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)

Regeneration of damaged periodontal tissues is mediated by periodontal cells, but a major sub-population comprises highly differentiated cells that do not renew. To overcome the loss of specialized cell types caused by disease, various therapeutic approaches including cell transplants have been developed to promote cell re-population in periodontal tissues. As previous transplantation studies used unlabeled cells, that are indistinguishable from host cells, it has been difficult to assess the contributions of transplanted cells to the healing processes. To track the fate and differentiation of rat periodontal cells transplanted into periodontal wounds, we used collagen-coated fluorescent beads as a permanent endocytosed marker, or cells constitutively expressing beta-galactosidase. We assessed osteogenic cell differentiation with immunohistochemical staining for osteopontin and bone sialoprotein. Cells were transplanted into periodontal wounds created in Sprague--Dawley male rats that are null for beta-galactosidase. Defects were allowed to heal spontaneously (controls), or were closed with collagen implants mixed with beta-galactosidase-positive (Lac-Z) periodontal cells, or closed with collagen implants mixed with periodontal cells loaded with fluorescent beads. Animals were killed at 1 and 2 weeks after surgery and tissues were prepared for morphometric assessment and immunostaining for osteopontin (OPN) and bone sialoprotein (BSP). Transplanted cells were easily distinguished by fluorescent beads or by beta-galactosidase-positive expression and were distributed throughout the regenerating periodontal ligament (PL) and alveolar bone. At 1 week after wounding, animals treated with beta-galactosidase-positive cells exhibited a slightly higher percentage of labeled cells in the PL compared with the fluorescent bead-labeled cell implant group (2% vs. 1% respectively; P > 0.2). At Week 2 percentages of labeled cells were slightly increased in the regenerating PL (approximately 3% for both groups, P > 0.2). In regenerating alveolar bone at 1 week, animals that were treated with beta-galactosidase-positive cells and fluorescent bead-loaded cells exhibited approximately 30% and 25% of labeled cells respectively. At 2 weeks after wounding there was an increase in the percentage of transplanted beta-galactosidase-positive cells (approximately 39% at week 2; P < 0.05), but not of transplanted cells with fluorescent beads (approximately 25% at week 2). In sites with transplanted cells there were higher percentages of OPN positive and BSP positive cells in nascent bone and more newly formed bone than in controls (>40%; P < 0.05). Transplantation of beta-galactosidase-positive cells or cells loaded with fluorescent beads is a useful method for assessing the fate and differentiation of periodontal cells in vivo. Fluorescent beads, however, are diluted at mitosis and this method underestimates the percentage of transplanted cells. As transplanted periodontal cells in both groups promoted regeneration of alveolar bone, cell transplantation could improve the restoration of periodontium destroyed by periodontitis.
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PMID:Transplantation of labeled periodontal ligament cells promotes regeneration of alveolar bone. 1116 14

The fidelity of poliovirus RNA-dependent RNA polymerase (3D(pol)) was determined using a system based on the fidelity of synthesis of the alpha-lac gene which codes for a subunit of beta-galactosidase. Synthesis products are screened for mutations by an alpha-complementation assay, in which the protein product from alpha-lac is used in trans to complement beta-galactosidase activity in bacteria that do not express alpha-Lac. Several polymerases have been analyzed by this approach allowing comparisons to be drawn. The assay included RNA synthesis by 3D(pol) on an RNA template that coded for the N-terminal region of alpha-Lac. The product of this reaction was used as a template for a second round of 3D(pol) synthesis and the resulting RNA was reverse transcribed to DNA by MMLV-RT. The DNA was amplified by PCR and inserted into a vector used to transform Escherichia coli. The bacteria were screened for beta-galactosidase activity by blue-white phenotype analysis with white or faint blue colonies scored as errors made during synthesis on alpha-lac. Results showed a mutation rate for 3D(pol) corresponding to approximately 4.5x10(-4) errors per base (one error in approximately 2200 bases). Analysis of mutations showed that base substitutions occurred with greater frequency than deletions and insertions.
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PMID:Determination of the mutation rate of poliovirus RNA-dependent RNA polymerase. 1122 80

The enzymatic access to nucleotide-activated oligosaccharides by a glycosidase-catalyzed transglycosylation reaction was explored. The nucleotide sugars UDP-GlcNAc and UDP-Glc were tested as acceptor substrates for beta-galactosidase from Bacillus circulans using lactose as donor substrate. The UDP-disaccharides Gal(beta1-4)GlcNAc(alpha1-UDP) (UDP-LacNAc) and Gal(beta1-4)Glc(alpha1-UDP) (UDP-Lac) and the UDP-trisaccharides Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP and Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP) were formed stereo- and regioselectively. Their chemical structures were characterized by 1H and 13C NMR spectroscopy and fast atom bombardment mass spectrometry. The synthesis in frozen solution at -5 degrees C instead of 30 degrees C gave significantly higher product yields with respect to the acceptor substrates. This was due to a remarkably higher product stability in the small liquid phase of the frozen reaction mixture. Under optimized conditions, at -5 degrees C and pH 4.5 with 500 mM lactose and 100 mM UDP-GlcNAc, an overall yield of 8.2% (81.8 micromol, 62.8 mg with 100% purity) for Gal(beta1-4)GlcNAc(alpha1-UDP) and 3.6% (36.1 micromol, 35 mg with 96% purity) for Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP) was obtained. UDP-Glc as acceptor gave an overall yield of 5.0% (41.3 micromol, 32.3 mg with 93% purity) for Gal(beta1-4)Glc(alpha1-UDP) and 1.6% (13.0 micromol, 12.2 mg with 95% purity) for Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP). The analysis of other nucleotide sugars revealed UDP-Gal, UDP-GalNAc, UDP-Xyl and dTDP-, CDP-, ADP- and GDP-Glc as further acceptor substrates for beta-galactosidase from Bacillus circulans.
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PMID:Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillus circulans. 1130 28

Our goal was to develop a system to study proteins that associate in vivo with the Moloney murine leukemia virus (M-MuLV) enhancer elements by the isolation of intact proviral chromatin. The M-MuLV long terminal repeats (LTRs) contain tandemly repeated transcriptional enhancer sequences consisting of smaller motifs that bind cellular DNA-binding proteins implicated in transcriptional regulation. The M-MuLV enhancers are also important for disease specificity and latency of disease induction. To enrich for proviral chromatin containing M-MuLV LTR sequences, an affinity purification scheme was employed that relies on the affinity of bacterial Lac repressor protein for Lac operator (LacO) DNA sequences. An infectious M-MuLV recombinant was constructed that contains bacterial LacO sequences inserted into a nonessential region downstream from the 5' LTR of the virus (M-MuLV-LacO). Nuclei from M-MuLV-LacO-infected cells were digested with PvuII (which will liberate an LTR fragment containing LacO sequences), and digested chromatin was leached from the nuclei in hypotonic buffer. M-MuLV-LacO chromatin was then recovered by binding to an affinity matrix consisting of a beta-galactosidase-Lac repressor fusion protein anchored to acrylamide beads by an anti-beta-galactosidase monoclonal antibody [7]. Specifically bound chromatin was eluted under physiological conditions by incubation with the galactose analog isopropyl-beta-D-thiogalactopyranoside. Southern blot analysis confirmed the specific enrichment of M-MuLV proviral chromatin by this method.
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PMID:Purification of Moloney murine leukemia virus chromatin from infected cells by an affinity method. 1138

The introduction of mammalian artificial chromosomes (ACs) into zygotes represents an alternative, more predictive technology for the production of recombinant proteins in transgenic animals. The aim of these experiments was to examine the effects of artificial chromosome microinjection into bovine pronuclei on embryo development and reporter gene expression. Bovine oocytes aspirated from 2-5 mm size follicles were matured in vitro for 22 hr. Mature oocytes were fertilized in vitro with frozen- thawed bull spermatozoa. Artificial chromosome carrying either beta-galactosidase (Lac-Z) gene or green fluorescence protein (GFP) gene were isolated by flow cytometry. A single chromosome was microinjected into one of the two pronuclei of bovine zygotes. Sham injected zygotes served as controls. Injected zygotes were cultured in G 1.2 medium for 7 days. Hatched blastocysts were cultured on blocked STO cell feeder layer for attachment and outgrowth of ICM and trophectoderm cells. The results showed a high zygote survival rate following LacZ-ACs microinjection (74%). However, the blastocyst development rate after 7 days of culture was significantly lower than that of sham injected zygotes (7.5 vs. 22%). Embryonic cells positive for Lac-Z gene were detected by PCR in three of nine outgrowth colonies. In addition, GFP gene expression was observed in 15 out of 85 (18%) embryos at the arrested 2-cell stage to blastocyst stage. Six blastocysts successfully outgrew, three outgrowths were GFP positive for up to 3 weeks in culture. We conclude that the methodology for artificial chromosome delivery into bovine zygotes could lead to viable blastocyst development, and reporter gene expression could be sustained during pre-implantation development.
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PMID:Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes. 1174 53

RET forms the catalytic component within the receptor complex that transmits signals from the GDNF family of neurotrophic factors. To study the mechanisms regulating the cell-type specific expression of this gene, we have cloned and characterised the murine c-ret locus. A cosmid contig comprising approximately 60 kb of the mouse genome encompassing the entire structural gene and flanking sequences have been isolated and the transcription initiation site identified and promoter characterised. The murine c-ret promoter lacks a TATA initiation motif and has GC enriched DNA sequences reminiscent of CpG islands. Analysis of transgenic mice lines bearing the Lac Z (beta-galactosidase) reporter gene under the control of 5' flanking sequences show modularity in the organisation of cis-regulatory domains within the locus. Cloned 5' flanking sequences comprise a distal regulatory domain directing Lac Z expression at the primitive streak, lateral mesoderm and facial ganglia and a proximal sensory neurones specific regulatory domain inducing Lac Z expression primarily within the developing somatosensory system. The spatial and temporal progression of transgene expression precisely recapitulates endogenous gene expression in developing sensory ganglia including its induction in postnatal Isolectin B4 binding nociceptive neurones.
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PMID:Flanking regulatory sequences of the locus encoding the murine GDNF receptor, c-ret, directs lac Z (beta-galactosidase) expression in developing somatosensory system. 1174 74

The first step in cellular entry of HIV involves binding of the viral envelope glycoprotein complex (gp120/gp41) to specific receptor molecules on the target cells. The cell-cell fusion (syncytium formation) between env expressing cells and CD4+ cells mimics the viral infection of the host cells. To search for anti-HIV substances preventing this process, we constructed the recombinant cell lines, HeLa/CD4/Lac-Z and HeLa/T-env/Tat for T-cell tropic (HIV-1(NL4-3)) system, and HOS/CD4/CCR5/Lac-Z and HeLa/M-env/Tat for macrophage tropic (HIV-1(SF162)) system. When each pair of cells were co-incubated for 20 hours, the multinuclear giant cells (syncytia) were formed and beta-galactosidase was expressed. These systems are less biohazardous because no infectious virus particles are used. Their validity in screening for anti-HIV substances which inhibit syncytium formation was confirmed using various known HIV entry inhibitors.
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PMID:A simple screening system for anti-HIV drugs: syncytium formation assay using T-cell line tropic and macrophage tropic HIV env expressing cell lines--establishment and validation. 1177 37

A special recombinant of Autographa californica multicapsid nuclear polyhedrosis virus (AcNPV) was designed to study the early histopathological events of baculovirus infection in Spodoptera exigua larvae. This recombinant contained a Drosophila melanogaster heat shock 70 promoter driving an Escherichia coli beta-galactosidase (Lac-Z) reporter gene to monitor the presence of early viral gene expression and a second reporter gene, the E. coli beta-glucuronidase (GUS) gene, under control of the very late AcNPV p10 promoter to monitor viral replication. In S. exigua larvae, permissive Spodoptera spp. cultured cells, and nonpermissive D. melanogaster cultured cells early viral gene expression was indicated by the appearance of Lac-Z as early as 3 hr p.i. Late viral gene expression was indicated by the appearance of GUS and occurred only in the permissive cultured cells and larvae. Early and late viral gene expression could be detected simultaneously using differential enzyme histochemistry. Analysis of infected S. exigua larvae revealed that midgut columnar cells and, at a low frequency, midgut regenerative cells were the primary sites of infection. Parental nucleocapsids were apparently transported through columnar cells to underlaying regenerative cells before virus replication and progeny production. Infection of tissues beside the midgut epithelium was not detected prior to viral replication within the midgut, suggesting that infection of the midgut is an important prelude to systemic infection.
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PMID:Passage of Autographa californica nuclear polyhedrosis virus through the midgut epithelium of Spodoptera exigua larvae. 1183 15

The late genes of the temperate phage of Pseudomonas aeruginosa are organized in an analogous fashion to the corresponding transcription units of the Escherichia coli P2 and P2-like phages. Sequence analysis of four putative late promoter regions, PP(phiCTX), PO(phiCTX), PV(phiCTX) and PF(phiCTX), reveals no similarity to sigma(70)-type promoters or promoter consensus sequences found in Pseudomonas, indicating the apparent need for a phage-encoded protein to control the expression of phiCTX late genes. To elucidate the mode of expression of the late genes, we fused the putative late promoter regions to the promoterless lacZalpha gene, which encodes the N-terminal part of beta-galactosidase as a reporter enzyme, in the promoter-probe vector pME4510. The candidate transactivator gene orf34 was cloned into expression vector pHA10, to generate the plasmid pHA34. The two recombinant plasmids were introduced together into E. coli XL1-Blue and P. aeruginosa PAO1S-Lac. Our results demonstrate that in phiCTX three late promoters (PP(phiCTX), PO(phiCTX), and PF(phiCTX)) are activated upon induction by IPTG in PAO1S-Lac carrying the cloned promoters and pHA34. Deletions and base-pair substitutions obtained by PCR-mediated mutagenesis demonstrated that two conserved sequences, TTGTAG-N(9)-cTACAa and GcCGCGCGCGCGgC, are essential for effective late gene expression. Whereas the late promoters were active in P. aeruginosa, only weak beta-galactosidase activity was obtained in E. coli.
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PMID:Organization and activation of the late promoters of phiCTX, a cytotoxin-converting phage from Pseudomonas aeruginosa. 1191 13

Estrogenicity was assessed using the Saccharomyces cerevisiae-based Lac-Z reporter assay and was reported as the logarithm of the inverse of the 50% molar beta-galactosidase activity (log[EC50(-1)]). In an effort to quantify the relationship between molecular structure of polycyclic aromatic hydrocarbons (PAHs) and estrogenic gene expression, a series of PAHs were evaluated. With noted exceptions, the results of these studies indicate that the initial two-dimensional structural warning for estrogenicity, the superpositioning of a hydroxylated aromatic system on the phenolic A-ring of 17-beta-estradiol, can be extended to the PAHs. This two-dimensional-alignment criterion correctly identified estrogenicity of 22 of the 29 PAHs evaluated. Moreover, the estrogenic potency of these compounds was directly related to the size of the hydrophobic backbone. The seven compounds classified incorrectly by this structural feature were either dihydroxylated naphthalenes or aromatic nitrogen-heterocyclic compounds; all such compounds were false positives. Results with dihydroxylated naphthalenes reveal derivatives that were nonestrogenic when superimposed on the phenolic A-ring of 17-beta-estradiol had the second hydroxyl group in the position of the C-ring or were catechol-like in structure. Structural alerts for nitrogen-heterocyclic compounds must take into account the position of the hydroxyl group and the in-ring nitrogen atom; compounds with the hydroxyl group and nitrogen atom involved with the same ring were observed to be nonactive.
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PMID:Xenoestrogenic gene expression: structural features of active polycyclic aromatic hydrocarbons. 1195 52


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