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)

Glutamic acid decarboxylase (GAD;E.C. 4.1.1.15) catalyzes the production of GABA, the major inhibitory neurotransmitter in the mammalian brain. We recently isolated a lambda gt-11 recombinant, lambda-GAD, that contains the cDNA for GAD from feline brain (Kaufman et al., 1986). Interestingly, the beta-galactosidase-GAD fusion protein encoded by lambda GAD is enzymatically active, catalyzing the conversion of glutamate to CO2 and GABA. Here we report the nucleotide sequence of feline GAD cDNA. It consists of 2265 bases, with a continuous open reading frame of 625 codons. The derived sequence contains the sequence Asn-Pro-His-Lys, which is identical to sequence at the pyridoxal phosphate-binding site of porcine DOPA decarboxylase (Bossa et al., 1977). The first ATG sequence in the open reading frame begins at nucleotide residue 118. The 585 codons 3' to this putative initiation site predict an amino acid composition, N-terminal residue, and molecular size consistent with published characterizations of GAD.
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PMID:Glutamic acid decarboxylase cDNA: nucleotide sequence encoding an enzymatically active fusion protein. 345 23

GABAA receptor channels (GABARs) composed of varying combinations of alpha 1, beta 1, and gamma 2S subunits were transiently expressed in mammalian cell lines. The whole-cell patch-clamp recording technique was used to determine which combinations of GABAR subunits produced functional receptor channels and whether assembly of GABAR subunits into receptor channels followed a random or preferred sequence. To identify rapidly cells expressing GABARs, mammalian cell lines were cotransfected with combinations of GABAR subunit cDNAs and the Escherichia coli beta-galactosidase gene as a transfection marker. Positively transfected cells were identified by staining with the enzyme substrate fluorescein di-beta-galactopyranoside. Using this technique, we confirmed that functional alpha 1 beta 1 and alpha 1 beta 1 gamma 2S GABARs were assembled in transfected mouse L929 fibroblast cells, but surprisingly, functional alpha 1 gamma 2S and beta 1 gamma 2S GABARs were not expressed. It was determined that after transient transfection, levels of expressed receptors varied little among individual cells permitting comparison of absolute whole-cell GABA-evoked current values. Whole-cell currents recorded from cells coexpressing alpha 1 beta 1 gamma 2S subunits were three to four times larger than those recorded from cells coexpressing alpha 1 beta 1 subunits, and they were always enhanced by coapplied diazepam. The increase in whole-cell current was due in part to the larger single-channel current of the alpha 1 beta 1 gamma 2S GABARs. GABARs comprised of alpha 1 beta 1 gamma 2S subunits were formed preferentially over GABARs of alpha 1 beta 1 subunits alone, since only after substantially increasing the ratio of the beta 1 expression vector over the alpha 1 and gamma 2S subunit expression vectors were alpha 1 beta 1 GABARs formed in the presence of the gamma 2S subunit. These findings suggest that assembly of GABARs from constituent subunits did not proceed randomly to form all possible combinations, but that certain subunit combinations were preferred intermediates during the assembly process.
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PMID:Assembly of GABAA receptor subunits: analysis of transient single-cell expression utilizing a fluorescent substrate/marker gene technique. 768 69

Studies of cell lineage in the rat cerebral cortex have provided new insights into the mechanisms of neuronal and glial determination. They have shown that clonally related cells, marked with retrovirus injection at embryonic day 16 (E16), express the same glial or neuronal phenotype, suggesting that separate progenitors for each of these cell phenotypes exist in the ventricular zone at that stage of corticogenesis. However, it is not known if such committed progenitors are present in the ventricular zone before E16. Another important question concerns which neurochemical features are shared by clonally related cells of the adult cerebral cortex. In this study we have addressed the first question by injecting a retroviral vector expressing beta-galactosidase into the telencephalic ventricles of rat embryos at different stages (E14-E19). In order to classify clonally related neurons in the cerebral cortex of these rats, we have used postembedding immunohistochemistry for the amino acid neurotransmitters glutamate, aspartate, and GABA. Glutamate and GABA immunoreactivity marked nonoverlapping populations of cells that corresponded to the pyramidal and nonpyramidal neuron types of the rat cerebral cortex. Clonally related neurons, marked by retrovirus injection at any day between E14 and E19, homogeneously expressed one or other phenotype and accordingly displayed glutamate or GABA immunoreactivity. This finding indicates that committed progenitor cells for pyramidal and nonpyramidal neurons are present in the ventricular zone before E16. To investigate whether lineage dictates other features in clonally related neurons, we performed an immunohistochemical analysis for the calcium-binding proteins calbindin, parvalbumin, and calretinin in clusters of clonally related nonpyramidal neurons. The same calcium-binding protein was rarely found in members of the same cluster, suggesting that lineage does not control the expression of calcium-binding proteins in cortical nonpyramidal neurons. As a result of examining a large number of clonally related neurons from brains injected at different ages, we observed remarkable differences in number and laminar distribution of pyramidal and nonpyramidal neurons marked with retrovirus. Clusters of nonpyramidal neurons were usually composed of two or three cells, and resided in the cortical layers that were just being generated at the time of injection. Clusters of pyramidal neurons were larger and dispersed in several layers in the earlier injections; their size and laminar distribution were progressively reduced for later injections. These observations suggest the existence of different mechanisms that generate the pyramidal and nonpyramidal neurons of the cerebral cortex.
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PMID:Lineage analysis reveals neurotransmitter (GABA or glutamate) but not calcium-binding protein homogeneity in clonally related cortical neurons. 790 3

Primary dissociated cultures of human fetal central nervous system cells were prepared and inoculated at different days in vitro with adenovirus that contained a reporter gene encoding beta-galactosidase. At various time intervals, the cultures were processed for characterization with X-gal histochemistry and additional immunostaining with neurofilament (NF), GABA and glial fibrillary acidic protein (GFA-P). We observed that NF (+) and GABA (+) neuronal as well as GFA-P (+) glial cells could express beta-galactosidase activity after inoculation. The labeling was detected up to 3 months after virus treatment. In addition, neurons cultivated for three months were found to be still permissive for virus infection. We can conclude that adenovirus may be considered as a potential vector to transfer genes to nerve cells.
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PMID:Adenovirus insertion encoding the Lac Z gene in human nervous cells in primary dissociated cultures. 798

The mechanisms involved in the targeting of proteins to different cytosolic compartments are still largely unknown. In this study we have investigated the targeting signal of the 65-kD isoform of glutamic acid decarboxylase (GAD65), a major autoantigen in two autoimmune diseases: Stiff-Man syndrome and insulin-dependent diabetes mellitus. GAD65 is expressed in neurons and in pancreatic beta-cells, where it is concentrated in the Golgi complex region and in proximity to GABA-containing vesicles. GAD65, but not the similar isoform GAD67 which has a more diffuse cytosolic distribution, is palmitoylated within its first 100 amino acids (a.a.). We have previously demonstrated that the domain corresponding to a.a. 1-83 of GAD65 is required for the targeting of GAD65 to the Golgi complex region. Here we show that this domain is sufficient to target an unrelated protein, beta-galactosidase, to the same region. Site-directed mutagenesis of all the putative acceptor sites for thiopalmitoylation within this domain did not abolish targeting of GAD65 to the Golgi complex region. The replacement of a.a. 1-29 of GAD67 with the corresponding a.a. 1-27 of GAD65 was sufficient to target the otherwise soluble GAD67 to the Golgi complex region. Conversely, the replacement of a.a. 1-27 of GAD65 with a.a. 1-29 of GAD67 resulted in a GAD65 protein that had a diffuse cytosolic distribution and was primarily hydrophilic, suggesting that targeting to the Golgi complex region is required for palmitoylation of GAD65. We propose that the domain corresponding to a.a. 1-27 of GAD65, contains a signal required for the targeting of GAD65 to the Golgi complex region.
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PMID:A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region. 803 38

An impairment of GABA(A)-receptor-mediated inhibitory neurotransmission has been implicated in the development of epileptic seizures. To determine whether seizures affect GABA(A)-receptor gene transcription in vivo, a transgenic mouse line carrying a lacZ-fusion gene driven by GABA(A)-receptor delta-subunit promoter and upstream sequences was subjected to pentylenetetrazol (PTZ)-induced seizures. After injection of a single convulsive dose of PTZ, the activity of the delta-subunit promoter, as monitored by beta-galactosidase immunohistochemistry, was increased selectively in neurons of layers II-IV of neocortex. In contrast, mice kindled by repeated administration of initially subconvulsive doses of PTZ did not show a change in transgene expression, even shortly after the last PTZ-induced seizure. These results show that transient changes in transcription of the GABA(A)-receptor delta-subunit gene occur after acute seizures, but not after kindling. The limited responsiveness of the GABA(A)-receptor delta-subunit promoter after repeated stimulation may reflect an inappropriate adaptation of cellular responses to recurrent excitation, thereby contributing to the development of seizure disorders.
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PMID:Activation of the GABA(A)-receptor delta-subunit gene promoter following pentylenetetrazole-induced seizures in transgenic mice. 942 23

GABA, which is present at high levels within the paraventricular nucleus (PVN) of the hypothalamus, has been implicated in neuroendocrine regulation. Here we use a transgenic mouse model expressing a human proenkephalin-beta-galactosidase fusion gene, in which both up-regulation and down-regulation of gene expression can be measured easily, to study the effects of GABA on basal and stress-induced gene expression within the PVN. This model has been shown previously to be appropriately physiologically regulated by stress within the PVN. Acute systemic administration of GABA, of aminooxyacetic acid, and of the selective GABA(B) receptor agonist, baclofen, induces transgene expression in the PVN. Chronic administration of aminooxyacetic acid inhibits both basal and stress-induced transgene expression. Acute or chronic administration of the selective GABA(A) agonist, muscimol, inhibits both basal and stress-induced expression of the transgene. Muscimol also inhibits transgene expression in hypothalamic slice cultures in which extrahypothalamic afferents are removed. It is surprising that, following chronic aminooxyacetic acid administration, the opiate antagonist naltrexone induces transgene expression in a manner similar to that observed with naloxone-precipitated opioid withdrawal and that expression is accompanied by a behavioral syndrome that partially mimicks opioid withdrawal. Taken together with our previous work, and using gene expression as our read-out, we conclude that transgene-expressing PVN neurons receive tonic inhibitory inputs mediated via GABA(A) receptors. Moreover, these inhibitory inputs can themselves be inhibited via GABA(B) and mu-opioid receptors.
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PMID:Proenkephalin gene regulation in the paraventricular nucleus by GABA: interactions with opioid systems in a transgenic model. 945 54

Defective herpes simplex virus (HSV) vectors containing glutamic acid decarboxylase (GAD) cDNAs, either GAD65 or GAD67, were used to examine GAD function and GABA synthesis in rat cortical astrocytes, CNS cells that do not endogenously synthesize GABA. GAD vector infection resulted in isoform-specific expression of GAD as determined by western blotting and immunohistochemistry. Astrocytes infected with a beta-galactosidase vector or uninfected expressed no GAD and contained no detectable GABA. GABA was detected in glial fibrillary acid protein-expressing cells after GAD65 vector infection. Significant amounts of GABA, as determined by HPLC, were synthesized in cultures infected with either GAD vector. The levels of GABA in GAD67 vector-infected cells were almost twofold higher than in GAD65 vector-infected cells. Vector infection did not alter levels of other intracellular amino acids. GABA was tonically released from astrocytes infected with the GAD67 vector, but no increase in release could be detected after treatment of the cells with K+, veratridine, glutamate, or bradykinin. The ability to transduce astrocytes so that they express GAD and thereby increase GABA levels provides a potential strategy for the treatment of neurologic disorders associated with hyperexcitable or diminished inhibitory activity.
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PMID:GABA synthesis in astrocytes after infection with defective herpes simplex virus vectors expressing glutamic acid decarboxylase 65 or 67. 983 28

Huntington's disease (HD) is a genetic disorder leading to the degeneration of striatal GABA-ergic output neurons. No treatment is currently available for this devastating disorder, although several neurotrophic factors, including brain-derived neurotrophic factor (BDNF), have been shown to be beneficial for striatal neuron survival. We analyzed the effect of adenovirus-mediated transfer of the BDNF gene in a model of HD. Using a stereological procedure, three groups of rats were given an intrastriatal injection of adenovirus encoding BDNF, beta-galactosidase, or sham surgery. Two weeks after treatment, the animals were lesioned with quinolinic acid (QUIN), a toxin that induces striatal neuron death by an excitotoxic process. One month after the lesion, histological study revealed that striatal neurons were protected only in rats treated with the BDNF adenovirus. Volume measurements showed that the QUIN-induced lesions were 55% smaller in the BDNF adenovirus-treated group than in the beta-galactosidase adenovirus-treated group (p < 0.05), and the sham-treated group (p < 0.05). To determine the survival of striatal GABA-ergic output neurons after the QUIN-induced lesion, we immunostained brain sections with DARPP-32, an antibody specific for striatal output neurons. Prior treatment with the BDNF adenovirus resulted in a cell survival of 64%, whereas that after beta-galactosidase treatment was 46% (p < 0.05), showing that the BDNF adenovirus protected the striatal neurons. These results indicate that transfer of the BDNF gene is of therapeutic value for Huntington's disease.
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PMID:Brain-derived neurotrophic factor-mediated protection of striatal neurons in an excitotoxic rat model of Huntington's disease, as demonstrated by adenoviral gene transfer. 1060 59

NPY exerts anxiolytic effects, which are mediated by activation of Y1 receptors in the amygdala. It has been shown that diazepam counteracts the anxiogenic effect of Y1 receptor antagonists, suggesting that NPYergic and GABAergic systems are coupled in the regulation of anxiety. We used a transgenic mouse model, expressing a mouse Y1 receptor-beta-galactosidase fusion gene (Y1R/LacZ), to study the effect of positive or negative modulators of GABA(A) receptors on Y1 receptor gene expression. Mice were treated for 14 days with diazepam (4 or 20 mg/kg), the anxiolytic beta-carboline-derivative abecarnil (0.3 or 6 mg/kg) and the anxiogenic beta-carboline FG7142 (20 mg/kg). Transgene expression was determined by quantitative analysis of beta-galactosidase histochemical staining in the medial amygdala and in the medial habenula as a control region. Chronic treatment with 20 mg/kg diazepam or 6 mg/kg abecarnil significantly increased, whereas FG 7142 decreased, transgene expression in the medial amygdala. A transient decrease in transgene expression was observed in the medial amygdala six hours after the acute treatment with 20 mg/kg FG 7142 but not with diazepam or abecarnil. No significant changes were observed in the medial habenula. These data suggest that modulation of GABA(A) receptor function may regulate Y1 receptor gene expression in medial amygdala.
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PMID:Chronic modulation of the GABA(A) receptor complex regulates Y1 receptor gene expression in the medial amygdala of transgenic mice. 1067 Apr 18


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