Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Insulin action is thought to be mediated by an inositol-, glucosamine- and galactose-containing oligosaccharide liberated by phosphodiesterase hydrolysis of a glycosyl-phosphatidylinositol. This oligosaccharide inhibits insulin biosynthesis and secretion in pancreatic islets. In the present study, two main glycolipids (peak I and II) were resolved by sequential TLC of lipids extracted from islet cells labelled with tritiated glucosamine, galactose or myristate. The two glycolipids displayed comparable sensitivity to beta-galactosidase but differed from one another by their sensitivity to phosphatidylinositol-specific phospholipase C. Moreover, structural heterogeneity within each peak was suggested by their partial resistance to nitrous acid deamination. These findings support the presence in islet cells of glycolipids similar to those currently considered as a possible postreceptor target for insulin in other cell types.
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PMID:Metabolic labelling and partial characterization of glycophospholipids in pancreatic islet cells. 165 34

An enzyme-linked immunosorbent assay (ELISA) for insulin was developed. Anti-insulin antibody was bound to the bottom of 96-well microtiter plates. Insulin conjugated to beta-galactosidase was used as a label and methyl umbilliferyl beta-D galactoside was used as an enzyme substrate. To estimate insulin, relative fluorescence was measured with a fluorescent microtiter plate reader. Unknowns from an insulin release experiment yielded results comparable to those obtained with our enzyme-immunoassay (EIA) and a conventional radioimmunoassay (RIA). The insulin ELISA is suitable for research purposes in which samples contain solutions of physiological salts and albumin, but not for samples containing serum. The insulin ELISA is as sensitive as the insulin RIA and has several advantages over the standard insulin RIA. These include (1) avoidance of hazards and inconvenience of handling radioactivity, (2) not requiring a separate test tube for each sample, (3) stability of the enzyme-labeled insulin (greater than 18 months), (4) short time period required for the assay (less than 6 hours), and (5) the possibility of long-term storage (at least 3 months) of antibody-coated microtiter plates.
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PMID:A rapid ELISA for measuring insulin in a large number of research samples. 265 25

Alterations in the cardiac tissue and serum acid hydrolase activities were studied in chronic streptozotocin-induced diabetes in rats. No changes were observed in total cardiac tissue homogenate lysosomal enzyme activities at 4 weeks of diabetes but there were significant alterations in the distribution of selected enzymes. Significant decreases in nonsedimentable beta-N-acetylglucosaminidase (NAG) and beta-galactosidase (Gal) activities were observed at 4 weeks of diabetes. At 8 weeks of the disease, decreased activities of NAG and Gal were observed in heart homogenates but no changes were apparent in alpha-mannosidase (Man) or acid phosphatase activities. Nonsedimentable activities of NAG and both sedimentable and nonsedimentable activities of Gal were decreased at 8 weeks. At 16 weeks of the diabetic condition, increased activities of NAG, Gal and acid phosphatase were observed. This increase at 16 weeks of the disease was due to an increase in sedimentable enzyme activity. At all times of diabetes, serum enzyme activities were significantly increased. Insulin treatment reversed all of the observed changes in tissue homogenates, but serum levels were not completely reversed. These results suggest that cardiac lysosomal hydrolases are probably only involved in the later stages of the diabetic cardiomyopathy when extensive ultrastructural derangements are evident. The present evidence also suggests that the heart may be a source of serum hydrolase activities.
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PMID:Alterations in heart and serum lysosomal activities in streptozotocin-induced diabetes. 295 2

We assayed plasma activities of beta-galactosidase, beta-hexosaminidase, alpha-fucosidase and alpha-galactosidase involved in degradation of the glycoprotein molecule in 110 insulin-dependent diabetics aged 3-1/2 to 19 years and compared them to a group of normal youngsters. We correlated the plasma enzyme activities with the duration, control and sequelae of insulin-dependent diabetes. Insulin-dependent diabetics had a significantly higher plasma activity of beta-hexosaminidase and alpha-mannosidase (p less than 0.01) and a significantly lower plasma activity of alpha-fucosidase and alpha-galactosidase (p less than 0.01). Of the 5 enzymes studied, only plasma beta-hexosaminidase correlated with fasting and postprandial blood sugar (p less than 0.01), cholesterol and triglycerides (p less than 0.05). Additionally, poor control of diabetes was also associated with a significantly higher plasma beta-hexosaminidase activity (p less than 0.01). Proteinuria or an abnormal Addis count suggestive of renal involvement was associated with various changes in plasma acidic hydrolases. These changes may be related to insulin deficiency rather than hyperglycemia and may be genetically determined.
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PMID:Plasma acidic glycohydrolases in insulin-dependent diabetes mellitus. 730 74

Hereditary diabetic mice (NSY) were inbred from original streptozotocin diabetic ICR mice for 8-9 generations using hyperglycemia as an index. The normoglycemic ICR mice were used as controls for the NSY line. The nonfasting blood sugar level of the NSY mice was 305 +/- 14 mg/100ml, while their immunoreactive insulin level was 30 +/- 4 microU/ml (the values of the controls were 165 +/- 12 mg/100 ml and 79 +/- 14 microU/ml, respectively). beta-N-Acetylglucosaminidase [EC 3.2.1.29], beta-galactosidase [EC 3.2.1.23], alpha-glucosidase [EC 3.2.1.21], and alpha-mannosidase [EC 3.2.1.24] activities were determined in the 1,000 X g supernatant of the liver and the kidney of control and streptozotocin diabetic ICR mice and their NSY line. In the kidneys of the insulinopenic NSY mice, the beta-galactosidase and alpha-mannosidase activities were significantly decreased. No significant changes were found in liver enzyme activities. Insulin treatment increased the kidney beta-galactosidase activity signficantly. The insulinopenic state, which caused a decrease in the glycosidase activities in the kidney, could induce retarded breakdown of glycoprotein.
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PMID:Glycosidase activities in the liver and kidney of hereditary diabetic mice. 739 Sep 71

Genetic manipulation of pancreatic islets before transplantation has the potential to alter cellular immunity as well as islet function. The purpose of this study was to examine the feasibility of gene transfer to islets, using replication-defective adenoviral vectors. Newborn mouse islets were infected with AdHCMVsp1LacZ vector encoding Escherichia coli beta-galactosidase (beta-gal). Islets were cocultured with vector, at virus-to-target cell ratios of 10:1, for 1 hr. Gene transfer was assessed by specific histochemical stain for beta-gal (X-gal). Islet DNA and RNA were analyzed by Southern and PCR for beta-gal and adeno sequences, and recombinant protein production by western and ONPG assays. Islet integrity after gene transfer was assessed by static incubations and transplantation to nondiabetic and to diabetic mice. Southern analysis and PCR confirmed the presence of E coli beta-galactosidase and the E4 adeno DNA in infected islets, but not in controls. Reverse-transcription PCR and western analysis demonstrated expression and protein production of inserted E coli beta-galactosidase, but not E4 message. Insulin release in response to static incubations was unimpaired in infected islets. Syngeneic islet grafts stained positively for insulin for up to 7 days. Transplanted, genetically manipulated islets functioned similarly to control islets in reversing murine drug-induced diabetes. Thus, gene transfer into islets can be accomplished using adenovirus-based vectors. The capacity of this virus to infect non-dividing cells allows insertion of cDNA into pancreatic islets, with potential application to the transplant setting.
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PMID:Efficient gene transfer to pancreatic islets mediated by adenoviral vectors. 783 50

Recent studies suggest that the ras-map kinase and PI3-kinase cascades converge. We sought to determine whether PI3-kinase is downstream of ras in insulin signaling in a classic insulin target cell. We generated a recombinant adenovirus encoding dominant negative ras by cloning the human H-ras cDNA with a ser to asn substitution at amino acid 17 (ras(asn17)) into the pACCMVpLpA vector and cotransfecting 293 cells with the pJM17 plasmid containing the adenoviral genome. Efficiency of gene transfer was assessed by infecting fully differentiated 3T3L1 adipocytes with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 70% of cells were infected. Infection of adipocytes with ras(asn17) resulted in 10-fold greater expression than endogenous ras. This high efficiency gene transfer allowed biochemical assays. Insulin stimulation of ras-GTP formation was inhibited in ras(asn17)-expressing cells. Map kinase gel mobility shift revealed that insulin (1 UM) or epidermal growth factor (100 ng/ml) resulted in the appearance of a hyperphosphorylated species of p42 map kinase in uninfected cells and those expressing beta-gal but not in cells expressing ras(asn17). In contrast, insulin increased IRS-1-associated PI3-kinase activity approximately 10-fold in control cells and high level overexpression of ras(asn17) did not impair this effect. Similarly, insulin and epidermal growth factor activation of total (no immunoprecipitation) PI3-kinase activity in both cytosol and total cellular membranes and insulin stimulation of glucose transport were not affected by expression of dominant negative ras. Thus, adenovirus-mediated gene transfer is effective for studying insulin signaling in fully differentiated insulin target cells. Inhibition of ras activation abolishes insulin-stimulated phosphorylation of map kinase but does not affect insulin stimulation of PI3-kinase activity. In normal cell physiology, PI3-kinase does not appear to be downstream of ras in mediating the actions of insulin.
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PMID:Adenovirus-mediated gene transfer of dominant negative ras(asn17) in 3T3L1 adipocytes does not alter insulin-stimulated P13-kinase activity or glucose transport. 899 89

Reduction of amylin content and secretion in rat islets was attempted by transduction with an adenovirus bearing a 0.2-kb fragment of rat amylin cDNA inserted in the antisense orientation (AdCMV-alpha amylin). Exposure of islets to AdCMV-alpha amylin at a multiplicity of infection (moi) of 200 (1.2 x 10(7) pfu/ml) reduced amylin mRNA levels by 37 +/- 5% (p < 0.005), whereas infection with an adenovirus expressing the reporter gene of beta-galactosidase (AdCMV-lacz) did not modify amylin expression. Transduction with the antisense construct was specifically associated with the decrease (30 +/- 6%; p < 0.001) in the amylin content. Insulin content was unaltered in AdCMV-alpha amylin islets compared to AdCMV-lacz-transduced or untransduced cells. Basal amylin secretion (2.8 mM glucose) was 36 +/- 3% (p < 0.005) lower in AdCMV-alpha amylin islets than in untransduced or AdCMV-lacz-transduced islets. In contrast, no difference in amylin secretion in response to high glucose concentrations (16.7 mM) was detected in AdCMV-alpha amylin-transduced islets. Thus, a reduction of amylin content and basal secretion in islet cells can be achieved by the adenovirus-mediated expression of antisense RNA.
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PMID:Reduction of islet amylin expression and basal secretion by adenovirus-mediated delivery of amylin antisense cDNA. 970 Sep 51

Constitutive activation of phosphoinositide 3-kinase (PI3K) stimulates glucose transport and GLUT4 glucose transporter translocation to the plasma membrane in adipocytes. To determine whether a direct interaction of PI3K with GLUT4-containing vesicles (hereafter called GLUT4 vesicles) is important for the effect of insulin on GLUT4 translocation, we targeted constitutively active PI3K to GLUT4 vesicles. We fused the inter-Src homology region 2 of the regulatory p85alpha subunit of PI3K (iSH2) either to a C-terminal sequence of GLUT4 (G4c, amino acids 406-509) or to this region and the N-terminal tail of GLUT4 (G4n, amino acids 1-19), resulting in the fusion proteins iSH2-G4c and G4n-iSH2-G4c, respectively. Coexpression of the fusion proteins or untargeted iSH2 with the catalytic p110alpha subunit of PI3K (p110) in 3T3-L1 adipocytes by adenovirus-mediated gene transfer increased total PI3K activity in homogenates 5.0-6.7-fold over nontransduced cells or cells transduced with adenovirus encoding beta-galactosidase. In contrast, PI3K activity in GLUT4 vesicles increased 11-13-fold with expression of either targeted construct and p110 but only 2-fold with the untargeted iSH2 and p110, indicating successful targeting of PI3K to GLUT4 vesicles. Both targeted and nontargeted constructs stimulated DNA synthesis to levels greater than insulin, demonstrating that both types of constructs had biologic activity in intact cells. Despite this, untargeted iSH2/p110 coexpression was more effective in stimulating 2-deoxyglucose uptake (6-fold) than either iSH2-G4c/p110 or G4n-iSH2-G4c/p110 coexpression (both 2-fold). Only iSH2/p110 coexpression led to a significant GLUT4 translocation to the plasma membrane. Insulin-stimulated glucose transport was unaffected by any construct. Thus, a direct interaction between PI3K and GLUT4 vesicles is either not required or not sufficient for GLUT4 translocation and stimulation of glucose transport.
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PMID:Targeting of constitutively active phosphoinositide 3-kinase to GLUT4-containing vesicles in 3T3-L1 adipocytes. 973 18

Transfer of genes with potential therapeutic utility to the pancreatic islets of Langerhans may enhance graft survival after islet transplantation. The aim of this study was to determine the optimal conditions for adenoviral-mediated gene transfer to the islets of Langerhans in the absence of vector-induced toxicity. Neonatal rat islets were transduced in groups of 25 with an adenoviral vector encoding beta-galactosidase (AdbetaGal) at doses of MOI 0, 10, 100 and 1000 pfu per islet cell. All experiments were performed in triplicate. Efficiency of gene transfer was determined by gross inspection and estimation of the percentage of beta-galactosidase positive cells after islet dispersion at 1, 4, 7 and 10 days post-transduction. Islet toxicity was assessed by measuring accumulated insulin levels at each time-point and by assessing static incubation insulin release at 3 and 10 days. Efficient dose-dependent gene transfer to the islets was documented at 1, 4, 7 and 10 days post-transduction. Transgene expression was relatively stable for the duration of the experiment. Insulin accumulation did not differ between transduced and non-transduced islets at each timepoint. Likewise, the insulin secretory response to glucose, obtained by dividing the insulin response to high glucose incubation by the insulin response to low glucose incubation was similar in transduced and non-transduced islets at 3 and 10 days at all doses studied. In summary, adenoviral-mediated transduction of islets results in dose dependent efficient gene transfer with relatively stable transgene expression in the absence of toxicity. This technology may be useful in the study of islet biology and also in the future in gene therapy approaches to the treatment of diabetes mellitus.
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PMID:Absence of toxicity associated with adenoviral-mediated transfer of the beta-galactosidase reporter gene to neonatal rat islets in vitro. 1046 38


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