Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sequential epitopes on the human insulin receptor recognized by polyclonal and monoclonal antibodies were investigated using a recombinant DNA technique. Short random fragments of receptor cDNA were cloned and expressed in Escherichia coli as beta-galactosidase fusion proteins by using the expression vector pUEX1. Immunoreactive peptides were detected by colony blotting and identified by sequencing the corresponding cDNA inserts. Eleven antigenic determinants were located with rabbit antisera, nine of these being on the alpha-subunit and two on the beta-subunit of which one was intracellular. Two human autoantibodies reacted with the alpha-subunit on blots, but no sequential epitopes could be located. In the rabbit sera, antibody reacting with these linear epitopes represented a substantial fraction (approximately 50%) of antibody reacting with reduced denatured receptor on blots, but a generally smaller fraction (5-40%) of antibody reacting with solubilized native receptor. Epitope-specific subfractions of antibodies were purified by binding to an elution from bacterial fusion proteins. All of these subfractions reacted with denatured receptor on nitrocellulose blots, but only three precipitated native receptor (epitopes between amino acids 190 and 231, 654 and 669, 954 and 982) and none inhibited insulin binding. (The numbering system used in this manuscript is that of Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf. L., Clauser, E., Ou, J., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A., and Rutter, W. J. (1985) Cell 40, 747-758). The binding sites of two monoclonal antibodies were also determined. One of these antibodies (83-14) is insulin-mimetic, but inhibits insulin binding and its epitope on the alpha-subunit (between amino acids 469 and 592) may contribute to the insulin binding site in the folded protein. The other antibody (18-44) binds close to the N terminus of the beta-subunit (amino acids 765-770) and does not inhibit insulin binding, but does mimic insulin action. The identification of epitopes therefore provides information on receptor conformation and allows structural domains to be identified which are involved in the functional effects of different antibodies.
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PMID:Identification of epitopes on the human insulin receptor reacting with rabbit polyclonal antisera and mouse monoclonal antibodies. 169 19

We recently reported marked differences in insulin binding properties in Chinese hamster ovarian cell mutants with genetic defects in protein N-glycosylation. To further characterize the role of insulin receptor carbohydrates, we have now studied the effect of lectins on [125I]insulin binding to wild type (WT) Chinese hamster ovarian cells and to two mutant cell lines: B4-2-1, to which insulin was previously shown to bind with higher affinity than normal, and Lec 1, to which insulin binds with much lower affinity. The results show that of four lectins that bound to WT cells; only wheat germ agglutinin and phytohemagglutinin-E competed with insulin binding to these cells, while Concanavalin A (ConA) and Erythrina cristagalli agglutinin (ECA) did not. After solubilization of the cells, however, a potent inhibition of insulin binding was also seen with ConA and ECA. This suggests that sugar determinants for ConA and ECA are present on the insulin receptor, but are not accessible at the surface of the cells. Mutant B4-2-1 cells, which are deficient in mannosylphosphoryldolichol synthase and beta-galactosidase, differed from WT cells in that ECA and ConA potently inhibited insulin binding in intact cells. This suggests that these lectin binding sites of or near the insulin receptor are more accessible at the cell surface in this mutant cell line. Mutant Lec 1 cells, deficient in N-acetylglucosaminyl-transferase I, cannot process N-linked carbohydrates from their oligomannose to their complex forms. In these cells, marked differences in the pattern of lectin inhibition were observed compared to that in WT or B4-2-1 cells. ConA exerted a strong inhibition of insulin binding to solubilized cell preparations. Its effect on intact cells was modest however, suggesting that in this mutant line exposure of the insulin receptor at the cell surface is not different from that in the WT cells. Neither ECA nor PHA inhibited [125I]insulin binding to either intact or solubilized Lec 1 cells, suggesting that the absence of sugar determinants for these two lectins may play a role in the very low insulin binding affinity previously reported in this cell line. In conclusion, these indirect studies with lectins suggest that the carbohydrate units of the insulin receptor are heterogeneous. While some may be important for proper exposure of the receptor at the cell surface, others may play a role in more intrinsic receptor properties.
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PMID:Lectins as probes of insulin receptor carbohydrate composition: studies in glycosylation mutants of Chinese hamster ovarian cells with altered insulin binding. 351 53

We characterized insulin receptors on a human lymphoblastoid cell line (IM-9) and studied their regulation using anti-receptor antibodies and fluorescence flow cytometry. The fluorescence intensity distribution of insulin receptors on cells was determined by incubating the cells with one of three different anti-receptor antisera (human serum B-9 containing polyclonal autoantibodies, serum from a rabbit with polyclonal antibodies, and a monoclonal antibody to the receptor produced in mouse hybridomas), followed by incubation with an appropriate fluorescein isothiocyanate-labeled second antibody and analysis on an Epics-V flow cytometer. All three anti-receptor antibodies specifically labeled the insulin receptors. The monoclonal antibody showed the highest level of labeling. Treatment of cells with proteolytic enzymes, such as trypsin or chymotrypsin, produced a dose-dependent loss of 125I-labeled insulin (125I-insulin) binding but a relatively small decrease in the binding of anti-receptor antibodies, suggesting that most antibody binding occurred in domains other than the insulin binding site. Treatment with glycosidic enzymes, such as neuraminidase and beta-galactosidase did not affect the binding of 125I-insulin, and fluorescence was actually enhanced by about 20% in the beta-galactosidase-treated cells. Exposure of IM-9 cells to insulin resulted in a reduction in the number of insulin receptors. Analysis of the down-regulated cells by immunofluorescence revealed a complete correlation between the percent binding of 125I-insulin and percent peak fluorescence. In all cases, receptors were lost proportionally from all cells, yielding a single symmetrical peak by fluorescence analysis. Exposure of IM-9 cells to anti-receptor antibodies at 37 degrees C for 16 hr also produced a down-regulation in the number of insulin receptors. Incubation with human antiserum B-9 caused a 95% loss of both 125I-insulin binding and peak fluorescence, while the monoclonal antibody resulted in a 50% loss of receptors. Incubation of cells with anti-receptor antibodies for 2 hr at 4 degrees C did not produce any receptor loss; however, the human anti-receptor antisera B-2 and B-9 inhibited the binding of the monoclonal anti-receptor antibody by about 50%, suggesting that these antisera contained autoantibodies directed at the monoclonal antibody binding site. These data indicate that insulin receptors can be regulated by both insulin and anti-receptor antibody and demonstrate the utility of immunofluorescence and flow cytometry as a tool for the study of the insulin receptor.
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PMID:Analysis of the insulin receptor by anti-receptor antibodies and flow cytometry. 639 Apr 39

The glycoproteinic nature of the insulin receptor was indicated using two different approaches: 1. [125I] insulin binding to soluble receptors from mouse liver was inhibited by digestion with beta-galactosidase or pretreatment with Ricinus communis I or concanavalin A. An other enzyme (neuraminidase) and lectins (wheat germ agglutinin, Dolichos biflorus) did not affect the binding reaction. These data confirmed that insulin directly interacts with the galactoglycoproteins of liver membranes. 2. The galactose oxidase-sodium boro[3H] hydride technique, previously used for labeling accessible membrane galactoglycoproteins, was again utilized to discern the components that interact with insulin. When liver membranes were equilibrated with 10-7 M insulin prior to labeling, the SDS gel radioactive profiles were specifically modified with two galactoglycoprotein of apparent molecular sizes 195 000 and 145 000, compatible with their participation in the insulin binding interaction. Membrane pretreatment with beta-galactosidase or Sophora japonica lectin reduced the labeling in most peaks, thus supporting the argument for labeling sensitivity. Preincubation of membranes with 10-7 M proinsulin slightly hindered labeling, while pretreatment with 10-7 M glucagon was ineffective, suggesting a specificity of the insulin effect. These data indicate that glycoprotein nature of the insulin receptor for two reasons: alteration of insulin binding after modification of the galactoglycoproteins, and alteration of galactoglycoprotein labeling after insulin binding. Two galactoglycoproteins, with apparent molecular weights 145 000 and 195 000, respectively, were identified and they are suggested to have insulin binding properties.
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PMID:Identification of liver cell membrane galactoglycoproteins involved in the process of insulin binding. 703 Mar 99

Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce insulin resistance in cultured cells as well as in animal models. The aim of this study was to map the in vivo mechanism whereby TNF-alpha contributes to the pathogenesis of impaired insulin signaling, using obese and lean Zucker rats in which TNF-alpha activity was inhibited through adenovirus-mediated gene transfer. We employed a replication-incompetent adenovirus-5 (Ad5) vector to endogenously express a TNF inhibitor (TNFi) gene, which encodes a chimeric protein consisting of the extracellular domain of the human 55-kDa TNF receptor joined to a mouse IgG heavy chain. Control animals consisted of rats infected with the same titer of adenovirus carrying the lac-z complementary DNA, encoding for beta-galactosidase. There was a significant reduction in plasma insulin and free fatty acid levels in TNFi obese rats 2 days following Ad5 administration. The peripheral insulin sensitivity index was 50% greater, whereas hepatic glucose output was completely suppressed during hyperinsulinemic glucose clamps in TNFi obese animals, with no differences observed between the two lean groups. The improvement in peripheral and hepatic sensitivity to insulin seen in the obese animals was independent of insulin receptor (IR) number and insulin binding affinity for IR. However, TNF-alpha neutralization led to a 2.5-fold increase in tyrosine phosphorylation of IR in skeletal muscle, whereas this was unchanged in liver. There was also a 4-fold increase in particulate protein tyrosine phosphatase activity of skeletal muscle in TNFi obese animals vs. beta-galactosidase controls, whereas protein tyrosine phosphatase activity in liver was unchanged. These results suggest that TNF-alpha is a mediator of insulin resistance in obesity and may modulate IR signaling in skeletal muscle and liver through different pathways. TNF-alpha may affect insulin action in the liver either at sites distal to the IR or indirectly, possibly because of increased provision of gluconeogenic substrates or altered counterregulation. In addition, the Ad5-mediated gene delivery system employed here provides an in vivo model that is efficient and economical for exploring mechanisms involved in TNF-alpha-induced insulin resistance in various genetic models of obesity-linked diabetes.
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PMID:An in vivo model for elucidation of the mechanism of tumor necrosis factor-alpha (TNF-alpha)-induced insulin resistance: evidence for differential regulation of insulin signaling by TNF-alpha. 983 30

Two Chinese hamster ovary (CHO) cell lines stably transfected with human insulin receptor cDNA, CHO-wt and CHO-mut, which express an equivalent number of normal and kinase-defective human insulin receptors, respectively, were used to assess the roles of insulin receptor tyrosine kinase activity in insulin-regulated gene expression. The effect of insulin on gene-33-promoter-driven chloramphenicol acetyltransferase (CAT), RSVLTR-driven beta-galactosidase (pRSVLTR-betagal) and SV40 late-promoter-driven hepatitis B surface antigen (pMLSV(2)HBsAg) were examined in CHO-wt and CHO-mut cells. Insulin-stimulated gene 33 promoter is 10- to 50-fold more effective in CHO-wt cells than that in parental CHO cells. However, no enhancement of insulin sensitivity of gene 33 promoter in CHO-mut cells relative to parental CHO cells was found. Similar phenomena were also observed, in that insulin regulated pRSVLTR-betagal and pMLSV(2)HBsAg in these three CHO lines. Our data indicated that the protein kinase activity of the insulin receptor is essential for the stimulatory activity of insulin toward the activities of different promoters. Copyright 1994 S. Karger AG, Basel
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PMID:Protein Kinase Activity of the Insulin Receptor Is Essential for Insulin-Regulated Gene Expression. 1172

Expression plasmids encoding either luciferase or beta-galactosidase were encapsulated in the interior of an "artificial virus" comprised of an 85 nm pegylated immunoliposome, which was targeted to the rhesus monkey brain in vivo with a monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIRMAb enables the liposome carrying the exogenous gene to undergo transcytosis across the blood-brain barrier and endocytosis across the neuronal plasma membrane following intravenous injection. The level of luciferase gene expression in the brain was 50-fold higher in the rhesus monkey as compared to the rat. Widespread neuronal expression of the beta-galactosidase gene in primate brain was demonstrated by both histochemistry and confocal microscopy. This approach makes feasible reversible adult transgenics in 24 hours.
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PMID:Global non-viral gene transfer to the primate brain following intravenous administration. 1257 13

Adaptation of the maternal uterine vasculature is essential for normal fetal and placental development in which angiogenesis is considered one of the most critical adaptive changes during pregnancy. Highly expressed in cytotrophoblasts and maternal endothelial cells during pregnancy, IGF-II promotes cell migration and regulates fetal and placental growth. We hypothesized that IGF-II regulates uterine angiogenesis during pregnancy. Both uterine vasculature and isolated uterine microvascular endothelial cells expressed high levels of IGF-II and IGF-II/mannose-6 phosphate receptor mRNA as shown by in situ hybridization. Physiological concentrations of IGF-II significantly increased vessel formation, as shown by a three-dimensional angiogenesis assay in vitro or a chicken chorionallantoic membrane assay in vivo. The angiogenic response of IGF-II could be reversed by the addition of beta-galactosidase or rabbit-antihuman IGF-II/M6P receptor antiserum, whereas blocking antibodies against IGF-I receptor or insulin receptor influenced IGF-II-induced sprout formation. IGF-II promoted migration of endothelial cells (10-250 ng/ml) tested in a modified Boyden chamber, but no stimulating effect on proliferation was observed. The application of several intracellular signal transduction molecules and their inhibitors indicated that protein kinase C and G(i) protein might play a role in the IGF-II-induced angiogenesis. Our results suggest an important angiogenic role of IGF-II in the vascular adaptation to pregnancy.
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PMID:Possible angiogenic roles of insulin-like growth factor II and its receptors in uterine vascular adaptation to pregnancy. 1455 59