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)

The differentiation potential of early mammalian myogenic cells was tested under clonal culture conditions. Cells were isolated from paraxial mesoderm and limb buds of transgenic mouse embryos at 9.5 days after conception and grown in culture at clonal density either on collagen-coated dishes or on various feeder cell layers. The transgene used contained a reporter gene encoding beta-galactosidase with a nuclear localization signal under the control of regulatory sequences from the gene for fast myosin light chain 3, so that beta-galactosidase staining indicated the presence of differentiated muscle cells. After 5 days in culture, the number and size of beta-galactosidase-positive (beta-gal+) clones were recorded. Cells isolated from somites I-V (the last five somites to have formed) or from unsegmented paraxial mesoderm did not give rise to any beta-gal+ clones. Cells isolated from somites VI-X or from the forelimb bud gave rise to beta-gal+ clones, but only on feeder cells. Cells from somites XI or older gave rise to beta-gal+ clones independently of the substrate. However, when cells isolated from unsegmented paraxial mesoderm or somites I-V were cultured with nontransgenic cells from the trunk (including neural tube and notochord), differentiation occurred on condition that the cells were in a three-dimensional aggregate, even though their specific position in the somite had been lost. By culturing explants ranging in size from 1 to < 100 cells in the presence of an inhibitor of cell division, we determined that a minimal number of 30-40 cells is required for mesodermal cells to differentiate.
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PMID:Myoblast differentiation during mammalian somitogenesis is dependent upon a community effect. 789 57

Alterations in the metabolic functions of trabecular meshwork (TM) cells are thought to be involved in the pathogenesis of primary open-angle glaucoma (POAG). In an investigation of this possibility, 30 trabeculectomy specimens from patients with POAG were examined histochemically for 11 lysosomal and membrane-bound enzymes. The patients ranged from 48 to 87 years in age. The degree of enzyme staining was compared with that of 15 age-matched controls obtained from an eye bank at less than 24 h after death. There was no history of eye disease in the controls. The enzymes examined were: dipeptidylpeptidases II and IV (DPPII and IV); beta-glucuronidase (beta-GLUC); acid-beta-galactosidase (s beta-GAL); N-acetyl-beta-D-glucosaminidase (NAG); nonspecific esterase (UE); acid phosphatase (SP); alkaline phosphatase (ALP); gamma-glutamyltransferase (GGT); and aminopeptidase A and M (APA and APM). Evaluation of the specimens was performed by two observers and by computer-aided optic densitometry. Results showed increased staining of SP, UE, GGT and APM in the pathological specimens as compared with the controls. SP and UE indicate phagocytic activity, APM is involved in collagen turnover and GGT participates in both drug detoxification and the breakdown of glutathione in the gamma-glutamyl cycle. Our observations show different hydrolase activities in the TM cells of human glaucomatous eyes as compared with normal values, suggesting that such metabolic differences may be related to the pathogenesis of POAG.
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PMID:Increased hydrolase activities in the human trabecular meshwork of glaucomatous eyes. 809 35

A system for targeting foreign DNA to epithelial cells in vitro has been developed by exploiting receptor-mediated endocytosis. The polymeric immunoglobulin receptor transports dimeric immunoglobulin A and immunoglobulin M through epithelial cells, including those of the respiratory tract, by binding the immunoglobulins at the basolateral surface and transporting them across the cell. Fab fragments of antibodies directed against the extracellular portion of the receptor, secretory component, are similarly transported. Anti-human secretory component Fab fragments were covalently linked to a polycation, and complexed to various expression plasmids. When bound to an expression plasmid containing the Escherichia coli lacZ gene ligated to the Rous sarcoma virus promoter, the complexes transfected HT29.74 human colon carcinoma cells induced to express polymeric immunoglobulin receptor, but not those lacking the receptor. Primary cultures of human tracheal epithelial cells grown on collagen gels, which induce the expression of polymeric immunoglobulin receptor, were also transfected with the complexes. From 5 to 66% of the respiratory epithelial cells had beta-galactosidase activity after treatment, comparable to the percentage of cultured human tracheal epithelial cells that express polymeric immunoglobulin receptor (8-35%). The addition of excess human secretory component (Fab ligand) to the culture medium at the time of transfection blocked the delivery of DNA. The expression plasmid, either alone, complexed to the polycation, or complexed to a carrier based on an irrelevant Fab fragment, was not effective in transfecting either cell type. This DNA carrier system introduces DNA specifically into epithelial cells that contain pIgR in vitro.
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PMID:Gene transfer into respiratory epithelial cells by targeting the polymeric immunoglobulin receptor. 822 56

To begin to assess the transcriptional mechanisms that regulate type I collagen gene expression in differentiating osteoblasts, we have sought to determine the minimal promoter sequences that confer osteoblast-specific expression to the alpha 2(I) collagen gene during murine development. Transgenic mice were generated harboring DNA constructs in which the -2000, -500, and -350 to +54 regions located upstream of the start of transcription were linked to the Escherichia coli beta-galactosidase reporter gene (LacZ). Histochemical staining using X-gal indicated that the -2000 lacZ transgene was strongly expressed in newly differentiated and fully functional osteoblasts at intramembranous and endochondral sites of ossification. The promoter was also active in osteocytes in regions of bone remodeling within alveolar bone. The temporal and spatial activity of this region of the promoter closely resembled the developmental patterns of expression of the endogenous alpha 2(I) collagen gene as determined by in situ hybridization. The cis-acting elements within the 500 and 350 bp segments of the alpha 2(I) collagen promoter also drove reporter gene expression in forming osteoblasts, although levels of transgene expression were not as marked as that seen with the 2000 bp promoter. Furthermore, the synthesis and secretion of TGF-beta 1 in osteogenic zones coincided with areas where the alpha 2(I) collagen promoter constructs were transcriptionally active. Since a nuclear factor 1 binding site present at -300 has been shown to mediate the effects of TGF-beta 1 on the alpha 2(I) collagen promoter, these data support a role for TGF-beta 1 in the control of this gene during development.
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PMID:Osteoblast-specific expression of the alpha 2(I) collagen promoter in transgenic mice: correlation with the distribution of TGF-beta 1. 823 83

The susceptibility of rodent hepatocytes to infection by mouse type C retroviruses was examined in vivo and in vitro and compared with the expression of two membrane proteins that function as transporters for the cationic amino acids CAT-1 and CAT-2. CAT-1 expression in rodents determines susceptibility to ecotropic retrovirus infection by serving as the virus receptor. Recently, it has been suggested that CAT-2 may be a receptor for amphotropic murine leukemia virus. In the present study, CAT-1 expression was observed in Hepa1, a cell line derived from a murine hepatoma, and in rat hepatocytes propagated on collagen monolayers in vitro but not in intact or regenerating rat liver in vivo. The expression of CAT-1 correlated with susceptibility to infection by an ecotropic retrovirus encoding beta-galactosidase. CAT-2 expression was observed in hepatocytes in vitro and in vivo, consistent with reports of infection of regenerating and cultured hepatocytes by amphotropic retroviruses. However, introduction of murine CAT-2 into nonpermissive Chinese hamster cells was not sufficient to confer susceptibility to amphotropic retrovirus infection, using a protocol that could easily demonstrate CAT-1-dependent infection by an ecotropic virus. Our data establish CAT-1 as a major determinant of ecotropic retrovirus infection in rodent hepatocytes and suggest that CAT-2 is not a receptor for viruses in the amphotropic subgroup.
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PMID:Retroviral infection and expression of cationic amino acid transporters in rodent hepatocytes. 838 31

We and others have previously shown that a 67-kD cell surface elastin/laminin-binding protein (EBP) is responsible for cell adhesion to elastin and laminin and for mediating the process of elastin fiber assembly, but the nature of this protein was unknown. In this report we provide evidence that a 67-kD catalytically inactive form of beta-galactosidase produced by alternative splicing demonstrates immunological and functional similarity and sequence homology to the 67-kD EBP, suggesting that the two might be the same. Antibody prepared to a synthetic peptide, N-Ac-GSPSAQDEASPL, corresponding to a frame-shift-generated sequence unique to the alternatively spliced form of human beta-galactosidase, also recognized sheep EBP both on Western blotting and in aortic tissue. Furthermore, this synthetic peptide (S-GAL) binds to elastin and laminin, but not to fibronectin, collagen I, or collagen III. Moreover, both tropoelastin and laminin which bind to S-GAL peptide affinity columns can be specifically eluted from them with an excess of free S-GAL peptides. In addition, sequence homology among this splice variant of human beta-galactosidase, sheep EBP, and NH2-terminal sequences of some elastases suggests that these proteins share a common ligand-binding motif that has not been previously recognized.
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PMID:The 67-kD elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of beta-galactosidase. 838 99

The purification by affinity chromatography of beta-galactosidase from strains carrying sdaA/lacZ gene fusions results in the copurification of L-serine deaminase 1. We conclude that sdaA is the structural gene for the latter enzyme. The purified L-serine deaminase 1 obtained after collagenase treatment of an sdaA-collagen-lacZ fusion differs from the native enzyme by the addition of several amino acids at the C-terminal. Like the enzyme in crude extracts, this purified enzyme is catalytically inactive, and is activated by incubation with iron and dithiothreitol.
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PMID:Use of gene fusions of the structural gene sdaA to purify L-serine deaminase 1 from Escherichia coli K-12. 843 13

The cysteine-rich trefoil motif of rat intestinal trefoil factor (rITF) was cloned and expressed in Escherichia coli. A 270-bp cDNA fragment including the signal sequence and the trefoil motif was cloned into the expression vector pAX5+ to direct the expression of a beta-galactosidase collagen-hinged fusion protein in E. coli. Cultures harbouring the recombinant plasmid produced a soluble novel protein with a molecular mass of 134.5 kDa, as predicted for the trefoil-motif-containing fusion protein. Purification of the rITF moiety was achieved by p-aminophenyl-thio-beta-D-galactoside(APTG)-affinity chromatography, collagenase digestion of the hybrid molecule, and removal of the beta-galactosidase-hinge molecule by a further APTG-affinity step. It was demonstrated that intrachain disulphide-bond formation in rITF occurred during the procedure, so no refolding steps were required. Analysis by immunoblotting revealed that the fusion protein and the cleaved trefoil-motif-containing protein were recognised by an antibody raised against the chemically synthesised peptide. The trefoil motif present in the fusion protein was used to localise putative trefoil-binding sites in sections of frozen rat tissue. Binding was demonstrated using the beta-galactosidase portion of the fusion protein as a reporter moiety, either directly with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside, or indirectly using a monoclonal antibody to beta-galactosidase and indirect immunohistochemistry. Binding sites were localised to the foveolar and surface epithelium of rat stomach, the collecting ducts of the kidney and within colonic crypts. The presence of a trefoil motif was necessary for binding. The use of beta-galactosidase fusion proteins for histochemical localisation of peptide-binding sites should prove more generally useful.
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PMID:Expression and purification of a trefoil peptide motif in a beta-galactosidase fusion protein and its use to search for trefoil-binding sites. 844 92

The low copy number plasmid R100 carries the pem region, consisting of two genes, pemI and pemK, which are required for stable maintenance of the plasmid. Here, to understand the regulation of the expression of the pem region, we constructed plasmids carrying either the pemI or the pemK gene, whose initiation codons were fused in frame with the lacZ gene, and examined their expression by assaying beta-galactosidase (LacZ) activity. The synthesis of both PemI and PemK proteins was found to be repressed coordinately in the presence of a plasmid carrying the entire pem region. This indicates that pemK and pemI cistrons form an operon, and that the expression of the operon is negatively regulated by its own products. We then conducted a gel retardation assay in vitro and found that the two pem products, each of which was obtained as a tripartite protein (PemI-collagen-LacZ and PemK-collagen-LacZ), bound cooperatively to a specific fragment containing the proximal region of the pem operon. The binding region, determined by DNase I footprinting analysis, included the promoter for the pem operon. This indicates that both PemI and PemK proteins bind to the promoter region to autoregulate their synthesis.
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PMID:Autoregulation by cooperative binding of the PemI and PemK proteins to the promoter region of the pem operon. 845 70

Biologic consequences of silicone implantation may include changes in host connective tissue metabolism. Lysosomal beta-galactosidase (beta-GAL) activity, which is a sensitive marker of fibrotic diseases and may be a useful marker of collagen turnover, was examined in the serum of rats with implanted silicones. No significant difference in spectrofluorometrically determined enzyme activity was demonstrated in rats subjected to dorsal submuscular pocket dissection without implantation and corresponding nonoperative controls. Rats with implanted solid silicone elastomer or free polydimethylsiloxane gel (both components obtained from mammary implant) revealed enhanced activity of serum beta-GAL. Higher enzyme activity was observed in animals with implanted silicone gel with a peak level of 2.73 +/- 0.08 pmol/30 min/ml 16 weeks after implantation. Increased collagen deposition and capsular thickness was demonstrated around implanted gel material as compared with that around elastomer shell. Animals with implanted absorbable and nonabsorbable materials, polyglactin and Teflon (polytetrafluoroethylene), respectively, after initial increase of beta-GAL activity demonstrated enzyme activity within the normal range. Findings indicate that there is enhanced lysosomal beta-GAL activity after silicone implantation in rats. Clinical relevance and its possible significance as a predictor or indicator of local or systemic fibrosis after silicone implantation seems worthy of further investigation.
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PMID:Enhanced activity of lysosomal beta-galactosidase after silicone implantation: an experimental study in rats. 850 83


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