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)

This article describes the production and characterization of cationic submicron particles constituted with Eudragit RS 100, plus different cationic surfactants, such as dioctadecyl-dimethyl-ammonium bromide (DDAB18) and diisobutyphenoxyethyl-dimethylbenzyl ammonium chloride (DEBDA), as a transport and delivery system for DNA/DNA and DNA/peptide nucleic acid (PNA) hybrids and PNA-DNA chimeras. Submicron particles could offer advantages over other delivery systems because they maintain unaltered physicochemical properties for long time periods, allowing long-term storage, and are suitable for industrial production. Submicron particles were characterized in terms of size, size distribution, morphology, and zeta potential. Moreover, the in vitro activity and ability of submicron particles to complex different types of nucleic acids were described. Finally, the ability of submicron particles to deliver functional genes to cells cultured in vitro was determined by a luciferase activity assay, demonstrating that submicron particles possess superior transfection efficiency with respect to commercially available, liposome-based transfection kits.
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PMID:Formulations for natural and peptide nucleic acids based on cationic polymeric submicron particles. 1846 54

The aim of our study was to assess the importance of the CXC chemokine and interleukin (IL)-8 in promoting the transition of prostate cancer (CaP) to the androgen-independent state. Stimulation of the androgen-dependent cell lines, LNCaP and 22Rv1, with exogenous recombinant human interleukin-8 (rh-IL-8) increased androgen receptor (AR) gene expression at the messenger RNA (mRNA) and protein level, assessed by quantitative polymerase chain reaction and immunoblotting, respectively. Using an androgen response element-luciferase construct, we demonstrated that rh-IL-8 treatment also resulted in increased AR transcriptional activity in both these cell lines, and a subsequent upregulation of prostate-specific antigen and cyclin-dependent kinase 2 mRNA transcript levels in LNCaP cells. Blockade of CXC chemokine receptor-2 signaling using a small molecule antagonist (AZ10397767) attenuated the IL-8-induced increases in AR expression and transcriptional activity. Furthermore, in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, coadministration of AZ10397767 reduced the viability of LNCaP and 22Rv1 cells exposed to bicalutamide. Our data show that IL-8 signaling increases AR expression and promotes ligand-independent activation of this receptor in two androgen-dependent cell lines, describing two mechanisms by which this chemokine may assist in promoting the transition of CaP to the androgen-independent state. In addition, our data show that IL-8-promoted regulation of the AR attenuates the effectiveness of the AR antagonist bicalutamide in reducing CaP cell viability.
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PMID:Interleukin-8 signaling promotes androgen-independent proliferation of prostate cancer cells via induction of androgen receptor expression and activation. 1848 23

This unit contains five protocols for assaying cell viability in vitro using primary neuronal cultures, including a novel method for use with transfected neurons. Three of the assays are based on the principle that cell death cascades alter membrane permeability. The lactate dehydrogenase (LDH) release assay measures the amount of the cytoplasmic enzyme released into the bathing medium, while the trypan blue and propidium iodide assays measure the ability of cells to exclude dye from their cytoplasm. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay measures the mitochondrial activity of viable cells by quantifying the conversion of the tetrazolium salt to its formazan product. Finally, the fifth assay details the measurement of luciferase expression as an indication of neuronal viability within a relatively small population of transfected neurons.
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PMID:Assessment of cell viability in primary neuronal cultures. 1863 99

Phenolic compounds are widely known for their roles as antioxidants and anti-inflammatory agents, as well as for their epidemiological association with reduced risks for certain types of diseases. In the present study, we used rabbit peripheral blood mononuclear cells (PBMCs) to evaluate possible artifacts that result from the reactivity of polyphenolics. We evaluated several common methods for cytotoxicity tests using nine polyphenolics, representing several major classes of tannins and their subunits. For three of those phenolics, we investigated whether or not the bioactivities of the phenolics were altered by spontaneous oxidation. Our study showed that many of the nine tested tannins interfered with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, which is commonly used to measure cell viability. A better method for determining cell viability is the luciferin/luciferase ATP assay, and using that method, we found that several tannins are toxic to PBMCs. We measured TNF-alpha production to assess possible anti-inflammatory activity, and found that only apigenin inhibited TNF-alpha production in LPS-stimulated cells (EC 50 1.0 microg/mL). The other polyphenolic compounds we tested either had no effect on TNF-alpha or increased its production. However, our data indicated that spontaneous oxidation altered the activity of phenolics, eliminating their toxicity. This study shows that the chemical reactivity of phenolics can significantly affect attempts to evaluate bioactivity in cultured cells and that particular attention should be paid to both methods for determining toxicity and to spontaneous oxidation of tannins during cell testing.
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PMID:Accurate assessment of the bioactivities of redox-active polyphenolics in cell culture. 1869 Jun 89

ABSTRACT Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove specific purine residues from the sarcin/ricin (S/R) loop of the large rRNA and arrest protein synthesis at the translocation step. In addition to their enzymatic activity, RIPs have been reputed to be potent antiviral agents against many plant, animal, and human viruses. We recently showed that pokeweed antiviral protein (PAP), an RIP from pokeweed, inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNAs and depurinating these RNAs at multiple sites downstream of the cap structure. In this study, we examined the activity of three different RIPs against capped and uncapped viral RNAs. PAP, Mirabilis expansa RIP (ME1), and the Saponaria officinalis RIP (saporin) depurinated the capped Tobacco mosaic virus and Brome mosaic virus RNAs, but did not depurinate the uncapped luciferase RNA, indicating that other type I RIPs besides PAP can distinguish between capped and uncapped RNAs. We did not detect depurination of Alfalfa mosaic virus (AMV) RNAs at multiple sites by PAP or ME1. Because AMV RNAs are capped, these results indicate that recognition of the cap structure alone is not sufficient for depurination of the RNA at multiple sites throughout its sequence. Furthermore, PAP did not cause detectable depurination of uncapped RNAs from Tomato bushy stunt virus (TBSV), Satellite panicum mosaic virus (SPMV), and uncapped RNA containing poliovirus internal ribosome entry site (IRES). However, in vitro translation experiments showed that PAP inhibited translation of AMV, TBSV, SPMV RNAs, and poliovirus IRES dependent translation. These results demonstrate that PAP does not depurinate every capped RNA and that PAP can inhibit translation of uncapped viral RNAs in vitro without causing detectable depurination at multiple sites. Thus, the cap structure is not the only determinant for inhibition of translation by PAP.
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PMID:Translation Inhibition of Capped and Uncapped Viral RNAs Mediated by Ribosome-Inactivating Proteins. 1894 81

To characterize the effects of cetyltriethylammonium bromide (CTAB) on the transcription of gp64 promoter from Bombyx mori nucleopolyhedrovirus (BmNPV), the plasmid pBmgp64Luc used in transient expression assay system was constructed by using the luciferase gene as a reporter under the control of BmNPV gp64 promoter. When the Bombyx mori cells (Bm-N) were transfected with the pBmgp64Luc, different treatments were undertaken. We found that the transient expression activity of luciferase could not be augmented directly by CTAB treatment alone, but could be enhanced more than 2 times by BmNPV treatment alone at a multiplicity of infection (MOI) of 0.5. Through co-treatment with 0.1 microg ml(-1) of CTAB and BmNPV at a MOI of 0.5, the enzymatic activity increased 5.21 times. We presumed that the stimulation of transcription of BmNPV gp64 promoter by CTAB was mediated by viral factors from BmNPV. In addition, the time curves of luciferase activity in cells transfected with pBmgp64Luc and transactivated by virus were observed.
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PMID:Cetyltriethylammonium bromide stimulating transcription of Bombyx mori nucleopolyhedrovirus gp64 gene promoter mediated by viral factors. 1900 60

An experimental study was undertaken to quantify the effects of cetyltriethylammonium bromide (CTAB) on the replication of Bombyx mori nucleopolyhedrovirus (BmNPV) and the transcriptionalactivity of BmNPV ie-1 promoter. The results demonstrated that the budded virus (BV) titer rose about 3.7-fold by adding CTAB to the culture media up to 0.1 mu g ml(-1) in infected Bm-N cells with a wild-type BmNPV. The transient expression level of luciferase driven by BmNPV ie-1 promoter was enhanced by more than 3-fold in the presence of 0.1 mu g ml(-1) of CTAB in uninfected insect cells via a transient expression system. Contrary to the rise in BV titer, the polyhedra inside the nucleus of infected cells dropped linearly from 4.0 x 10(6) ml(-1) down to 2.1 x 10(6) ml(-1) with in a range of CTAB concentrations from 0 to 0.25 mu g ml(-1). The same trend in expression level of beta -galactosidase or phytase was given when the Bm-N cells or fifth-instar silkworm larvae infected with a recombinant BmNPV containing the beta -galactosidase or phytase reporter gene driven by the polyhedrin promoter. We deduced that CTAB appeared to affect the virus bi-phasic life cycle stages and production pathways, resulting in an enhancement in BV production and a suppression of occluded virus (OV) production and expression of foreign genes controlled by the polyhedrin promoter.
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PMID:Effects of cetyltriethylammonium bromide on the replication of Bombyx mori nucleopolyhedrovirus. 1900 1

The luciferase gene expression of lipoplexes, a liposome containing luciferase plasmid (pCMVLuc), in HeLa cell lines, was investigated. Cationic liposomes were prepared by the chloroform film method with sonication. The lipoplex was formed by loading the liposome with pCMVLuc. The lipoplex with an optimal weight ratio of dimethyl dioctadecyl ammonium bromide (DDAB)/pCMVLuc protected from DNaseI was determined by an agarose gel electrophoresis. The selected lipoplexes were assayed for luciferaase activity by using a luminometer. The effect on cell proliferation was evaluated by WST-1 assay. The highest luciferase activity of 1.5 x 10(6) RLU was observed in the cholesterol (Chol)/DDAB (2:1 molar ratio) lipoplex at the DDAB/pCMVLuc weight ratio of 10:1 at 48 hours, which was about 10, 100, and 1,000 times higher than the DDAB, L-alpha-dipalmitoyl phosphatidylcholine (DPPC)/Chol/DDAB (1:2:1 molar ratio), and DPPC/Chol/DDAB (2:2:1 molar ratio) lipoplexes, respectively. The liposome with the smallest particle size was obtained from the cationic liposome composed of DPPC/Chol/DDAB (7:1:1 molar ratio) with the zeta potential of 7.17 +/- 0.73. The optimal weight ratio of DDAB/pCMVLuc that protected pCMVLuc from DNaseI digestion was 4:1 in the DDAB formulation. The Chol/DDAB (2:1 molar ratio) lipoplex with the DDAB/pCMVLuc of 10:1 showed the highest luciferase activity of 1.5 x 10(6) RLU and the highest cytotoxicity as well. DPPC/Chol/DDAB (1:1:1 molar ratio)-lipoplex (DDAB/pCMVLuc = 14:1), which had the amount of DPPC and cholesterol not exceeding 33 and 50% mol, respectively, gave the lower gene expression of about 4 times, but lower cytoxicity of about 14 times, than the Chol/DDAB lipoplex (2:1 molar ratio) and was considered to be the most suitable formulation. The results from this study can be applied as a model for the development of a gene-therapeutic dosage form.
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PMID:Expression of luciferase plasmid (pCMVLuc) entrapped in DPPC/cholesterol/DDAB liposomes in HeLa cell lines. 1951

This paper reviews different techniques for analyzing the transfection efficiencies and cytotoxicities of dendriplexes-complexes of nucleic acids with dendrimers. Analysis shows that three plasmids are mainly used in transfection experiments: plasmid DNA encoding luciferase from the firefly Photinus pyralis, beta-galactosidase, or green fluorescent protein. The effective charge ratio of transfection does not directly correlate with the charge ratio obtained from gel electrophoresis, zeta-potential or ethidium bromide intercalation data. The most popular cells for transfection studies are human embryonic kidney cells (HEK293), mouse embryonic cells (NIH/3T3), SV40 transformed monkey kidney fibroblasts (COS-7) and human epithelioid cervical carcinoma cells (HeLa). Cellular uptake is estimated using fluorescently-labeled dendrimers or nucleic acids. Transfection efficiency is measured by the luciferase reporter assay for luciferase, X-Gal staining or beta-galactosidase assay for beta-galactosidase, and confocal microscopy for green fluorescent protein. Cytotoxicity is determined by the MTT test and lactate dehydrogenase assays. On the basis of the papers reviewed, a standard essential set of techniques for characterizing dendriplexes was constructed: (1) analysis of size and shape of dendriplexes in dried/frozen state by electron or atomic force microscopy; (2) analysis of charge/molar ratio of complexes by gel electrophoresis or ethidium bromide intercalation assay or zeta-potential measurement; (3) analysis of hydrodynamic diameter of dendriplexes in solution by dynamic light scattering. For the evaluation of transfection efficiency the essential techniques are (4) luciferase reporter assay, beta-galactosidase assay or green fluorescent protein microscopy, and (5) cytotoxicity by the MTT test. All these tests allow the transfection efficiencies and cytotoxicities of different kinds of dendrimers to be compared.
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PMID:How to study dendriplexes II: Transfection and cytotoxicity. 1981 39

The luminescence of Cypridina luciferin-luciferase solution is inhibited by illumination from a carbon arc of 15,000 foot candles in between 1 and 2 seconds. The blue to violet rays are the effective ones, the limits lying somewhere around 4,600 A. u. to 3,800 A. u. The luciferin, not the luciferase, is the substance affected by the light. The effect is partially reversible in the dark. The chemiluminescences obtained by oxidizing phosphorus, lophin, and chlorphenylmagnesium bromide are not inhibited by light under the above conditions.
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PMID:THE INHIBITION OF CYPRIDINA LUMINESCENCE BY LIGHT. 1987 69


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