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 flux in rat hepatic ratio of adenosine triphosphate levels to adenosine diphosphate levels (ATP/ADP) during the onset and progression of paracetamol-induced cell injury both in vivo and in vitro were investigated and compared. Leakage of lactate dehydrogenase (LDH) and potassium (K+), and mg water/mg dry weight quantified cell injury. ATP and ADP levels were determined using the luciferin-luciferase bioluminescence assay. For in vitro studies, liver slices obtained from phenobarbitone-induced rats were exposed to 10 mM paracetamol for 120 min (T0-T120) and, then incubated without paracetamol up to a further 240 min (T120-T360). For in vivo studies, groups of four phenobarbitone-induced rats received i.p. injections of 800 mg/kg paracetamol. ATP/ADP ratios fall upon exposure to paracetamol both in vitro and in vivo. However, unlike the in vitro situation where the fall in ATP/ADP ratios precedes and accompanies the progression of cell injury, the in vivo fall in ATP/ADP ratios is shown to occur as cell injury measurements begin to recover to control levels. However, despite these differences classic paracetamol-induced centrilobular necrosis is observed to occur both in vitro and in vivo. This study demonstrates that the liver slice model is a simple and useful technique to investigate the underlying mechanisms of paracetamol-induced cell injury.
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PMID:Comparison of paracetamol-induced hepatotoxicity in the rat in vivo with progression of cell injury in vitro in rat liver slices. 983 56

The formulation of cationic polymers of polyethylenimine (PEI) with plasmid DNA has been optimized to deliver genes into the Xenopus tadpole brain in vivo. Using intraventricular microinjections of 1 microl (containing 0.5 to 1 microg DNA) we show that the linear, low molecular weight polymer, 22 kDa PEI was significantly more efficient than a branched 25 kDa polymer. Complexes bearing a slightly positive net charge (formed with a ratio of 6 PEI amines per DNA phosphate) provided the best levels of transfection. Transgene expression was DNA-dose dependent and was maintained over 6 days, the time course of the experiment. Spatial distribution was examined using a beta-galactosidase construct and neurones expressing this transgene were found spread throughout the brain. The possibility of using this technique to evaluate physiological regulations was approached by examining the effects of tri-iodothyronine (T3), on transcription from the mammalian TRH and Krox-24 promoter sequences. Adding physiological concentrations of T3 to the aquarium water significantly reduced transcription from the rat TRH promoter whilst the same treatment increased transcription from a mouse Krox-24 -luciferase construct. Thus, PEI-DNA transfection provides a versatile and easily applied method for following physiological regulations at the transcriptional level in the tadpole brain.
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PMID:T3-dependent physiological regulation of transcription in the Xenopus tadpole brain studied by polyethylenimine based in vivo gene transfer. 987 14

Bone sialoprotein (BSP) and osteopontin (OPN) are two major noncollagenous matrix proteins in mineralized connective tissue that have discrete roles in bone matrix formation, mineralization, and remodeling. The osteotropic secosteroid, 1,25-dihydroxyvitamin D3, a potent regulator of bone remodeling required for normal bone development, has been shown to exert differential effects on OPN and BSP expression by bone cells in vitro. To investigate these effects in vivo, we induced vitamin D3 deficiency in a transgenic mouse line (rBSP2.7Luc) that has a 2.7 kb rat BSP promoter linked to a luciferase reporter gene in its genome. Pregnant rBSP2.7Luc mice were fed vitamin D3-deficient food and demineralized water for 6 weeks. Their offspring were weaned at 3 weeks of age and then fed vitamin D-deficient food for an additional week. The control group were fed normal rodent pellets and water during the entire experimental procedure. Bone tissues from 40, 4-week-old offspring in each group were analyzed for BSP, OPN and luciferase expression. Vitamin D3-deficient mice displayed a rachitic phenotype that included reduced size and malformation of bones. Assays of the BSP promoter transgene in calvariae, mandibles, and tibiae of the rachitic mice showed increases in luciferase activity of 3.1-, 1.9-, and 4.6-fold, respectively, when compared with control littermates. Semiquantitative reverse transcriptase polymerase chain reaction assays of BSP mRNA revealed increases of 7-, 74-, and 66-fold, respectively, in the same rachitic bones, while OPN mRNA was reduced 12.5-fold in calvariae and 2-fold in tibiae and mandibles. In situ hybridization using mouse cRNA probes revealed that the increased BSP expression and decreased OPN expression in the vitamin D3-deficient mice was primarily in osteoblastic cells on the surface of calvariae and endosteal spaces of alveolar bone, on newly formed epiphyseal bone, and in cementoblasts and in hypertrophic chondrocytes. These studies are the first to show that BSP and OPN are differentially regulated by vitamin D3 in vivo, reflecting the diverse roles of these protein in bone remodeling. Moreover, the increased expression of the BSP transgene in the rachitic mice demonstrates that vitamin D3 regulation of BSP expression is mediated, in part, by element(s) within the 2.7 kb promoter region.
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PMID:Altered expression of bone sialoproteins in vitamin D-deficient rBSP2.7Luc transgenic mice. 993 76

A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r2 = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.
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PMID:Real-time monitoring of Escherichia coli O157:H7 adherence to beef carcass surface tissues with a bioluminescent reporter. 1010 75

We recently suggested that prolonged deregulated expression of AP-1 activity in colonic cells by bile acids may contribute to tumour promotion in the colon. In the present study, using two human colon carcinoma cell lines, HT-29 and HCT 116, transiently transfected with the AP-1-luciferase reporter construct, we showed that the bile acids, deoxycholate, chenodeoxycholate, ursodeoxycholate and lithocholate, induced AP-1-dependent gene transcription in a dose-dependent manner, whereas cholate was without effect. The greatest effect was observed with deoxycholate, and the ability of this bile acid to induce reporter gene activity was significantly correlated with its ability to induce cell proliferation (r = 0.91, P = 0.01). Cholesterol and the long chain fatty acids, myristate, palmitate and stearate, had no effect on AP-1-dependent gene transcription, whereas the short chain fatty acid, butyrate, exhibited a marked effect. Mindful of the fact that the concentrations of lumenal components that are actually in or entering the epithelial cells in the colon are presumably lower than lumenal values, we considered it of interest to determine the effect of dilution on the capacity of human faecal water to induce AP-1 activity and also cell proliferation. We demonstrated that diluted lipid extracts, from all of the faecal water samples examined, significantly induced AP-1-dependent gene transcription in the colonic cells, and that this effect differed markedly between the extracts. We confirmed that the faecal water lipid extracts, at the same dilution at which they increased AP-1 activity, significantly induced proliferation in the same cell line. These data suggest that lipid components of human faecal water, which is in direct contact with the colon epithelium and may be physiologically more active than the solid phase, can activate AP-1, a transcription factor whose activation has been associated with the promotion of neoplastic transformation.
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PMID:Effects of colonic lumenal components on AP-1-dependent gene transcription in cultured human colon carcinoma cells. 1035 75

Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum.
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PMID:Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida. 1046 70

Freeze-drying of three different forms of gene delivery systems was performed using a controlled two-step drying process and 10% sucrose as lyoprotectant. Complexes of pCMVL plasmid with transferrin-conjugated polyethylenimine, adenovirus-enhanced transferrinfection consisting of pCMVL/transferrin-polylysine complexes linked to inactivated adenovirus particles, and a recombinant, E1-defective adenovirus expressing a luciferase reporter gene were tested. Three weeks after freeze-drying the reagents were rehydrated with water and tested for transfection activity. Luciferase gene expression levels were retained at high levels in all three systems, in contrast to reagents stored in solution. The use of the lyoprotectant was essential. In the absence of sucrose the transfection activities dropped by a factor of 100-1000. The data suggest freeze-drying as a useful method for stabilization and storage of standardized batches of transfection agents.
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PMID:Stabilization of gene delivery systems by freeze-drying. 1047 20

Walleye dermal sarcoma virus (WDSV) is a retrovirus aetiologically associated with a multifocal skin tumour of walleye. Tumours synchronously develop on 27% of fish and regress seasonally; their severity is influenced by water temperature. To functionally characterize the LTR of WDSV, the LTR was fused to the luciferase reporter gene. WDSV LTR was found to be transcriptionally active in both fish and mammalian cells. WDSV LTR deletion mutants were constructed to identify specific regions that were functionally important in modulating viral gene expression and in temperature responsiveness. The 5' end 60 bp, which contain a putative ecdysone-response element also present in another fish retrovirus, positively modulated transcription from the WDSV LTR at 25 degrees C, but not at lower temperatures. A 13 bp region (nt -288 to -275) comprising a putative activator protein-1 element was necessary for maintaining WDSV LTR activity at all temperatures. In marked contrast to the short direct repeats found in mammalian retroviral LTRs, five 5 bp direct repeats (nt -336 and -272) were found to negatively regulate transcription from the WDSV LTR. A region spanning nt -440 to -218 stimulated the activity of a heterologous mammalian promoter in an orientation-dependent manner, modestly in fish cells (1.3- to 2-fold), but markedly (3.7- to 5.1-fold) in mammalian cells. Our results strongly suggest that the putative promoter elements present in the WDSV LTR function differentially in a temperature-specific manner and that complex interactions between these elements modulate WDSV LTR activity in response to temperature changes.
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PMID:Functional characterization of a piscine retroviral promoter. 1056 36

Non-viral gene therapy is a potential treatment to many incurable retinal diseases. To fulfill this promise, plasmid DNA must be delivered to the retinal target cells. We evaluated the efficacy of synthetic DNA complexing compounds in transfecting primary human retinal pigment epithelial (RPE) cells in vitro. Fetal human RPE cells were cultured with or without extracellular matrix (ECM), produced using calf corneal endothelial cells. Plasmids encoding nuclear localizing beta galactosidase or luciferase (pRSVLuc, pCLuc4, pSV2Luc) were complexed in water at various +/- charge ratios using cationic lipids (Lipofectin, DOTAP, DOGS), polyethylene imines (25 and 750 kDa), and with degraded 6th generation starburst polyamidoamine dendrimers. Luciferase was quantified using a luminometric assay and beta galactosidase with X-gal staining. Toxicities of transfections were evaluated with the MTT-assay. Using beta galactosidase as the reporter gene naked DNA did not transfect RPE cells at measurable levels whereas 1-5% of the cells expressed histochemically detectable amounts of the gene after transfection with cationic lipid DNA complexes. In RPE cells, Rous sarcoma virus and cytomegalovirus (CMV) were more efficient promoters than SV40 in driving luciferase expression, and CMV was chosen for further experiments. At optimal complex charge ratios, expression levels of luciferase were > 10(9) light units/mg protein after transfection using dendrimers and PEI25, while transfection mediated with the other carriers resulted in luciferase expression levels of 10(7)-10(9) light units/mg protein or less. In general, dendrimers and large molecular weight PEI were less toxic than cationic lipids or PEI25 to RPE cells. Serum and ECM decreased gene expression to the RPE cells with all carriers. Despite low percentage of transfected cells the transgene expression per RPE cell is high, important feature in the retinal tissue with small dimensions, in particular in the case of secreted gene products. Degraded dendrimers and high molecular weight PEI exhibited the best combination of high activity and low toxicity in RPE cell transfection.
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PMID:Gene delivery and expression in human retinal pigment epithelial cells: effects of synthetic carriers, serum, extracellular matrix and viral promoters. 1075 12

Anti-sense oligonucleotides are potential therapeutic agents that are used to block protein expression from mRNA. To assess the essential properties for an efficient cellular delivery system of phosphorothioate oligonucleotides (PS-ODNs), different cationic carriers were compared. The carriers were complexed with oligonucleotides at various +/- charge ratios in MES-Hepes buffer. Cationic polymers, polylysines (PLL, mean MWs 4000, 20000, 200000 kDa), polyethyleneimines (PEI, mean MWs 25 and 800 kDa) and fractured sixth-generation polyamidoamine dendrimer (PAMAM) were tested for ODN delivery into a D 407 cell line (human retinal pigment epithelial cells) with stably transfected luciferase gene. Anti-sense ODN was directed against the luciferase gene, and the anti-sense effect was determined using a luminometric method. Lipid-based vehicles included DOTAP, DOTAP/DOPE (1/1 by mol), DOTAP/Chol (1/1 by mol), DOTAP/DOPE/Chol (2/1/1 by mol), DOGS and Cytofectin GS/DOPE (2/1 by mol). Additionally a membrane-active peptide JTS-1 (NH(2) -GLFEALLELLESLWELLLEA-COOH) was added to the complexes containing DOTAP, PEI or PLL. In D 407 and CV-1 cells, the anti-sense effect was seen only with lipid-based carriers with a membrane-active component (DOPE or JTS-1). The polymeric systems were ineffective. The effect of the complexation medium was further studied on CV-1 cells. Complexes were prepared in either water, MES-Hepes buffer or cell growth medium (DMEM). Complexes prepared in water were generally most effective and the greater activity is probably due to the smaller complex size. Complex sizes differed greatly in buffer and DMEM, especially in the case of DOPE containing complexes. In conclusion, lipid carrier with a membrane active component and small complex size are required for an efficient cellular delivery of phosphorothioate oligonucleotides.
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PMID:A lipid carrier with a membrane active component and a small complex size are required for efficient cellular delivery of anti-sense phosphorothioate oligonucleotides. 1076 96


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