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)

Alpha-synuclein is a presynaptic protein which is implicated in some neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, multiple systems atrophy, and Hallervorden-Spatz disease, and its overexpression contributes to the loss of dopaminergic neurons. Although the role of alpha-synuclein in these paradigms has been widely documented, its exact function is still elusive. And the dysfunction of the transcription factor nuclear factor (NF-kappaB) also exists in many neurodegenerative diseases. In this reason the purpose of this study was to investigate the molecular mechanism of alpha-synuclein's toxicity and its association with NF-kappaB by MTT assay, Western blot method, and luciferase assay. Results showed that overexpressed alpha-synuclein and 1-methyl-4-phenylpyridinium (MPP(+)) suppressed the SH-SY5Y cell viability and attenuate NF-kappaB-mediated luciferase expression significantly. Moreover, the impairment function was enhanced with the increase of alpha-synuclein protein level. We also found that overexpressed alpha-synuclein localized both in the cytoplasms and nuclei, down-regulated the anti-apoptotic Bcl-2 expression and up-regulated the pro-apoptotic glycogen synthase kinase 3beta (GSK3beta) protein level. In conclusion, all these findings mentioned above suggested that alpha-synuclein shared some toxic functional homology with neurotoxin MPP(+), and the proapoptotic effects of alpha-synuclein might be mediated at least in part by the impairment of NF-kappaB signaling pathway which involves GSK3beta.
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PMID:Overexpressed alpha-synuclein regulated the nuclear factor-kappaB signal pathway. 1771 23

The development of functional profiling technologies provides opportunity for high-throughput functional genomics studies. We describe a cell-based screening system to identify novel human genes associated with cell proliferation. The method integrates luciferase reporter gene activity, fluorescence stain, automated microscopy and cellular phenotype assays. We successfully used the system to screen 409 novel human genes cloned by our lab and found that 27 genes significantly up-regulated promoter-Renilla luciferase reporter plasmid (pRL) activity. Among them, five genes, TRAF3IP3, ZNF306, ZNF250, SGOL1, and ZNF434, were determined through morphological observation, calcein AM fluorescence stain, MTT assay and cell cycle analysis to be associated with cell proliferation. Furthermore, we showed that the gene TRAF3IP3, which initially was identified to specifically interact with TRAF3, stimulated cell growth by modulating the c-Jun N-terminal kinase (JNK) pathway, and RNAi of TRAF3IP3 confirmed that the effect was physiological and necessary. In summary, we integrated a rapid and efficient system for screening novel growth regulatory genes. Using the new screening system we identified five genes associated with cell proliferation for the first time.
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PMID:Identification of five human novel genes associated with cell proliferation by cell-based screening from an expressed cDNA ORF library. 1786 42

beta-Catenin, the chief oncogenic component of the canonical Wnt pathway, is known to be involved in a variety of cancers, including hepatocellular carcinoma (HCC). Although the mechanism of beta-catenin activation in HCC is multifactorial, it is indisputably implicated at various stages of hepatocarcinogenesis, making it an attractive therapeutic target. Here we investigate the effect of small interfering RNA-mediated beta-catenin knockdown on the growth and survival of human hepatoma cell lines with (HepG2) and without (Hep3B) beta-catenin mutations. Transfection of HepG2 and Hep3B cells with human beta-catenin (CTNNB1) small interfering RNA resulted in a significant beta-catenin decrease, as confirmed by Western blot analyses and immunofluorescence, also leading to decreased expression of known target genes such as cyclin D1 and glutamine synthetase. The decrease in beta-catenin activity was confirmed by TOPflash reporter luciferase assay. The functional impact of diminished beta-catenin was exhibited as temporal decrease in tumor cell viability by the MTT assay. A concomitant decrease in tumor cell proliferation was also evident with [(3)H]thymidine incorporation and verified with soft agar assays. Thus, beta-catenin is essential for the survival and growth of hepatoma cells independent of mutations in the beta-catenin gene and provide a proof of principle for the significance of the therapeutic inhibition of beta-catenin in HCC.
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PMID:siRNA-mediated beta-catenin knockdown in human hepatoma cells results in decreased growth and survival. 1803 Mar 63

Nicotinic acetylcholine receptors (nAChR) are expressed on normal bronchial epithelial and nonsmall cell lung cancer (NSCLC) cells and are involved in cell growth regulation. Nicotine induced cell proliferation. The purpose of this study was to determine if interruption of autocrine nicotinic cholinergic signaling might inhibit A549 NSCLC cell growth. For this purpose alpha-Cobratoxin (alpha-CbT), a high affinity alpha7-nAChR antagonist was studied. Cell growth decrease was evaluated by Clonogenic and MTT assays. Evidence of apoptosis was identified staining cell with Annexin-V/PI. Characterization of the basal NF-kappaB activity was done using the Trans-AM NF-kappaB assay colorimetric kit. "In vivo" antitumour activity was evaluated in orthotopically transplanted nude mice monitored by In vivo Imaging System technology. alpha-CbT caused concentration-dependent cell growth decrease, mitochondrial apoptosis caspases-9 and 3-dependent, but caspase-2 and p53-independent and down-regulation of basal high levels of activated NF-kappaB. alpha-CbT treatment determines a significant reduction of tumor growth in nude mice orthotopically engrafted with A549-luciferase cells (4.6% of living cells vs. 31% in untreated mice). No sign of toxicity was reported related to treatment. These findings suggest that alpha7-nAChR antagonists namely alpha-CbT may be useful adjuvant for treatment of NSCLC and potentially other cancers.
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PMID:Natural agents targeting the alpha7-nicotinic-receptor in NSCLC: a promising prospective in anti-cancer drug development. 1806 32

Novel biodegradable poly(disulfide amine)s with defined structure, high transfection efficiency, and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. Michael addition between N, N'-cystaminebisacrylamide (CBA) and three N-Boc protected diamines ( N-Boc-1,2-diaminoethane, N-Boc-1,4-diaminobutane, and N-Boc-1,6-diaminohexane) followed by N-Boc deprotection under acidic condition resulted in final cationic polymers with disulfide bonds, tertiary amine groups in main chains, and pendant primary amine groups in side chains. Polymer structures were confirmed by 1H NMR, and their molecular weights were in the range 3.3-4.7 kDa with narrow polydispersity (1.12-1.17) as determined by size exclusion chromatography (SEC). Acid-base titration assay showed that the poly(disulfide amine)s possessed superior buffering capacity to branched PEI 25 kDa in the pH range 7.4-5.1, which may facilitate the escape of DNA from the endosomal compartment. Gel retardation assay demonstrated that significant polyplex dissociation was observed in the presence of 5.0 mM DTT within 1 h, suggesting rapid DNA release in the reduction condition such as cytoplasm due to the cleavage of disulfide bonds. Genetic transfections mediated by these poly(disulfide amine)s were side-chain spacer length dependent. The poly(disulfide amine) with a hexaethylene spacer, poly(CBA-DAH), had comparable transfection efficiency to bPEI 25 kDa in the tested cell lines, i.e., 293T cells, Hela cells, and NIH3T3 cells. This same poly(disulfide amine) mediated 7-fold higher luciferase expression than bPEI 25 kDa in C2C12 cells (mouse myoblast cell line), a cell line difficult to transfect with many cationic polymers. Furthermore, MTT assay indicated that all three poly(disulfide amine)s/pDNA polyplexes were significantly less toxic than bPEI/pDNA complexes.
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PMID:Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity. 1831 39

Herein, a novel series of multivalent polycationic beta-cyclodextrin "click clusters" with discrete molecular weight have been synthesized, characterized, and examined as therapeutic pDNA carriers. The materials were creatively designed based on a beta-cyclodextrin core to impart a biocompatible multivalent architecture and oligoethyleneamine arms to facilitate pDNA binding, encapsulation, and cellular uptake. An acetylated-per-azido-beta-cyclodextrin (4) was reacted with series of alkyne dendrons (7a-e) (containing one to five ethyleneamine units) using copper-catalyzed 1,3-dipolar cycloaddition, to form a series of click clusters (9a-e) bearing 1,2,3-triazole linkers. Gel electrophoresis experiments, dynamic light scattering, and transmission electron microscopy revealed that the macromolecules bind and compact pDNA into spherical nanoparticles in the size range of 80-130 nm. The polycations protect pDNA against nuclease degradation, where structures 9c, 9d, and 9e did not allow pDNA degradation in the presence of serum for up to 48 h. The cellular uptake profiles were evaluated in Opti-MEM and demonstrate that all the click clusters efficiently deliver Cy5-labeled pDNA into HeLa and H9c2 (2-1) cells, and compounds 9d and 9e yielded efficacy similar to that of the positive controls, Jet-PEI and Superfect. Furthermore, the luciferase gene delivery experiments revealed that the level of reporter gene expression increased with an increase in oligoethyleneamine number within the cluster arms. The cytotoxicity profiles of these materials were evaluated by protein, MTT, and LDH assays, which demonstrate that all the click clusters remain nontoxic within the expected dosage range while the positive controls, Jet PEI and Superfect, were highly cytotoxic. In particular, 9d and 9e were the most effective and promising polycationic vehicles to be further optimized for future systemic delivery experiments.
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PMID:Polycationic beta-cyclodextrin "click clusters": monodisperse and versatile scaffolds for nucleic acid delivery. 1833 83

To analyze the function of kir3dl1 core promoter and study the possible regulation mechanism of kir3dl1 gene expression, a kir3dl1 promoter-luciferase reporter vector was constructed and the promoter activity was evaluated in the K562 cell line. A core promoter fragment of the kir3dl1 5'-untranslated region was amplified by PCR. PCR products were cloned into BglII/NcoI-digested pGL3-basic reporter vector; the polycationic compound SuperFect-reporter vector complexes were transferred into K562 cells. The Dual-Luciferase Reporter Assay System was used to quantitate the reporter vector luciferase activity. MTT method was used to measure the influence of SuperFect-DNA complexes on the survival rate of K562 cells. The results indicated that a 254-bp promoter fragment of kir3dl1 gene was successfully constructed and cloned into the pGL3-basic reporter vector, which was authenticated by BglII/NcoI digestion and DNA sequencing. The luciferase activity of the minimal promoter construct was significantly higher than that of the pGL3-Basic promoter in K562 cells. Transiently transfected cells presented continuously optimal luciferase activity and relative luciferase activity up to 3 days. The cell activity was between 76% and 92%. It is concluded that a kir3dl1 promoter-luciferase reporter vector is successfully constructed, the transfection system used in this study can effectively transfer gene in K562 cells. The kir3dl1 core promoter possesses higher activity in K562 cells, and can promote significantly expression of luciferase reporter gene in K562 cells.
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PMID:[Construction of kir3dl1 promoter expression vector and its activity in K562 cell line]. 1842 44

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

Supplemental oxygen, used to treat pulmonary insufficiency in newborns, contributes to the development of bronchopulmonary dysplasia (BPD). Cytochrome P4501A enzymes are induced by hyperoxia in animal models, but their role in human systems is unknown. Here we investigated the molecular mechanisms of induction of CYP1A1 by hyperoxia in human lung cell lines. Three human lung cell lines were exposed to hyperoxia (95% O2) for 0-72 h, and CYP1A1 activities, apoprotein contents, and mRNA levels were determined. Hyperoxia significantly induced CYP1A1 activity and protein contents (2-4 fold), and mRNA levels (30-40 fold) over control in each cell line. Transfection of a CYP1A1 promoter/luciferase reporter construct, followed by hyperoxia (4-72 h), showed marked (2-6 fold) induction of luciferase expression. EMSA and siRNA experiments strongly suggest that the Ah receptor (AHR) is involved in the hyperoxic induction of CYP1A1. MTT reduction assays showed attenuation of cell injury with the CYP1A1 inducer beta-naphthoflavone (BNF). Our results strongly suggest that hyperoxia transcriptionally activates CYP1A1 expression in human lung cell lines by AHR-dependent mechanisms, and that CYP1A1 induction is associated with decreased toxicity. This novel finding of induction of CYP1A1 in the absence of exogenous AHR ligands could lead to novel interventions in the treatment of BPD.
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PMID:Regulation of cytochrome P4501A1 expression by hyperoxia in human lung cell lines: Implications for hyperoxic lung injury. 1882 9


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