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 effect of N6,O2'-dibutyryl adenosine 3',5'-cyclic-monophosphate (dbcAMP) on the mobilization of calcium (Ca2+), inorganic phosphate (Pi) and lysosomal enzymes was studied in a bone culture system for 24 h using half calvaria from 6--7 day-old mice. DbcAMP inhibited spontaneous as well as parathyroid hormone-stimulated mineral mobilization. DbcAMP in a concentration of 5 x 10(-4)M also reduced the activities of beta-glucuronidase, beta-galactosidase and acid phosphatase found in the media while the activities of lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were not affected. It is concluded that cAMP is not a stimulator but an inhibitor of bone resorption within the culture period studied (24 h) and that the cyclic nucleotide might interfere with release processes involved in bone resorption.
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PMID:Inhibitory effect of dibutyryl cyclic AMP on the release of calcium, inorganic phosphate and lysosomal enzymes from calvarial bones cultured for 24 hours. 22 6

Human parathyroid hormone (PTH) has been expressed in Escherichia coli as a cro-beta-galactosidase-hPTH fusion protein under temperature-sensitive control of the lambda phage PR promoter. The lacZ gene has been truncated to a different extent revealing an optimal length of the prokaryotic peptide portion between 199 and 407 amino acid residues. Up to 250 mg of pure fusion protein have been obtained from 1-liter E. coli culture by stepwise solubilization with urea. The linkage between the prokaryotic and the eukaryotic protein moiety consists of an Asp-Pro peptide bond and therefore is easily cleavable by acid treatment. A simple procedure for the purification of the hormone is described. The resulting recombinant hormone reacts with anti-PTH antibodies and stimulates renal adenylate cyclase identically to bovine or human PTH.
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PMID:Expression of human parathyroid hormone in Escherichia coli. 252 44

The effect of indomethacin on bone resorption was studied in an organ culture system, using calvarial bones from 6--7-day-old mice. It was found that indomethacin inhibited spontaneous bone resorption, as estimated by decreased release of 45Ca, Ca2+ and Pi. Indomethacin reduced the release of beta-glucuronidase, beta-galactosidase and beta-N-acetylglucosaminidase, diminished glucose consumption and lactate production, but showed no effect on the release of lactate dehydrogenase. No inhibitory effect of indomethacin on the release of 45Ca stimulated by parathyroid hormone, prostaglandin E2 or 1 alpha(OH)D3 could be registered. 5,8,11,14-eicosatetraynoic acid, an inhibitor of both cyclo- and lipoxygenase pathway of arachidonate metabolism, reduced the spontaneous release of 45Ca, whereas the selective lipoxygenase inhibitor 5,8,11-eicosatriynoic acid was without effect. The results presented indicate that indomethacin may have an inhibitory effect upon the osteoclasts, probably by decreased metabolism of arachidonic acid via the cyclo-oxygenase pathway. A possible relationship between this finding and the pathogenesis of rapid destruction of articular bone in osteoarthritic patients treated with indomethacin is discussed.
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PMID:Indomethacin inhibits bone resorption and lysosomal enzyme release from bone in organ culture. 745 22

Human parathyroid hormone (hPTH) has been bacterially expressed in bioreactors as cro-beta-galactosidase-hPTH fusion protein. We have developed a large-scale purification scheme that exploits the pH-dependent differential solubility of hPTH and a two-step chromatographic procedure. We demonstrate that in a number of assay systems, the recombinant material obtained by this procedure is biologically active.
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PMID:Large-scale preparation and biological activity of recombinant human parathyroid hormone. 775 67

Degradable matrices containing expression plasmid DNA [gene-activated matrices (GAMs)] were implanted into segmental gaps created in the adult rat femur. Implantation of GAMs containing beta-galactosidase or luciferase plasmids led to DNA uptake and functional enzyme expression by repair cells (granulation tissue) growing into the gap. Implantation of a GAM containing either a bone morphogenetic protein-4 plasmid or a plasmid coding for a fragment of parathyroid hormone (amino acids 1-34) resulted in a biological response of new bone filling the gap. Finally, implantation of a two-plasmid GAM encoding bone morphogenetic protein-4 and the parathyroid hormone fragment, which act synergistically in vitro, caused new bone to form faster than with either factor alone. These studies demonstrate for the first time that repair cells (fibroblasts) in bone can be genetically manipulated in vivo. While serving as a useful tool to study the biology of repair fibroblasts and the wound healing response, the GAM technology may also have wide therapeutic utility.
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PMID:Stimulation of new bone formation by direct transfer of osteogenic plasmid genes. 865 Jan 65

The effect of amino acid substitution at the P(3) and P(4) subsites on the cleavage activity of a recombinant secretory-type Kex2 protease (Kex2-660) was investigated using recombinant fusion proteins. They were constructed from a truncated Escherichia coli beta-galactosidase (beta G-117S) followed by human parathyroid hormone amino acids 1-34 [hPTH(1-34)] with a junction designed to allow cleavage with Kex2-660. Kex2-660 preferentially cleaved the fusion protein with Arg-His at the P(3)-P(4) subsite, whereas it poorly cleaved the fusion proteins with Val-Asp, Gly-His, Cys-His and Leu-His compared with the original sequence, Val-Lys. As for Asp and Glu at P(4), they precluded the cleavage almost completely. Some of the substitutions were investigated kinetically using the fusion proteins and peptide-substrate mimics corresponding to the 15 amino acids contained in the junction. The substitution Val-Lys to Arg-His resulted in a 2-fold increase in k(cat) and the introduction of Asp at P(4) of the peptide substrate resulted in a large increase in K(m) and a change in the optimum pH. The ordered cleavage of the fusion protein was attained by introducing the two dibasic sites, Arg-His and Asp-His, at P(4)-P(3). The fusion protein was cleaved with Kex2 only at the former site in 3.0 M urea at pH 8.0 and the liberated peptides containing the latter site could be cleaved further with Kex2 at pH 6.5 once purified in urea-free solution. Thus Kex2 exhibited extended substrate specificity beyond P(2)-P(1) in the cleavage of the fusion proteins, although it depended on the reaction conditions.
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PMID:Effect of amino acid substitution at the P(3) and P(4) subsites of fusion proteins on kex2 protease activity. 1091 38

Precursor cells, isolated from bone marrow, can develop into various cell types and may contribute to skeletal growth, remodeling, and repair. The D1 cell line was cloned from a multipotent mouse bone marrow stromal precursor and has osteogenic, chondrogenic, and adipogenic properties. The osteogenic phenotype of these precursor cells is relevant to the process of fracture healing and osteointegration of prosthetic implants. The D1 cells were labeled genetically using a replication incompetent retroviral vector encoding beta-galactosidase, an enzyme which is used as a marker. Labeled cells are readily identifiable by staining with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside and by flow cytometry, and retain the desired osteogenic characteristics in vivo as shown by von Kossa staining, alkaline phosphatase assay, an increase in cyclic adenosine monophosphate in response to parathyroid hormone, osteocalcin messenger ribonucleic acid production, and bone formation in diffusion chambers. In addition, the cells cloned from marrow stroma repopulate the marrow of host mice, persist for several weeks, and retain their osteogenic potential ex vivo. The data suggest that such cells may be used to replenish the number of osteoprogenitors in marrow, which appear to decrease with age, thereby leading to recovery from bone loss and improved bone growth and repair. Labeling these cells creates a model in which to study the potential of such cells to participate in fracture repair, ingrowth around prosthetic implants, treatment of osteoporosis, and to explore the possibility of gene delivery to correct mutations or defects in metabolism that are responsible for certain skeletal abnormalities.
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PMID:Pluripotential mesenchymal cells repopulate bone marrow and retain osteogenic properties. 1103 62

The activator protein-1 (AP-1) and runt domain binding (Runx/RD/Cbfa) sites and their respective binding proteins, c-Fos/c-Jun and Runx2 (Cbfa1), regulate the rat matrix metalloproteinase-13 (MMP-13) promoter in both parathyroid hormone (PTH)-treated and differentiating osteoblastic cells in culture. To determine the importance of these regulatory sites in the expression of MMP-13 in vivo, transgenic mice containing either wild-type (-456 or -148) or AP-1 and Runx/RD/Cbfa sites mutated (-148A3R3) MMP-13 promoters fused with the E. coli lacZ reporter were generated. The wild-type transgenic lines expressed higher levels of bacterial beta-galactosidase in bone, teeth, and skin compared to the mutant and non-transgenic lines. Next, we investigated if overexpression of Runx2 directed by the MMP-13 promoter regulated expression of bone specific genes in vivo, and whether this causes morphological changes in these animals. Real time RT-PCR experiments identified increased mRNA expression of bone forming genes and decreased MMP-13 in the tibiae of transgenic mice (14 days and 6 weeks old). Histomorphometric analyses of the proximal tibiae showed increased bone mineralization surface, mineral apposition rate, and bone formation rate in the transgenic mice which appears to be due to decreased osteoclast number. Since MMP-13 is likely to play a role in recruiting osteoclasts to the bone surface, decreased expression of MMP-13 may cause reduced osteoclast-mediated bone resorption, resulting in greater bone formation in transgenic mice. In summary, we show here that the 148 bp upstream of the MMP-13 transcriptional start site is sufficient and necessary for gene expression in bone, teeth, and skin in vivo and the AP-1 and Runx/RD/Cbfa sites are likely to regulate this. Overexpression of Runx2 by these regulatory elements appears to alter the balance between the bone formation-bone resorption processes in vivo.
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PMID:Overexpression of Runx2 directed by the matrix metalloproteinase-13 promoter containing the AP-1 and Runx/RD/Cbfa sites alters bone remodeling in vivo. 1663 21

Genetically altered mice are an important tool for biomedical research. Several transgenic mice have been created in which activation of the transgene results in production of beta-galactosidase that can be detected by histological means. While preservation and subsequent visualization of enzyme activity in soft tissues can be complicated, it is particularly difficult in bone specimens, especially those that have been decalcified. For these studies, we examined the bones of parathyroid hormone-related peptide (PTHrP) knock-in mice in which expression of PTHrP resulted in beta-galactosidase production. During the past decade, several studies have demonstrated the importance of PTHrP in bone. Thus, it is important to preserve and detect beta-galactosidase enzymatic activity in bone for these studies. We demonstrate here that beta-galactosidase was visualized better in slides with bone sections taken from PTHrP knock-in mice when bones were frozen and sectioned compared to bones that were embedded in plastic and sectioned using a microtome. Importantly, we were able to visualize beta-galactosidase in plastic embedded bones when specimens were fixed, stained (X-gal), embedded in plastic, and then sectioned rather than being fixed, embedded in plastic, sectioned, then stained.
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PMID:Beta-galactosidase detection as an indicator of endogenous PTHrP in cartilage. 1856 83

Telomere-dependent replicative senescence is one of the mechanisms that limit the number of population doublings of normal human cells. By overexpression of telomerase, cells of various origins have been successfully immortalized without changing the phenotype. While a limited number of telomerase-immortalized cells of epithelial origin are available, none of renal origin has been reported so far. Here we have established simple and safe conditions that allow serial passaging of renal proximal tubule epithelial cells (RPTECs) until entry into telomere-dependent replicative senescence. As reported for other cells, senescence of RPTECs is characterized by arrest in G1 phase, shortened telomeres, staining for senescence-associated beta-galactosidase, and accumulation of gamma-H2AX foci. Furthermore, ectopic expression of the catalytic subunit of telomerase (TERT) was sufficient to immortalize these cells. Characterization of immortalized RPTEC/TERT1 cells shows characteristic morphological and functional properties like formation of tight junctions and domes, expression of aminopeptidase N, cAMP induction by parathyroid hormone, sodium-dependent phosphate uptake, and the megalin/cubilin transport system. No genomic instability within up to 90 population doublings has been observed. Therefore, these cells are proposed as a valuable model system not only for cell biology but also for toxicology, drug screening, biogerontology, as well as tissue engineering approaches.
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PMID:hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics. 1871 36


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