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

Glutamate decarboxylase (GAD; E.C. 4.1.1.15) converts glutamate to gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the vertebrate central nervous system. This report describes the isolation of a GAD complementary DNA clone by immunological screening of a lambda gt-11 brain complementary DNA expression library. The fusion protein produced by this clone catalyzes the conversion of glutamate to GABA and carbon dioxide, confirming its identity as GAD. Antibodies to beta-galactosidase remove GAD enzymatic activity from solution, showing that this activity is associated with the fusion protein. In immunoblotting experiments all three available antisera to GAD reacted with the fusion polypeptide and with two major polypeptides (molecular size, 60,000 and 66,000 daltons) in brain extracts.
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PMID:Brain glutamate decarboxylase cloned in lambda gt-11: fusion protein produces gamma-aminobutyric acid. 351 61

A human insulinoma cDNA library was constructed in the expression plasmid vector pUEX1. The clone pUEX1Ins12 was selected by means of hybridization with an insulin probe. It codes for full size amino acid sequence preproinsulin. The bacterial strain pUEX3Ins8 producing proinsulin as beta-galactosidase fusion protein was obtained for the use of recombinant protein as an antigen in an ELISA to detect serum antibodies in subjects with IDDM. Recombinant clones containing the middle, N- and C-terminal domains of the GAD65, the major autoantigen in IDDM, were constructed in pVEX1. These clones may become important tools to study the nature of GAD autoreactivity in IDDM. The clone pHICEO.9 was selected from the human insulinoma cDNA library by immunoscreening with total human insulinoma protein antibodies. This clone expresses the C-terminal fragment of human cholesterol esterase/lipase containing its antigenic determinant and can be used for blood lipase determination. Four clones containing cDNA inserts (0.47-1.42 kb) without any significant homologies to the known sequences in the Gene Bank were obtained by means of statistic selection.
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PMID:[Study on structural gene expression in human insulinoma]. 774 51

Transplantation of genetically engineered cells can provide sustained focal delivery of naturally occurring molecules, including neurotransmitters and growth factors. We have engineered immortalized mouse cortical neurons and glia to deliver GABA by driving GAD(65) expression. Engineered cell lines showed GAD(65) mRNA expression, enzymatic activity, and GABA release. In vitro, basal flux of GABA was approximately 20% of total cellular GABA. We transplanted these GABA-producing cells bilaterally into either the anterior or the posterior substantia nigra of 43 rats. The rats were subsequently kindled through an electrode placed in the entorhinal cortex. GABA-producing cells, but not beta-galactosidase-producing cells, affected kindling rates. The number of stimulations needed to reach the first stage-5 seizure and to achieve full kindling differed significantly between the anterior and posterior transplantation sites when GAD(65)-producing cells were transplanted but not when beta-galactosidase-producing cells were transplanted. Our data show that transplanted engineered cells can make and release GABA at physiologically meaningful concentrations.
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PMID:Conditionally immortalized cell lines, engineered to produce and release GABA, modulate the development of behavioral seizures. 1068 70

We have engineered conditionally-immortalized mouse astrocytes to express beta-galactosidase or GAD(65) in a tetracycline-controlled fashion. The engineered cell lines, BASlinbetagal and BASlin65, divide at 33 degrees C but cease division at 39 degrees C. We carried out morphological and biochemical analyses to further understand GABA production and release, and to determine the suitability of these cells for transplantation. Using the BASlinbetagal cell line, we showed a dramatic regulation of beta-galactosidase expression by tetracycline. The BASlin65 cell line showed functional GAD(65) enzymatic activity and GABA production, both of which were suppressed by growth in the presence of tetracycline. When cultured in the absence of tetracycline, BASlin65 cells have a total GABA content equal to or greater than other GABA-ergic cell lines. Immunofluorescence microscopy revealed that GAD(65) had a distinct perinuclear localization and punctate staining pattern. GABA, on the other hand, showed diffuse staining throughout the cytoplasm. BASlin65 cells not only synthesize GABA, they also release it into the extracellular environment. Their ability to produce and release significant amounts of GABA in a tetracycline-regulated manner makes BASlin65 cells a useful cellular model for the study of GABA production and release. Furthermore, their non-tumorigenicity makes them excellent candidates for transplantation into specific regions of the brain to provide a localized and regulatable source of GABA to the local neuronal circuitry.
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PMID:Conditionally-immortalized astrocytic cell line expresses GAD and secretes GABA under tetracycline regulation. 1079 32

Long-term solid-organ allografts typically develop diffuse arterial intimal lesions (graft arterial disease; GAD), consisting of smooth-muscle cells (SMC), extracellular matrix and admixed mononuclear leukocytes. GAD eventually culminates in vascular stenosis and ischemic graft failure. Although the exact mechanisms are unknown, chronic low-level alloresponses likely induce inflammatory cells and/or dysfunctional vascular wall cells to secrete growth factors that promote SMC intimal recruitment, proliferation and matrix synthesis. Although prior work demonstrated that the endothelium and medial SMCs lining GAD lesions in cardiac allografts are donor-derived, the intimal SMC origin could not be determined. They are generally presumed to originate from the donor media, leading to interventions that target donor medial SMC proliferation, with limited efficacy. However, other reports indicate that allograft vessels may contain host-derived endothelium and SMCs (refs. 8,9). Moreover, subpopulations of bone-marrow and circulating cells can differentiate into endothelium, and implanted synthetic vascular grafts are seeded by host SMCs and endothelium. Here we used murine aortic transplants to formally identify the source of SMCs in GAD lesions. Allografts in beta-galactosidase transgenic recipients showed that intimal SMCs derived almost exclusively from host cells. Bone-marrow transplantation of beta-galactosidase--expressing cells into aortic allograft recipients demonstrated that intimal cells included those of marrow origin. Thus, smooth-muscle--like cells in GAD lesions can originate from circulating bone--marrow-derived precursors.
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PMID:Host bone-marrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy. 1138 13

In this paper we analyse the expression pattern of a zebrafish dlx4/6 enhancer/reporter construct in embryonic transgenic mice. We show that the pattern of LacZ/beta-galactosidase in cells that tangentially migrate from the ganglionic eminences to the cerebral cortex is identical to that of various subpallial markers, namely Dlx and GAD genes, that are known to label this population. Because beta-galactosidase activity persists long after expression of the Dlx genes and the transgene becomes undetectable, we were able to analyse the beta-galactosidase-positive cell population of the mature cortex through X-gal staining and immunohistochemistry. We show that this population is largely identical with the adult cortical and hippocampal interneuron population, providing further evidence for their subpallial origin.
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PMID:Expression from a Dlx gene enhancer marks adult mouse cortical GABAergic neurons. 1173 34

Local application of GABA-potentiating agents can prevent or reduce the development and maintenance of behavioral seizures induced by limbic kindling in rats. Microinjection and lesion studies suggest that the transition zone between anterior and posterior piriform cortex (PC), termed here central PC, is a potential target for transplantation of GABA-producing cells. In the present study, we transplanted conditionally immortalized mouse cortical neurons, engineered with the GABA-synthesizing enzyme GAD(65), to the central PC of rats. Suspensions of 1.5 x 10(5) cells in 1 microl were transplanted bilaterally. Control animals received transplantation of beta-galactosidase (beta-gal)-expressing cells. All rats were subsequently kindled through a chronically implanted electrode placed in the basolateral amygdala. The pre- and postkindling threshold currents for eliciting behavioral seizures were determined before and after kindling. We found the prekindling partial seizure threshold to be significantly increased by about 200% in the rats that received the GABA-producing cells compared to rats receiving beta-gal-producing transplants. After kindling, the seizure threshold tended to be higher by 100% in rats that received GABA-producing cells, although the difference from controls was not statistically significant. GABA-producing transplants had no significant effect on the rate of amygdala kindling, but the latency to the first generalized seizure during kindling was significantly increased in animals receiving GABA-producing cells. The transplanted cells showed long-term GAD(65) expression as verified immunohistologically after termination of the experiments. The findings substantiate and extend previous findings that the central PC is part of the anatomical substrate that facilitates propagation from partial to generalized seizures. The data demonstrate that genetically engineered cells have the potential to raise seizure thresholds when transplanted to the central PC.
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PMID:Genetically engineered GABA-producing cells demonstrate anticonvulsant effects and long-term transgene expression when transplanted into the central piriform cortex of rats. 1209 95

Plasmid-DNA gene-gun immunization may be an efficient approach for investigating the role of skin dendritic cells (DCs) in type 1 diabetes (T1D) pathogenesis and the significance of the presentation of peptides that mimic autoantigenic epitopes in aggravating or modulating the autoimmune reaction. Gene-gun immunization has been described as producing long-lasting immune responses elicited by skin DCs, especially Langerhans cells (LCs). Therefore, we tested the immune response and diabetes modulation in nonobese diabetic (NOD) mice and in control BALB/c mice, by gene-gun administration of plasmid-DNA encoding (1) human 65 kDa glutamic acid decarboxylase (hGAD65) mimicking the crucial mouse autoantigen GAD65 (similarity of 95.7%) or (2) beta-galactosidase (betaGAL) as a negative control. Expression of GAD and betaGAL in skin of pc-GAD- and pc-LacZ-injected mice, respectively, was confirmed. It was surprising that both pc-LacZ-injected BALB/c and NOD mice exhibited a betaGAL-specific Th1 immune response: spleen cells of pc-LacZ mice proliferated specifically to betaGAL (P < 10(-4)) and secreted significant amounts of IFNgamma (P < 10(-4)). pc-LacZ mice also developed a betaGAL-specific Th1-related (IgG2a/2c) and Th2-related (IgG1) humoral response. Although pc-GAD BALB/c mice showed Th2-related GAD-specific IgG1 production and a significant secretion of IL4 (P < .03), pc-GAD NOD mice did not generate either an antibody response or a T cell response specific to GAD. Moreover, gene-gun immunization encoding hGAD65 did not clearly modulate diabetes onset in NOD mice. This absence of detectable GAD-specific response may implicate skin DC deficiencies in NOD mice. The gene-gun technique could thus provide an interesting model for studying skin DC abnormalities in NOD mice and their potential implication of presenting mimetic peptides that modulate the autoimmune response in T1D.
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PMID:Gene-gun biolistic immunization encoding glutamic acid decarboxylase: a model for studying Langerhans cell abnormalities and mimicry in the nonobese diabetic mouse. 1612 2