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)

The functional role of glutamate decarboxylase (GAD) and its product GABA in pancreatic islets has remained elusive. Mouse beta-cells express the larger isoform GAD67, whereas human islets express only the smaller isoform GAD65. We have generated two lines of transgenic mice expressing human GAD65 in pancreatic beta-cells (RIP7-hGAD65, Lines 1 and 2) to study the effect that GABA generated by this isoform has on islet cell function. The ascending order of hGAD65 expression and/or activity in beta-cells was Line 1 heterozygotes < Line 2 heterozygotes < Line 1 homozygotes. Line 1 heterozygotes have normal glucose tolerance, whereas Line 1 homozygotes and Line 2 heterozygotes exhibit impaired glucose tolerance and inhibition of insulin secretion in vivo in response to glucose. In addition, fasting levels of blood glucose are elevated and insulin is decreased in Line 1 homozygotes. Pancreas perfusion experiments suggest that GABA generated by GAD65 may function as a negative regulator of first-phase insulin secretion in response to glucose by affecting a step proximal to or at the K(ATP)(+) channel.
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PMID:Increased expression of GAD65 and GABA in pancreatic beta-cells impairs first-phase insulin secretion. 1095 Aug 38

This study examined whether changes in the levels of the messenger RNAs (mRNAs) encoding the gamma-aminobutyric acid (GABA) synthesizing enzymes, glutamate decarboxylase (GAD)65 and GAD67 and transforming growth factor-alpha (TGFalpha) in the hypothalamus are correlated with the arrest of pulsatile GnRH release during infancy in the agonadal male monkey. This experiment also provided the opportunity to examine changes in hypothalamic GnRH gene expression during this critical phase of primate development. Male rhesus monkeys were castrated at 1 week of age: four were killed 4-7 weeks after orchidectomy while pulsatile GnRH release was robust as reflected by high circulating LH levels, and four were killed at 12-15 months of age after establishing that pulsatile GnRH release had been arrested. GAD65, GAD67, TGFalpha, and GnRH mRNA levels were estimated using RNase protection assays employing homologous probes and the results were expressed relative to cyclophilin mRNA levels. GnRH peptide was measured by RIA. GAD65 and GAD67 mRNA levels in the hypothalamus of juveniles were significantly greater than those in neonatal monkeys. On the other hand, hypothalamic TGFalpha and GnRH mRNA (and peptide) levels in agonadal neonate and juvenile monkeys were indistinguishable. These results indicate that the molecular concomitants associated with bringing the hypothalamic GnRH pulse generator into check in agonadal neonatal males are not a mirror image of those previously reported at the time this neuronal network is reactivated at puberty when TGFalpha and GnRH gene expression increase and GAD65 and GAD67 mRNA levels remain unchanged. Thus, the neurobiological mechanism that reactivates pulsatile GnRH release at puberty is likely to involve more than a simple reversal of that underlying inhibition of the same network in late infancy.
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PMID:Changes in hypothalamic gene expression associated with the arrest of pulsatile gonadotropin-releasing hormone release during infancy in the agonadal male rhesus monkey (Macaca mulatta). 1096 98

GABA is synthesized by glutamate decarboxylase (GAD), which has two forms, GAD65 and GAD67. To elucidate the molecular mechanisms of mouse GAD65 (mGAD65) gene expression, we isolated and characterized the mGAD65 gene. The mGAD65 gene was found to be divided into 16 exons and spread over 75 kb. The sequence of the first exon and the 5'-flanking region indicated the presence of potential neuron-specific cis-regulatory elements. We used transgenic mice to examine the expression pattern conferred by a 9.2-kb promoter-proximal DNA fragment of the mGAD65 gene fused to the bacterial lacZ reporter gene. Transgenic mice showed high beta-galactosidase activity specifically in brain and testis. They also showed characteristic patterns of transgene expression in olfactory bulb, cerebellar cortex, and spinal cord, a similar expression pattern to that of endogenous mGAD65. However, no transgene expression was observed in the ventral thalamus or hypothalamus, in which high mGAD65 gene expression levels have been observed. These results suggest that the 9.2-kb DNA fragment of the mGAD65 gene is associated with its tissue-specific expression and its targeted expression in GABAergic neurons of specific brain regions but that additional regulatory elements are necessary to obtain fully correct expression.
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PMID:Structure of the mouse glutamate decarboxylase 65 gene and its promoter: preferential expression of its promoter in the GABAergic neurons of transgenic mice. 1098 22

In adult brain, the inhibitory GABAergic neurons utilize two distinct molecular forms of the GABA-synthesizing enzyme glutamate decarboxylase (GAD), GAD65 and GAD67. During embryonic development, two truncated forms of GAD67 are also expressed (GAD25 and GAD44), which are translated from two embryonic-specific splice variants of GAD67 messenger RNA. It has recently been established that the excitatory dentate granule cells, in addition to the neurotransmitter glutamate, also contain low levels of GABA and GAD67, which are increased after limbic seizures. To study the seizure-induced activation of glutamate decarboxylase, we investigated the expression of both embryonic and adult glutamate decarboxylase messenger RNAs in the adult rat hippocampus after kainic acid administration by semi-quantitative reverse transcription-coupled polymerase chain reaction, in situ hybridization and immunoblotting. We observed a rapid induction of the embryonic glutamate decarboxylase messenger RNA in the granule cells of dentate gyrus. The expression of embryonic glutamate decarboxylase transcripts, identified here as the splice variant that contains exon 7/B, peaked at about 2h after kainic acid injection and gradually returned to nearly basal levels by 24h. Strikingly, this transient induction of embryonic glutamate decarboxylase messenger RNA was not accompanied by concomitant synthesis of its corresponding protein product GAD25. In contrast, the adult GAD67 messenger RNA and protein were both clearly up-regulated in granule cells, albeit with a certain delay, reaching a maximum around 4-6h after kainic acid injection and gradually returned to control levels by 24h. GAD65 remained unchanged at both messenger RNA and protein levels during the studied period. These characteristic and highly reproducible changes in the synthesis of glutamate decarboxylases indicate that GAD67 is the predominant form of glutamate decarboxylases involved in the elevated synthesis of GABA during seizures and suggest that the transient induction of the embryonic GAD67 messenger RNA that contains exon 7/B, but not GAD25 protein, may exert a role solely in the subsequent up-regulation of adult GAD67 transcription. Expression of the messenger RNA encoding for an alternatively spliced, truncated form of the GABA-synthesizing enzyme glutamate decarboxylase was detected in dentate granule cells briefly after kainic acid-induced seizures. Just as during embryonic development, expression of the alternatively spliced messenger RNA was transient and followed by transcription of its adult form, indicating a possible recapitulation of an embryonic program of gene expression in adult granule cells after epileptic seizures.
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PMID:Differential regulation of adult and embryonic glutamate decarboxylases in rat dentate granule cells after kainate-induced limbic seizures. 1100 67

Amino acid sequences of E. coli glutamate decarboxylase (GADa) and those of 36 GAD of different origin were compared by pairwise alignment using computer program CLUSTAL. GADalpha and plant enzymes showed 59.8-67.8% subunit homology, GADalpha and other bacterial GAD--49.8-77.6%, whereas GADalpha and animal enzymes--13.9-58.8%. Two PLP domains exhibited higher homology comparing to that of the whole subunit in the case of GAD67, plant (68.4-73.9%), and bacterial (46.7-83.2%) enzymes. The alignment of PLP-domains of 37 GAD, three group II decarboxylases, and two pyridoxal enzymes with known 3D structures (bacterial ORD and mAAT from chicken heart) allowed us to reveal conserved residues of the active sites. Their functional role is discussed. Modelling of the PLP-binding sites in active centers for GADalpha and human brain GAD67 was done using the Swiss-PdbViewer homology modelling program. Although the homology between GADalpha and GAD67 is rather low, structural similarity of their active sites allows us to consider here a functional convergence. Thus, glutamate decarboxylation by GADalpha may be helpful for understanding general mechanism of this reaction.
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PMID:Structure of glutamate decarboxylase and related PLP-enzymes: computer-graphical studies. 1102 57

In normal development, there are dramatic changes in both the level and the laminar pattern of expression of the two forms of glutamate decarboxylase (GAD67, GAD65), the synthetic enzyme for gamma-aminobutyric acid (GABA). We have used antibodies to determine whether these normal postnatal changes in the expression of the two GADs depend on visual input by comparing normal and dark-reared cat visual cortex. Western blot analysis showed no significant differences in the levels of expression of the two enzymes between rearing conditions at either 5 or 20 weeks. Immunohistochemistry was used to compare the laminar distribution of the GADs in the two rearing conditions. At 1 week of age, both GAD67 and GAD65 immunoreactivity is concentrated in deep layers of visual cortex. At 5 and 20 weeks in both rearing conditions, GAD67-stained cells bodies were distributed rather uniformly across all cortical layers. GAD65 primarily labeled puncta (synaptic terminals) and these were also distributed rather uniformly across all visual cortical layers in both rearing conditions. Counts of GAD67-positive cell bodies and GAD65-positive puncta also revealed no differences between the rearing conditions. Thus, both GAD67, which produces the basal pool of GABA, and GAD65, which is specialized to respond to short-term increases in demand in synaptic terminals, developed normal levels of expression and normal intracellular and laminar distributions in the absence of visual input. Physiological studies suggest immaturity in the GABA system of dark-reared visual cortex. The present results indicate that such abnormalities are not due to presynaptic alterations in GABA synthetic enzymes.
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PMID:Comparison of the expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) in the visual cortex of normal and dark-reared cats. 1117 87

Glutamate decarboxylase (GAD), is a key enzyme in the central nervous system (CNS) that synthesizes the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) from glutamate. Our previous phylogenetic studies on the evolution of this enzyme indicates that there are at least two distinct forms: GAD65 and GAD67. They are the products of separate genes and probably derive from a common ancestral GAD gene following gene duplication prior to the emergence of the teleosts more than 200 Myr ago. Furthermore, a third GAD-like molecule, GAD3, discovered in the armed grenadier, Coryphaenoides (Nematonurus) armatus, is equally divergent from both GAD65 and GAD67. Specimens of C. (N.) armatus were collected by trawl at a depth of 4,000 m in the Porcupine Seabight (Northeastern Atlantic), and brains dissected and frozen for RNA extraction. All three GAD forms are found in the cerebellum, telencephalon and hypothalamus. Semiquantitative PCR analysis showed that males and females have similar levels of expression of GAD67 and GAD3 in the tissues studied. Independent of the sex examined, the levels of expression of GAD65 and GAD67 in the cerebellum were approximately half that in the telencephalon. GAD3 levels were approximately 30% higher in the cerebellum than in either the telencephalon or hypothalamus. In contrast to GAD67 and GAD3, hypothalamic expression of GAD65 mRNA is 1.8 times higher (p < 0.05) in males than in females. These data indicate that the expression of GAD65, a key enzyme for the synthesis of GABA is sexually dimorphic in females and males of C. (N.) armatus.
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PMID:Sexually dimorphic expression of glutamate decarboxylase mRNA in the hypothalamus of the deep sea armed grenadier, Coryphaenoides (Nematonurus) armatus. 1125 19

We investigated the effect of hypoxia on glutamate metabolism and uptake in rat pheochromocytoma (PC12) cells. Various key enzymes relevant to glutamate production, metabolism and transport were coordinately regulated by hypoxia. PC12 cells express two glutamate-metabolizing enzymes, glutamine synthetase (GS) and glutamate decarboxylase (GAD), as well as the glutamate-producing enzyme, phosphate-activated glutaminase (PAG). Exposure to hypoxia (1% O(2)) for 6 h or longer increased expression of GS mRNA and protein and enhanced GS enzymatic activity. In contrast, hypoxia caused a significant decrease in expression of PAG mRNA and protein, and also decreased PAG activity. In addition, hypoxia led to an increase in GAD65 and GAD67 protein levels and GAD enzymatic activity. PC12 cells express three Na(+)-dependent glutamate transporters; EAAC1, GLT-1 and GLAST. Hypoxia increased EAAC1 and GLT-1 protein levels, but had no effect on GLAST. Chronic hypoxia significantly enhanced the Na(+)-dependent component of glutamate transport. Furthermore, chronic hypoxia decreased cellular content of glutamate, but increased that of glutamine. Taken together, the hypoxia-induced changes in enzymes related to glutamate metabolism and transport are consistent with a decrease in the extracellular concentration of glutamate. This may have a role in protecting PC12 cells from the cytotoxic effects of glutamate during chronic hypoxia.
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PMID:Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells. 1125 12

Neural cultures derived from differentiating embryonic stem (ES) cells are a potentially powerful in vitro model of neural development. We show that neural cells derived from mouse ES cells express mRNAs characteristic of GABAergic neurons. The glutamate decarboxylase genes (Gad1 and Gad2), required for GABA synthesis and the vesicular inhibitory amino acid transporter (Viaat) gene, required for GABA vesicular packaging are activated in the ES-derived cultures. Nearly half of the ES-derived neurons express the GAD67 protein, the product of the Gad1 gene. Building on these results we show that Gad1-lacZ "knockin" reporter ES cell lines can be used to easily monitor Gad1 expression patterns and expression levels during ES differentiation. We also demonstrate that the ES-derived neural progenitors can be infected with retroviruses or transfected with plasmids via lipofection. These experiments outline the basic strategies and methods required for studies of GABAergic gene expression and regulation in ES-derived neuronal cultures.
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PMID:Neuronal development of embryonic stem cells: a model of GABAergic neuron differentiation. 1139 54

The recombinant forms of the two human isozymes of glutamate decarboxylase, GAD65 and GAD67, are potently and reversibly inhibited by molecular oxygen (Ki = 0.46 and 0.29 mM, respectively). Inhibition of the vesicle-associated glutamate decarboxylase (GAD65) by molecular oxygen is likely to result in incomplete filling of synaptic vesicles with gamma-aminobutyric acid (GABA) and may be a contributing factor in the genesis of oxygen-induced seizures. Under anaerobic conditions, nitric oxide inhibits both GAD65 and GAD67 with comparable potency to molecular oxygen (Ki = 0.5 mM). Two forms of porcine cysteine sulfinic acid decarboxylase (CSADI and CSADII) are also sensitive to inhibition by molecular oxygen (Ki = 0.30 and 0.22 mM, respectively) and nitric oxide (Ki = 0.3 and 0.2 mM, respectively). Similar inhibition of glutamate decarboxylase and cysteine sulfinic acid decarboxylase by two different radical-containing compounds (O2 and NO) is consistent with the notion that these reactions proceed via radical mechanisms.
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PMID:Oxygen-induced seizures and inhibition of human glutamate decarboxylase and porcine cysteine sulfinic acid decarboxylase by oxygen and nitric oxide. 1145 99


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