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:4.1.1.15 (glutamate decarboxylase)
2,169 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Levels of mRNAs encoding for the two isoforms of glutamate decarboxylase, GAD67 and GAD65, were quantified in the rat striatum and cerebral cortex following single injections of the non-competitive NMDA receptor antagonist MK-801. Acute MK-801 induced a dose-dependent decrease in GAD65 mRNA levels in ventral sectors of the dorsal striatum. No statistically significant changes in GAD67 mRNA levels were measured in the same conditions. In the frontal and cingulate cortex, GAD65 but not GAD67 mRNA levels were significantly decreased after a single high dose of MK-801 (0.1 mg kg-1). These results suggest that NMDA receptors are involved in the transcriptional regulation of the GABA synthesizing enzyme, GAD65.
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PMID:MK-801 decreases striatal and cortical GAD65 mRNA levels. 854 90

The effects of in vivo administration of dopamine receptor agonists or antagonists on the mRNA levels encoding for the two isoforms of glutamate decarboxylase, GAD65 and GAD67, and for preproenkephalin were studied in regions of the rat dorsal striatum by radioactive in situ hybridization histochemistry. Changes in striatal mRNA levels after drug treatment were quantified by computerized densitometry on X-ray films. Chronic administration of the dopamine receptor agonist apomorphine or the D1 dopamine receptor agonist SKF-38393 resulted in increased GAD65 mRNA levels in the dorsomedial, ventromedial, dorsolateral and ventrolateral sectors of the striatum. Apomorphine or SKF-38393 treatment did not induce significant effects on GAD67 and preproenkephalin mRNA levels in striatum. On the other hand, chronic administration of the D2 dopamine receptor agonist quinpirole significantly decreased GAD67 in the dorsolateral and ventrolateral and preproenkephalin in the ventrolateral sectors of the striatum. Quinpirole treatment did not induce significant changes in GAD65 mRNA levels. Chronic administration of the dopamine receptor antagonist haloperidol resulted in a significant increase in GAD67 and preproenkephalin mRNA levels in the dorsomedial, dorsolateral and ventrolateral striatal sectors. Chronic treatment with the D2/D3 dopamine receptor antagonist sulpiride resulted in a significant increase in GAD67 in the ventromedial and ventrolateral and PPE in the dorsomedial and ventrolateral striatal sectors. Haloperidol or sulpiride did not induce significant changes in striatal GAD65 mRNA levels. Chronic administration of the D1 dopamine receptor antagonist SCH-23390 had no significant effect on GAD67, GAD65 or preproenkephalin mRNA levels. In the present experimental conditions, stimulation of dopamine receptors with apomorphine or SKF-38393 resulted in increased GAD65 mRNA levels whereas blockade of dopamine receptors with haloperidol or sulpiride resulted in increased GAD67 mRNA levels. These results indicate that striatal GAD65 and GAD67 mRNA levels are differentially regulated by dopamine receptor subtypes.
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PMID:Differential regulation of mRNA levels encoding for the two isoforms of glutamate decarboxylase (GAD65 and GAD67) by dopamine receptors in the rat striatum. 875 Aug 62

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease in which cytokines are thought to play an important role in beta-cell destruction and immune regulation. A major target of beta-cell autoimmunity in IDDM is the enzyme glutamate decarboxylase (GAD). We hypothesized that cytokines in the insulitis lesion modulate the synthesis of GAD. This may, in turn, modify the rate of beta-cell destruction. Accordingly we cultured rat islets in the presence and absence of cytokines, and measured synthesis of both isoforms of GAD, GAD65 and GAD67, by [35S]methionine incorporation and immunoprecipitation with a rabbit antiserum that recognizes both GAD65 and GAD67. Incubation of islets with interleukin (IL)-1 beta (1 ng/ml, 24 h), tumour necrosis factor alpha (TNF-alpha; 200 units/ml, 24 h) or interferon gamma (IFN-gamma; 500 units/ml, 72 h) significantly decreased the synthesis of both GAD65 and GAD67, but reduced neither total protein synthesis nor insulin accumulation in the medium or content. Incubation of islets for 24 h in IFN-alpha (1000 units/ml), TNF-beta (50 ng/ml), IL 2 (1000 units/ml), IL-4 (100 ng/ml), IL-6 (10 ng/ml), IL-10 (20 ng/ml), IL-12 (10 ng/ml) or transforming growth factor beta 2 (TGF-beta 2; 5 ng/ml) did not significantly alter GAD65 or GAD67 synthesis. Inhibition of GAD65 and GAD67 protein synthesis by IL-1 beta, TNF-alpha or IFN-gamma was reversed by co-incubation with the nitric oxide synthase inhibitor, NG-monomethyl arginine (NMMA). Expression of both GAD65 and GAD67 mRNA, measured by RNase protection assay, was also decreased by IL-1 beta and completely restored to baseline levels by NMMA. Thus the synthesis of both isoforms of islet GAD is selectively decreased in the presence of IL-1 beta, TNF-alpha or IFN-gamma by a NO-mediated mechanism, probably at the level of cytokine gene transcription. As GAD autoimmunity has been previously shown to have a pathogenic role in an animal model of IDDM, its inhibition by cytokines might limit the immune response, thereby regulating the rate of beta-cell destruction in IDDM.
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PMID:Cytokine regulation of glutamate decarboxylase biosynthesis in isolated rat islets of Langerhans. 876 Mar 54

The subunit structure of brain glutamate decarboxylase in cerebellum was investigated by using gel electrophoresis and antisera that specifically recognize the individual isoforms of brain glutamate decarboxylase (termed GAD65 and GAD67). The antisera were prepared against peptides that corresponded to amino acid sequences specific to each isoform. Each antiserum reacted specifically with the appropriate peptide in an ELISA and with the appropriate form of GAD on immunoblots. Nondenaturing gradient gel electrophoresis indicated that GAD is principally multimeric with monomeric forms comprising < 3% of the total. Immunoprecipitation and immunoaffinity chromatography experiments were performed with antisera W624 and W883, which were prepared against peptides specific to GAD65 and GAD67, respectively. Immunoprecipitates prepared from cerebellar supernatants with W624 contained both GAD65 and GAD67, whereas some GAD67 was left in the supernatant. In a similar manner, immunoprecipitates prepared with W883 contained both GAD65 and GAD67, whereas some GAD65 remained in the supernatant. In addition, immunoaffinity columns prepared with either W624 or W883 retained both GAD65 and GAD67 even after extensive washing. These results are consistent with the presence of heteromultimers of GAD65 and GAD67 in cerebellum in addition to homomers of each form.
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PMID:Heteromers of glutamate decarboxylase isoforms occur in rat cerebellum. 878 39

The effect of dopaminergic denervation, alone or followed by chronic intermittent L-DOPA administration, on the levels of mRNAs encoding for the two isoforms of the GABA-synthesizing enzyme, glutamate decarboxylase (GAD65 and GAD67), were measured by in-situ hybridization in the caudate and putamen of macaque monkeys. When compared to control monkeys, the level of GAD67 mRNA was increased in the putamen and caudate of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. On the other hand, GAD65 mRNA labeling in MPTP-treated monkeys was not significantly different from the controls. In MPTP-treated monkeys that received L-DOPA, a significant increase in both GAD67 and GAD65 mRNA levels was measured in the putamen when compared to control or MPTP-treated monkeys. The results suggest that the dyskinetic effect of L-DOPA is paralleled by an increased GABAergic activity in the striatum.
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PMID:L-DOPA regulates glutamate decarboxylases mRNA levels in MPTP-treated monkeys. 880 32

Immunohistochemical methods were used to determine the earliest times of detection for two forms of glutamate decarboxylase (GAD67 and GAD65) in the embryonic and early postnatal rat hippocampal formation and to determine whether their distribution patterns differed from each other and from those of the adult. Both GAD67- and GAD65-containing neurons were observed as early as embryonic day 17 (E17)-E18 in the hippocampus and E19 in the dentate gyrus, and this was substantially earlier than GAD had been detected previously in the hippocampal formation. The two GAD isoforms displayed very similar distribution patterns, but these patterns were distinctly different from those of the adult. From E17 to E20, GAD67 and GAD65 were expressed in neuronal cell bodies throughout the hippocampal and dentate marginal zones (future dendritic layers), and relatively few existed within the principal cell body layers, where GAD-positive neurons are frequently concentrated in the adult. At E21 to postnatal day 1 (P1), there was a sudden shift from a predominance of GAD-containing cell bodies within the developing dendritic regions to a meshwork of GAD-positive processes with terminal-like varicosities in these same regions. This pattern also contrasted with that of the adult, in which GAD-labeled terminals are highly concentrated in the principal cell layers. Electron microscopic observations of the GAD-labeled processes at P1 confirmed their axon-like appearance and demonstrated that the immunoreactivity was consistently localized in vesicle-filled regions that were often closely apposed to and, in some instances, established synaptic contacts with dendritic profiles. The present identification of an early abundance of GAD-containing structures in the hippocampal formation and the marked change in their distribution during development complement recent observations of developmental changes in the functioning of the GABA system and provide additional support for the early involvement of this neurotransmitter system in hippocampal development.
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PMID:Prominent expression of two forms of glutamate decarboxylase in the embryonic and early postnatal rat hippocampal formation. 882 30

In the present study, we have investigated the developmental expression of the transmitter-synthesizing enzymes choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in rat medial septal neurons by using in situ hybridization histochemistry. In addition, we have employed immunostaining for ChAT and the calcium-binding protein parvalbumin, known to be contained in septohippocampal GABAergic neurons. A large number of GAD67 mRNA-expressing neurons were already observed in the septal complex on embryonic day (E) 17, the earliest time point studied. During later developmental stages, there was mainly an increase in the intensity of labeling. Neurons expressing ChAT mRNA were first recognized at E 20, and their number slowly increased during postnatal development of the septal region. The adult pattern of ChAT mRNA-expressing neurons was observed around postnatal day (P) 16. By using a monoclonal ChAT antibody, the first immunoreactive cells were not seen before P 8. Similarly, the first weakly parvalbumin-immunoreactive neurons were seen in the septal complex by the end of the 1st postnatal week. These results indicate that in situ hybridization histochemistry may be an adequate method to monitor the different development of transmitter biosynthesis in cholinergic and GABAergic septal neurons. Moreover, the late onset of ChAT mRNA expression would be compatible with a role of target-derived factors for the differentiation of the cholinergic phenotype.
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PMID:Development of cholinergic and GABAergic neurons in the rat medial septum: different onset of choline acetyltransferase and glutamate decarboxylase mRNA expression. 886 26

The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA-producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells. Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells. Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations. These results indicate that dentate granule cells normally contain two "fast-acting" amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects.
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PMID:Basal expression and induction of glutamate decarboxylase and GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus. 888 46

The gamma-aminobutyric acid (GABA)-synthesizing enzyme glutamate decarboxylase (GAD) was studied during development of the chick telencephalon. By means of reverse-phase HPLC analysis, we showed that GABA indeed accumulates during embryogenesis, whereas the levels of glutamate, the substrate for GAD, are more or less unchanged up to later developmental stages. The enzyme activity increased approximately 25-fold from embryonic day 3 to embryonic day 17. Immunoblotting data revealed that two GAD proteins, of approximately 65 and 67 kDa, were present during the period investigated. Furthermore, Northern blot analysis with probes obtained from rat cDNA sequences, as well as a chicken-specific probe for GAD65 generated by means of reverse transcriptase-polymerase chain reaction (RT-PCR), strengthened the interpretation that the chick embryo expresses genes corresponding to GAD65 and GAD67. The rat probes recognized transcript sizes of 3.9 kb (GAD65) and 5.6 kb (GAD67), sizes which are different from those of the rat brain (Erlander et al., Neuron, 7, 91-100, 1991). Sequencing of the RT-PCR products revealed a high level of homology (82% at the nucleotide level) between the mammalian and chick GAD65 genes. Taken together, these findings suggest that the chick embryo expresses two GAD genes during embryogenesis. The functional properties of each gene product remain to be investigated.
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PMID:Two glutamate decarboxylase forms corresponding to the mammalian GAD65 and GAD67 are expressed during development of the chick telencephalon. 892 2


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