(alpha)11 proteins. Here we report that murine G(alpha)11 is most abundant in fat and that expression of the constitutively active form of G(alpha)11 [G(alpha)11(Q209L)] in 3T3-L1 adipocytes causes recruitment of GLUT4 to the plasma membrane and stimulation of 2-deoxyglucose uptake. In contrast to the action of insulin on GLUT4, the effects of endothelin 1 and G(alpha)11 were not inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM. Signaling by insulin, endothelin 1, or G(alpha)11(Q209L) also mobilized cortical F-actin in cultured adipocytes. Importantly, GLUT4 translocation caused by all three agents was blocked upon disassembly of F-actin by latrunculin B, suggesting that the F-actin polymerization caused by these agents may be required for their effects on GLUT4. Remarkably, expression of a dominant inhibitory form of the actin-regulatory GTPase ARF6 [ARF6(T27N)] in cultured adipocytes selectively inhibited both F-actin formation and GLUT4 translocation in response to endothelin 1 but not insulin. These data indicate that ARF6 is a required downstream element in endothelin 1 signaling through G(alpha)11 to regulate cortical actin and GLUT4 translocation in cultured adipocytes, while insulin action involves different signaling pathways.

A role for kinesin in insulin-stimulated GLUT4 glucose transporter translocation in 3T3-L1 adipocytes.

Insulin regulates glucose uptake in adipocytes and muscle by stimulating the movement of sequestered glucose transporter 4 (GLUT4) proteins from intracellular membranes to the cell surface. Here we report that optimal insulin-mediated GLUT4 translocation is dependent upon both microtubule and actin-based cytoskeletal structures in cultured adipocytes. Depolymerization of microtubules and F-actin in 3T3-L1 adipocytes causes the dispersion of perinuclear GLUT4-containing membranes and abolishes insulin action on GLUT4 movements to the plasma membrane. Furthermore, heterologous expression in 3T3-L1 adipocytes of the microtubule-binding protein hTau40, which impairs kinesin motors that move toward the plus ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with the hypothesis that both the actin and microtubule-based cytoskeleton, as well as a kinesin motor, direct the translocation of GLUT4 to the plasma membrane in response to insulin.

ADP-ribosylation factor 6 as a target of guanine nucleotide exchange factor GRP1.

The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol(3,4,5)P(3), an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol(3,4,5)P(3) may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.

Influence of protein conditioning films on binding of a bacterial polysaccharide adhesin from Hyphomonas MHS-3.

A putative polysaccharide adhesin which mediates non-specific attachment of Hyphomonas MHS-3 (MHS-3) to hydrophilic substrata has been isolated and partially characterized. A polysaccharide-enriched portion of the extracellular polymeric substance (EPS(P)) from MHS-3 was separated into four fractions using high performance size exclusion chromatography (HPSEC). Comparison of chromatograms of EPS(P) from MHS-3 and a reduced adhesion strain (MHS-3 rad) suggested that one EPS(P) fraction, which consisted of carbohydrate, served as an adhesin. Adsorption of this fraction to germanium (Ge) was investigated using attenuated total reflection Fourier transform infrared (ATR/FT-IR) spectrometry. Binding curves indicated that the isolated fraction had a relatively high affinity for Ge when ranked against an adhesive protein from Mytilis edulis, mussel adhesive protein (MAP) and an acidic polysaccharide (alginate from Macrocystis pyrifera). Spectral features were used to identify the fraction as a polysaccharide previously reported to adsorb preferentially out of the EPS(P) mixture. Conditioning the Ge substratum with either bovine serum albumin (BSA) or MAP decreased the adsorption of the adhesive polysaccharide significantly. Conditioning Ge with these proteins also decreased adhesion of whole cells.

Adhesion of biofilms to inert surfaces: A molecular level approach directed at the marine environment.

Protein/ligand interactions involved in mediating adhesion between microorganisms and biological surfaces have been well-characterized in some cases (e.g. pathogen/host interactions). The strategies microorganisms employ for attachment to inert surfaces have not been so clearly elucidated. An experimental approach is presented which addresses the issues from the point of view of molecular interactions occurring at the interface.

Structure, function and immunochemistry of bacterial exopolysaccharides.

There has been much written on bacterial exopolysaccharides (EPS) and their role in virulence. Less has been published regarding EPS in free living species. This review focuses on that subject, emphasizing their functions in the environment and the use of antibody probes to study them.

Postmortem metabolism of short-finned squid muscle (Illex illecebrosus).

Postmortem biochemical changes were examined in the mantle muscle of the short-finned squid (Illex illecebrosus) in relation to the physical events associated with rigor. Unlike mammalian muscle, the major muscle phosphagen is arginine phosphate rather than creatine phosphate. Arginine phosphate levels did not change dramatically during the progress of rigor development. ATP depletion was found to be closely related to glycogen depletion as is often observed in mammalian muscle. The postmortem accumulation of octopine was related to the initial muscle glycogen content at death but a significant lag in its production was observed. The postmortem conversion of glucose to glucose-6-phosphate appeared to be the rate-limiting step in the overall conversion of glycogen to octopine. The intermediates found in the postmortem catabolism of squid muscle ATP were ADP, AMP, IMP Ino and Hx. Unlike most vertebrate fishes, AMP was found to accumulate in squid before conversion to IMP whereas accumulations of IMP and Ino were less than those normally found in vertebrate muscle.