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:1.14.14.3 (luciferase)
38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of bacterial luciferase from Photobacterium phosphoreum with reduced flavin was investigated using various 8-substituted FMNH2 analogs. Flavins tested were FMNH2 and FMNH2 substituted at the 8 position with HO-, CH3O-, C2H5O-, Cl-, Br-, I-, H2N-, (CH3)HN-, and (ch3)2n. 8-ch30-, c2h5o-, cl-, and Br-FMNH2 showed luminescent activity in the luciferase reaction with emission peaks at various wavelengths. 8-HO- and I-FMNH2 were competitive inhibitors toward FMNH2 in the luminescent reaction. 8-Amino analogs of FMNH2 showed no luminescent or inhibitor activity. The dissociation constant of the luciferase-FMNH2 analog complex was determined kinetically as a substrate or inhibitor constant. A contribution of the imino group at position 5 in the isoalloxazine ring to the FMNH2 binding to luciferase was suggested by a Hammett plot of the dissociation constants.
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PMID:Studies on luciferase from Photobacterium phosphoreum. XI. Interaction of 8-substituted FMNH2 with luciferase. 73 95

The possible role of cyclic AMP (cAMP) in the regulation of the vasopressin (VP) gene was tested in two cellular expression systems: one cell line with endogenous VP expression and the other which was transiently with a VP promoter-luciferase fusion gene. 8,Bromo-cAMP stimulated the VP mRNA content about 4-fold in the human VP-expressing small cell lung carcinoma cell line GLC-8. The luciferase activity in P19 embryonal carcinoma cells which were transiently transfected with -174 to +44 of the 5'-flanking region of the human VP gene linked to the firefly luciferase gene, was stimulated about 2-fold by the cAMP analogue. The results indicate that cAMP plays a role in the upregulation of the VP gene and hence point to several putative nucleotide motives in the promoter functionally conferring this response.
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PMID:Vasopressin gene expression is stimulated by cyclic AMP in homologous and heterologous expression systems. 217 21

2-Bromo[1-14C]1-decanal was synthesized as an affinity labeling probe for the aliphatic aldehyde site of Vibrio harveyi luciferase. In the presence of excess amounts of this probe, the inactivation of bacterial luciferase occurred following apparent first order kinetics. This inactivation was markedly retarded in the presence of decanal but neither butanal (a very poor aldehyde substrate) nor FMN (a reaction product derived from reduced FMN) showed any significant protective effect. Upon mixing luciferase with the affinity labeling probe, a noncovalent complex was formed prior to the covalent attachment. At pH 6 and 23 degrees C, the dissociation constant for the binding step and the rate constant for the covalent modification step were determined to be 23 microM and 1 min-1, respectively. The displacement of a bound aldehyde substrate by this probe added secondarily was also demonstrated. The inactivation of luciferase was correlated with both the incorporation of about 1.2 molecules of the probe and the loss of 0.8 to 1.1 cysteinyl residues/luciferase alpha beta dimer. The presence of an essential sulfhydryl group at the aldehyde site of luciferase has thus been demonstrated. This sulfhydryl group was a constituent residue of the alpha subunit and was near the alpha beta subunit interface. This residue appears to be the same essential cysteinyl group previously identified by chemical modification (Nicoli, M.Z., Meighen, E.A., and Hastings, J.W. (1974) J. Biol. Chem. 249, 2385-2392). The labeled luciferase did not exhibit any significant binding for the reduced FMN substrate.
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PMID:Affinity labeling of the aldehyde site of bacterial luciferase. 654 53

Acridine dyes and other DNA-intercalating agents such as ethidium bromide, theophylline, and caffeine induce luminescence in dark variants (K variants) different luminous species of bacteria, as well as in their wild-type luminous cells, prior to induction. The increase in luminescence appears 10-20 min after addition of these agents and is inhibited by chloramphenicol or rifampicin. Addition of these agents affects the synthesis of both luciferase and aldehyde-synthesizing enzymes. It is hypothesized that these agents, through their intercalation into DNA, cause configurational changes resulting in derepressed transcription of the luminescence operon.
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PMID:Acridine dyes and other DNA-intercalating agents induce the luminescence system of luminous bacteria and their dark variants. 694 43

The use of cationic lipids for gene transfer to airway epithelia has shown promise in in vitro and in vivo studies. However, previous studies have used a wide variety of different lipid preparations and different formulations. Few studies have been designed to optimize the variables involved in transfection, and none have been focused on airway epithelia. Therefore we examined variables that affect cationic lipid-mediated transfection of HeLa cells and of airway epithelial cells grown on permeable filter supports at the air-liquid interface. To quantitate expression of cDNA, we assayed expression of luciferase. We found that the ratio of DNA to lipid was an important variable that determined transfection efficiency. In both HeLa and airway epithelial cells, the optimum charge ratio of cationic lipid to anionic DNA was approximately 1.25, consistent with the notion that a positively charged complex facilitates interaction with the negatively charged cell membrane. After testing a series of readily available cationic lipids, we found that 1,2-dimyristyloxypropyl-N,N-dimethyl-hydroxyethyl ammonium bromide (DMRIE)/dioleoyl phosphatidylethandamine (DOPE) appeared to show good efficacy. The concentration of DNA and cationic lipid also played an important role: in HeLa cells the optimum concentration of cationic lipid was approximately 5 microM and in airway epithelial cells was approximately 15 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Optimization of cationic lipid-mediated gene transfer to airway epithelia. 763 13

In patients with Alzheimer's disease, hippocampal cells are among the first neuronal cells of the brain to degenerate. Both rat primary hippocampal neurons and cells of the clonal mouse hippocampal cell line HT22 express endogenous functional glucocorticoid receptors (GRs), as shown by transient transfection of cells with a luciferase reporter plasmid containing GR-responsive elements. The influence of activated GRs on oxidative stress-induced neuronal cell death in vitro was investigated employing these hippocampal model systems. Two oxidative stressors were investigated, the free radical-inducing Alzheimer's disease-associated amyloid beta-protein, which is toxic to hippocampal neurons, and the excitatory amino acid glutamate, which induces oxidative cell death in HT22 cells via an increase in intracellular peroxides. Cellular viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test and trypan exclusion staining, followed by microscopical cell counting. Glucocorticoids strongly increased the vulnerability of the hippocampal cells to amyloid beta-protein and glutamate. This increase could be blocked by the specific GR antagonist RU486. Our data suggest that changes in hippocampal GR homeostasis and regulation may render hippocampal neurons more vulnerable to oxidative stress-induced neuronal degeneration.
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PMID:Glucocorticoids enhance oxidative stress-induced cell death in hippocampal neurons in vitro. 897 91

Cationic lipid-mediated transfection of the alveolar epithelium in vivo will require exposure of plasmid DNA and cationic lipids to endogenous surfactant lipids and proteins in the alveolar space. Effects of pulmonary surfactant and of surfactant constituents on transfection in vitro of two respiratory epithelial cell lines (MLE-15 and H441) with a plasmid encoding the luciferase reporter gene were studied using two cationic lipid formulations: 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide/cholesterol (DMRIE/C) and 1,2-dioleoyl-3-trimethylammonium propane/dioleoyl phosphatidylethanolamine (DOTAP/DOPE). Gene expression, as assessed by luciferase activity, decreased as increasing concentrations of natural surfactant were added to cationic lipid-DNA complexes. Incorporation of phospholipids DOPC/DOPG or surfactant proteins SP-B or SP-C in the cationic lipid formulation inhibited transfection. A fluorescent lipid mixing assay was used to determine the effects of surfactant proteins SP-B and SP-C on mixing between cationic lipid-DNA complexes and surfactant lipid vesicles. Mixing between DOPC/DOPG vesicles and cationic lipid-DNA complexes in the absence of added proteins amounted to 10-20%. Addition of SP-B or SP-C increased the mixing of DOPC/DOPG vesicles with DOTAP/DOPE-DNA complexes, but not DMRIEC-DNA complexes. These results demonstrate that pulmonary surfactant lipids and proteins inhibit transfection with cationic lipid-DNA complexes in vitro, and may therefore represent a barrier to gene transfer in the lung.
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PMID:Pulmonary surfactant inhibits cationic liposome-mediated gene delivery to respiratory epithelial cells in vitro. 905 18

LMH-2A is an estrogen-responsive avian hepatoma cell line whose susceptibility to cationic-lipid-mediated transfection is poorly described. 3 beta[N-N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DCC) requires a one-step synthesis, and can be used to formulate transfection-grade liposomes when combined with dioleoylphosphatidyl-ethanolamine (DOPE) 1/1 (wt/wt). Luciferase activities in LMH-2A cells were 8.5-fold and 87.5-fold greater than those in HepG2 and FTO2B cells, respectively, following DCC-liposome-mediated transfection with a reporter consisting of the human cytomegalovirus immediate-early promoter (CMV), joined to Photinus pyralis luciferase (L) cDNA, designated pCMVL. Using pCMVL, N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimun bromide) (BMOP)/DOPE 1/1 (wt/wt), at a 7.5:1 ratio with DNA, produced luciferase activities that were 2.9-fold higher than those of DCC-liposomes, at an optimal 10:1 lipid:DNA ratio. At optimal lipid:DNA ratios, commercially available liposomes, Transfectam, Lipofectamine, and Lipofectin, produced luciferase activities that were 1.39, 1.03, and 0.47-fold those of DCC-liposomes. The effect of 0, 10, 100, or 500 nM/L 17 beta-estradiol on the expression of pCMVL and a second luciferase reporter containing the -593/+48 promoter region of the estrogen-responsive avian apo VLDL-II gene, designated pApoL, was tested in cells cultured in the presence or absence of 10% chicken serum. The CMV promoter supported a high level of expression in LMH-2A cells that was unaffected by serum alone, but was weakly responsive to estrogen. Estrogen responses of both reporters reached a plateau at 10 nM/L. Estrogen increased the expression of pApoL 24-fold and 79-fold in the absence and presence of serum, respectively. The -593/+48 region of the apo VLDL-II promoter may not contain previously reported negative insulin response elements, but chicken serum contains factors that enhance estrogen responsiveness of this region.
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PMID:Transfection of avian LMH-2A hepatoma cells with cationic lipids. 918 23

The pericellular proteoglycan biglycan is among the major secretory products of osteoblasts and articular chondrocytes but the regulatory agents and signal transduction pathways that ultimately lead to alterations in biglycan gene expression are poorly defined. We report here on the transcriptional up-regulation of biglycan in MG-63 osteosarcoma cells by agents that increase intracellular cAMP levels. Transfection of these cells with biglycan promoter luciferase reporter fusion genes and subsequent treatment with forskolin or the cAMP analog 8-Bromo-cAMP resulted in an up to 3.8-fold stimulation of biglycan promoter activity. This effect could be prevented with the compound KT5720, a specific inhibitor of the cAMP-dependent protein kinase. Up-regulation of transcription is also reflected at the level of mRNA expression, since biglycan mRNA steady state levels in MG-63 cells increased approximately 2-fold after 24 hours of forskolin treatment. These data suggest that elevated levels of intracellular cAMP increase transcription from the biglycan promoter in bone cells and implicate for the first time the cAMP/protein kinase A signal transduction pathway in the regulation of biglycan gene expression.
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PMID:Biglycan gene promoter activity in osteosarcoma cells is regulated by cyclic AMP. 919 8

We have investigated the morphology and transfection activity of cationic liposome-DNA complexes (CLDC) under conditions relevant to both in vivo and in vitro studies. Moreover we have attempted to establish structure-function relationships relevant for high transfection activities under both conditions. CLDC were composed of dimethyldioctadecylammonium bromide with either 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol) interacting either with pre-condensed DNA or with uncondensed plasmid DNA. Furthermore for steric stabilization 1% poly(ethylene glycol)-phospholipid conjugate was added to CLDC containing Chol and plasmid DNA. The in vivo studies were carried out in mice following i.v. injection, and the in vitro studies were performed on SK-BR-3 human breast cancer cells in the presence of media with serum. The morphology of the CLDC, monitored by freeze-fracture electron microscopy, was investigated after mixing with mouse serum or the medium where the cells were kept. The substitution of DOPE with Chol, and the addition of N-[omega-methoxypoly(oxyethylene)-alpha-oxycarbonyl-DSPE+ ++ are producing CLDC which are stabilized with respect to time and serum, and are relatively small (100-300 nm). These stabilized complexes show high expression of a marker gene in mouse lungs reaching expression values up to 10 ng luciferase per mg tissue protein, but relatively low expression in SK-BR-3 cells in vitro. Additionally, some of the complexes containing pre-condensed DNA look like 'map-pin' structures showing heads of the size of liposomes and short, stiff and tapering tails. The in vivo transfection activity of these preparations is highest. Similar complexes containing DOPE rather than Chol as helper lipid precipitate in the presence of serum and especially of cell medium and convert into hexagonal lipid (HII) phase. Such complexes, despite their high transfection activity in vitro, show very little transfection activity in vivo. These comparisons may help us to understand the fundamental difference between in vitro and in vivo activity of CLDC: high in vitro transfection activity seems to be associated with hexagonal lipid precipitates whereas high in vivo activity seems to be related with small, stabilized complexes, which in our case also exhibit some protrusions (map-pin structures).
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PMID:Ultrastructural characterization of cationic liposome-DNA complexes showing enhanced stability in serum and high transfection activity in vivo. 976 90


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