Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0011849 (diabetes)
 277,896 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

cDNAs coding for the full-length human 65 and 67 kDa glutamic acid decarboxylases (GAD65 and GAD67) were amplified from pancreas and hippocampus cDNA libraries by polymerase chain reaction, respectively. Both cDNAs were inserted into a baculovirus vector which mediated highly efficient expression of the human GAD65 and GAD67 with histidine-hexapeptides as affinity ligands at their C-termini in Spodoptera frugiperda (Sf9) cells. The recombinant GAD proteins were purified to homogeneity by affinity chromatography using a metal-chelating matrix. The infected Sf9 insect cells expressed the recombinant human GAD65 and GAD67 with natural-like conformations, as confirmed by measurement of their enzyme activities as well as their fully restored autoantigenicities. Immunoprecipitation of metabolically labeled infected Sf9 cells demonstrated the autoantigenic potential of the recombinant GAD proteins. The practicability of using recombinant GAD65 and GAD67 derived from the baculovirus expression system for the development of an immunoassay for the diagnosis of insulin-dependent diabetes mellitus is discussed.
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PMID:Baculovirus-mediated expression of human 65 kDa and 67 kDa glutamic acid decarboxylases in SF9 insect cells and their relevance in diagnosis of insulin-dependent diabetes mellitus. 837 Jun 67

Homogenates of pancreatic islets catalyzed breakdown of L-glutamate to GABA with a rate of 0.24 +/- 0.04 nmol.min-1 x mg-1 protein at 37 degrees C. The formation of GABA was stimulated by addition of pyridoxal phosphate in the range 0.05-1 microM (0.97 +/- 0.02 nmol.min-1 x mg protein-1 at a saturating cofactor concentration), which indicates that the process was catalyzed by glutamic acid decarboxylase. The half-maximal effect was obtained with 0.1 microM PLP. Kinetic analyses of the results showed that the Vmax and Km for the reaction were 1.12 nmol.min-1 x mg protein-1 and 0.66 mM, respectively. The pH optimum was 7.0. Subcellular fractionation revealed that 51% of GAD activity was present in the cytosol, 17% in microsomes, 9% in secretory granules, 5% in mitochondria, and 11% in cell debris. Comparison of the kinetic properties of the cytosolic and microsomal forms of the enzyme showed that their Km for glutamate was the same, but that the cytosolic GAD had a lower Km for PLP. GABA synthesis in the nominal absence of PLP was enhanced by malate (twofold increase at 5 mM) and citrate (threefold increase at 5 mM), but was unaffected by ATP and chloride. However, if the islet homogenate was prepared and incubated in the presence of PLP, neither malate nor citrate influenced enzyme activity. Aspartate and AOA were powerful inhibitors of glutamate breakdown. Freshly isolated islets contained approximately 4 mM GABA, whereas the concentration was < 0.1 mM in whole pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)
Diabetes 1993 Oct
PMID:GABA production in rat islets of Langerhans. 837 91

We investigated the presence of autoantibodies to baculovirus-expressed human recombinant 65- and 67-kD isoforms of glutamate decarboxylase (GAD65 and GAD67) in insulin-dependent diabetes mellitus (IDDM). In the immunoprecipitation test using [35S]methionine-labeled GADs antibodies to GAD65 were detected in 13/15 (87%) islet cell antibody (ICA)-positive and in 1/35 (2.9%) ICA-negative first-degree relatives of patients with IDDM, in 6/11 (54.5%) ICA-positive nondiabetic schoolchildren, and in 35/50 (70%) patients with newly diagnosed IDDM. GAD67 antibodies were positive only in five (33%) of the ICA-positive relatives (P < 0.05) and in nine (18%) IDDM patients at onset (P < 0.00001). After onset of IDDM antibodies to GAD65 and GAD67 declined but were still positive in 25 and 9.4% of subjects with long-standing IDDM (> 10 yr). In all study groups antibodies to GAD67 were only detected in GAD65 antibody-positive sera. An immunotrapping enzyme activity assay for GAD65 antibodies was positive in 64/75 (85.3%) of sera that were GAD antibody positive in the immunoprecipitation test (r = 0.870, P < 0.0001). In two (2.7%) sera GAD65 antibodies that block GAD enzyme activity were found. Our data suggest that antibodies to GAD65 but not to GAD67 represent sensitive markers for preclinical and overt IDDM. The immunotrapping assay here described represents a valuable technique for specific and sensitive screening for GAD antibodies.
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PMID:Prevalence of autoantibodies to the 65- and 67-kD isoforms of glutamate decarboxylase in insulin-dependent diabetes mellitus. 837 91

Cytoplasmic islet cell antibodies are well-established predictive markers of IDDM. Although target molecules of ICA have been suggested to be gangliosides, human monoclonal ICA of the immunoglobulin G class (MICA 1-6) produced from a patient with newly diagnosed IDDM recognized glutamate decarboxylase as a target antigen. Here we analyzed the possible heterogeneity of target antigens of ICA by subtracting the GAD-specific ICA staining from total ICA staining of sera. This was achieved 1) by preabsorption of ICA+ sera with recombinant GAD65 and/or GAD67 expressed in a baculovirus system and 2) by ICA analysis of sera on mouse pancreas, as GAD antibodies do not stain mouse islets in the immunofluorescence test. We show that 24 of 25 sera from newly diagnosed patients with IDDM recognize islet antigens besides GAD. In contrast, GAD was the only islet antigen recognized by ICA from 7 sera from patients with stiff man syndrome. Two of these sera, however, recognized antigens besides GAD in Purkinje cells. In patients with IDDM, non-GAD ICA were diverse. One group, found in 64% of the sera, stained human and mouse islets, whereas the other group of non-GAD ICA was human specific. Therefore, mouse islets distinguish two groups of non-GAD ICA and lack additional target epitopes of ICA besides GAD. Longitudinal analysis of 6 sera from nondiabetic ICA+ individuals revealed that mouse-reactive ICA may appear closer to clinical onset of IDDM in some individuals. Mouse-reactive ICAs, however, remained absent in 36% of the patients at diagnosis of IDDM.
Diabetes 1993 Nov
PMID:Cytoplasmic islet cell antibodies recognize distinct islet antigens in IDDM but not in stiff man syndrome. 840 7

Individuals with or at risk for insulin-dependent diabetes (IDD) frequently have autoantibodies against an islet cell cytoplasmic (ICA) antigen thought to be a sialoglycolipid. However, we now report that preabsorption of ICA-positive sera with recombinant glutamate decarboxylase (human GAD 65 and/or GAD 67) reduced or blocked the ICA reactivity of 5/18 (27%) new-onset IDD patients and 7/18 (39%) prediabetics. Interestingly, nondiabetic subjects with ICA of > or = 5 yr in duration had GAD-reactive ICA significantly more often (16/24, 67%, P < 0.04) than the diabetic groups. ICA reactivity to GAD was not related to serum ICA titer nor the age of the individual, and in all cases tested was blocked by GAD 65 or GAD 67 with equivalent efficiency. The ICA observed in 21/25 (84%) IDD patients with ICA long after clinical onset of disease (9-42 yr) was reactive to GAD. A natural history analysis of three individuals showed conversions from ICA which was reactive to GAD to a non-GAD-reactive ICA nearer to their clinical onsets of IDD. This study further defines the autoantigens reactive to ICA, and suggests that, whereas ICA that are not reactive to GAD may identify an advanced and more prognostic lesion, GAD-reactive ICA may typify the early or inductive lesion that may or may not progress to clinically significant beta cell injury.
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PMID:Islet cell cytoplasmic autoantibody reactivity to glutamate decarboxylase in insulin-dependent diabetes. 842 31

The classification of adults with diabetes mellitus can be invalidated by patients who initially present as NIDDM but who later become frankly insulin dependent. In some of these, the pathogenesis could be similar to that in IDDM, namely autoimmune destruction of the pancreatic beta-cells. We studied 102 patients > 35 yr of age at diabetes onset who had initially been nonketotic and non-insulin-dependent for > or = 6 mo. They were classified according to glucagon-stimulated C-peptide levels into an insulin-deficient group (n = 33) and a non-insulin-deficient group (n = 69). We measured antibodies to GAD, islet cell cytoplasm, thyroid antigens, and gastric parietal cells in both groups. Anti-GAD was significantly higher in the insulin deficient group, 76% (25 of 33), than in the non-insulin deficient group, 12% (8 of 69), and this difference was substantially greater than that shown for ICAs. Thus, in a proportion of adults who present with NIDDM, a slowly evolving autoimmune insulitis can be revealed by testing for anti-GAD. This could have important connotations not only for early intervention, but also for the correct classification of diabetes.
Diabetes 1993 Feb
PMID:Antibodies to glutamic acid decarboxylase reveal latent autoimmune diabetes mellitus in adults with a non-insulin-dependent onset of disease. 842 74

The GABA synthesizing enzyme GAD is a prominent islet cell autoantigen in type I diabetes. The two forms of GAD (GAD64 and GAD67) are encoded by different genes in both rats and humans. By in situ hybridization analysis of rat and human pancreases, expression of both genes was detected in rat islets, whereas only GAD64 mRNA was detected in human islets. Immunocytochemical analysis of rat and human pancreatic sections or isolated islets with antibodies to GAD64 and GAD67 in combination with antibodies to insulin, glucagon, or SRIF confirmed that a GAD64 and GAD67 expression were beta-cell specific in rat islets. In contrast, only GAD64 was detected in human islets and was, in addition to beta-cells, also surprisingly localized to some alpha-cells, delta-cells, and PP-cells. In long-term (4 wk) monolayer cultures of newborn rat islet cells, GAD64 expression remained beta-cell specific as observed in vivo, whereas GAD67 was localized not only to the beta-cells but also in the alpha-cells and delta-cells. A small but distinct fraction of GAD positive cells in these monolayer cultures did not accumulate GABA immunoreactivity, which may indicate cellular heterogeneity with respect to GABA catabolism or GAD enzyme activity. In a rat insulinoma cell line (NHI-6F) producing both glucagon and insulin depending on the culture conditions, GAD64 expression was detected only in cultures in which the insulin producing phenotype dominated. In conclusion, these data demonstrate that the two GAD isoforms are differentially expressed in rat and human islets but also that the expression differs according to culture conditions. These findings emphasize the need to consider both the species and culture conditions of islets.
Diabetes 1993 Mar
PMID:Differential expression of glutamic acid decarboxylase in rat and human islets. 843 19

Plasmids containing cDNA for the rat 67- and 65-kD isoforms of glutamate decarboxylase (GAD-67 and GAD-65) were expressed in COS-cells, and lysates of [35S]methionine-labeled cells were used for immunoprecipitations. Sera from 38 patients with type 1 (insulin-dependent) diabetes mellitus, which precipitated a 64-kD antigen from rat islets, reacted with recombinant GAD-65 in relation to their anti-64-kD titers. The eight strongest sera also precipitated recombinant GAD-67, suggesting that certain epitopes are common to both isoforms. Subsequently, [35S]methionine-labeled GAD-65 was purified from COS cell lysates and employed in a binding assay with 50 sera of patients with recent onset of type 1 diabetes mellitus. 38 sera (76%) precipitated labeled GAD-65 with titers that correlated with islet cell antibodies (ICA), determined in a standard immunofluorescence assay. 2 sera were GAD positive but ICA negative, 4 were positive only for ICA, and 6 were negative for both GAD and ICA, as were the sera of 20 controls. The data illustrate that antibodies against GAD-65 are present in a majority of patients with type 1 diabetes mellitus and that autoantibodies against other islet cell antigens also exist. The radioligand-binding assay, which is convenient and sensitive for detecting GAD antibodies, will facilitate the screening of individuals with autoimmune islet cell disease.
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PMID:Demonstration of GAD-65 as the main immunogenic isoform of glutamate decarboxylase in type 1 diabetes and determination of autoantibodies using a radioligand produced by eukaryotic expression. 848 75

One of the paradoxes of insulin-dependent diabetes mellitus is that the destruction of the pancreatic islets' endocrine cells is restricted to the insulin-producing beta cells, whereas the main autoantibodies, islet cell antibodies (ICA), are directed against all endocrine islet cells. GAD has recently been proposed as the main target of the humoral and cellular autoimmune attack to the islets, and since in rat pancreas this enzyme was expressed only in the beta cells, this provided an explanation for the cell specificity of the destructive process. The finding of GAD-positive cells in the islets of two diabetic patients, one of whom had completely lost the beta cells, led us to study in detail the distribution of GAD in normal human islet cells using a panel of GAD antisera and the double indirect immunofluorescence technique on cryostat sections, monolayer cultures and cytosmears. The results showed that GAD is present not only in the cytoplasm of beta cells but also in 69% of the alpha and 27% of the delta cells. GAD was not present, however, on the surface of the islet cells. These results suggest that the cellular distribution of GAD can not by itself explain the selectivity of beta cell destruction in insulin-dependent diabetes mellitus.
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PMID:Expression of glutamic acid decarboxylase (GAD) in the alpha, beta and delta cells of normal and diabetic pancreas: implications for the pathogenesis of type I diabetes. 851 74

We determined the prevalence of antibodies to glutamic acid decarboxylase (anti-GAD) in Japanese diabetic patients. Anti-GAD were detected by RIP Anti-GAD Hoechst, which is a new sensitive radioimmunoassay (RIA) kit using purified pig brain GAD as the antigen. One thousand nine hundred Japanese patients were collected by the Study Group for Antibodies to GAD. The prevalence of anti-GAD in the subjects of this study was: 35.4% (326/921) in all patients with IDDM, 50.3% (96/191) in patients with IDDM less than 1-year duration, 4.3% (29/680) in NIDDM, 37.9% (39/103) in slowly progressive IDDM, 10.5% (4/38) in gestational diabetes mellitus, 0% (0/27) in impaired glucose tolerance, 4.8% (6/124) in the school children with glycosuria, 2.1% (1/47) in the relatives of IDDM and 5.0% (1/20) in neurological diseases without diabetes. The prevalence in normal subjects was 2.2% (7/323). Anti-GAD are frequently detected by the RIA kit in patients with IDDM of short duration and this assay may be useful for population screening for IDDM and for better understanding of its pathogenesis.
Diabetes Res Clin Pract 1995 Jun
PMID:Antibodies to GAD in Japanese diabetic patients: a multicenter study. 852 98


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