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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat superior cervical ganglion neurons require the presence of nerve growth factor (NGF) to develop and survive in culture. If NGF is removed from the culture medium, then the neurons die of programmed cell death. We investigated the potential role of Ca2+ and reactive oxygen species in this process. We found that overexpression of human wild-type copper/zinc-superoxide dismutase in cultured superior cervical ganglion neurons, using an adenovirus-based vector, substantially protected the cells from the effects of NGF withdrawal, although overexpression of the Ca(2+)-binding protein calbindin D28k or the enzyme beta-galactosidase did not. We also observed that treatment of the cells with the cytokine transforming growth factor-beta 1, which has been shown to protect neurons against oxidative injury, delayed cell death produced by NGF withdrawal. These data suggest a role for reactive oxygen species in triggering programmed cell death of rat sympathetic neurons upon growth factor withdrawal.
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PMID:Expression of human copper/zinc-superoxide dismutase inhibits the death of rat sympathetic neurons caused by withdrawal of nerve growth factor. 760 46

Exposure of PC12-VG cells to an extremely low frequency magnetic field (ELFMF) enhanced the beta-galactosidase gene expression stimulated by treatment of the cells with forskolin. The enhancing effect of the ELFMF was inhibited by treatment of the cells with a specific inhibitor of PKC, calphostin C, as well as with the Ca2+ entry blockers nifedipin and dantrolen. Enhancement appeared within the first hour of a 4h forskolin treatment when the ELFMF was given at different times during culture. We speculate that exposure of PC12-VG cells to an ELFMF during the early response to forskolin treatment affects cell signal transduction, resulting in enhanced gene expression.
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PMID:Enhancement of beta-galactosidase gene expression in rat pheochromocytoma cells by exposure to extremely low frequency magnetic fields. 761 92

Endothelial cells, because of their proximity to the blood stream, provide an attractive system for gene transfer and delivery of gene products that control foci of vascular disease processes. We describe a simple, new methodology to achieve highly efficient transformation of cultured human endothelial cells derived from umbilical veins (HUVEC). A plasmid pCH110 containing coding region for beta-galactosidase driven by SV 40 early promoter region was employed to transfect HUVEC. The developed protocol exploits the role of apolipoprotein E (Apo E) in the metabolism of Apo E-containing lipoproteins and its high affinity binding to LDL receptors. DNA transfection of cultured HUVEC was carried out using standard transfection methods including calcium phosphate precipitation, polybrene mediated transfection, and lipofection. The new methodology of transfecting HUVEC employed Apo E adsorbed lipofection reagent-DNA complex, and was found to be the most efficient procedure to transform HUVEC in comparison to the standard methods used in this study.
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PMID:High-efficiency transformation of human endothelial cells by Apo E-mediated transfection with plasmid DNA. 766 41

The interrelationship between autoepitopes, DNA-binding domains, and C-reactive protein (CRP)-binding domains on a histone H1 molecule was examined using fusion proteins of beta-galactosidase and truncated histone H1 molecules. At least two CRP-binding sites were detected on a histone H1 molecule. Site 1 was composed of approximately 25 amino acids and calcium ion was required for the binding of CRP. Site 2, composed of approximately 20 amino acids and not requiring calcium ion, was identical or located very close to a DNA-binding domain and an epitope of anti-histone H1 autoantibodies in SLE sera. These data suggest that, at physiological ionic strength, histone H1 of either free or immune-complexed form could bind to CRP via site 1. These data are discussed with respect to the possible role of CRP in the handling and clearance of immune complexes in patients with systemic lupus erythematosus.
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PMID:Interrelationship between autoepitope, DNA-binding domain, and CRP-binding domain on a histone H1 molecule. 767 45

Efforts to develop an improved assay for plasma and tissue transglutaminase have led us to a convenient, sensitive microtiter plate assay for coagulation factor XIII using human fibrinogen as an immobilized substrate. Factor XIII was activated in the presence of calcium, thrombin, and immobilized fibrinogen and then assayed by adding biotinylcadaverine. The reaction was terminated by adding EDTA and the level of incorporated biotin was measured with streptavidin-beta-galactosidase. In this assay, the analytical range for human platelet factor XIII was 0.01-100 ng and 1-100 ng for guinea pig liver transglutaminase. Fibrinogen-coated plates gave more than 100-fold increase in sensitivity compared with N,N-dimethylcasein-coated microtiter plates. The intraassay coefficient of variation was less than 5% (n = 12) and interassay less than 6% (n = 4). The sensitivity of this assay reduced the volumes of plasma samples required and consequently eliminated the need to remove fibrinogen from such test samples. As expected, factor XIII activity could be inhibited by putrescine and antibodies against factor XIII as well as by a monoclonal antibody that bound to the carboxyl terminus of human fibrin gamma-chains. The assay provided a sensitive, simple, and rapid method for measuring factor XIII.
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PMID:A microtiter assay for factor XIII using fibrinogen and biotinylcadaverine as substrates. 769 7

IL-2-mediated T cell proliferation is a critical early event in the inflammatory process. Formation of the NFAT-1 transcriptional complex on the IL-2 promoter is essential for IL-2 transcription. Using a cell line that is stably transfected with a trimer of the NFAT-1 regulatory element linked to a lac-Z reporter gene, we screened for inhibitors of NFAT-1-mediated beta-galactosidase activity. WIN 61058 and WIN 53071 were identified as microM inhibitors. These compounds also inhibited beta-galactosidase mRNA levels. Similar inhibition of NFAT-1-mediated gene expression was observed in a second cell line, which is stably transfected with NFAT-1 regulatory elements linked to the reporter gene for sCD8. At 10 microM, both compounds inhibited IL-2 mRNA and protein levels in the NFAT-1-linked lac-Z transfectants, and in human lymphocytes. Both compounds inhibited the mixed lymphocyte reaction, and this inhibition was reversed by exogenous IL-2. WIN 53071 inhibited IL-2 production induced in the calcium-dependent PMA and ionomycin pathway. Conversely, calcium-independent anti-CD28 Ab and PMA-induced IL-2 production was resistant. Both compounds altered the NFAT-1 transcriptional complex, causing its retarded mobility on gels. By these functional criteria, we believe we have identified two structurally distinct, novel inhibitors of NFAT-1-mediated transcription.
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PMID:Functional characterization of novel IL-2 transcriptional inhibitors. 770 10

Muscular dysgenesis (mdg) is a recessive lethal mutation in the mouse which drastically affects skeletal muscle development during embryonic life. Physiologically, the disease is characterized by a complete paralysis resulting from a lack of excitation-contraction coupling. Existing electrophysiological, biochemical, and genetic evidence shows that mdg/mdg mice express a basic alteration of L-type voltage-sensitive Ca2+ channels in skeletal muscle. Studies on mdg/mdg myotubes in primary culture have shown that +/+ fibroblasts or +/+ Schwann cells may fuse with them and correct their functional deficiency by genetic complementation. As the spontaneous formation of heterocaryons is thought to be an exclusive property of myoblasts, we asked whether fibroblasts may have changed their properties before fusion occurred. We used primary cells issued from sciatic nerves dissected from newborn transgenic mice carrying the pHuDes1-nls-LacZ transgene (Des-LacZ cells) as non-muscle cells. These cells were mainly fibroblasts (80%) positive for Thy1.1 and Schwann cells positive for S100. The cultures were negative for myogenic markers (desmin, troponin T), did not form myotubes long-term, and did not display significant activation of the muscle reporter gene (pHuDes1-nls-LacZ). After a few days in coculture with dysgenic or normal myotubes, the muscle reporter gene (beta-galactosidase) was detected both within dysgenic myotubes, correlating with the restoration of normal contractile activity, and normal myotubes. As well as confirming that fusion takes place, this shows that Des-LacZ cells nuclei incorporated into recipient myotubes express their own myogenic genes. Moreover, individual mononucleated Des-LacZ cells expressing beta-galactosidase were observed, indicating that myogenic genes were being expressed before fusion. This suggests a mechanism of myotube driven myogenic recruitment of cells during the in vitro myogenesis. Analysis of the distribution of the induced Des-LacZ cells (positive for beta-galactosidase) in compartmentalized muscle cocultures showed that in the presence of dysgenic myotubes, these cells were equally distributed in both myotube free and enriched areas, whereas in the presence of normal myotubes, the positive cells remained in close vicinity of the myotubes. This difference could be explained by the fact that the dysgenic phenotype might include release of the induction process from its normal controls. Our results are consistent with the idea of a transcellular mechanism triggering myogenic differentiation in non-muscle cells, and that myotubes themselves are able to drive myogenic recruitment of cells during the in vitro myogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Myotube driven myogenic recruitment of cells during in vitro myogenesis. 773 31

Optimal conditions for formation of calcium phosphate-DNA precipitates and for chromaffin cell transfection by the calcium phosphate method were examined. A relationship was observed between turbidity of calcium phosphate solutions and the ability of calcium phosphate-DNA mixtures to give efficient transfection of bovine chromaffin cells. Under optimal conditions up to 35% of chromaffin cells in cultures transfected with plasmid DNA encoding human proenkephalin or Escherichia coli beta-galactosidase expressed the respective proteins. Important factors for transfection were the pH (6.95) and buffer employed for calcium phosphate-DNA precipitate formation, the amount and type of DNA, and the absence of serum in the cultures. Additionally, phosphate and calcium concentrations in the culture medium during incubation of cells with DNA are critical. Optimal conditions for transfection of chromaffin cells were also useful for transfection of clonal BSC-40 cells, an African green monkey kidney cell line. These results suggest that the optimal conditions described here for chromaffin cells may have broad applicability to other cell types. In addition, the results suggest that it is possible to optimize the solutions used for transfection conditions by monitoring calcium phosphate formation.
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PMID:Optimization of calcium phosphate transfection for bovine chromaffin cells: relationship to calcium phosphate precipitate formation. 779 20

Current assays for functional activation of Gs-coupled receptors usually involve quantitation of adenylyl cyclase or measurement of cAMP concentration by radioimmunoassay. The activation of Gq-coupled receptors is commonly assayed by measurement of the production of inositol triphosphate or diacylglycerol from phosphatidylinositol 4,5-bisphosphate or of changes in intracellular calcium. These assays generally require large numbers of cells (10(5)-10(6)) and/or the use of radioactive materials. We have developed a rapid nonradioactive colorimetric assay that utilizes a beta-galactosidase (lacZ) gene fused to five copies of the cyclic AMP response element (CRE) to detect the activation of CRE-binding protein that results from an increase in intracellular cAMP or calcium. This assay can be performed using as few as 30,000 cells in a 96-well format with the end products measured simultaneously in a microplate reader. Consequently, a single individual can readily assay 1000 samples a day. Using this assay, the fold increase in beta-galactosidase activity was similar in magnitude to increases in cAMP or adenylyl cyclase activity and was approximately linear from 0.01 to 0.27 fmol/cell of intracellular cAMP. Furthermore, pharmacological characterization of one of the melanocortin receptors, mMC5-R, using this assay resulted in a similar order of potency for several melanocortin peptides to that obtained with a commonly used adenylyl cyclase enzyme assay. This assay is also useful for the characterization of Gq-coupled receptors as is demonstrated here using cells transfected with the mouse bombesin receptor. The large-scale capacity of this assay makes it an excellent method for screening molecules of interest acting on Gs- and Gq-coupled receptors.
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PMID:A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways. 779 37

A cDNA clone encoding the calcium-binding subunit of calcineurin, calcineurin B, was isolated from a bovine brain library by immunoscreening. The 841 bp cDNA has a 56 bp 5'-noncoding region, an open reading frame of 510 bp, and a 275 bp 3'-noncoding sequence. The deduced amino acid sequence of bovine calcineurin B differs from the previously reported protein sequence (Aitken et al., 1984) by three residues. The sequence contained additional valine at the carboxyl terminus and substitutions of Met-11 and Ser-153 (the positions according to Aitken et al., 1984) by cysteine. The amino acid sequence of bovine calcineurin B was found to be identical to that of human calcineurin B sequence (Guerini et al., 1989). In fact, 97.1% homology was observed between the coding regions of human and bovine calcineurin B. In addition, a very high homology of 95.2% was observed for the 3'-noncoding region while the 5'-noncoding region showed 58.9% homology. The beta-galactosidase fusion protein, having the apparent molecular weight of 29 kDa, was detected on Western blots by subunit B-specific monoclonal antibody (Matsui et al., 1985). Northern analysis revealed that there is a single calcineurin B transcript in bovine brain which is 2.3 kb in length. This is in agreement with the observation of only one immunologically detectable subunit B protein in bovine brain (Matsui et al., 1985).
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PMID:Isolation and characterization of a cDNA clone coding for the calcium-binding subunit of calcineurin from bovine brain: an identical amino acid sequence to the human protein. 780 16


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