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)

DNA can be compacted using polyethylene glycol-substituted poly-L-lysine into discrete unimolecular (with respect to DNA) nanoparticles with minor diameter < 20 nm that are stable in normal saline for at least 23 months at 4 degrees C. We compared the activity of firefly luciferase in lungs of C57BL/6 mice that received 100 microg compacted plasmid in 25 microl saline (shown to be the optimal dose) via intratracheal or intranasal instillation with levels in animals given 100 microg naked plasmid or in untreated mice. Mice dosed with compacted DNA nanoparticles had peak activity of luciferase in lung at 2 days postinstillation, which declined in log-linear fashion with a half-life of 1.4 days. Luciferase activity in animals dosed with naked DNA was 200-fold less. Addition of polyethylene glycol to the complex was necessary for efficient gene transfer and animals that received DNA compacted with unmodified poly-L-lysine did not exhibit luciferase activity above background. Immunohistochemical staining for bacterial beta-galactosidase 2 days after administration of a compacted lacZ expression plasmid (n = 8) revealed expression predominantly in the dependent portions of the right lungs of mice, in alveolar and airway epithelial cells, though macrophages and sometimes endothelial cells also were transfected. No staining for beta-galactosidase was observed in uninjected animals (n = 4) or those dosed with naked lacZ plasmid (n = 7). Tissue survey for transgene expression shows expression only in lung and trachea following intranasal administration. Stable compacted DNA nanoparticles transfer exogenous genes to airway epithelium and show promise for lung gene therapy.
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PMID:Transfection of airway epithelium by stable PEGylated poly-L-lysine DNA nanoparticles in vivo. 1466 96

Reporter enzymes such as firefly luciferase or beta-galactosidase of Escherichia coli are frequently used to study transcriptional activity of genes and to investigate the effects of novel compounds on gene or transcription factor activity. It is generally assumed that the activity of these enzymes is unaffected by the treatment conditions. Therefore, this factor is not considered when interpreting the data obtained. Biologically active compounds such as sesquiterpene lactones (SLs) have also been tested in reporter gene assays for their influence on gene expression. Here we show in in vitro and ex vivo experiments that SLs inhibit firefly luciferase activity probably by direct targeting of the enzyme while beta-galactosidase remains almost completely unaffected. The loss of luciferase activity after SL treatment could be an effect of their sulfhydryl-modifying potency and the subsequent alteration of the enzyme's tertiary structure. These results demonstrate that the effect of the test substance on the reporter enzyme used should be taken into consideration when the transcriptional effect of novel compounds is investigated.
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PMID:Sesquiterpene lactones inhibit luciferase but not beta-galactosidase activity in vitro and ex vivo. 1511 90

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive production of extracellular matrix (ECM) and understood to develop under the influence of certain growth factors. Connective tissue growth factor (CTGF) is a cysteine-rich mitogenic peptide that is implicated in various fibrotic disorders and induced in fibroblasts after activation with transforming growth factor-beta (TGF-beta). To better understand the mechanisms of persistent fibrosis seen in SSc, we previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, TGF-beta transiently induced subcutaneous fibrosis and serial injections of CTGF after TGF-beta caused persistent fibrosis. To further define the mechanisms of skin fibrosis induced by TGF-beta and CTGF in vivo, we investigated in this study, the effects of growth factors on the promoter activity of the proalpha2 (I) collagen (COL1A2) gene in skin fibrosis. For this purpose, we utilized transgenic reporter mice harboring the -17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase gene or a bacterial beta-galactosidase gene. Serial injections of CTGF after TGF-beta resulted in a sustained elevation of COL1A2 mRNA expression and promoter activity compared with consecutive injection of TGF-beta alone on day 8. We also demonstrated that the number of fibroblasts with activated COL1A2 transcription was increased by serial injections of CTGF after TGF-beta in comparison with the injection of TGF-beta alone. Furthermore, the serial injections recruited mast cells and macrophages. The number of mast cells reached a maximum on day 4 and remained relatively high up to day 8. In contrast to the kinetics of mast cells, the number of macrophages was increased on day 4 and continued to rise during the subsequent consecutive CTGF injections until day 8. These results suggested that CTGF maintains TGF-beta-induced skin fibrosis by sustaining COL1A2 promoter activation and increasing the number of activated fibroblasts. The infiltrated mast cells and macrophages may also contribute to the maintenance of fibrosis.
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PMID:Connective tissue growth factor causes persistent proalpha2(I) collagen gene expression induced by transforming growth factor-beta in a mouse fibrosis model. 1560 79

Nucleic acid drugs have great potential to treat many devastating aliments, but their application has been hindered by the lack of efficacious and nontoxic delivery vehicles. Here, a new library of poly(glycoamidoamine)s (D1-D4, G1-G4, and M1-M4) has been synthesized by polycondensation of esterified d-glucaric acid (D), dimethyl-meso-galactarate (G), and d-mannaro-1,4:6,3-dilactone (M) with diethylenetriamine (1), triethylenetetramine (2), tetraethylenepentamine (3), and pentaethylenehexamine (4). The stereochemistry of the carbohydrate hydroxyl groups and the number of amine units have been systematically changed in an effort to examine how the polymer chemistry affects the plasmid DNA (pDNA) binding affinity, the compaction of pDNA into nanoparticles (polyplexes), the material cytotoxicity, and the efficacy of nucleic acid delivery. The polymers with four secondary amines (D4, G4, and M4) between the carbohydrates were found to have the highest pDNA binding affinity and the galactarate polymers generally yielded the smallest polyplexes. Delivery studies with pDNA containing the firefly luciferase or beta-galactosidase reporter genes in BHK-21, HeLa, and HepG2 cells demonstrated that all of the poly(glycoamidoamine)s deliver pDNA without cytotoxicity. Polymers D4, G4, and M4 displayed the highest delivery efficiency, where G4 was found to be a particularly effective delivery vehicle. Heparin competition assays indicated that this may be a result of the higher pDNA binding affinity displayed by G4 as compared to D4 and M4. Polyplexes formed by polymers with weaker pDNA affinities may dissociate at the cell surface due to interactions with negatively charged glycosaminoglycans, which would cause a decrease in the number of polyplexes that are endocytosed.
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PMID:Hydroxyl stereochemistry and amine number within poly(glycoamidoamine)s affect intracellular DNA delivery. 1574 Jan 38

Luciferase reporter constructs are an accurate method of assessing gene promoter activity and vectors constitutively expressing luciferase are useful in quantifying transfection efficiency. Common methodologies for examining the induction of the heat shock (stress) response require exposure of cells transfected with luciferase-expressing vectors to a mild heat stress. Here we re-examine the under-recognised phenomenon that luciferase is exquisitely sensitive to small temperature changes. In cells subjected to mild heat exposure following transfection with both luciferase and beta-galactosidase reporter vectors, a marked reduction in luciferase activity was observed compared with beta-galactosidase activity. On exposing recombinant firefly luciferase to small increases in temperature in vitro, a time and temperature dependent decrease in luciferase activity was demonstrated. Loss of luciferase activity following mild heat exposure will result in misinterpretation of reporter activity. This vastly underappreciated effect is worthy of further emphasis and luciferase reporter vectors should be used with caution in protocols that involve exposure to temperatures outside the physiological range.
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PMID:Firefly luciferase terminally degraded by mild heat exposure: implications for reporter assays. 1644 36

We generated a sequential reporter-enzyme luminescence (SRL) technology for in vivo detection of beta-galactosidase (beta-gal) activity. The substrate, a caged D-luciferin-galactoside conjugate, must first be cleaved by beta-gal before it can be catalyzed by firefly luciferase (FLuc) to generate light. As a result, luminescence is dependent on beta-gal activity. Using this technology, constitutive beta-gal activity in engineered cells and inducible tissue-specific beta-gal expression in transgenic mice can now be visualized noninvasively over time. A substantial advantage of beta-gal as a bioluminescent probe is that the enzyme retains full activity outside of cells, unlike FLuc, which requires intracellular cofactors. As a result, antibodies conjugated to the recombinant beta-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo. Thus, coupling the properties of FLuc to the advantages of beta-gal permits bioluminescent imaging applications that previously were not possible.
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PMID:Luminescent imaging of beta-galactosidase activity in living subjects using sequential reporter-enzyme luminescence. 1655 35

Replication of human immunodeficiency virus type 1 (HIV-1) is controlled by a variety of viral and host proteins. The viral protein Tat acts in concert with host cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR) through a specific interaction with a 59-residue stem-loop RNA known as the trans-activation responsive element (TAR). Inhibitors of Tat-TAR recognition are expected to block transcription and suppress HIV-1 replication. In previous studies, we showed that 2'-O-methyl (OMe) oligonucleotide mixmers containing locked nucleic acid (LNA) residues are powerful steric block inhibitors of Tat-dependent trans-activation in a HeLa cell reporter system. Here we compare OMe/LNA mixmer oligonucleotides with oligonucleotides containing tricyclo-DNAs and their mixmers with OMe residues in four different assays: (1) binding to the target TAR RNA, (2) Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract, (3) trans-activation inhibition in HeLa cells containing a stably integrated firefly luciferase reporter gene under HIV-1 LTR control, and (4) an anti-HIV beta-galactosidase reporter assay of viral infection. Although tricyclo-DNA oligonucleotides bound TAR RNA more weakly, they were as good as OMe/LNA oligonucleotides in suppressing in vitro transcription and trans-activation in HeLa cells when delivered by cationic lipid. No inhibition of in vitro transcription and trans-activation in HeLa cells was observed for tricyclo-DNA/OMe mixmers, even though their affinities to TAR RNA were strong and their cell distributions did not differ from oligonucleotides containing all or predominantly tricyclo-DNA residues. Tricyclo-DNA 16-mer showed sequence-specific inhibition of beta-galactosidase expression in an anti-HIV HeLa cell reporter assay.
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PMID:Tricyclo-DNA containing oligonucleotides as steric block inhibitors of human immunodeficiency virus type 1 tat-dependent trans-activation and HIV-1 infectivity. 1746 63

A DNA cartridge encoding Photinus pyralis luciferase (luc), lacZ homology extensions and an excisable marker was constructed to facilitate the conversion of Escherichia coli lacZ fusions to luc fusions by lambda Red-mediated recombination. This tool was used to transform a cspA::lacZ strain into a luminescent biosensor for C-group translational inhibitors. Comparison of cspA::lacZ and cspA::luc cells showed native firefly luciferase to be a more rapid and sensitive reporter than beta-galactosidase for chloramphenicol detection. To evaluate the usefulness of a red-shifted variant of P. pyralis luciferase (LucR1) for biosensor development, a single copy translational fusion between the SOS-inducible sulA promoter and the lucR1 gene was inserted at the malP site of the E. coli chromosome. The sulA::lucR1 fusion allowed high signal detection of the quinolone ofloxacin to levels as low as 15% of the minimum inhibitory concentration and could be combined with a cspA::lacZ fusion to yield a biosensor suitable for the independent and dual detection of chloramphenicol and ofloxacin.
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PMID:Stress-activated bioluminescent Escherichia coli sensors for antimicrobial agents detection. 1789 48

The promoter regions of the Bombyx mori HSC70-4 and B. mori TCTP genes characterized previously were used for the construction of a series of constitutive gene expression systems active in cultured cells. The relative abilities of these promoters were evaluated by comparing those of a silkworm actin A3 (BmActin3) promoter, which is used widely as the first choice. A series of constitutive expression systems constructed were assayed for the transcription efficiency by connecting four reporter cDNAs, firefly luciferase, 3GFP, Ds-Red, and beta-galactosidase gene using the Gateway LR reaction. The insertion of an intron enhancer into the site between the TCTP promoter and gene increased the transcription of the BmTCTP promoter by 10-fold. The insertion of the IE-1 gene and HR3 enhancer to the all three promoters were found to increase the transcription up to 560 times.
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PMID:Construction of gene expression systems in insect cell lines using promoters from the silkworm, Bombyx mori. 1792 82

This unit describes two nonisotopic systems for reporter gene activity in cells transfected with the firefly luciferase expression plasmid or the beta-galactosidase expression plasmid. Both of these chemiluminescent assays have the advantages of high sensitivity and broad linear range. In the chemiluminescent detection procedures given in this unit, both luciferase activity and beta-galactosidase activity can be measured with either a luminometer or a scintillation counter.
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PMID:Nonisotopic assays for reporter gene activity. 1826 86


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