Increased expression of the SNARE accessory protein Munc18c in lipid-mediated insulin resistance

Fatty acids inhibit insulin-mediated glucose metabolism in skeletal muscle, an effect largely attributed to defects in insulin-mediated glucose transport. Insulin-resistant mice transgenic for the overexpression of lipoprotein lipase (LPL) in skeletal muscle were used to examine the molecular mechanism(s) in more detail. Using DNA gene chip array technology, and confirmation by RT-PCR and Western analysis, increases in the yeast Sec1p homolog Munc18c mRNA and protein were found in the gastrocnemius muscle of transgenic mice, but not other tissues. Munc18c has been previously demonstrated to impair insulin-mediated glucose transport in mammalian cells in vitro. Of interest, stably transfected C2C12 cells overexpressing LPL not only demonstrated increases in Munc18c mRNA and protein but also in transcription rates of the Munc18c gene. jlr To confirm the relevance of fatty acid metabolism and insulin resistance to the expression of Munc18c in vivo, a 2-fold increase in Munc18c protein was demonstrated in mice fed a high-fat diet for 4 weeks. Together, these data are the first to implicate in vivo increases in Munc18c as a potential contributing mechanism to fatty acid-induced insulin resistance.

Early pregnancy factor treatment suppresses the inflammatory response and adhesion molecule expression in the spinal cord of SJL/J mice with experimental autoimmune encephalomyelitis and the delayed-type hypersensitivity reaction to trinitrochlorobenzene in normal BALB/c mice

Early pregnancy factor (EPF) is a secreted protein, present in serum during early pregnancy and essential for maintaining viability of the embryo. It is a homologue of chaperonin 10 (Cpn10) but, unlike Cpn10, it has an extracellular role. EPF has immunosuppressive and growth regulatory properties. Previously we have reported the preparation of recombinant EPF (rEPF) and shown that treatment with rEPF will suppress clinical signs of MBP-EAE in Lewis rats and PLP-EAE in SJL/J mice. In the present study, these findings have been extended to investigate possible mechanisms involved in the action of EPF. Following treatment of mice with rEPF from the day of inoculation, there were fewer infiltrating CD3+ and CD4+ cells in the parenchyma of the spinal cord during the onset of disease and after the initial episode, compared with mice treated with vehicle. Expression of the integrins LFA-1, VLA-4 and Mac-1 and of members of the immunoglobulin superfamily of adhesion molecules ICAM-1 and VCAM-1 was suppressed in the central nervous system (CNS) following rEPF treatment. The expression of PECAM-1 was not affected. To determine if rEPF suppressed T cell activation in the periphery, the delayed-type hypersensitivity (DTH) reaction of normal BALB/c mice to trinitrochlorobenzene (TNCB) following treatment with rEPF was studied. The results showed that treatment with rEPF suppressed the DTH reaction, demonstrating the ability of EPF to downregulate the cell-mediated immune response. These results indicate that suppression of immunological mechanisms by rEPF plays a major role in the reduction of clinical signs of disease in experimental autoimmune encephalomyelitis (EAE). (C) 2003 Elsevier Science B.V. All rights reserved.

Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10

Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. ReIB activation is required for myeloid DC differentiation. Here, we show that antigen-exposed DCs in which ReIB function is inhibited lack cell surface CD40, prevent priming of immunity, and suppress previously primed immune responses. DCs generated from CD40-deficient mice similarly confer suppression. Regulatory CD4(+) T cells induced by the DCs transfer antigen-specific Infectious tolerance to primed recipients in an interleukin10-dependent fashion. Thus CD40, regulated by ReIB activity, determines the consequences of antigen presentation by myeloid DCs. These observations have significance for autoimmune immunotherapy and suggest a mechanism by which peripheral tolerance might be constitutively maintained by RelB(-) CD40(-) DCs.

GRIP domain-mediated targeting of two new coiled-coil proteins, GCC88 and GCC185, to subcompartments of the trans-Golgi network

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the traps-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of Hela cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the traps-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.

Wall mediated transport in confined spaces: Exact theory for low density

We present a theory for the transport of molecules adsorbed in slit and cylindrical nanopores at low density, considering the axial momentum gain of molecules oscillating between diffuse wall reflections. Good agreement with molecular dynamics simulations is obtained over a wide range of pore sizes, including the regime of single-file diffusion where fluid-fluid interactions are shown to have a negligible effect on the collective transport coefficient. We show that dispersive fluid-wall interactions considerably attenuate transport compared to classical hard sphere theory.

Synthesis of novel 2-amino-1,3-thiazole-5-carboxylates utilising ultrasonic and thermally mediated aminolysis

Novel 2-amino-1,3-thiazole-5-carboxylates have been synthesised in high yield by unprecedented ultrasonic and thermally mediated nucleophilic displacement of bromide from ethyl 2-bromo-1,3-thiazole-5-carboxylate by primary, secondary and aryl amines.