Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The transcriptional regulation of the murine gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was studied by beta-galactosidase histochemistry in transgenic mice carrying fusion genes between progressively longer portions of the 5'-upstream regulatory region of GAD67 and E. coli lacZ. No expression was detected in brains of mice carrying 1.3 kb of upstream sequences including a housekeeping and two conventional promoters, and two negative regulatory elements with homology to known silencers. In mice carrying the same portion of the promoter region plus the first intron, lacZ expression in the adult central nervous system was found in few, exclusively neuronal sites. The number of correctly stained GABAergic centres increased dramatically with increasing the length of the 5'-upstream region included in the construct which suggests that multiple putative spatial enhancers are located in this region. Their action is influenced by epigenetic mechanisms that may be due to site-of-integration and transgene copy-number effects. Additional cis-acting elements are needed to obtain fully correct expression in all GABAergic neurons of the adult central nervous system.
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PMID:Regulation of cell-type specific expression of lacZ by the 5'-flanking region of mouse GAD67 gene in the central nervous system of transgenic mice. 975 66

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

We have developed defective herpes simplex virus 1 (HSV-1) vectors, based on amplicon plasmids with a replication-deficient mutant, as helper for the transfer of the glutamic acid decarboxylase (GAD67) or beta-galactosidase (beta-gal) gene as control directed by HCMV promoter into neuronal-like cells (PC12) and primary neurons. GAD67 protein was detected immunochemically, while GAD67 activity in virus-producing and nonproducing cell lines was detected enzymatically or by GABA release. Infection with GAD67-expressing amplicon vectors enhanced the resistance of PC12 cells to H(2)O(2). This protection was related to increased energy metabolism, as shown by MTT reduction and ATP level, and involved the GABA shunt, as shown by the reduction in ATP level seen in the presence of gamma-vinyl GABA (GVG), a specific GABA transaminase inhibitor. Level of glutathione (GSH), which requires ATP for its synthesis, was increased by the GAD67 transgene. The activity of glucose-6-phosphate dehydrogenase involved in the maintenance of the NADPH that can be used for the regeneration of the GSH pool, was increased by infection with amplicon vectors. Thus, replication-deficient HSV-1 and the GAD67 transgene have complementary neuroprotective effects and infection with GAD67-expressing amplicon vectors was able to protect nondifferentiated cortical neurons from glutamate toxicity mediated by oxidative stress. Such defective GAD67-expressing HSV-1, as neurotropic vector, should be helpful in neurodegenerative diseases implicating alterations of energy metabolism and oxidative stress in neuronal cells expressing GABA transaminase.
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PMID:Enhancement of neuronal protection from oxidative stress by glutamic acid decarboxylase delivery with a defective herpes simplex virus vector. 1463 8

AML1/Runx1 (Runx1) is a mammalian transcription factor that plays critical roles in regulating the differentiation of a number of different cell types. In the present study, we have utilized mice expressing beta-galactosidase (beta-gal) under the control of the Runx1 promoter to characterize the spatiotemporal expression pattern of Runx1 during retinogenesis. Expression of beta-gal was first detected at embryonic day 13.5 in post-mitotic cells located in the inner retina and overlapped with expression of the early amacrine and ganglion cell marker protein Islet1. During subsequent developmental stages, the number of beta-gal-positive cells increased in a central-to-peripheral gradient until late embryogenesis but then decreased in the early post-natal retina. beta-gal-positive cells were located primarily in the ganglion cell layer by late embryonic/early post-natal stages and were identified as a subpopulation of displaced amacrine cells by the continued expression of Islet1, as well as Pax6, and the coexpression of the amacrine cell subtype-specific markers choline acetyltransferase, calretinin and the 65-kDa isoform of glutamic acid decarboxylase. These findings identify Runx1 as a novel marker for a restricted amacrine cell subtype and suggest a role for this gene in regulating the post-mitotic development of these cells.
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PMID:Runx1 expression defines a subpopulation of displaced amacrine cells in the developing mouse retina. 1602 91