In order to test the hypothesis that this covalent modification is a relevant step in insulin action, we have examined the relationship between disulfide binding of insulin and several insulin bioeffects, using sulfhydryl group blocking reagents as probes.The half-time of disappearance of disulfide linked insulin-receptor complexes (I-(S-S)-R) was rapid (4.5 min), consistent with their hypothesized role.A cell-impermeable reagent 5,5'-dithiobis(nitrobenzoic acid) (DTNB) had no effect on specific insulin binding but caused a dose-dependent decrease in both I-(S-S)-R and insulin-stimulated glucose transport.DTNB also inhibited the effect of insulin to stimulate glucose oxidation and to inhibit epinephrinestimulated cyclic AMP production.In cultured IM-9 lymphocytes, insulin-induced down regulation of its receptor was decreased by 75% in the presence of 1 m M DTNB.Receptor antibodies stimulated adipocyte glucose transport maximally but their effect, unlike that of insulin, was not inhibited by DTNB.These findings suggest that receptor sulfhydryl groups are required for insulin action and support the notion that their interchange with insulin is a necessary step in activation of postreceptor pathways.There is increasing evidence for the role of biological disulfides and disulfide-sulfhydryl interchange reactions in metabolic events (Freedman, 1979;Vauquelin and Maguire, 1980;Mukherjee and Mukherjee, 1981;Gilbert, 1982).Given that insulin is a dipeptide with one intrachain and two interchain disulfide bonds, it is reasonable to propose that its action might involve disulfide-sulfhydryl interactions, as first suggested by Cadenas et al. (1961) andFong et al. (1962).In the 1970s, Czech and co-workers postulated that activation of glucose transport by insulin involved the oxidation
The role of the epidermal growth factor (EGF) receptor in cell invasion by Salmonella typhimurium was examined in vitro and in vivo by using waved-2 mice which express an EGF receptor with reduced kinase activity. S. typhimurium invaded fibroblasts from waved-2 mice as efficiently as fibroblasts from wild-type control animals. In vivo, S. typhimurium both invaded the gastrointestinal tract and penetrated through to the spleen of waved-2 mice. Our studies suggest that the EGF receptor has only a limited role, if any, in cell invasion by S. typhimurium.
Platform technologies (PT) are techniques or tools that enable a range of scientific investigations and are critical to today's advanced technology research environment. Once installed, they require specialized staff for their operations, who in turn, provide expertise to researchers in designing appropriate experiments. Through this pipeline, research outputs are raised to the benefit of the researcher and the host institution. Platform facilities provide access to instrumentation and expertise for a wide range of users beyond the host institution, including other academic and industry users. To maximize the return on these substantial public investments, this wider access needs to be supported. The question of support and the mechanisms through which this occurs need to be established based on a greater understanding of how PT facilities operate. This investigation was aimed at understanding if and how platform facilities across the Bio21 Cluster meet operating costs. Our investigation found: 74% of platforms surveyed do not recover 100% of direct operating costs and are heavily subsidized by their home institution, which has a vested interest in maintaining the technology platform; platform managers play a major role in establishing the costs and pricing of the facility, normally in a collaborative process with a management committee or institutional accountant; and most facilities have a three-tier pricing structure recognizing internal academic, external academic, and commercial clients.
Human placenta and IM-9 lymphocytes contain subpopulations of atypical insulin receptors which differ from classical insulin receptors in their higher binding affinity for insulin-like growth factors I and II (IGF-I and IGF-II). Both types of insulin receptors may be derived from different but related genes, or may represent alternative post-translational modifications of the same gene product. To test these possibilities, we have examined the IGF binding characteristics of the human insulin receptors expressed in Chinese hamster ovary (CHO) cells which had been stably transfected with cloned human insulin receptor cDNA (CHO-T cells). The parent CHO cells contained 3 × 103 rodent insulin receptors/cell, and the CHO-T cells, 2.0 × 106 human insulin receptors/cell. Competition binding studies showed that the binding of [125I]IGF-I and [125I]multiplication stimulating activity (MSA/rat IGF-II) to parent CHO cells was primarily to type I and II IGF receptors, which cross-react poorly or not at all with insulin. However, competition binding studies with CHOT cells showed that [125I]IGF-I binding was displaced 60-70%, and [125I]MSA binding, 50-55%, by low concentrations of insulin (20 ng/ml) and no further by higher concentrations of insulin (500 ng/ml). The insulin-insensitive IGF binding sites corresponded to the rodent type I and II IGF receptors; the insulinsensitive IGF binding sensitive sites resembled the human atypical insulin receptors in that they bound IGF-I and MSA with moderately high affinity and reacted with insulin, MSA, and IGF-I in that order of potency. Atypical insulin receptors were also demonstrated by insulin-sensitive [125I]IGF-I and [125I]MSA binding to solubilized CHO-T proteins adsorbed to microtiter wells coated with monoclonal antibodies specific for the human insulin receptor. These results suggest that atypical human insulin receptors are generated by differential post-translational processing of the same gene product as classical human insulin receptors. (Endocrinology127: 1301–1309, 1990)
The structure of naturally-formed covalent disulphide-linked complexes between insulin and its receptor was examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. To prevent destabilization of disulphide bonds at alkaline pH the standard discontinuous electrophoresis conditions were changed to a continuous buffer system at pH 7.0. 125I-insulin was first bound to either rat adipocytes or human placental membranes for 10 min at 37 degrees C. After washing, non-dissociable radioactivity was extracted from cells or membranes in Triton X-100 and immunoprecipitated with an antiserum (B-2) to the insulin receptor. Electrophoresis of the immune precipitate revealed the two smaller of the three reported species of native insulin receptor (Mr values approx. 350 000, 290 000 and 260 000); in addition, a species of Mr 200 000 was also frequently observed in adipocytes. When non-dissociable 125I-insulin was chemically crosslinked to adipocytes or placental membranes, prior to solubilization and immunoprecipitation, all three species of the native receptor were labelled; after reduction, only a single species of Mr 130000 was observed. These findings indicate that disulphide exchange of insulin occurs with the Mr 130000 (alpha) binding subunit within partially reduced species of the native, oligomeric receptor. The degree of disulphide binding of insulin could therefore depend on the relative abundance of partially reduced receptor species and on the redox state of the cell membrane.
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