Tibetan Firefly Luciferase with Low Temperature Adaptation.

Science.gov (United States)

Mitani, Yasuo; Futahashi, Ryo; Liu, Zichao; Liang, Xingcai; Ohmiya, Yoshihiro

2017-03-01

Fireflies are widespread all over the world and a numerous numbers of luciferases have been isolated and characterized. In this study, we identified and characterized the luciferase and luciferase-like genes from a Tibetan firefly collected in Shangri-La, China. The altitude of this area is more than 3300 m. We saw this Tibetan firefly flying with strong luminescence after sunset at ~10°C. We analyzed the transcriptome of Tibetan firefly using head, thorax, abdomen (without light organ), and light organ tissue by RNA sequencing. We identified one luciferase gene, which was almost identical to luciferase from fireflies Pyrocoelia species, and expressed specifically in the light organ. Interestingly, the optimal temperature of the Tibetan firefly recombinant luciferase was 10°C. The K m for D-luciferin and ATP of the recombinant luciferase was 23 and 154 μm, respectively. The optimal pH was around 7.0-7.5. The emission peak was 556 nm at pH 8.0, while it shifted to 606 nm at pH 6.0. We also found a luciferase-like gene with 43% identical amino acids to the Tibetan firefly luciferase, which was scarcely expressed in any portion of the adult body. No luciferase activity was detected for this luciferase-like protein. © 2016 The American Society of Photobiology.

Development of a dual luciferase activity and fluorescamine protein assay adapted to a 384 micro-well plate format: Reducing variability in human luciferase transactivation cell lines aimed at endocrine active substances

Science.gov (United States)

Brennan, Jennifer; Tillitt, Donald E.

2018-01-01

There is a need to adapt cell bioassays to 384-well and 1536-well formats instead of the traditional 96-well format as high-throughput screening (HTS) demands increase. However, the sensitivity and performance of the bioassay must be re-verified in these higher micro-well plates, and verification of cell health must also be HT (high-throughput). We have adapted two commonly used human breast luciferase transactivation cell bioassays, the recently re-named estrogen agonist/antagonist screening VM7Luc4E2 cell bioassay (previously designated BG1Luc4E2) and the androgen/glucocorticoid screening MDA-kb2 cell bioassay, to 384-well formats for HTS of endocrine-active substances (EASs). This cost-saving adaptation includes a fast, accurate, and easy measurement of protein amount in each well via the fluorescamine assay with which to normalize luciferase activity of cell lysates without requiring any transfer of the cell lysates. Here we demonstrate that by accounting for protein amount in the cell lysates, antagonistic agents can easily be distinguished from cytotoxic agents in the MDA-kb2 and VM7Luc4E2 cell bioassays. Additionally, we demonstrate via the fluorescamine assay improved interpretation of luciferase activity in wells along the edge of the plate (the so-called “edge effect”), thereby increasing usable wells to the entire plate, not just interior wells.

Two panels of steroid receptor luciferase reporter cell lines for compound profiling

Czech Academy of Sciences Publication Activity Database

Sedlák, David; Paguio, A.; Bartůněk, Petr

2011-01-01

Roč. 14, č. 2 (2011), s. 248-266 ISSN 1386-2073 R&D Projects: GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z50520514 Keywords : nuclear hormone receptor * steroid receptor * cell-based luciferase reporter assay Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.785, year: 2011

Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase

Energy Technology Data Exchange (ETDEWEB)

Yoshida, Wataru; Kezuka, Aki; Murakami, Yoshiyuki; Lee, Jinhee; Abe, Koichi [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588 (Japan); Motoki, Hiroaki; Matsuo, Takafumi; Shimura, Nobuaki [System Instruments Co., Ltd., 776-2 Komiya-cho, Hachioji, Tokyo 192-0031 (Japan); Noda, Mamoru; Igimi, Shizunobu [Division of Biomedical Food Research, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Ikebukuro, Kazunori, E-mail: ikebu@cc.tuat.ac.jp [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588 (Japan)

2013-11-01

Graphical abstract: -- Highlights: •Zif268 fused to luciferase was used for E. coli O157, Salmonella and coliform detection. •Artificial zinc finger protein fused to luciferase was constructed for Norovirus detection. •An analyzer that automatically detects PCR products by zinc finger protein fused to luciferase was developed. •Target pathogens were specifically detected by the automatic analyzer with zinc finger protein fused to luciferase. -- Abstract: An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268–luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF–luciferase fusion protein. By means of the automatic analyzer with ZF–luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0 × 10 to 1.0 × 10{sup 6} copies.

Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase

International Nuclear Information System (INIS)

Yoshida, Wataru; Kezuka, Aki; Murakami, Yoshiyuki; Lee, Jinhee; Abe, Koichi; Motoki, Hiroaki; Matsuo, Takafumi; Shimura, Nobuaki; Noda, Mamoru; Igimi, Shizunobu; Ikebukuro, Kazunori

2013-01-01

Graphical abstract: -- Highlights: •Zif268 fused to luciferase was used for E. coli O157, Salmonella and coliform detection. •Artificial zinc finger protein fused to luciferase was constructed for Norovirus detection. •An analyzer that automatically detects PCR products by zinc finger protein fused to luciferase was developed. •Target pathogens were specifically detected by the automatic analyzer with zinc finger protein fused to luciferase. -- Abstract: An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268–luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF–luciferase fusion protein. By means of the automatic analyzer with ZF–luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0 × 10 to 1.0 × 10 6 copies

Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.

Science.gov (United States)

Masser, Anna E; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan; Andréasson, Claes

2016-05-01

Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd.

Use of luciferase probes to measure ATP in living cells and animals.

Science.gov (United States)

Morciano, Giampaolo; Sarti, Alba Clara; Marchi, Saverio; Missiroli, Sonia; Falzoni, Simonetta; Raffaghello, Lizzia; Pistoia, Vito; Giorgi, Carlotta; Di Virgilio, Francesco; Pinton, Paolo

2017-08-01

ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.

Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

International Nuclear Information System (INIS)

Wang Lei; Sasai, Ken; Akagi, Tsuyoshi; Tanaka, Shinya

2008-01-01

The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development

Third-generation Ah receptor-responsive luciferase reporter plasmids: amplification of dioxin-responsive elements dramatically increases CALUX bioassay sensitivity and responsiveness.

Science.gov (United States)

He, Guochun; Tsutsumi, Tomoaki; Zhao, Bin; Baston, David S; Zhao, Jing; Heath-Pagliuso, Sharon; Denison, Michael S

2011-10-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like chemicals are widespread and persistent environmental contaminants that produce diverse toxic and biological effects through their ability to bind to and activate the Ah receptor (AhR) and AhR-dependent gene expression. The chemically activated luciferase expression (CALUX) system is an AhR-responsive recombinant luciferase reporter gene-based cell bioassay that has been used in combination with chemical extraction and cleanup methods for the relatively rapid and inexpensive detection and relative quantitation of dioxin and dioxin-like chemicals in a wide variety of sample matrices. Although the CALUX bioassay has been validated and used extensively for screening purposes, it has some limitations when screening samples with very low levels of dioxin-like chemicals or when there is only a small amount of sample matrix for analysis. Here, we describe the development of third-generation (G3) CALUX plasmids with increased numbers of dioxin-responsive elements, and stable transfection of these new plasmids into mouse hepatoma (Hepa1c1c7) cells has produced novel amplified G3 CALUX cell bioassays that respond to TCDD with a dramatically increased magnitude of luciferase induction and significantly lower minimal detection limit than existing CALUX-type cell lines. The new G3 CALUX cell lines provide a highly responsive and sensitive bioassay system for the detection and relative quantitation of very low levels of dioxin-like chemicals in sample extracts.

Third-Generation Ah Receptor–Responsive Luciferase Reporter Plasmids: Amplification of Dioxin-Responsive Elements Dramatically Increases CALUX Bioassay Sensitivity and Responsiveness

Science.gov (United States)

He, Guochun; Tsutsumi, Tomoaki; Zhao, Bin; Baston, David S.; Zhao, Jing; Heath-Pagliuso, Sharon; Denison, Michael S.

2011-01-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like chemicals are widespread and persistent environmental contaminants that produce diverse toxic and biological effects through their ability to bind to and activate the Ah receptor (AhR) and AhR-dependent gene expression. The chemically activated luciferase expression (CALUX) system is an AhR-responsive recombinant luciferase reporter gene–based cell bioassay that has been used in combination with chemical extraction and cleanup methods for the relatively rapid and inexpensive detection and relative quantitation of dioxin and dioxin-like chemicals in a wide variety of sample matrices. Although the CALUX bioassay has been validated and used extensively for screening purposes, it has some limitations when screening samples with very low levels of dioxin-like chemicals or when there is only a small amount of sample matrix for analysis. Here, we describe the development of third-generation (G3) CALUX plasmids with increased numbers of dioxin-responsive elements, and stable transfection of these new plasmids into mouse hepatoma (Hepa1c1c7) cells has produced novel amplified G3 CALUX cell bioassays that respond to TCDD with a dramatically increased magnitude of luciferase induction and significantly lower minimal detection limit than existing CALUX-type cell lines. The new G3 CALUX cell lines provide a highly responsive and sensitive bioassay system for the detection and relative quantitation of very low levels of dioxin-like chemicals in sample extracts. PMID:21775728

Transgenic Mouse Model Harboring the Transcriptional Fusion Ccl20-Luciferase as a Novel Reporter of Pro-Inflammatory Response

Science.gov (United States)

Crispo, Martina; Van Maele, Laurye; Tabareau, Julien; Cayet, Delphine; Errea, Agustina; Ferreira, Ana María; Rumbo, Martin; Sirard, Jean Claude

2013-01-01

The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo. PMID:24265691

Cellular Immune Response Against Firefly Luciferase After Sleeping Beauty–Mediated Gene Transfer In Vivo

Science.gov (United States)

Podetz-Pedersen, Kelly M.; Vezys, Vaiva; Somia, Nikunj V.; Russell, Stephen J.

2014-01-01

Abstract The Sleeping Beauty (SB) transposon system has been shown to mediate new gene sequence integration resulting in long-term expression. Here the effectiveness of hyperactive SB100X transposase was tested, and we found that hydrodynamic co-delivery of a firefly luciferase transposon (pT2/CaL) along with SB100X transposase (pCMV-SB100X) resulted in remarkably sustained, high levels of luciferase expression. However, after 4 weeks there was a rapid, animal-by-animal loss of luciferase expression that was not observed in immunodeficient mice. We hypothesized that this sustained, high-level luciferase expression achieved using the SB100X transposase elicits an immune response in pT2/CaL co-administered mice, which was supported by the rapid loss of luciferase expression upon challenge of previously treated animals and in naive animals adoptively transferred with splenocytes from previously treated animals. Specificity of the immune response to luciferase was demonstrated by increased cytokine expression in splenocytes after exposure to luciferase peptide in parallel with MHC I–luciferase peptide tetramer binding. This anti-luciferase immune response observed following continuous, high-level luciferase expression in vivo clearly impacts its use as an in vivo reporter. As both an immunogen and an extremely sensitive reporter, luciferase is also a useful model system for the study of immune responses following in vivo gene transfer and expression. PMID:25093708

Luciferase-Specific Coelenterazine Analogues for Optical Contamination-Free Bioassays.

Science.gov (United States)

Nishihara, Ryo; Abe, Masahiro; Nishiyama, Shigeru; Citterio, Daniel; Suzuki, Koji; Kim, Sung Bae

2017-04-19

Spectral overlaps among the multiple optical readouts commonly cause optical contamination in fluorescence and bioluminescence. To tackle this issue, we created five-different lineages of coelenterazine (CTZ) analogues designed to selectively illuminate a specific luciferase with unique luciferase selectivity. In the attempt, we found that CTZ analogues with ethynyl or styryl groups display dramatically biased bioluminescence to specific luciferases and pHs by modifying the functional groups at the C-2 and C-6 positions of the imidazopyradinone backbone of CTZ. The optical contamination-free feature was exemplified with the luciferase-specific CTZ analogues, which illuminated anti-estrogenic and rapamycin activities in a mixture of optical probes. This unique bioluminescence platform has great potential for specific and high throughput imaging of multiple optical readouts in bioassays without optical contamination.

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

DEFF Research Database (Denmark)

Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper

2011-01-01

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized...... relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means...

A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the LD50 of polychlorinated biphenyls in avian species

International Nuclear Information System (INIS)

Manning, Gillian E.; Farmahin, Reza; Crump, Doug; Jones, Stephanie P.; Klein, Jeff; Konstantinov, Alex; Potter, Dave; Kennedy, Sean W.

2012-01-01

Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase reporter gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 reporter gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R 2 ≥ 0.87, p < 0.0001). Thus, the LRG assay in combination with the knowledge of a species' AHR1 LBD sequence can be used to predict PCB-induced embryolethality in potentially any avian species of interest without the use of lethal methods on a large number of individuals. -- Highlights: ► PCB embryolethality in birds can be predicted from a species' AHR1 genotype. ► The reporter gene assay is useful for predicting species sensitivity to PCBs. ► The relative potency of PCBs does not appear to differ between AHR1 genotypes. ► Contamination of PCB 105 and PCB 118 did not affect their relative

A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the LD{sub 50} of polychlorinated biphenyls in avian species

Energy Technology Data Exchange (ETDEWEB)

Manning, Gillian E., E-mail: gmann017@uottawa.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Farmahin, Reza, E-mail: mfarm070@uottawa.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Crump, Doug, E-mail: doug.crump@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Jones, Stephanie P., E-mail: stephanie.jones@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Klein, Jeff, E-mail: jeffery@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3M5 (Canada); Konstantinov, Alex, E-mail: alex@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3M5 (Canada); Potter, Dave, E-mail: dpotter@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3M5 (Canada); Kennedy, Sean W., E-mail: sean.kennedy@ec.gc.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada)

2012-09-15

Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase reporter gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 reporter gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R{sup 2} ≥ 0.87, p < 0.0001). Thus, the LRG assay in combination with the knowledge of a species' AHR1 LBD sequence can be used to predict PCB-induced embryolethality in potentially any avian species of interest without the use of lethal methods on a large number of individuals. -- Highlights: ► PCB embryolethality in birds can be predicted from a species' AHR1 genotype. ► The reporter gene assay is useful for predicting species sensitivity to PCBs. ► The relative potency of PCBs does not appear to differ between AHR1 genotypes. ► Contamination of PCB 105 and PCB 118 did not affect

Uptake kinetics and biodistribution of C-14-D-luciferin-a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction : impact on bioluminescence based reporter gene imaging

NARCIS (Netherlands)

Berger, Frank; Paulmurugan, Ramasamy; Bhaumik, Srabani; Gambhir, Sanjiv Sam

2008-01-01

Purpose Firefly luciferase catalyzes the oxidative decarboxylation of D-luciferin to oxyluciferin in the presence of cofactors, producing bioluminescence. This reaction is used in optical bioluminescence-based molecular imaging approaches to detect the expression of the firefly luciferase reporter

Luciferase-Zinc-Finger System for the Rapid Detection of Pathogenic Bacteria.

Science.gov (United States)

Shi, Chu; Xu, Qing; Ge, Yue; Jiang, Ling; Huang, He

2017-08-09

Rapid and reliable detection of pathogenic bacteria is crucial for food safety control. Here, we present a novel luciferase-zinc finger system for the detection of pathogens that offers rapid and specific profiling. The system, which uses a zinc-finger protein domain to probe zinc finger recognition sites, was designed to bind the amplified conserved regions of 16S rDNA, and the obtained products were detected using a modified luciferase. The luciferase-zinc finger system not only maintained luciferase activity but also allowed the specific detection of different bacterial species, with a sensitivity as low as 10 copies and a linear range from 10 to 10 4 copies per microliter of the specific PCR product. Moreover, the system is robust and rapid, enabling the simultaneous detection of 6 species of bacteria in artificially contaminated samples with excellent accuracy. Thus, we envision that our luciferase-zinc finger system will have far-reaching applications.

Excited-State Dynamics of Oxyluciferin in Firefly Luciferase

KAUST Repository

Snellenburg, Joris J.; Laptenok, Sergey P.; DeSa, Richard J.; Naumov, Pance; Solntsev, Kyril M.

2016-01-01

The color variations of light emitted by some natural and mutant luciferases are normally attributed to collective factors referred to as microenvironment effects; however, the exact nature of these interactions between the emitting molecule (oxyluciferin) and the active site remains elusive. Although model studies of noncomplexed oxyluciferin and its variants have greatly advanced the understanding of its photochemistry, extrapolation of the conclusions to the real system requires assumptions about the polarity and proticity of the active site. To decipher the intricate excited-state dynamics, global and target analysis is performed here for the first time on the steady-state and time-resolved spectra of firefly oxyluciferin complexed with luciferase from the Japanese firefly (Luciola cruciata). The experimental steady-state and time resolved luminescence spectra of the oxyluciferin/luciferase complex in solution are compared with the broadband time-resolved firefly bioluminescence recorded in vivo. The results demonstrate that de-excitation of the luminophore results in a complex cascade of photoinduced proton transfer processes and can be interpreted by the pH dependence of the emitted light. It is confirmed that proton transfer is the central event in the spectrochemistry of this system for which any assignment of the pH dependent emission to a single chemical species would be an oversimplification.

Excited-State Dynamics of Oxyluciferin in Firefly Luciferase

KAUST Repository

Snellenburg, Joris J.

2016-11-23

The color variations of light emitted by some natural and mutant luciferases are normally attributed to collective factors referred to as microenvironment effects; however, the exact nature of these interactions between the emitting molecule (oxyluciferin) and the active site remains elusive. Although model studies of noncomplexed oxyluciferin and its variants have greatly advanced the understanding of its photochemistry, extrapolation of the conclusions to the real system requires assumptions about the polarity and proticity of the active site. To decipher the intricate excited-state dynamics, global and target analysis is performed here for the first time on the steady-state and time-resolved spectra of firefly oxyluciferin complexed with luciferase from the Japanese firefly (Luciola cruciata). The experimental steady-state and time resolved luminescence spectra of the oxyluciferin/luciferase complex in solution are compared with the broadband time-resolved firefly bioluminescence recorded in vivo. The results demonstrate that de-excitation of the luminophore results in a complex cascade of photoinduced proton transfer processes and can be interpreted by the pH dependence of the emitted light. It is confirmed that proton transfer is the central event in the spectrochemistry of this system for which any assignment of the pH dependent emission to a single chemical species would be an oversimplification.

Characterization of CG6178 gene product with high sequence similarity to firefly luciferase in Drosophila melanogaster.

Science.gov (United States)

Oba, Yuichi; Ojika, Makoto; Inouye, Satoshi

2004-03-31

This is the first identification of a long-chain fatty acyl-CoA synthetase in Drosophila by enzymatic characterization. The gene product of CG6178 (CG6178) in Drosophila melanogaster genome, which has a high sequence similarity to firefly luciferase, has been expressed and characterized. CG6178 showed long-chain fatty acyl-CoA synthetic activity in the presence of ATP, CoA and Mg(2+), suggesting a fatty acyl adenylate is an intermediate. Recently, it was revealed that firefly luciferase has two catalytic functions, monooxygenase (luciferase) and AMP-mediated CoA ligase (fatty acyl-CoA synthetase). However, unlike firefly luciferase, CG6178 did not show luminescence activity in the presence of firefly luciferin, ATP, CoA and Mg(2+). The enzymatic properties of CG6178 including substrate specificity, pH dependency and optimal temperature were close to those of firefly luciferase and rat fatty acyl-CoA synthetase. Further, phylogenic analyses strongly suggest that the firefly luciferase gene may have evolved from a fatty acyl-CoA synthetase gene as a common ancestral gene.

The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells.

Science.gov (United States)

Markova, Svetlana V; Larionova, Marina D; Gorbunova, Darya A; Vysotski, Eugene S

2017-10-01

The bioluminescence of a marine copepod Metridia longa is determined by a small secreted coelenterazine-dependent luciferase that uses coelenterazine as a substrate of enzymatic reaction to generate light (λ max =480nm). To date, four different isoforms of the luciferase differing in size, sequences, and properties have been cloned by functional screening. All of them contain ten conserved Cys residues that suggests up to five SS intramolecular bonds per luciferase molecule. Whereas the use of copepod luciferases as bioluminescent reporters in biomedical research in vivo is growing from year to year, their application for in vitro assays is still limited by the difficulty in obtaining significant amounts of luciferase. The most cost-effective host for producing recombinant proteins is Escherichia coli. However, prokaryotic and eukaryotic cells maintain the reductive environment in cytoplasm that hinders the disulfide bond formation and consequently the proper folding of luciferase. Here we report the expression of the MLuc7 isoform of M. longa luciferase in E. coli cells and the efficient procedure for refolding from inclusion bodies yielding a high-active monomeric protein. Furthermore, in a set of identical experiments we demonstrate that bioluminescent and structural features of MLuc7 produced in bacterial cells are identical to those of MLuc7 isoform produced from culture medium of insect cells. Although the yield of high-purity protein is only 6mg/L, the application of E. coli cells to produce the luciferase is simpler and more cost-effective than the use of insect cells. We expect that the suggested technology of Metridia luciferase production allows obtaining of sufficient amounts of protein both for the development of novel in vitro analytical assays with the use of MLuc7 as a label and for structural studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity.

Directory of Open Access Journals (Sweden)

Dmitry Akhmedov

Full Text Available The cAMP response element binding protein (CREB is induced during fasting in the liver, where it stimulates transcription of rate-limiting gluconeogenic genes to maintain metabolic homeostasis. Adenoviral and transgenic CREB reporters have been used to monitor hepatic CREB activity non-invasively using bioluminescence reporter imaging. However, adenoviral vectors and randomly inserted transgenes have several limitations. To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc. cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Strikingly, CREB was markedly stimulated in liver, but not in skeletal muscle, after overnight voluntary wheel-running exercise, uncovering differential regulation of CREB in these tissues under catabolic states. The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

Combined image guided monitoring the pharmacokinetics of rapamycin loaded human serum albumin nanoparticles with a split luciferase reporter

Science.gov (United States)

Wang, Fu; Yang, Kai; Wang, Zhe; Ma, Ying; Gutkind, J. Silvio; Hida, Naoki; Niu, Gang; Tian, Jie

2016-02-01

Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery carriers upon administration in the blood circulation, which complicates the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction with near infrared (NIR) fluorophores to investigate the respective PK profiles of a drug and its carrier in a biodegradable drug delivery system. In this system, a prototype hydrophobic agent, rapamycin (Rapa), was encapsulated into human serum albumin (HSA) to form HSA Rapa nanoparticles, which were then labeled with Cy5 fluorophore to facilitate the fluorescence imaging of HSA carrier. Meanwhile, we employed transgenetic HN12 cells that were modified with a split luciferase reporter, whose bioluminescence function is regulated by Rapa, to reflect the PK profile of the encapsulated agent. It was interesting to discover that there existed an obvious inconsistency of PK behaviors between HSA carrier and rapamycin in vitro and in vivo through near infrared fluorescence imaging (NIFRI) and bioluminescence imaging (BLI) after treatment with Cy5 labeled HSA Rapa. Nevertheless, HSA Rapa nanoparticles manifested favorable in vivo PK and tumor suppression efficacy in a follow-up therapeutic study. The developed strategy of combining a molecular reporter and a fluorophore in this study could be extended to other drug delivery systems to provide profound insights for non-invasive real-time evaluation of PK profiles of drug-loaded nanoparticles in pre-clinical studies.Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery

Luciferase-Specific Coelenterazine Analogues for Optical Contamination-Free Bioassays

OpenAIRE

Ryo Nishihara; Masahiro Abe; Shigeru Nishiyama; Daniel Citterio; Koji Suzuki; Sung Bae Kim

2017-01-01

Spectral overlaps among the multiple optical readouts commonly cause optical contamination in fluorescence and bioluminescence. To tackle this issue, we created five-different lineages of coelenterazine (CTZ) analogues designed to selectively illuminate a specific luciferase with unique luciferase selectivity. In the attempt, we found that CTZ analogues with ethynyl or styryl groups display dramatically biased bioluminescence to specific luciferases and pHs by modifying the functional groups ...

Luciferase inactivation in the luminous marine bacterium Vibrio harveyi.

Science.gov (United States)

Reeve, C A; Baldwin, T O

1981-06-01

Luciferase was rapidly inactivated in stationary-phase cultures of the wild type of the luminous marine bacterium Vibrio harveyi, but was stable in stationary-phase cultures of mutants of V. harveyi that are nonluminous without exogenous aldehyde, termed the aldehyde-deficient mutants. The inactivation in the wild type was halted by cell lysis and was slowed or stopped by O2 deprivation or by addition of KCN and NaF or of chloramphenicol. If KCN and NaF or chloramphenicol were added to a culture before the onset of luciferase inactivation, then luciferase inactivation did not occur. However, if these inhibitors were added after the onset of luciferase inactivation, then luciferase inactivation continued for about 2 to 3 h before the inactivation process stopped. The onset of luciferase inactivation in early stationary-phase cultures of wild-type cell coincided with a slight drop in the intracellular adenosine 5'-triphosphate (ATP) level from a relatively constant log-phase value of 20 pmol of ATP per microgram of soluble cell protein. Addition of KCN and NaF to a culture shortly after this drop in ATP caused a rapid decrease in the ATP level to about 4 pmol of ATP per microgram whereas chloramphenicol added at this same time caused a transient increase in ATP level to about 25 pmol/microgram. The aldehyde-deficient mutant (M17) showed a relatively constant log-phase ATP level identical with that of the wild-type cells, but rather than decreasing in early stationary phase, the ATP level increased to a value twice that in log-phase cells. We suggest that the inactivation of luciferase is dependent on the synthesis of some factor which is produced during stationary phase and is itself unstable, and whose synthesis is blocked by chloramphenicol or cyanide plus fluoride.

Super RLuc8: A novel engineered Renilla luciferase with a red-shifted spectrum and stable light emission.

Science.gov (United States)

Rahnama, Somaieh; Saffar, Behnaz; Kahrani, Zahra Fanaei; Nazari, Mahboobeh; Emamzadeh, Rahman

2017-01-01

Renilla luciferase is a bioluminescent enzyme which is broadly used as a reporter protein in molecular biosensors. In this study, a novel luciferase with desired light emission wavelength and thermostability is reported. The results indicated that the new luciferase, namely super RLuc8, had a red-shifted spectrum and showed stable light emission. Super RLuc8 showed a 10-fold (p-value=0.0084) increase in the thermostability at 37°C after 20min incubation, in comparison to the native enzyme. The optimum temperature of the mutant increased from 30 to 37°C. Molecular dynamics simulation analysis indicated that the increased thermostability was most probably caused by a better structural compactness and more local rigidity in the regions out of the emitter site. Copyright © 2016 Elsevier Inc. All rights reserved.

Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling

Directory of Open Access Journals (Sweden)

Elisa Fanunza

2018-02-01

Full Text Available The interferon (IFN system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24 is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE. Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z′- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

In Vivo Determination of Vitamin D Function Using Transgenic Mice Carrying a Human Osteocalcin Luciferase Reporter Gene

Directory of Open Access Journals (Sweden)

Tomoko Nakanishi

2013-01-01

Full Text Available Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation. We established transgenic mouse line expressing luciferase under the control of a 10-kb osteocalcin enhancer/promoter sequence. Using these transgenic mice, we evaluated the active forms of vitamins D2 and D3 for their bone morphogenetic function by in vivo bioluminescence. As the result, strong activity for ossification was observed with 1α,25-hydroxyvitamin D3. Our mouse system can offer a feasible detection method for assessment of osteogenic activity in the development of functional foods and medicines by noninvasive screening.

A transgenic Plasmodium falciparum NF54 strain that expresses GFP-luciferase throughout the parasite life cycle.

Science.gov (United States)

Vaughan, Ashley M; Mikolajczak, Sebastian A; Camargo, Nelly; Lakshmanan, Viswanathan; Kennedy, Mark; Lindner, Scott E; Miller, Jessica L; Hume, Jen C C; Kappe, Stefan H I

2012-12-01

Plasmodium falciparum is the pathogenic agent of the most lethal of human malarias. Transgenic P. falciparum parasites expressing luciferase have been created to study drug interventions of both asexual and sexual blood stages but luciferase-expressing mosquito stage and liver stage parasites have not been created which has prevented the easy quantification of mosquito stage development (e.g. for transmission blocking interventions) and liver stage development (for interventions that prevent infection). To overcome this obstacle, we have created a transgenic P. falciparum NF54 parasite that expresses a GFP-luciferase transgene throughout the life cycle. Luciferase expression is robust and measurable at all life cycle stages, including midgut oocyst, salivary gland sporozoites and liver stages, where in vivo development is easily measurable using humanized mouse infections in conjunction with an in vivo imaging system. This parasite reporter strain will accelerate testing of interventions against pre-erythrocytic life cycle stages. Copyright © 2012 Elsevier B.V. All rights reserved.

Cloning and characterization of luciferase from a Fijian luminous click beetle.

Science.gov (United States)

Mitani, Yasuo; Futahashi, Ryo; Niwa, Kazuki; Ohba, Nobuyoshi; Ohmiya, Yoshihiro

2013-01-01

Luminous click beetle is distributed almost exclusively in Central and South America with a single genus in Melanesia. Among these click beetles, the description of Melanesian species has been fragmentary, and its luciferase gene and phylogenetic relation to other click beetles still remain uncertain. We collected a living luminous click beetle, Photophorus jansonii in Fiji. It emits green-yellow light from two spots on the pronotum and has no ventral luminous organ. Here, we cloned a luciferase gene from this insect by RT-PCR. The deduced amino acid sequence showed high identity of ~85% to the luciferases derived from other click beetle species. The luciferase of the Fijian click beetle was produced as a recombinant protein to characterize its biochemical properties. The Km for D-luciferin and ATP were 173 and 270 μm, respectively. The luciferase was pH-insensitive and the spectrum measured at pH 8.0 showed a peak at 559 nm, which was in the range of green-yellow light as seen in the luminous spot of the living Fijian click beetle. The Fijian click beetle luciferase was assigned to the Elateridae clade by a phylogenetic analysis, but it made a clearly different branch from Pyrophorus group examined in this study. © 2013 The American Society of Photobiology.

The location and nature of general anesthetic binding sites on the active conformation of firefly luciferase; a time resolved photolabeling study.

Directory of Open Access Journals (Sweden)

Sivananthaperumal Shanmugasundararaj

Full Text Available Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP-driven production of light. We have used time-resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [(3H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC-MSMS revealed a similar conformation-dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies.

A Multisampling Reporter System for Monitoring MicroRNA Activity in the Same Population of Cells

Directory of Open Access Journals (Sweden)

Pei-Chen Huang

2009-01-01

Full Text Available MicroRNAs (miRNAs downregulate gene expression by binding to the partially complementary sites in the 3′ untranslated region (UTR of target mRNAs. Several methods, such as Northern blot analysis, quantitative real-time RT-PCR, microarray, and the luciferase reporter system, are commonly used to quantify the relative level or activity of miRNAs. The disadvantage of these methods is the requirement for cell lysis, which means that several sets of wells/dishes of cells must be prepared to monitor changes in miRNA activity in time-course studies. In this study, we developed a multisampling reporter system in which two secretable bioluminescence-generating enzymes are employed, one as a reporter and the other as an internal control. The reporters consist of a pair of vectors containing the Metridia luciferase gene, one with and one without a duplicated miRNA targeting sequence at their 3′UTR, while the other vector coding for the secreted alkaline phosphatase gene is used as an internal control. This method allows miRNA activity to be monitored within the same population of cells over time by withdrawing aliquots of the culture medium. The practicability and benefits of this system are addressed in this report.

Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the beta subunit.

Science.gov (United States)

Campbell, Zachary T; Weichsel, Andrzej; Montfort, William R; Baldwin, Thomas O

2009-07-07

Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic alpha subunit and a homologous but noncatalytic beta subunit. Despite decades of enzymological investigation, structural evidence defining the active center has been elusive. We report here the crystal structure of V. harveyi luciferase bound to flavin mononucleotide (FMN) at 2.3 A. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. This structure also provides the first data specifying the conformations of a mobile loop that is crystallographically disordered in both prior crystal structures [(1995) Biochemistry 34, 6581-6586; (1996) J. Biol. Chem. 271, 21956 21968]. This loop appears to be a boundary between solvent and the active center. Within this portion of the protein, a single contact was observed between Phe272 of the alpha subunit, not seen in the previous structures, and Tyr151 of the beta subunit. Substitutions at position 151 on the beta subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH(2)). These findings partially address the long-standing question of how the beta subunit stabilizes the active conformation of the alpha subunit, thereby participating in the catalytic mechanism.

The cAMP-dependent protein kinase inhibitor H-89 attenuates the bioluminescence signal produced by Renilla Luciferase.

Directory of Open Access Journals (Sweden)

Katie J Herbst

2009-05-01

Full Text Available Investigations into the regulation and functional roles of kinases such as cAMP-dependent protein kinase (PKA increasingly rely on cellular assays. Currently, there are a number of bioluminescence-based assays, for example reporter gene assays, that allow the study of the regulation, activity, and functional effects of PKA in the cellular context. Additionally there are continuing efforts to engineer improved biosensors that are capable of detecting real-time PKA signaling dynamics in cells. These cell-based assays are often utilized to test the involvement of PKA-dependent processes by using H-89, a reversible competitive inhibitor of PKA.We present here data to show that H-89, in addition to being a competitive PKA inhibitor, attenuates the bioluminescence signal produced by Renilla luciferase (RLuc variants in a population of cells and also in single cells. Using 10 microM of luciferase substrate and 10 microM H-89, we observed that the signal from RLuc and RLuc8, an eight-point mutation variant of RLuc, in cells was reduced to 50% (+/-15% and 54% (+/-14% of controls exposed to the vehicle alone, respectively. In vitro, we showed that H-89 decreased the RLuc8 bioluminescence signal but did not compete with coelenterazine-h for the RLuc8 active site, and also did not affect the activity of Firefly luciferase. By contrast, another competitive inhibitor of PKA, KT5720, did not affect the activity of RLuc8.The identification and characterization of the adverse effect of H-89 on RLuc signal will help deconvolute data previously generated from RLuc-based assays looking at the functional effects of PKA signaling. In addition, for the current application and future development of bioluminscence assays, KT5720 is identified as a more suitable PKA inhibitor to be used in conjunction with RLuc-based assays. These principal findings also provide an important lesson to fully consider all of the potential effects of experimental conditions on a cell

Aerosol delivery of Akt controls protein translation in the lungs of dual luciferase reporter mice.

Science.gov (United States)

Tehrani, A M; Hwang, S-K; Kim, T-H; Cho, C-S; Hua, J; Nah, W-S; Kwon, J-T; Kim, J-S; Chang, S-H; Yu, K-N; Park, S-J; Bhandari, D R; Lee, K-H; An, G-H; Beck, G R; Cho, M-H

2007-03-01

Lung cancer has emerged as a leading cause of cancer death in the world; however, most of the current conventional therapies are not sufficiently effective in altering the progression of disease. Therefore, development of novel treatment approaches is needed. Although several genes and methods have been used for cancer gene therapy, a number of problems such as specificity, efficacy and toxicity reduce their application. This has led to re-emergence of aerosol gene delivery as a noninvasive method for lung cancer treatment. In this study, nano-sized glucosylated polyethyleneimine (GPEI) was used as a gene delivery carrier to investigate the effects of Akt wild type (WT) and kinase deficient (KD) on Akt-related signaling pathways and protein translation in the lungs of CMV- LucR-cMyc-IRES-LucF dual reporter mice. These mice are a powerful tool for the discrimination between cap-dependent/-independent protein translation. Aerosols containing self-assembled nano-sized GPEI/Akt WT or GPEI/Akt KD were delivered into the lungs of reporter mice through nose-only-inhalation-chamber with the aid of nebulizer. Aerosol delivery of Akt WT caused the increase of protein expression levels of Akt-related signals, whereas aerosol delivery of Akt KD did not. Furthermore, dual luciferase activity assay showed that aerosol delivery of Akt WT enhanced cap-dependent protein translation, whereas a reduction in cap-dependent protein translation by Akt KD was observed. Our results clearly showed that targeting Akt may be a good strategy for prevention as well as treatment of lung cancer. These studies suggest that our aerosol delivery is compatible for in vivo gene delivery which could be used as a noninvasive gene therapy in the future.

Using a split luciferase assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins.

Science.gov (United States)

Saw, Wan Ting; Matsuda, Zene; Eisenberg, Roselyn J; Cohen, Gary H; Atanasiu, Doina

2015-11-15

Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion. Copyright © 2015 Elsevier Inc. All rights reserved.

A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Shi, Junwei; Zhang, Huan; Fang, Liurong; Xi, Yongqiang; Zhou, Yanrong; Luo, Rui; Wang, Dang; Xiao, Shaobo; Chen, Huanchun

2014-01-01

Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis

A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

Energy Technology Data Exchange (ETDEWEB)

Shi, Junwei; Zhang, Huan; Fang, Liurong; Xi, Yongqiang; Zhou, Yanrong; Luo, Rui; Wang, Dang, E-mail: wangdang511@126.com; Xiao, Shaobo; Chen, Huanchun

2014-10-03

Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.

F-18 Labeled Diabody-Luciferase Fusion Proteins for Optical-ImmunoPET

Energy Technology Data Exchange (ETDEWEB)

Wu, Anna M. [Univ. of California, Los Angeles, CA (United States)

2013-01-18

The goal of the proposed work is to develop novel dual-labeled molecular imaging probes for multimodality imaging. Based on small, engineered antibodies called diabodies, these probes will be radioactively tagged with Fluorine-18 for PET imaging, and fused to luciferases for optical (bioluminescence) detection. Performance will be evaluated and validated using a prototype integrated optical-PET imaging system, OPET. Multimodality probes for optical-PET imaging will be based on diabodies that are dually labeled with 18F for PET detection and fused to luciferases for optical imaging. 1) Two sets of fusion proteins will be built, targeting the cell surface markers CEA or HER2. Coelenterazine-based luciferases and variant forms will be evaluated in combination with native substrate and analogs, in order to obtain two distinct probes recognizing different targets with different spectral signatures. 2) Diabody-luciferase fusion proteins will be labeled with 18F using amine reactive [18F]-SFB produced using a novel microwave-assisted, one-pot method. 3) Sitespecific, chemoselective radiolabeling methods will be devised, to reduce the chance that radiolabeling will inactivate either the target-binding properties or the bioluminescence properties of the diabody-luciferase fusion proteins. 4) Combined optical and PET imaging of these dual modality probes will be evaluated and validated in vitro and in vivo using a prototype integrated optical-PET imaging system, OPET. Each imaging modality has its strengths and weaknesses. Development and use of dual modality probes allows optical imaging to benefit from the localization and quantitation offered by the PET mode, and enhances the PET imaging by enabling simultaneous detection of more than one probe.

Design and introduction of a disulfide bridge in firefly luciferase: increase of thermostability and decrease of pH sensitivity.

Science.gov (United States)

Imani, Mehdi; Hosseinkhani, Saman; Ahmadian, Shahin; Nazari, Mahboobeh

2010-08-01

The thermal sensitivity and pH-sensitive spectral properties of firefly luciferase have hampered its application in a variety of fields. It is proposed that the stability of a protein can be increased by introduction of disulfide bridge that decreases the configurational entropy of unfolding. A disulfide bridge is introduced into Photinus pyralis firefly luciferase to make two separate mutant enzymes with a single bridge. Even though the A103C/S121C mutant showed remarkable thermal stability, its specific activity decreased, whereas the A296C/A326C mutant showed tremendous thermal stability, relative pH insensitivity and 7.3-fold increase of specific activity. Moreover, the bioluminescence emission spectrum of A296C/A326C was resistant against higher temperatures (37 degrees C). Far-UV CD analysis showed slight secondary structure changes for both mutants. Thermal denaturation analysis showed that conformational stabilities of A103C/S121C and A296C/A326C are more than native firefly luciferase. It is proposed that since A296 and A326 are situated in the vicinity of the enzyme active site microenvironment in comparison with A103 and S121, the formation of a disulfide bridge in this region has more impact on enzyme kinetic characteristics.

Bioluminescence of beetle luciferases with 6'-amino-D-luciferin analogues reveals excited keto-oxyluciferin as the emitter and phenolate/luciferin binding site interactions modulate bioluminescence colors.

Science.gov (United States)

Viviani, Vadim R; Neves, Deimison Rodrigues; Amaral, Danilo Trabuco; Prado, Rogilene A; Matsuhashi, Takuto; Hirano, Takashi

2014-08-19

Beetle luciferases produce different bioluminescence colors from green to red using the same d-luciferin substrate. Despite many studies of the mechanisms and structural determinants of bioluminescence colors with firefly luciferases, the identity of the emitters and the specific active site interactions responsible for bioluminescence color modulation remain elusive. To address these questions, we analyzed the bioluminescence spectra with 6'-amino-D-luciferin (aminoluciferin) and its 5,5-dimethyl analogue using a set of recombinant beetle luciferases that naturally elicit different colors and different pH sensitivities (pH-sensitive, Amydetes vivianii λmax=538 nm, Macrolampis sp2 λmax=564 nm; pH-insensitive, Phrixotrix hirtus λmax=623 nm, Phrixotrix vivianii λmax=546 nm, and Pyrearinus termitilluminans λmax=534 nm), a luciferase-like enzyme (Tenebrionidae, Zophobas morio λmax=613 nm), and mutants of C311 (S314). The green-yellow-emitting luciferases display red-shifted bioluminescence spectra with aminoluciferin in relation to those with D-luciferin, whereas the red-emitting luciferases displayed blue-shifted spectra. Bioluminescence spectra with 5,5-dimethylaminoluciferin, in which enolization is blocked, were almost identical to those of aminoluciferin. Fluorescence probing using 2-(4-toluidino)naphthalene-6-sulfonate and inference with aminoluciferin confirm that the luciferin binding site of the red-shifted luciferases is more polar than in the case of the green-yellow-emitting luciferases. Altogether, the results show that the keto form of excited oxyluciferin is the emitter in beetle bioluminescence and that bioluminescence colors are essentially modulated by interactions of the 6'-hydroxy group of oxyluciferin and basic moieties under the influence of the microenvironment polarity of the active site: a strong interaction between a base moiety and oxyluciferin phenol in a hydrophobic microenvironment promotes green-yellow emission, whereas a more polar

Probing the emitter site of Renilla luciferase using small organic molecules; an attempt to understand the molecular architecture of the emitter site.

Science.gov (United States)

Salehi, Farajollah; Emamzadeh, Rahman; Nazari, Mahboobeh; Rasa, Seyed Mohammad Mahdi

2016-12-01

Renilla luciferase is a sensitive enzyme and has wide applications in biotechnology such as drug screening. Previous studies have tried to show the catalytic residues, nevertheless, the accurate architecture and molecular behavior of its emitter site remains uncharacterized. In this study, the activity of Renilla luciferase, in the presence of two small organic molecules including dimethyl sulfoxide (DMSO) and isopropanol was considered and the structure was studied by circular dichroism (CD) and fluorescence spectroscopy. Moreover, the interaction of small organic molecules with the Renilla luciferase was studied using molecular dynamics simulations. Kinetics studies showed that at low concentration of DMSO (16.6-66mM) and isopropanol (19.3-76mM) the K m changed and a competitive inhibition pattern was observed. Moreover, spectroscopy studies reveled that the changes of activity of Renilla luciferase in the presence of low concentrations of small organic molecules was not associated with structural collapse or severe changes in the enzyme conformation. Molecular dynamics simulations indicated that DMSO and isopropanol, as probing molecules, were both able to bind to the emitter site and remained with the residues of the emitter site. Based on the probing data, the architecture of the emitter site in the "non-binding" model was proposed. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

Science.gov (United States)

Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

2012-06-01

There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

Development of a luciferase-based reporter system to monitor Bifidobacterium breve UCC2003 persistence in mice

Directory of Open Access Journals (Sweden)

Hill Colin

2008-09-01

Full Text Available Abstract Background Probiotics such as bifidobacteria have been shown to maintain a healthy intestinal microbial balance and help protect against infections. However, despite these benefits, bifidobacteria still remain poorly understood at the biochemical, physiological and especially the genetic level. Herein we describe, for the first time, the development of a non-invasive luciferase-based reporter system for real-time tracking of Bifidobacterium species in vivo. Results The reporter vector pLuxMC1 is based on the recently described theta-type plasmid pBC1 from B. catenatulatum 1 and the luxABCDE operon from pPL2lux 2. Derivatives of pLuxMC1, harbouring a bifidobacterial promoter (pLuxMC2 as well as a synthetically derived promoter (pLuxMC3 3 placed upstream of luxABCDE, were constructed and found to stably replicate in B. breve UCC2003. The subsequent analysis of these strains allowed us to assess the functionality of pLuxMC1 both in vitro and in vivo. Conclusion Our results demonstrate the potential of pLuxMC1 as a real-time, non-invasive reporter system for Bifidobacterium. It has also allowed us, for the first time, to track the colonisation potential and persistence of this probiotic species in real time. An interesting and significant outcome of the study is the identification of the caecum as a niche environment for B. breve UCC2003 within the mouse gastrointestinal tract (GI tract.

Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells.

Science.gov (United States)

Kreuzer, J; Denger, S; Reifers, F; Beisel, C; Haack, K; Gebert, J; Kübler, W

1996-07-01

Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and

Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta

DEFF Research Database (Denmark)

Hermanrud, Christina; Ryner, Malin; Luft, Thomas

2016-01-01

a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays......Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach...... to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines...

Firefly luciferase gene contains a cryptic promoter

Czech Academy of Sciences Publication Activity Database

Vopálenský, V.; Mašek, T.; Horváth, Ondřej; Vicenová, B.; Mokrejš, M.; Pospíšek, M.

2008-01-01

Roč. 14, č. 9 (2008), s. 1720-1729 ISSN 1355-8382 Grant - others:GAČR(CZ) GA204/03/1487; GAČR(CZ) GA301/07/0607; Mšk(CZ) LC06066 Program:LC Institutional research plan: CEZ:AV0Z50520514 Keywords : luciferase * cryptic promoter * hepatitis C virus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.018, year: 2008

Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag.

Science.gov (United States)

Rathnayaka, Tharangani; Tawa, Minako; Nakamura, Takashi; Sohya, Shihori; Kuwajima, Kunihiro; Yohda, Masafumi; Kuroda, Yutaka

2011-12-01

Gaussia luciferase (GLuc) is the smallest known bioluminescent protein and is attracting much attention as a potential reporter protein. However, its 10 disulfide bond forming cysteines have hampered the efficient production of recombinant GLuc and thus limited its use in bio-imaging application. Here, we demonstrate that the addition of a short solubility enhancement peptide tag (SEP-Tag) to the C-terminus of GLuc (GLuc-C9D) significantly increased the fraction of soluble protein at a standard expression temperature. The expression time was much shorter, and the final yield of GLuc-C9D was significantly higher than with our previous pCold expression system. Reversed phase HPLC indicated that the GLuc-C9D variant folded with a single disulfide bond pattern after proper oxidization. Further, the thermal denaturation of GLuc-C9D was completely reversible, and its secondary structure content remained unchanged until 40°C as assessed by CD spectroscopy. The (1)H-NMR spectrum of GLuc indicated sharp well dispersed peaks typical for natively folded proteins. GLuc-C9D bioluminescence activity was strong and fully retained even after incubation at high temperatures. These results suggest that solubilization using SEP-Tags can be useful for producing large quantities of proteins containing multiple disulfide bonds. Copyright © 2011. Published by Elsevier B.V.

Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

Directory of Open Access Journals (Sweden)

TELISSA C. KASSAR

Full Text Available ABSTRACT Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV expressing Gaussia luciferase (GLuc (YFV-GLuc. We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967, indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.

A mouse model of pulmonary metastasis from spontaneous osteosarcoma monitored in vivo by Luciferase imaging.

Directory of Open Access Journals (Sweden)

Silvia Miretti

Full Text Available BACKGROUND: Osteosarcoma (OSA is lethal when metastatic after chemotherapy and/or surgical treatment. Thus animal models are necessary to study the OSA metastatic spread and to validate novel therapies able to control the systemic disease. We report the development of a syngeneic (Balb/c murine OSA model, using a cell line derived from a spontaneous murine tumor. METHODOLOGY: The tumorigenic and metastatic ability of OSA cell lines were assayed after orthotopic injection in mice distal femur. Expression profiling was carried out to characterize the parental and metastatic cell lines. Cells from metastases were propagated and engineered to express Luciferase, in order to follow metastases in vivo. PRINCIPAL FINDINGS: Luciferase bioluminescence allowed to monitor the primary tumor growth and revealed the appearance of spontaneous pulmonary metastases. In vivo assays showed that metastasis is a stable property of metastatic OSA cell lines after both propagation in culture and luciferase trasduction. When compared to parental cell line, both unmodified and genetically marked metastatic cells, showed comparable and stable differential expression of the enpp4, pfn2 and prkcd genes, already associated to the metastatic phenotype in human cancer. CONCLUSIONS: This OSA animal model faithfully recapitulates some of the most important features of the human malignancy, such as lung metastatization. Moreover, the non-invasive imaging allows monitoring the tumor progression in living mice. A great asset of this model is the metastatic phenotype, which is a stable property, not modifiable after genetic manipulation.

Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation.

Science.gov (United States)

Li, Jian-Feng; Bush, Jenifer; Xiong, Yan; Li, Lei; McCormack, Matthew

2011-01-01

Protein-protein interactions (PPIs) constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC) as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs) and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.

Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation.

Directory of Open Access Journals (Sweden)

Jian-Feng Li

Full Text Available Protein-protein interactions (PPIs constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.

The nature of the GRE influences the screening for GR-activity enhancing modulators.

Directory of Open Access Journals (Sweden)

Karen Dendoncker

Full Text Available Glucocorticoid resistance (GCR, i.e. unresponsiveness to the beneficial anti-inflammatory activities of the glucocorticoid receptor (GR, poses a serious problem in the treatment of inflammatory diseases. One possible solution to try and overcome GCR, is to identify molecules that prevent or revert GCR by hyper-stimulating the biological activity of the GR. To this purpose, we screened for compounds that potentiate the dexamethasone (Dex-induced transcriptional activity of GR. To monitor GR transcriptional activity, the screen was performed using the lung epithelial cell line A549 in which a glucocorticoid responsive element (GRE coupled to a luciferase reporter gene construct was stably integrated. Histone deacetylase inhibitors (HDACi such as Vorinostat and Belinostat are two broad-spectrum HDACi that strongly increased the Dex-induced luciferase expression in our screening system. In sharp contrast herewith, results from a genome-wide transcriptome analysis of Dex-induced transcripts using RNAseq, revealed that Belinostat impairs the ability of GR to transactivate target genes. The stimulatory effect of Belinostat in the luciferase screen further depends on the nature of the reporter construct. In conclusion, a profound discrepancy was observed between HDACi effects on two different synthetic promoter-luciferase reporter systems. The favorable effect of HDACi on gene expression should be evaluated with care, when considering them as potential therapeutic agents. GEO accession number GSE96649.

Effects of Epigenetic Modulation on Reporter Gene Expression: Implications for Stem Cell Imaging

Science.gov (United States)

Krishnan, Manickam; Park, Jinha M.; Cao, Feng; Wang, Dongxu; Paulmurugan, Ramasay; Tseng, Jeffrey R.; Gonzalgo, Mark L.; Gambhir, Sanjiv S.; Wu, Joseph C.

2013-01-01

Tracking stem cell localization, survival, differentiation, and proliferation following transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3–8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage-1 (843±28) to passage-20 (250±10) to passage-40 (44±3) to passage-60 (3±1 RLU/µg) (P<0.05 vs. passage-1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared to trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator) (P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 weeks compared to 1 week for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation. PMID:16246867

Use of nuclear receptor luciferase-based bioassays to detect endocrine active chemicals in a biosolids-biochar amended soil.

Science.gov (United States)

Anderson, Carolyn G; Joshi, Geetika; Bair, Daniel A; Oriol, Charlotte; He, Guochun; Parikh, Sanjai J; Denison, Michael S; Scow, Kate M

2017-08-01

Biosolids are a potentially valuable source of carbon and nutrients for agricultural soils; however, potential unintended impacts on human health and the environment must be considered. Virtually all biosolids contain trace amounts endocrine-disrupting chemicals derived from human use of pharmaceuticals and personal care products (PPCPs). One potential way to reduce the bioavailability of PPCPs is to co-apply biosolids with biochar to soil, because biochar's chemical (e.g., aromaticity) and physical properties (e.g., surface area) give it a high affinity to bind many organic chemicals in the environment. We developed a soil-specific extraction method and utilized a luciferase-based bioassay (CALUX) to detect endocrine active chemicals in a biosolids-biochar co-amendment soil greenhouse study. Both biochar (walnut shell, 900 °C) and biosolids had positive impacts on carrot and lettuce biomass accumulation over our study period. However, the walnut shell biochar stimulated aryl hydrocarbon receptor activity, suggesting the presence of potential endocrine active chemicals in the biochar. Since the biochar rate tested (100 t ha -1 ) is above the average agronomic rate (10-20 t ha -1 ), endocrine effects would not be expected in most environmental applications. The effect of high temperature biochars on endocrine system pathways must be explored further, using both quantitative analytical tools to identify potential endocrine active chemicals and highly sensitive bioanalytical assays such as CALUX to measure the resulting biological activity of such compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

In-vivo quantification of primary microRNA processing by Drosha with a luciferase based system

International Nuclear Information System (INIS)

Allegra, Danilo; Mertens, Daniel

2011-01-01

Research highlights: → Posttranscriptional regulation of miRNA processing is difficult to quantify. → Our in-vivo processing assay can quantify Drosha cleavage in live cells. → It is based on luciferase reporters fused with pri-miRNAs. → The assay validates the processing defect caused by a mutation in pri-16-1. → It is a sensitive method to quantify pri-miRNA cleavage by Drosha in live cells. -- Abstract: The RNAse III Drosha is responsible for the first step of microRNA maturation, the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is finely regulated and influences the amount of mature microRNA in a cell. We describe in the present work a method to quantify Drosha processing activity in-vivo, which is applicable to any microRNA. With respect to other methods for measuring Drosha activity, our system is faster and scalable, can be used with any cellular system and does not require cell sorting or use of radioactive isotopes. This system is useful to study regulation of Drosha activity in physiological and pathological conditions.

Luciferase genes cloned from the unculturable luminous bacteroid symbiont of the Caribbean flashlight fish, Kryptophanaron alfredi.

Science.gov (United States)

Haygood, M G; Cohn, D H

1986-01-01

Light organs of anomalopid (flashlight) fish contain luminous bacteroids that have never been cultured and, consequently, have been difficult to study. We have characterized the luciferase (lux) region of DNA extracted from light organs of the Caribbean flashlight fish Kryptophanaron alfredi by hybridization of cloned Vibrio harveyi lux genes to restriction-endonuclease-digested, light organ DNA. Comparison of the hybridization pattern of light organ DNA with that of DNA of a putative symbiotic isolate provides a method for identifying the authentic luminous symbiont regardless of its luminescence, and was used to reject one such isolate. Light organ DNA was further used to construct a cosmid clone bank and the luciferase genes were isolated. Unlike other bacterial luciferase genes, the genes were not expressed in Escherichia coli. When placed under the control of the E. coli trp promoter, the genes were transcribed but no luciferase was detected, suggesting a posttranscriptional block to expression.

Human CRF2 α and β splice variants: pharmacological characterization using radioligand binding and a luciferase gene expression assay

International Nuclear Information System (INIS)

Ardati, A.; Goetschy, V.; Gottowick, J.; Henriot, S.; Deuschle, U.; Kilpatrick, G.J.; Valdenaire, O.

1999-01-01

Corticotropin releasing factor (CRF) receptors belong to the super-family of G protein-coupled receptors. These receptors are classified into two subtypes (CRF 1 and CRF 2 ). Both receptors are positively coupled to adenylyl cyclase but they have a distinct pharmacology and distribution in brain. Two isoforms belonging to the CRF 2 subtype receptors, CRF 2α and CRF 2β , have been identified in rat and man. The neuropeptides CRF and urocortin mediate their actions through this CRF G protein-coupled receptor family. In this report, we describe the pharmacological characterization of the recently identified hCRF 2β receptor. We have used radioligand binding with [ 125 I]-tyr 0 -sauvagine and a gene expression assay in which the firefly luciferase gene expression is under the control of cAMP responsive elements. Association kinetics of [ 125 I]-tyr 0 -sauvagine binding to the hCRF 2β receptor were monophasic while dissociation kinetics were biphasic, in agreement with the kinetics results obtained with the hCRF 2α receptor. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically and with parameters obtained with the hCRF 2α receptor. A non-hydrolysable GTP analog, Gpp(NH)p, reduced the high affinity binding of [ 125 I]-tyr 0 -sauvagine to both hCRF 2 receptor isoforms in a similar manner. The rank order of potency of CRF agonist peptides in competition experiments was identical for both hCRF 2 α-helical CRF (9-41) oCRF). Similarly, agonist potency was similar for the two isoforms when studied using the luciferase gene reporter system. The peptide antagonist α-helical CRF (9-41) exhibited a non-competitive antagonism of urocortin-stimulated luciferase expression with both hCRF 2 receptor isoforms. Taken together, these results indicate that the pharmacological profiles of the CRF 2 splice variants are identical. This indicates that the region of the N-terminus that varies

Step-wise addition of disulfide bridge in firefly luciferase controls color shift through a flexible loop: a thermodynamic perspective.

Science.gov (United States)

Nazari, Mahboobeh; Hosseinkhani, Saman; Hassani, Leila

2013-02-01

Multi-color bioluminescence is developed using the introduction of single/double disulfide bridges in firefly luciferase. The bioluminescence reaction, which uses luciferin, Mg(2+)-ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by the luciferase and emits visible light. The bioluminescence color of firefly luciferases is determined by the luciferase sequence and assay conditions. It has been proposed that the stability of a protein may increase through the introduction of a disulfide bridge that decreases the configurational entropy of unfolding. Single and double disulfide bridges are introduced into Photinus pyralis firefly luciferase to make separate mutant enzymes with a single/double bridge (C(81)-A(105)C, L(306)C-L(309)C, P(451)C-V(469)C; C(81)-A(105)C/P(451)C-V(469)C, and A(296)C-A(326)C/P(451)C-V(469)C). By introduction of disulfide bridges using site-directed mutagenesis in Photinus pyralis luciferase the color of emitted light was changed to red or kept in different extents. The bioluminescence color shift occurred with displacement of a critical loop in the luciferase structure without any change in green emitter mutants. Thermodynamic analysis revealed that among mutants, L(306)C-L(309)C shows a remarkable stability against urea denaturation and also a considerable increase in kinetic stability and a clear shift in bioluminescence spectra towards red.

Corneal NF-kappaB activity is necessary for the retention of transparency in the cornea of UV-B-exposed transgenic reporter mice.

Science.gov (United States)

Alexander, George; Carlsen, Harald; Blomhoff, Rune

2006-04-01

To determine the dynamics of Nuclear Factor-kappaB (NF-kappaB) in murine corneal pathology and the role of NF-kappaB in maintaining corneal clarity after ultraviolet B radiation insult, transgenic mice containing NF-kappaB-luciferase reporter were exposed to LPS (bacterial lipopolysaccharide), TNF-alpha (Tumor Necrosis Factor-alpha) or 4 kJ m(-2) UV-B radiation. NF-kappaB decoy oligonucleotides were also administered in some of the UV-B experiments. Following various exposure times, the mice were sacrificed and whole eyes or corneal tissues were obtained. Whole eyes were examined for scattering using a point-source optical imaging technique. Tissue homogenates were examined for luciferase activity using a luminometer. TNF-alpha and LPS-injected NF-kappaB-luciferase transgenic mice demonstrated 3-10-fold increases in cornea NF-kappaB with peak activities at 4 and 6 hr post-injection, respectively. Mice exposed to 4 kJ m(-2) UV-B exhibited a 3-fold increase in NF-kappaB activity 4 hr post-exposure. The administration of NF-kappaB-decoy oligonucleotides to mice had the effect of reducing UV-B-induced NF-kappaB activity in the cornea and significantly increasing the amount of light scattering in UV-B exposed corneas 7 days post-UV-B exposure when compared to sham injected mice. These results indicate that NF-kappaB is activated in cornea in pathologies that involves increased plasma levels of LPS and TNF-alpha, as well as direct UV-B exposure, and suggest that NF-kappaB activation play an essential part in the corneal healing process.

Constant pH Accelerated Molecular Dynamics Investigation of the pH Regulation Mechanism of Dinoflagellate Luciferase.

Science.gov (United States)

Donnan, Patrick H; Ngo, Phong D; Mansoorabadi, Steven O

2018-01-23

The bioluminescence reaction in dinoflagellates involves the oxidation of an open-chain tetrapyrrole by the enzyme dinoflagellate luciferase (LCF). The activity of LCF is tightly regulated by pH, where the enzyme is essentially inactive at pH ∼8 and optimally active at pH ∼6. Little is known about the mechanism of LCF or the structure of the active form of the enzyme, although it has been proposed that several intramolecularly conserved histidine residues in the N-terminal region are important for the pH regulation mechanism. Here, constant pH accelerated molecular dynamics was employed to gain insight into the conformational activation of LCF induced by acidification.

a positive control plasmid for reporter gene assay

African Journals Online (AJOL)

STORAGESEVER

2008-07-04

Jul 4, 2008 ... qualification as a positive control for luciferase reporter gene assays. Key words: Reporter gene plasmid, luciferase assay, cytomegalovirus promoter/enhancer, human melanoma cell line. INTRODUCTION. Reporter genes, often called reporters, have become a precious tool in studies of gene expression ...

A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability.

Science.gov (United States)

Larsson, Marie C; Lerm, Maria; Ängeby, Kristian; Nordvall, Michaela; Juréen, Pontus; Schön, Thomas

2014-11-01

The intracellular (IC) effect of drugs against Mycobacterium tuberculosis (Mtb) is not well established but increasingly important to consider when combining current and future multidrug regimens into the best possible treatment strategies. For this purpose, we developed an IC model based on a genetically modified Mtb H37Rv strain, expressing the Vibrio harvei luciferase (H37Rv-lux) infecting the human macrophage like cell line THP-1. Cells were infected at a low multiplicity of infection (1:1) and subsequently exposed to isoniazid (INH), ethambutol (EMB), amikacin (AMI) or levofloxacin (LEV) for 5days in a 96-well format. Cell viability was evaluated by Calcein AM and was maintained throughout the experiment. The number of viable H37Rv-lux was determined by luminescence and verified by a colony forming unit analysis. The results were compared to the effects of the same drugs in broth cultures. AMI, EMB and LEV were significantly less effective intracellularly (MIC90: >4mg/L, 8mg/L and 2mg/L, respectively) compared to extracellularly (MIC90: 0.5mg/L for AMI and EMB; 0.25mg/L for LEV). The reverse was the case for INH (IC: 0.064mg/L vs EC: 0.25mg/L). In conclusion, this luciferase based method, in which monitoring of cell viability is included, has the potential to become a useful tool while evaluating the intracellular effects of anti-mycobacterial drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.

Science.gov (United States)

Paul, T A; Burns, J C; Shike, H; Getchell, R; Bowser, P R; Whitlock, K E; Casey, J W

2001-06-01

A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.

Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

NARCIS (Netherlands)

Legler, J.; Dennekamp, M.; Vethaak, A.D.; Brouwer, A.; Koeman, J.H.; Burg, van der B.; Murk, A.J.

2002-01-01

Sediments may be the ultimate sink for persistent (xeno-) estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The

Human CRF{sub 2} {alpha} and {beta} splice variants: pharmacological characterization using radioligand binding and a luciferase gene expression assay

Energy Technology Data Exchange (ETDEWEB)

Ardati, A. [Rhone-Poulenc Rorer, Cardiovascular Biology, NW4, 500 Arcola Road, Collegeville, PA (United States); Goetschy, V.; Gottowick, J.; Henriot, S.; Deuschle, U.; Kilpatrick, G.J. [Central Nervous System, Pharma Division, F. Hoffmann-La Roche AG, CH-4070 Basel (Switzerland); Valdenaire, O. [Cardiovascular Research, Pharma Division, F. Hoffmann-La Roche AG, CH-4070 Basel (Switzerland)

1999-03-14

Corticotropin releasing factor (CRF) receptors belong to the super-family of G protein-coupled receptors. These receptors are classified into two subtypes (CRF{sub 1} and CRF{sub 2}). Both receptors are positively coupled to adenylyl cyclase but they have a distinct pharmacology and distribution in brain. Two isoforms belonging to the CRF{sub 2} subtype receptors, CRF{sub 2{alpha}} and CRF{sub 2{beta}}, have been identified in rat and man. The neuropeptides CRF and urocortin mediate their actions through this CRF G protein-coupled receptor family. In this report, we describe the pharmacological characterization of the recently identified hCRF{sub 2{beta}} receptor. We have used radioligand binding with [{sup 125}I]-tyr{sup 0}-sauvagine and a gene expression assay in which the firefly luciferase gene expression is under the control of cAMP responsive elements. Association kinetics of [{sup 125}I]-tyr{sup 0}-sauvagine binding to the hCRF{sub 2{beta}} receptor were monophasic while dissociation kinetics were biphasic, in agreement with the kinetics results obtained with the hCRF{sub 2{alpha}} receptor. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically and with parameters obtained with the hCRF{sub 2{alpha}} receptor. A non-hydrolysable GTP analog, Gpp(NH)p, reduced the high affinity binding of [{sup 125}I]-tyr{sup 0}-sauvagine to both hCRF{sub 2} receptor isoforms in a similar manner. The rank order of potency of CRF agonist peptides in competition experiments was identical for both hCRF{sub 2}{alpha}-helical CRF{sub (9-41)}oCRF). Similarly, agonist potency was similar for the two isoforms when studied using the luciferase gene reporter system. The peptide antagonist {alpha}-helical CRF{sub (9-41)} exhibited a non-competitive antagonism of urocortin-stimulated luciferase expression with both hCRF{sub 2} receptor isoforms. Taken together, these results indicate that the

Plasmodium falciparum transfected with ultra bright NanoLuc luciferase offers high sensitivity detection for the screening of growth and cellular trafficking inhibitors.

Directory of Open Access Journals (Sweden)

Mauro F Azevedo

Full Text Available Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.

Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: Comparisons with AHR1-mediated reporter gene activity and in ovo toxicity

Energy Technology Data Exchange (ETDEWEB)

Manning, Gillian E., E-mail: gmann017@uottawa.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Mundy, Lukas J., E-mail: lukas.mundy@ec.gc.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Crump, Doug, E-mail: doug.crump@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Jones, Stephanie P., E-mail: stephanie.jones@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Chiu, Suzanne, E-mail: suzanne.chiu@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Klein, Jeff, E-mail: jeffery@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada); Konstantinov, Alex, E-mail: alex@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada); Potter, Dave, E-mail: dpotter@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada); Kennedy, Sean W., E-mail: sean.kennedy@ec.gc.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada)

2013-01-01

Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species. -- Highlights: ► The chicken isn't the most sensitive species to CYP1A induction by PCB 105 and 118. ► The relative potency of PCBs differs between avian species. ► EROD activity was correlated with luciferase activity from the LRG assay. ► EROD activity was a better predictor of

Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: Comparisons with AHR1-mediated reporter gene activity and in ovo toxicity

International Nuclear Information System (INIS)

Manning, Gillian E.; Mundy, Lukas J.; Crump, Doug; Jones, Stephanie P.; Chiu, Suzanne; Klein, Jeff; Konstantinov, Alex; Potter, Dave; Kennedy, Sean W.

2013-01-01

Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species. -- Highlights: ► The chicken isn't the most sensitive species to CYP1A induction by PCB 105 and 118. ► The relative potency of PCBs differs between avian species. ► EROD activity was correlated with luciferase activity from the LRG assay. ► EROD activity was a better predictor of toxicity than CYP

A novel reporter system for neutralizing and enhancing antibody assay against dengue virus.

Science.gov (United States)

Song, Ke-Yu; Zhao, Hui; Jiang, Zhen-You; Li, Xiao-Feng; Deng, Yong-Qiang; Jiang, Tao; Zhu, Shun-Ya; Shi, Pei-Yong; Zhang, Bo; Zhang, Fu-Chun; Qin, E-De; Qin, Cheng-Feng

2014-02-18

Dengue virus (DENV) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. Antibody plays distinct roles in controlling DENV infections. Neutralizing antibody is protective against DENV infection, whereas sub-neutralizing concentration of antibody can increase DENV infection, termed antibody-dependent enhancement (ADE). Plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies. In this study, a novel reporter virus-based system was developed for measuring neutralization and ADE activity. A stable Renilla luciferase reporter DENV (Luc-DENV) that can produce robust luciferase signals in BHK-21 and K562 cells were used to establish the assay and validated against traditional plaque-based assay. Luciferase value analysis using various known DENV-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. The newly developed assay was finally validated with clinical samples from infected animals and individuals. This reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for DENV antibodies.

[Effect of transcription activity regulated by VNTR-ZNF and -14C/T variants in the promoter region of ATP-binding cassette transporter 1 in HepG2 cells].

Science.gov (United States)

Gao, Shenxia; Zhao, Lili; Zhang, Ying; Mao, Yongmin

2016-10-01

To explore the effect of VNTR-ZNF and -14C/T variants of the promoter region of the ABCA1 gene on the transcription activity of genes in vitro. The recombinants were constructed by ligating DNA fragment containing VNTR-ZNF ACCCC inserted/deleted allele with or without -14C/T substitution fragments with a PGL2-basic vector containing luciferase reporter gene. The recombinants were then transfected into HepG2 cells using the cationic lipid method. After 48 h, transfected cells were collected and used to detect the luciferase activity. Luciferase activity of PGL2-ZNF-ACCCCDel was greater than that of PGL2-ZNF-ACCCCIns. Luciferase activity of PGL2-ZNFDel-14C was greater than that of PGL2-ZNFDel-14T, PGL2-ZNFIns-14C, PGL2-ZNFIns-14T. Compared with the insertion type, the ACCCC-deleted type of VNTR-ZNF can significantly enhance the transcription activity of ABCA1. And co-transfection of -14 C allele can further enhance this activity.

Steady-state fluorescence and phosphorescence spectroscopic studies of bacterial luciferase tryptophan mutants.

Science.gov (United States)

Li, Z; Meighen, E A

1994-09-01

Bacterial luciferase, which catalyzes the bioluminescence reaction in luminous bacteria, consists of two nonidentical polypeptides, α and β. Eight mutants of luciferase with each of the tryptophans replaced by tyrosine were generated by site-directed mutagenesis and purified to homogeneity. The steady-state tryptophan fluorescence and low-temperature phosphorescence spectroscopic properties of these mutants were characterized. In some instances, mutation of only a single tryptophan residue resulted in large spectral changes. The tryptophan residues conserved in both the α and the β subunits exhibited distinct fluorescence emission properties, suggesting that these tryptophans have different local enviroments. The low-temperature phosphorescence data suggest that the tryptophans conserved in bot the α and the β subunits are not located at the subunit interface and/or involved in subunit interactions. The differences in the spectral properties of the mutants have provided useful information on the local environment of the individual tryptophan residues as well as on the quaternary structure of the protein.

Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals.

Science.gov (United States)

Gabriel, Gabriele V M; Viviani, Vadim R

2016-12-01

Most luminescent biosensors for heavy metals are fluorescent and rely on intensity measurements, whereas a few are ratiometric and rely on spectral changes. Bioluminescent biosensors for heavy metals are less common. Firefly luciferases have been coupled to responsive promoters for mercury and arsenium, and used as light on biosensors. Firefly luciferase bioluminescence spectrum is naturally sensitive to heavy metal cations such as zinc and mercury and to pH. Although pH sensitivity of firefly luciferases was shown to be useful for ratiometric estimation of intracellular pH, its potential use for ratiometric estimation of heavy metals was never considered. Using the yellow-emitting Macrolampis sp2 firefly luciferase and site-directed mutagenesis, we show that the residues H310 and E354 constitute two critical sites for metal sensitivity that can be engineered to increase sensitivity to zinc, nickel, and mercury. A linear relationship between cation concentration and the ratio of bioluminescence intensities at 550 and 610 nm allowed, for the first time, the ratiometric estimation of heavy metals concentrations down to 0.10 mM, demonstrating the potential applicability of firefly luciferases as enzymatic and intracellular ratiometric metal biosensors.

Highly specific expression of luciferase gene in lungs of naive nude mice directed by prostate-specific antigen promoter

International Nuclear Information System (INIS)

Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng

2005-01-01

PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10 9 PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases

The PPARγ ligand ciglitazone regulates androgen receptor activation differently in androgen-dependent versus androgen-independent human prostate cancer cells

International Nuclear Information System (INIS)

Moss, Patrice E.; Lyles, Besstina E.; Stewart, LaMonica V.

2010-01-01

The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPARγ ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPARγ ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPARγ ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPARγ. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPARγ and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.

Activation of cAMP-dependent signaling pathway induces mouse organic anion transporting polypeptide 2 expression.

Science.gov (United States)

Chen, Chuan; Cheng, Xingguo; Dieter, Matthew Z; Tanaka, Yuji; Klaassen, Curtis D

2007-04-01

Rodent Oatp2 is a hepatic uptake transporter for such compounds as cardiac glycosides. In the present study, we found that fasting resulted in a 2-fold induction of Oatp2 expression in liver of mice. Because the cAMP-protein kinase A (PKA) signaling pathway is activated during fasting, the role of this pathway in Oatp2 induction during fasting was examined. In Hepa-1c1c7 cells, adenylyl cyclase activator forskolin as well as two cellular membrane-permeable cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, induced Oatp2 mRNA expression in a time- and dose-dependent manner. These three chemicals induced reporter gene activity in cells transfected with a luciferase reporter gene construct containing a 7.6-kilobase (kb) 5'-flanking region of mouse Oatp2. Transient transfection of cells with 5'-deletion constructs derived from the 7.6-kb Oatp2 promoter reporter gene construct, as well as 7.6-kb constructs in which a consensus cAMP response element (CRE) half-site CGTCA (-1808/-1804 bp) was mutated or deleted, confirms that this CRE site was required for the induction of luciferase activity by forskolin. Luciferase activity driven by the Oatp2 promoter containing this CRE site was induced in cells cotransfected with a plasmid encoding the protein kinase A catalytic subunit. Cotransfection of cells with a plasmid encoding the dominant-negative CRE binding protein (CREB) completely abolished the inducibility of the reporter gene activity by forskolin. In conclusion, induction of Oatp2 expression in liver of fasted mice may be caused by activation of the cAMP-dependent signaling pathway, with the CRE site (-1808/-1804) and CREB being the cis- and trans-acting factors mediating the induction, respectively.

Dioxin-like activity of brominated dioxins as individual compounds or mixtures in in vitro reporter gene assays with rat and mouse hepatoma cell lines.

Science.gov (United States)

Suzuki, G; Nakamura, M; Michinaka, C; Tue, N M; Handa, H; Takigami, H

2017-10-01

In vitro reporter gene assays detecting dioxin-like compounds have been developed and validated since the middle 1990's, and applied to the determination of dioxin-like activities in various samples for their risk management. Data on characterizing the potency of individual brominated dioxins and their activity in mixture with chlorinated dioxins are still limited on the cell-based assay. This study characterized the dioxin-like activities of the 32 brominated dioxins, such as polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans (PBDFs), coplanar polybrominated biphenyls, mixed halogenated dibenzo-p-dioxins and dibenzofurans (PXDFs), as a sole component or in a mixture by DR-CALUX (dioxin-responsive chemically activated luciferase expression) using the rat hepatoma H4IIE cell line and XDS-CALUX (xenobiotic detection systems-chemically activated luciferase expression) assays using the mouse hepatoma H1L6.1 cell line. The 2,3,7,8-TCDD-relative potencies (REPs) of most of the brominated dioxins were within a factor of 10 of the WHO toxicity equivalency factor (WHO-TEF) for the chlorinated analogues. The REPs of a few PXDFs were an order of magnitude higher than the corresponding WHO-TEFs, indicating their toxicological importance. Results with reconstituted mixtures suggest that the activity of brominated and chlorinated dioxins in both CALUX assays was dose-additive. Thus, obtained results indicated the applicability of the CALUX assays as screening tools of brominated dioxins together with their chlorinated analogues. Copyright © 2017 Elsevier Ltd. All rights reserved.

A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands.

Directory of Open Access Journals (Sweden)

Takako Noguchi

Full Text Available In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR, respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that

Quantitative and Dynamic Imaging of ATM Kinase Activity.

Science.gov (United States)

Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

The PPAR{gamma} ligand ciglitazone regulates androgen receptor activation differently in androgen-dependent versus androgen-independent human prostate cancer cells

Energy Technology Data Exchange (ETDEWEB)

Moss, Patrice E.; Lyles, Besstina E.; Stewart, LaMonica V., E-mail: lstewart@mmc.edu

2010-12-10

The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPAR{gamma} ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPAR{gamma} ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPAR{gamma} ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPAR{gamma}. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPAR{gamma} and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.

Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors

Directory of Open Access Journals (Sweden)

Pierre Danhier

2015-12-01

Full Text Available Here we have developed a hypoxia response element driven imaging strategy that combined the hypoxia-driven expression of two optical reporters with different half-lives to detect temporal changes in hypoxia and hypoxia inducible factor (HIF activity. For this purpose, human prostate cancer PC3 cells were transfected with the luciferase gene fused with an oxygen-dependent degradation domain (ODD-luc and a variant of the enhanced green fluorescent protein (EGFP. Both ODD-luciferase and EGFP were under the promotion of a poly-hypoxia-response element sequence (5xHRE. The cells constitutively expressed tdTomato red fluorescent protein. For validating the imaging strategy, cells were incubated under hypoxia (1% O2 for 48 hours and then reoxygenated. The luciferase activity of PC3-HRE-EGFP/HRE-ODD-luc/tdtomato cells detected by bioluminescent imaging rapidly decreased after reoxygenation, whereas EGFP levels in these cells remained stable for several hours. After in vitro validation, PC3-HRE-EGFP/HRE-ODD-luc/tdtomato tumors were implanted subcutaneously and orthotopically in nude male mice and imaged in vivo and ex vivo using optical imaging in proof-of-principle studies to demonstrate differences in optical patterns between EGFP expression and bioluminescence. This novel "timer" imaging strategy of combining the short-lived ODD-luciferase and the long-lived EGFP can provide a time frame of HRE activation in PC3 prostate cancer cells and will be useful to understand the temporal changes in hypoxia and HIF activity during cancer progression and following treatments including HIF targeting strategies.

Determination of relative assay response factors for toxic chlorinated and brominated dioxins/furans using an enzyme immunoassay (EIA) and a chemically-activated luciferase gene expression cell bioassay (CALUX).

Science.gov (United States)

Samara, Fatin; Gullett, Brian K; Harrison, Robert O; Chu, Andrew; Clark, George C

2009-04-01

Determination of toxic activity requires knowledge of both the concentration and toxicity to evaluate the risk for adverse human health and environmental effects. A chemically-activated luciferase gene expression cell bioassay system (CALUX) and an antibody-based method enzyme immunoassay (EIA) were used to detect the dioxin-like response of several polybrominated, polychlorinated, and polybrominated/chlorinated dibenzo-p-dioxins/furans (PBDDs/Fs, PCDDs/Fs, and PBCDDs/Fs, respectively). It has been suggested that the biological activity of the brominated and mixed bromo/chloro compounds is similar to their chlorinated analogues (measured by binding to the Ah receptor). PBDD/F, PCDD/F, and PBCDD/F laboratory standards exhibited biological activity ranging over three orders of magnitude. The highest relative potency (REP) values from CALUX analysis, when compared to 2,3,7,8-TCDD, were 2,3,7,8-TBDD at 0.99 (+/-0.07), 1,2,3,7,8-PeCDD at 0.69, and 2-Br-3,7,8-TriCDD at 0.72 (+/-0.02). Cross-reactivities were calculated using EIA for several PBDDs/Fs and PBCDDs. The highest percent cross-reactivity was found for 2,3,7,8-TBDD at 138 (+/-34%), and 2,3,7-TriBDD at 84 (+/-36%).

Application of luciferase assay for ATP to antimicrobial drug susceptibility

Science.gov (United States)

Chappelle, E. W.; Picciolo, G. L.; Vellend, H.; Tuttle, S. A.; Barza, M. J.; Weinstein, L. (Inventor)

1977-01-01

The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium. The amount of ATP is assayed in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture. The sample of the cultured bacterium is subjected to an antibiotic agent. The amount of bacterial adenosine triphosphate is assayed after treatment with the antibiotic by measuring the luminescent light resulting from the reaction. The ATP index is determined from the values obtained from the assay procedures.

A Noninvasive In Vitro Monitoring System Reporting Skeletal Muscle Differentiation.

Science.gov (United States)

Öztürk-Kaloglu, Deniz; Hercher, David; Heher, Philipp; Posa-Markaryan, Katja; Sperger, Simon; Zimmermann, Alice; Wolbank, Susanne; Redl, Heinz; Hacobian, Ara

2017-01-01

Monitoring of cell differentiation is a crucial aspect of cell-based therapeutic strategies depending on tissue maturation. In this study, we have developed a noninvasive reporter system to trace murine skeletal muscle differentiation. Either a secreted bioluminescent reporter (Metridia luciferase) or a fluorescent reporter (green fluorescent protein [GFP]) was placed under the control of the truncated muscle creatine kinase (MCK) basal promoter enhanced by variable numbers of upstream MCK E-boxes. The engineered pE3MCK vector, coding a triple tandem of E-Boxes and the truncated MCK promoter, showed twentyfold higher levels of luciferase activation compared with a Cytomegalovirus (CMV) promoter. This newly developed reporter system allowed noninvasive monitoring of myogenic differentiation in a straining bioreactor. Additionally, binding sequences of endogenous microRNAs (miRNAs; seed sequences) that are known to be downregulated in myogenesis were ligated as complementary seed sequences into the reporter vector to reduce nonspecific signal background. The insertion of seed sequences improved the signal-to-noise ratio up to 25% compared with pE3MCK. Due to the highly specific, fast, and convenient expression analysis for cells undergoing myogenic differentiation, this reporter system provides a powerful tool for application in skeletal muscle tissue engineering.

Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase.

Directory of Open Access Journals (Sweden)

Patrizia Pellegatti

2008-07-01

Full Text Available There is growing awareness that tumour cells build up a "self-advantageous" microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP.Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours.Our results show that ATP in the tumour interstitium is in the hundreds micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.

Probing Bioluminescence Resonance Energy Transfer in Quantum Rod-Luciferase Nanoconjugates.

Science.gov (United States)

Alam, Rabeka; Karam, Liliana M; Doane, Tennyson L; Coopersmith, Kaitlin; Fontaine, Danielle M; Branchini, Bruce R; Maye, Mathew M

2016-02-23

We describe the necessary design criteria to create highly efficient energy transfer conjugates containing luciferase enzymes derived from Photinus pyralis (Ppy) and semiconductor quantum rods (QRs) with rod-in-rod (r/r) microstructure. By fine-tuning the synthetic conditions, CdSe/CdS r/r-QRs were prepared with two different emission colors and three different aspect ratios (l/w) each. These were hybridized with blue, green, and red emitting Ppy, leading to a number of new BRET nanoconjugates. Measurements of the emission BRET ratio (BR) indicate that the resulting energy transfer is highly dependent on QR energy accepting properties, which include absorption, quantum yield, and optical anisotropy, as well as its morphological and topological properties, such as aspect ratio and defect concentration. The highest BR was found using r/r-QRs with lower l/w that were conjugated with red Ppy, which may be activating one of the anisotropic CdSe core energy levels. The role QR surface defects play on Ppy binding, and energy transfer was studied by growth of gold nanoparticles at the defects, which indicated that each QR set has different sites. The Ppy binding at those sites is suggested by the observed BRET red-shift as a function of Ppy-to-QR loading (L), where the lowest L results in highest efficiency and furthest shift.

A Luciferase Reporter Gene Assay to Measure Ebola Virus Viral Protein 35-Associated Inhibition of Double-Stranded RNA-Stimulated, Retinoic Acid-Inducible Gene 1-Mediated Induction of Interferon β.

Science.gov (United States)

Cannas, Valeria; Daino, Gian Luca; Corona, Angela; Esposito, Francesca; Tramontano, Enzo

2015-10-01

During Ebola virus (EBOV) infection, the type I interferon α/β (IFN-α/β) innate immune response is suppressed by EBOV viral protein 35 (VP35), a validated drug target. Identification of EBOV VP35 inhibitors requires a cellular system able to assess the VP35-based inhibitory functions of viral double-stranded RNA (dsRNA) IFN-β induction. We established a miniaturized luciferase gene reporter assay in A549 cells that measures IFN-β induction by viral dsRNA and is dose-dependently inhibited by VP35 expression. When compared to influenza A virus NS1 protein, EBOV VP35 showed improved inhibition of viral dsRNA-based IFN-β induction. This assay can be used to screen for EBOV VP35 inhibitors. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

Science.gov (United States)

Nuñez, S B; Medin, J A; Braissant, O; Kemp, L; Wahli, W; Ozato, K; Segars, J H

1997-03-14

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

Downstream reporter gene imaging for signal transduction pathway of dopamine type 2 receptor

International Nuclear Information System (INIS)

Le, Uyenchi N.; Min, Jung Joon; Moon, Sung Min; Bom, Hee Seung

2004-01-01

The Dopamine 2 receptor (D2R) signal pathway regulates gene expression by phosphorylation of proteins including cAMP reponse element-binding protein (CREB), a transcription factor. In this study, we developed a reporter strategy using the GAL4 fusion CREB to assess the phosphorylation of CREB, one of the targets of the D2R signal transduction pathway. We used three plasmids: GAL4 fusion transactivator (pCMV-CREB), firefly luciferase reporter with GAL4 binding sites (pG5-FLUC), and D2R plasmid (pCMV-D2R). Group 1 293T cells were transiently transfected with pCMV-CREB and pG5-FLUC, and group 2 cells were transfected with all three plasmids. Transfected cells were stimulated with different concentrations of dopamine (0-200 M). For animal studies, group 1 and 2 cells (1x10 6 ) were subcutaneously injected on the left and right thigh of six nude mice, respectively. Dopamine stimiulation was performed with intraperitoneal injection of L-DOPA incombination with carbidopa, a peripheral DOPA decarboxylase inhibitor. Bioluminescence optical imaging studies were performed before and after L-DOPA injection. In cell culture studies, group 1 cells showed strong luciferase activity which implies direct activation of the signaling pathway due to growth factors contained in culture medium. Group 2 cells showed strong luciferase activity and a further increase after administration of dopamine. In animal studies, group 1 and 2 cells showed bioluminescence signal before L-DOPA injection, but signal from group 2 cells significantly increased 12 h after L-DOPA injection. The signal from group 1 cells disappeared thereafter, but group 2 cells continued to show signal until 36 h of L-DOPA injection. This study demonstrates imaging of the D2R signal transduction pathway and should be useful for noninvasive imaging of downstream effects of G-coupled protein pathways

Detailed assessment of gene activation levels by multiple hypoxia-responsive elements under various hypoxic conditions.

Science.gov (United States)

Takeuchi, Yasuto; Inubushi, Masayuki; Jin, Yong-Nan; Murai, Chika; Tsuji, Atsushi B; Hata, Hironobu; Kitagawa, Yoshimasa; Saga, Tsuneo

2014-12-01

HIF-1/HRE pathway is a promising target for the imaging and the treatment of intractable malignancy (HIF-1; hypoxia-inducible factor 1, HRE; hypoxia-responsive element). The purposes of our study are: (1) to assess the gene activation levels resulting from various numbers of HREs under various hypoxic conditions, (2) to evaluate the bidirectional activity of multiple HREs, and (3) to confirm whether multiple HREs can induce gene expression in vivo. Human colon carcinoma HCT116 cells were transiently transfected by the constructs containing a firefly luciferase reporter gene and various numbers (2, 4, 6, 8, 10, and 12) of HREs (nHRE+, nHRE-). The relative luciferase activities were measured under various durations of hypoxia (6, 12, 18, and 24 h), O2 concentrations (1, 2, 4, 8, and 16 %), and various concentrations of deferoxamine mesylate (20, 40, 80, 160, and 320 µg/mL growth medium). The bidirectional gene activation levels by HREs were examined in the constructs (dual-luc-nHREs) containing firefly and Renilla luciferase reporter genes at each side of nHREs. Finally, to test whether the construct containing 12HRE and the NIS reporter gene (12HRE-NIS) can induce gene expression in vivo, SPECT imaging was performed in a mouse xenograft model. (1) gene activation levels by HREs tended to increase with increasing HRE copy number, but a saturation effect was observed in constructs with more than 6 or 8 copies of an HRE, (2) gene activation levels by HREs increased remarkably during 6-12 h of hypoxia, but not beyond 12 h, (3) gene activation levels by HREs decreased with increasing O2 concentrations, but could be detected even under mild hypoxia at 16 % O2, (4) the bidirectionally proportional activity of the HRE was confirmed regardless of the hypoxic severity, and (5) NIS expression driven by 12 tandem copies of an HRE in response to hypoxia could be visualized on in vivo SPECT imaging. The results of this study will help in the understanding and assessment of

A Luciferase-Expressing Leishmania braziliensis Line That Leads to Sustained Skin Lesions in BALB/c Mice and Allows Monitoring of Miltefosine Treatment Outcome.

Directory of Open Access Journals (Sweden)

Adriano C Coelho

2016-05-01

Full Text Available Leishmania braziliensis is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying L. braziliensis pathogenesis or response to chemotherapy in vivo due to the natural resistance of most mouse strains to infection with these parasites. The aim of this work was to develop an experimental set up that could be used to assess drug efficacy against L. braziliensis. The model was tested using miltefosine.A L. braziliensis line, originally isolated from a cutaneous leishmaniasis patient, was passaged repeatedly in laboratory rodents and further genetically manipulated to express luciferase. Once collected from a culture of parasites freshly transformed from amastigotes, 106 wild type or luciferase-expressing stationary phase promastigotes were inoculated subcutaneously in young BALB/c mice or golden hamsters. In both groups, sustained cutaneous lesions developed at the site of inoculation, no spontaneous self- healing being observed 4 months post-inoculation, if left untreated. Compared to the wild type line features, no difference was noted for the luciferase-transgenic line. Infected animals were treated with 5 or 15 mg/kg/day miltefosine orally for 15 days. At the end of treatment, lesions had regressed and parasites were not detected. However, relapses were observed in animals treated with both doses of miltefosine.Here we described experimental settings for a late-healing model of cutaneous leishmaniasis upon inoculation of a luciferase-expressing L. braziliensis line that can be applied to drug development projects. These settings allowed the monitoring of the transient efficacy of a short-term miltefosine administration.

Identification and Functional Analysis of Gene Regulatory Sequences Interacting with Colorectal Tumor Suppressors

DEFF Research Database (Denmark)

Dahlgaard, Katja; Troelsen, Jesper

2018-01-01

Several tumor suppressors possess gene regulatory activity. Here, we describe how promoter and promoter/enhancer reporter assays can be used to characterize a colorectal tumor suppressor proteins’ gene regulatory activity of possible target genes. In the first part, a bioinformatic approach...... of the quick and efficient In-Fusion cloning method, and how to carry out transient transfections of Caco-2 colon cancer cells with the produced luciferase reporter plasmids using polyethyleneimine (PEI). A plan describing how to set up and carry out the luciferase expression assay is presented. The luciferase...... to identify relevant gene regulatory regions of potential target genes is presented. In the second part, it is demonstrated how to prepare and carry out the functional assay. We explain how to clone the bioinformatically identified gene regulatory regions into luciferase reporter plasmids by the use...

Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta.

Science.gov (United States)

Hermanrud, Christina; Ryner, Malin; Luft, Thomas; Jensen, Poul Erik; Ingenhoven, Kathleen; Rat, Dorothea; Deisenhammer, Florian; Sørensen, Per Soelberg; Pallardy, Marc; Sikkema, Dan; Bertotti, Elisa; Kramer, Daniel; Creeke, Paul; Fogdell-Hahn, Anna

2016-03-01

Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the "Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk" (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519 ng/mL for LUCIMU, 814 ng/mL for LUCRH, and 320 ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low

PET/CT imaging of human somatostatin receptor 2 (hsstr2) as reporter gene for gene therapy

International Nuclear Information System (INIS)

Hofmann, M.; Gazdhar, A.; Weitzel, T.; Schmid, R.; Krause, T.

2006-01-01

Localized information on region-selective gene expression in small animals is widely obtained by use of reporter genes inducing light emission. Using these reporter genes for imaging deep inside the human body fluorescent probes are hindered by attenuation, scattering and possible fluorescence quenching. This can be overcome by use of radio-peptide receptors as reporter genes. Therefore, the feasibility of the somatostatin receptor 2 expression vector system for expression imaging was checked against a control vector containing luciferase gene. For in vivo transduction of vector DNA into the rat forelimb muscles the in vivo electroporation technique was chosen because of its high regio-selectivity. The gene expression was imaged by high-sensitive CCD camera (luciferase activity) and by PET/CT using a Ga-68-DOTATOC as radio peptide probe. The relative sstr2 expression was enhanced by gene transduction at maximum to a factor of 15. The PET/CT images could be fully quantified. The above demonstrated feasibility of radio-peptide PET/CT reporter gene imaging may serve in the future as a tool for full quantitative understanding of regional gene expression, especially in large animals and humans

PET/CT imaging of human somatostatin receptor 2 (hsstr2) as reporter gene for gene therapy

Energy Technology Data Exchange (ETDEWEB)

Hofmann, M. [Molecular Imaging and Therapy Group (MIT-Bern), Clinic of Nuclear Medicine, Inselspital, Medical School Bern (Switzerland)]. E-mail: Michael.Hofmann@insel.ch; Gazdhar, A. [Division of Pulmonary Medicine, University Hospital Bern (Switzerland); Weitzel, T. [Molecular Imaging and Therapy Group (MIT-Bern), Clinic of Nuclear Medicine, Inselspital, Medical School Bern (Switzerland); Schmid, R. [Division of Thoracic Surgery, University Hospital Bern (Switzerland); Krause, T. [Molecular Imaging and Therapy Group (MIT-Bern), Clinic of Nuclear Medicine, Inselspital, Medical School Bern (Switzerland)

2006-12-20

Localized information on region-selective gene expression in small animals is widely obtained by use of reporter genes inducing light emission. Using these reporter genes for imaging deep inside the human body fluorescent probes are hindered by attenuation, scattering and possible fluorescence quenching. This can be overcome by use of radio-peptide receptors as reporter genes. Therefore, the feasibility of the somatostatin receptor 2 expression vector system for expression imaging was checked against a control vector containing luciferase gene. For in vivo transduction of vector DNA into the rat forelimb muscles the in vivo electroporation technique was chosen because of its high regio-selectivity. The gene expression was imaged by high-sensitive CCD camera (luciferase activity) and by PET/CT using a Ga-68-DOTATOC as radio peptide probe. The relative sstr2 expression was enhanced by gene transduction at maximum to a factor of 15. The PET/CT images could be fully quantified. The above demonstrated feasibility of radio-peptide PET/CT reporter gene imaging may serve in the future as a tool for full quantitative understanding of regional gene expression, especially in large animals and human000.

Rescue the failed half-ZFN by a sensitive mammalian cell-based luciferase reporter system.

Directory of Open Access Journals (Sweden)

Weifeng Zhang

Full Text Available ZFN technology is a powerful research tool and has been used for genome editing in cells lines, animals and plants. The generation of functional ZFNs for particular targets in mammalian genome is still challenging for an average research group. The modular-assembly method is relatively fast, easy-to-practice but has a high failure rate. Some recent studies suggested that a ZFP with low binding activity might be able to form a working ZFN pair with another binding active half-ZFP. In order to unveil the potential ZFP candidates among those with low binding activities, this paper established a highly sensitive mammalian cell-based transcriptional reporter system to assess the DNA binding activities of ZFPs by inserting multiple copies of ZFN target sequence fragment (TSF of an interested gene (e. g., hPGRN or hVEGF. Our results showed that this system increased the screening sensitivity up to 50-fold and markedly amplified the differences in the binding activities between different ZFPs. We also found that the targeted chromosomal gene repair efficiency of each hPGRN or hVEGF ZFN pair was in proportion with the combination of the binding activities of the ZFL (Left zinc finger and ZFR (Right zinc finger. A hPGRN ZFR with low binding ability was able to form a biological active ZFN if combined with a hPGRN ZFL with relatively high binding ability. Lastly, site-specific genome editing by hPGRN ZFNs generated by this system was confirmed by sequencing, and the PGRN knock-out cell line showed significantly decreased cell growth compared with the control. Our system will provide a valuable tool for further optimizing the nucleases with regard to specificity and cytotoxicity.

Development of a Positive-readout Mouse Model of siRNA Pharmacodynamics

Directory of Open Access Journals (Sweden)

Mark Stevenson

2013-01-01

Full Text Available Development of RNAi-based therapeutics has the potential to revolutionize treatment options for a range of human diseases. However, as with gene therapy, a major barrier to progress is the lack of methods to achieve and measure efficient delivery for systemic administration. We have developed a positive-readout pharmacodynamic transgenic reporter mouse model allowing noninvasive real-time assessment of siRNA activity. The model combines a luciferase reporter gene under the control of regulatory elements from the lac operon of Escherichia coli. Introduction of siRNA targeting lac repressor results in increased luciferase expression in cells where siRNA is biologically active. Five founder luciferase-expressing and three founder Lac-expressing lines were generated and characterized. Mating of ubiquitously expressing luciferase and lac lines generated progeny in which luciferase expression was significantly reduced compared with the parental line. Administration of isopropyl β-D-1-thiogalactopyranoside either in drinking water or given intraperitoneally increased luciferase expression in eight of the mice examined, which fell rapidly when withdrawn. Intraperitoneal administration of siRNA targeting lac in combination with Lipofectamine 2000 resulted in increased luciferase expression in the liver while control nontargeting siRNA had no effect. We believe a sensitive positive readout pharmacodynamics reporter model will be of use to the research community in RNAi-based vector development.

Activity report

International Nuclear Information System (INIS)

1990-11-01

The Department of Physics and Measurement Technology, Biology and Chemistry (IFM) presents every year a progress report containing a brief description of activities in research and education within the department. The report is intended as an information for colleagues and institutions. The present report contains activities for the academic year July 1989 to June 1990

Luminopsins integrate opto- and chemogenetics by using physical and biological light sources for opsin activation

Science.gov (United States)

Berglund, Ken; Clissold, Kara; Li, Haofang E.; Wen, Lei; Park, Sung Young; Gleixner, Jan; Klein, Marguerita E.; Lu, Dongye; Barter, Joseph W.; Rossi, Mark A.; Augustine, George J.; Yin, Henry H.; Hochgeschwender, Ute

2016-01-01

Luminopsins are fusion proteins of luciferase and opsin that allow interrogation of neuronal circuits at different temporal and spatial resolutions by choosing either extrinsic physical or intrinsic biological light for its activation. Building on previous development of fusions of wild-type Gaussia luciferase with channelrhodopsin, here we expanded the utility of luminopsins by fusing bright Gaussia luciferase variants with either channelrhodopsin to excite neurons (luminescent opsin, LMO) or a proton pump to inhibit neurons (inhibitory LMO, iLMO). These improved LMOs could reliably activate or silence neurons in vitro and in vivo. Expression of the improved LMO in hippocampal circuits not only enabled mapping of synaptic activation of CA1 neurons with fine spatiotemporal resolution but also could drive rhythmic circuit excitation over a large spatiotemporal scale. Furthermore, virus-mediated expression of either LMO or iLMO in the substantia nigra in vivo produced not only the expected bidirectional control of single unit activity but also opposing effects on circling behavior in response to systemic injection of a luciferase substrate. Thus, although preserving the ability to be activated by external light sources, LMOs expand the use of optogenetics by making the same opsins accessible to noninvasive, chemogenetic control, thereby allowing the same probe to manipulate neuronal activity over a range of spatial and temporal scales. PMID:26733686

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

Science.gov (United States)

Mofford, David M; Adams, Spencer T; Reddy, G S Kiran Kumar; Reddy, Gadarla Randheer; Miller, Stephen C

2015-07-15

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

High-throughput screening of effective siRNAs using luciferase-linked chimeric mRNA.

Directory of Open Access Journals (Sweden)

Shen Pang

Full Text Available The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.

Estrogen receptor and PI3K/Akt signaling pathway involvement in S-(-equol-induced activation of Nrf2/ARE in endothelial cells.

Directory of Open Access Journals (Sweden)

Ting Zhang

Full Text Available S-(-equol, a natural product of the isoflavone daidzein, has been reported to offer cytoprotective effects with respect to the cardiovascular system, but how this occurs is unclear. Interestingly, S-(-equol is produced by the human gut, suggesting a role in physiological processes. We report that treatment of human umbilical vein endothelial cells and EA.hy926 cells with S-(-equol induces ARE-luciferase reporter gene activity that is dose and time dependent. S-(-equol (10-250 nM increases nuclear factor-erythroid 2-related factor 2 (Nrf2 as well as gene products of Nrf2 target genes heme oxygenase-1 (HO-1 and NAD(PH (nicotinamide-adenine-dinucleotide-phosphate quinone oxidoreductase 1 (NQO1. Endothelial cells transfected with an HA-Nrf2 expression plasmid had elevated HA-Nrf2, HO-1, and NQO1 in response to S-(-equol exposure. S-(-equol treatment affected Nrf2 mRNA only slightly but significantly increased HO-1 and NQO1 mRNA. The pretreatment of cells with specific ER inhibitors or PI3K/Akt (ICI182,780 and LY294002 increased Nrf2, HO-1, and NQO1 protein, impaired nuclear translocation of HA-Nrf2, and decreased ARE-luciferase activity. Identical experiments were conducted with daidzein, which had effects similar to S-(-equol. In addition, DPN treatment (an ERβ agonist induced the ARE-luciferase reporter gene, promoting Nrf2 nuclear translocation. Cell pretreatment with an ERβ antagonist (PHTPP impaired S-(-equol-induced Nrf2 activation. Pre-incubation of cells followed by co-treatment with S-(-equol significantly improved cell survival in response to H2O2 or tBHP and reduced apoptotic and TUNEL-positively-stained cells. Notably, the ability of S-(-equol to protect against H2O2-induced cell apoptosis was attenuated in cells transfected with an siRNA against Nrf2. Thus, beneficial effects of S-(-equol with respect to cytoprotective antioxidant gene activation may represent a novel strategy to prevent and treat cardiovascular diseases.

Ethanol extract of Portulaca Oleracea L. reduced the carbon tetrachloride induced liver injury in mice involving enhancement of NF-κB activity

Science.gov (United States)

Shi, Hongguang; Liu, Xuefeng; Tang, Gusheng; Liu, Haiyan; Zhang, Yinghui; Zhang, Bo; Zhao, Xuezhi; Wang, Wanyin

2014-01-01

Acute hepatic injury causes high morbidity and mortality world-wide. Management of severe acute hepatic failure continues to be one of the most challenging problems in clinical medicine. In present study, carbon tetrachloride (CCl4) was used to induce acute liver damage in mice and the protective effects of ethanol extract of Portulaca Oleracea L. (PO) were examined. The aminotransferase activities were biochemical estimated and the liver damage was tested by morphological histological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The role of PO on the activity of NF-κB was determined by luciferase reporter gene assay and immunohistochemistry. The level of p-p65 was tested by western blot. Our results showed that PO administration on mice would decrease the serum aminotransferase level and reduced the liver histological damage. We also found that nuclear translocation of p65 was enhanced in liver tissues of mice treated with PO compared with control animals. In addition, in cultured hepatic cells, PO increased the NF-κB luciferase reporter gene activity and upregulated the level of phosphorylation of p65, but had no effects on mice liver SOD activity and MDA level. Collectively, PO attenuated CCl4 induced mice liver damage by enhancement of NF-κB activity. PMID:25628785

Inhibition of metastatic tumor growth in mouse lung by repeated administration of polyethylene glycol-conjugated catalase: quantitative analysis with firefly luciferase-expressing melanoma cells.

Science.gov (United States)

Hyoudou, Kenji; Nishikawa, Makiya; Umeyama, Yukari; Kobayashi, Yuki; Yamashita, Fumiyoshi; Hashida, Mitsuru

2004-11-15

To develop a novel and effective approach to inhibit tumor metastasis based on controlled delivery of catalase, we first evaluated the characteristics of the disposition and proliferation of tumor cells. Then, we examined the effects of polyethylene glycol-conjugated catalase (PEG-catalase) on tumor metastasis. On the basis of the results obtained, PEG-catalase was repetitively administered to completely suppress the growth of tumor cells. Murine melanoma B16-BL6 cells were stably transfected with firefly luciferase gene to obtain B16-BL6/Luc cells. These cells were injected intravenously into syngeneic C57BL/6 mice. PEG-catalase was injected intravenously, and the effect was evaluated by measuring the luciferase activity as the indicator of the number of tumor cells. At 1 hour after injection of B16-BL6/Luc cells, 60 to 90% of the injected cells were recovered in the lung. The numbers decreased to 2 to 4% at 24 hours, then increased. An injection of PEG-catalase just before inoculation significantly reduced the number of tumor cells at 24 hours. Injection of PEG-catalase at 1 or 3 days after inoculation was also effective in reducing the cell numbers. Daily dosing of PEG-catalase greatly inhibited the proliferation and the number assayed at 14 days after inoculation was not significantly different from the minimal number observed at 1 day, suggesting that the growth had been markedly suppressed by the treatment. These findings indicate that sustained catalase activity in the blood circulation can prevent the multiple processes of tumor metastasis in the lung, which could lead to a state of tumor dormancy.

A PXR reporter gene assay in a stable cell culture system: CYP3A4 and CYP2B6 induction by pesticides.

Science.gov (United States)

Lemaire, Géraldine; de Sousa, Georges; Rahmani, Roger

2004-12-15

A stable hepatoma cell line expressing the human pregnane X receptor (hPXR) and the cytochrome P4503A4 (CYP3A4) distal and proximal promoters plus the luciferase reporter gene was developed to assess the ability of several xenobiotic agents to induce CYP3A4 and CYP2B6. After selection for neomycin resistance, one clone, displaying high luciferase activity in response to rifampicin (RIF), was isolated and the stable expression of hPXR was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Dose-response curves were generated by treating these cells with increasing concentrations of RIF, phenobarbital (PB), clotrimazole (CLOT) or 5beta-pregnane-3,20-dione (5beta-PREGN). The effective concentrations for half maximal response (EC50) were determined for each of these compounds. RIF was the most effective compound, with maximal luciferase activity induced at 10 microM. The agonist activities of PXR-specific inducers measured using our stable model were consistent with those measured in transient transfectants. The abilities of organochlorine (OC), organophosphate (OP) and pyrethroid pesticides (PY) to activate hPXR were also assessed and found to be consistent with the abilities of these compounds to induce CYP3A4 and CYP2B6 in primary culture of human hepatocytes. These results suggest that CYP3A4 and CYP2B6 regulation through PXR activation by persistent pesticides may have an impact on the metabolism of xenobiotic agents and endogenous steroid hormones. Our model provides a useful tool for studying hPXR activation and for identifying agents capable of inducing CYP3A4 and CYP2B6.

Metastasizing, Luciferase Transduced MAT-Lu Rat Prostate Cancer Models: Follow up of Bolus and Metronomic Therapy with Doxorubicin as Model Drug

International Nuclear Information System (INIS)

Jantscheff, Peter; Esser, Norbert; Geipel, Andreas; Woias, Peter; Ziroli, Vittorio; Goldschmidtboing, Frank; Massing, Ulrich

2011-01-01

The most fatal outcomes of prostate carcinoma (PCa) result from hormone-refractory variants of the tumor, especially from metastatic spread rather than from primary tumor burden. The goal of the study was to establish and apply rat MAT-Lu prostate cancer tumor models for improved non-invasive live follow up of tumor growth and metastasis by in vivo bioluminescence. We established luciferase transduced MAT-Lu rat PCa cells and studied tumor growth and metastatic processes in an ectopic as well as orthotopic setting. An intravenous bolus treatment with doxorubicin was used to demonstrate the basic applicability of in vivo imaging to follow up therapeutic intervention in these models. In vitro analysis of tissue homogenates confirmed major metastatic spread of subcutaneous tumors into the lung. Our sensitive method, however, for the first time detects metastasis also in lymph node (11/24), spleen (3/24), kidney (4/24), liver (5/24), and bone tissue (femur or spinal cord - 5/20 and 12/20, respectively). Preliminary data of orthotopic implantation (three animals) showed metastatic invasion to investigated organs in all animals but with varying preference (e.g., to lymph nodes). Intravenous bolus treatment of MAT-Lu PCa with doxorubicin reduced subcutaneous tumor growth by about 50% and the number of animals affected by metastatic lesions in lymph nodes (0/4), lung (3/6) or lumbar spine (0/2), as determined by in vivo imaging and in vitro analysis. Additionally, the possible applicability of the luciferase transduced MAT-Lu model(s) to study basic principles of metronomic therapies via jugular vein catheter, using newly established active microport pumping systems, is presented

Metastasizing, Luciferase Transduced MAT-Lu Rat Prostate Cancer Models: Follow up of Bolus and Metronomic Therapy with Doxorubicin as Model Drug

Energy Technology Data Exchange (ETDEWEB)

Jantscheff, Peter, E-mail: jantscheff@tumorbio.uni-freiburg.de [Tumour Biology Center, Clinical Research, Department Lipids & Liposomes, Breisacher Str.117, D-79106 Freiburg (Germany); Esser, Norbert [ProQinase GmbH, Breisacher Str. 117, D-79106 Freiburg (Germany); Geipel, Andreas; Woias, Peter [Laboratory for Design of Microsystems, Department of Microsystems Engineering (IMTEK), Georges-Köhler-Allee 106, D-79110 Freiburg (Germany); Ziroli, Vittorio [Tumour Biology Center, Clinical Research, Department Lipids & Liposomes, Breisacher Str.117, D-79106 Freiburg (Germany); Goldschmidtboing, Frank [Laboratory for Design of Microsystems, Department of Microsystems Engineering (IMTEK), Georges-Köhler-Allee 106, D-79110 Freiburg (Germany); Massing, Ulrich [Tumour Biology Center, Clinical Research, Department Lipids & Liposomes, Breisacher Str.117, D-79106 Freiburg (Germany)

2011-06-17

The most fatal outcomes of prostate carcinoma (PCa) result from hormone-refractory variants of the tumor, especially from metastatic spread rather than from primary tumor burden. The goal of the study was to establish and apply rat MAT-Lu prostate cancer tumor models for improved non-invasive live follow up of tumor growth and metastasis by in vivo bioluminescence. We established luciferase transduced MAT-Lu rat PCa cells and studied tumor growth and metastatic processes in an ectopic as well as orthotopic setting. An intravenous bolus treatment with doxorubicin was used to demonstrate the basic applicability of in vivo imaging to follow up therapeutic intervention in these models. In vitro analysis of tissue homogenates confirmed major metastatic spread of subcutaneous tumors into the lung. Our sensitive method, however, for the first time detects metastasis also in lymph node (11/24), spleen (3/24), kidney (4/24), liver (5/24), and bone tissue (femur or spinal cord - 5/20 and 12/20, respectively). Preliminary data of orthotopic implantation (three animals) showed metastatic invasion to investigated organs in all animals but with varying preference (e.g., to lymph nodes). Intravenous bolus treatment of MAT-Lu PCa with doxorubicin reduced subcutaneous tumor growth by about 50% and the number of animals affected by metastatic lesions in lymph nodes (0/4), lung (3/6) or lumbar spine (0/2), as determined by in vivo imaging and in vitro analysis. Additionally, the possible applicability of the luciferase transduced MAT-Lu model(s) to study basic principles of metronomic therapies via jugular vein catheter, using newly established active microport pumping systems, is presented.

Metastasizing, Luciferase Transduced MAT‑Lu Rat Prostate Cancer Models: Follow up of Bolus and Metronomic Therapy with Doxorubicin as Model Drug

Directory of Open Access Journals (Sweden)

Peter Woias

2011-06-01

Full Text Available The most fatal outcomes of prostate carcinoma (PCa result from hormone-refractory variants of the tumor, especially from metastatic spread rather than from primary tumor burden. The goal of the study was to establish and apply rat MAT-Lu prostate cancer tumor models for improved non-invasive live follow up of tumor growth and metastasis by in vivo bioluminescence. We established luciferase transduced MAT-Lu rat PCa cells and studied tumor growth and metastatic processes in an ectopic as well as orthotopic setting. An intravenous bolus treatment with doxorubicin was used to demonstrate the basic applicability of in vivo imaging to follow up therapeutic intervention in these models. In vitro analysis of tissue homogenates confirmed major metastatic spread of subcutaneous tumors into the lung. Our sensitive method, however, for the first time detects metastasis also in lymph node (11/24, spleen (3/24, kidney (4/24, liver (5/24, and bone tissue (femur or spinal cord - 5/20 and 12/20, respectively. Preliminary data of orthotopic implantation (three animals showed metastatic invasion to investigated organs in all animals but with varying preference (e.g., to lymph nodes. Intravenous bolus treatment of MAT-Lu PCa with doxorubicin reduced subcutaneous tumor growth by about 50% and the number of animals affected by metastatic lesions in lymph nodes (0/4, lung (3/6 or lumbar spine (0/2, as determined by in vivo imaging and in vitro analysis. Additionally, the possible applicability of the luciferase transduced MAT-Lu model(s to study basic principles of metronomic therapies via jugular vein catheter, using newly established active microport pumping systems, is presented.

Relationship of the luminous bacterial symbiont of the Caribbean flashlight fish, Kryptophanaron alfredi (family Anomalopidae) to other luminous bacteria based on bacterial luciferase (luxA) genes.

Science.gov (United States)

Haygood, M G

1990-01-01

Flashlight fishes (family Anomalopidae) have light organs that contain luminous bacterial symbionts. Although the symbionts have not yet been successfully cultured, the luciferase genes have been cloned directly from the light organ of the Caribbean species, Kryptophanaron alfredi. The goal of this project was to evaluate the relationship of the symbiont to free-living luminous bacteria by comparison of genes coding for bacterial luciferase (lux genes). Hybridization of a lux AB probe from the Kryptophanaron alfredi symbiont to DNAs from 9 strains (8 species) of luminous bacteria showed that none of the strains tested had lux genes highly similar to the symbiont. The most similar were a group consisting of Vibrio harveyi, Vibrio splendidus and Vibrio orientalis. The nucleotide sequence of the luciferase alpha subunit gene luxA) of the Kryptophanaron alfredi symbiont was determined in order to do a more detailed comparison with published luxA sequences from Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi. The hybridization results, sequence comparisons and the mol% G + C of the Kryptophanaron alfredi symbiont luxA gene suggest that the symbiont may be considered as a new species of luminous Vibrio related to Vibrio harveyi.

Phytol directly activates peroxisome proliferator-activated receptor α (PPARα) and regulates gene expression involved in lipid metabolism in PPARα-expressing HepG2 hepatocytes

International Nuclear Information System (INIS)

Goto, Tsuyoshi; Takahashi, Nobuyuki; Kato, Sota; Egawa, Kahori; Ebisu, Shogo; Moriyama, Tatsuya; Fushiki, Tohru; Kawada, Teruo

2005-01-01

The peroxisome proliferator-activated receptor (PPAR) is one of the indispensable transcription factors for regulating lipid metabolism in various tissues. In our screening for natural compounds that activate PPAR using luciferase assays, a branched-carbon-chain alcohol (a component of chlorophylls), phytol, has been identified as a PPARα-specific activator. Phytol induced the increase in PPARα-dependent luciferase activity and the degree of in vitro binding of a coactivator, SRC-1, to GST-PPARα. Moreover, the addition of phytol upregulated the expression of PPARα-target genes at both mRNA and protein levels in PPARα-expressing HepG2 hepatocytes. These findings indicate that phytol is functional as a PPARα ligand and that it stimulates the expression of PPARα-target genes in intact cells. Because PPARα activation enhances circulating lipid clearance, phytol may be important in managing abnormalities in lipid metabolism

Adaptation of Clostridium difficile toxin A for use as a protein translocation system

Energy Technology Data Exchange (ETDEWEB)

Kern, Stephanie M. [Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202 (United States); Feig, Andrew L., E-mail: afeig@chem.wayne.edu [Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202 (United States)

2011-02-25

Research highlights: {yields} Catalytic domain of TcdA was replaced by a luciferase reporter. {yields} Each functional domain retains activity in the context of the fusion protein. {yields} We provide evidence that reporter proteins are delivered into vero cells. {yields} System releases cargo into the cytosol, providing a powerful new biotechnology tool. -- Abstract: A cellular delivery system is a useful biotechnology tool, with many possible applications. Two derivatives of Clostridium difficile toxin A (TcdA) have been constructed (GFP-TcdA and Luc-TcdA), by fusing reporter genes to functional domains of TcdA, and evaluated for their ability to translocate their cargo into mammalian cells. The cysteine protease and receptor binding domains of TcdA have been examined and found to be functional when expressed in the chimeric construct. Whereas GFP failed to internalize in the context of the TcdA fusion, significant cellular luciferase activity was detected in vero cell lysates after treatment with Luc-TcdA. Treatment with bafilomycin A1, which inhibits endosomal acidification, traps the luciferase activity within endosomes. To further understand these results, clarified lysates were subjected to molecular weight sieving, demonstrating that active luciferase was released from Luc-TcdA after translocation and internal processing.

Adaptation of Clostridium difficile toxin A for use as a protein translocation system

International Nuclear Information System (INIS)

Kern, Stephanie M.; Feig, Andrew L.

2011-01-01

Research highlights: → Catalytic domain of TcdA was replaced by a luciferase reporter. → Each functional domain retains activity in the context of the fusion protein. → We provide evidence that reporter proteins are delivered into vero cells. → System releases cargo into the cytosol, providing a powerful new biotechnology tool. -- Abstract: A cellular delivery system is a useful biotechnology tool, with many possible applications. Two derivatives of Clostridium difficile toxin A (TcdA) have been constructed (GFP-TcdA and Luc-TcdA), by fusing reporter genes to functional domains of TcdA, and evaluated for their ability to translocate their cargo into mammalian cells. The cysteine protease and receptor binding domains of TcdA have been examined and found to be functional when expressed in the chimeric construct. Whereas GFP failed to internalize in the context of the TcdA fusion, significant cellular luciferase activity was detected in vero cell lysates after treatment with Luc-TcdA. Treatment with bafilomycin A1, which inhibits endosomal acidification, traps the luciferase activity within endosomes. To further understand these results, clarified lysates were subjected to molecular weight sieving, demonstrating that active luciferase was released from Luc-TcdA after translocation and internal processing.

DNA Nanoparticles: Detection of Long-Term Transgene Activity in Brain using Bioluminescence Imaging

Directory of Open Access Journals (Sweden)

David M. Yurek

2011-09-01

Full Text Available In this study, we used bioluminescence imaging (BLI to track long-term transgene activity following the transfection of brain cells using a nonviral gene therapy technique. Formulations of deoxyribonucleic acid (DNA combined with 30-mer lysine polymers (substituted with 10 kDa polyethylene glycol form nanoparticles that transfect brain cells in vivo and produce transgene activity. Here we show that a single intracerebral injection of these DNA nanoparticles (DNPs into the rat cortex, striatum, or substantia nigra results in long-term and persistent luciferase transgene activity over an 8- to 11-week period as evaluated by in vivo BLI analysis, and single injections of DNPs into the mouse striatum showed stable luciferase transgene activity for 1 year. Compacted DNPs produced in vivo signals 7- to 34-fold higher than DNA alone. In contrast, ex vivo BLI analysis, which is subject to less signal quenching from surrounding tissues, demonstrated a DNP to DNA alone ratio of 76- to 280-fold. Moreover, the ex vivo BLI analysis confirmed that signals originated from the targeted brain structures. In summary, BLI permits serial analysis of luciferase transgene activity at multiple brain locations following gene transfer with DNPs. Ex vivo analysis may permit more accurate determination of relative activities of gene transfer vectors.

miR-582-5p is upregulated in patients with active tuberculosis and inhibits apoptosis of monocytes by targeting FOXO1.

Science.gov (United States)

Liu, Yanhua; Jiang, Jing; Wang, Xinjing; Zhai, Fei; Cheng, Xiaoxing

2013-01-01

Macrophage apoptosis is a host innate defense mechanism against tuberculosis (TB). In this study, we found that percentage of apoptotic cells in peripheral blood monocytes from patients with active TB was lower than that from healthy controls (pmicroRNAs can modulate apoptosis of monocytes, we investigated differentially expressed microRNAs in patients with active TB. miR-582-5p was mainly expressed in monocytes and was upregulated in patients with active TB. The apoptotic percentage of THP-1 cells transfected with miR-582-5p mimics was significantly lower than those transfected with negative control of microRNA mimics (pmicroRNA mimics were transfected into THP-1 cells. RT-PCR and western blot analysis showed that the miR-582-5p could suppress both FOXO1 mRNA and protein expression. Co-transfection of miR-582-5p and FOXO1 3'UTR-luciferase reporter vector into cells demonstrated that significant decrease in luciferase activity was only found in reporter vector that contained a wild type sequence of FOXO1 3'UTR, suggesting that miR-582-5p could directly target FOXO1. In conclusion, miR-582-5p inhibited apoptosis of monocytes by down-regulating FOXO1 expression and might play an important role in regulating anti-M. tuberculosis directed immune responses.

Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1).

Science.gov (United States)

Ahmad, Muhammad Khairi; Tabana, Yasser M; Ahmed, Mowaffaq Adam; Sandai, Doblin Anak; Mohamed, Rafeezul; Ismail, Ida Shazrina; Zulkiflie, Nurulisa; Yunus, Muhammad Amir

2017-12-01

A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones. Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing. NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.

Environmental Application of Reporter-Genes Based Biosensors for Chemical Contamination Screening

Directory of Open Access Journals (Sweden)

Matejczyk Marzena

2014-12-01

Full Text Available The paper presents results of research concerning possibilities of applications of reporter-genes based microorganisms, including the selective presentation of defects and advantages of different new scientific achievements of methodical solutions in genetic system constructions of biosensing elements for environmental research. The most robust and popular genetic fusion and new trends in reporter genes technology – such as LacZ (β-galactosidase, xylE (catechol 2,3-dioxygenase, gfp (green fluorescent proteins and its mutated forms, lux (prokaryotic luciferase, luc (eukaryotic luciferase, phoA (alkaline phosphatase, gusA and gurA (β-glucuronidase, antibiotics and heavy metals resistance are described. Reporter-genes based biosensors with use of genetically modified bacteria and yeast successfully work for genotoxicity, bioavailability and oxidative stress assessment for detection and monitoring of toxic compounds in drinking water and different environmental samples, surface water, soil, sediments.

Evaluation of the Luciferase Assay-Based In Vitro Elicitation Test for Serum IgE

Directory of Open Access Journals (Sweden)

Ryosuke Nakamura

2012-01-01

Results: The area under the ROC curves was highest in the EXiLE test (0.977, followed by CAP-FEIA (0.926 and degranulation (0.810. At an optimal cutoff range (1.648-1.876 calculated from the ROC curve of the EXiLE test, sensitivity and specificity were 0.944 and 0.917, respectively. A 95% positive predictive value was given at a cutoff level of 2.054 (fold increase in luciferase expression by logistic regression analysis. Conclusions: In contrast to in vivo tests, the EXiLE test appears to be a useful tool in diagnosing patients suspected of having IgE-dependent EW allergy without the risk of severe systemic reactions.

LANSCE Activity Report

Energy Technology Data Exchange (ETDEWEB)

Amy Robinson; Audrey Archuleta; Barbara Maes; Dan Strottman; Earl Hoffman; Garth Tietjen; Gene Farnum; Geoff Greene; Joyce Roberts; Ken Johnson; Paul Lewis; Roger Pynn; Stan Schriber; Steve Sterbenz; Steve Wender; Sue Harper

1999-02-01

The Los Alamos Neutron Science Center Activity Report describes scientific and technological progress and achievements in LANSCE Division during the period of 1995 to 1998. This report includes a message from the Division Director, an overview of LANSCE, sponsor overviews, research highlights, advanced projects and facility upgrades achievements, experimental and user program accomplishments, news and events, and a list of publications. The research highlights cover the areas of condensed-matter science and engineering, accelerator science, nuclear science, and radiography. This report also contains a compact disk that includes an overview, the Activity Report itself, LANSCE operations progress reports for 1996 and 1997, experiment reports from LANSCE users, as well as a search capability.

Activation of the Nrf2/ARE pathway via S-alkylation of cysteine 151 in the chemopreventive agent-sensor Keap1 protein by falcarindiol, a conjugated diacetylene compound

International Nuclear Information System (INIS)

Ohnuma, Tomokazu; Nakayama, Shinji; Anan, Eisaburo; Nishiyama, Takahito; Ogura, Kenichiro; Hiratsuka, Akira

2010-01-01

Under basal conditions, the interaction of the cytosolic protein Kelch-like ECH-associated protein 1 (Keap1) with the transcription factor nuclear factor-E2-related factor 2 (Nrf2) results in a low level of expression of cytoprotective genes whose promoter region contains the antioxidant response element (ARE). In response to oxidants and electrophiles, Nrf2 is stabilized and accumulates in the nucleus. The mechanism for this effect has been proposed to involve thiol-dependent modulation of Keap1, leading to loss of its ability to negatively regulate Nrf2. We previously reported that falcarindiol (heptadeca-1,9(Z)-diene-4,6-diyne-3,8-diol), which occurs in Apiaceae and the closely related Araliaceae plants, causes nuclear accumulation of Nrf2 and induces ARE-regulated enzymes. Here, we report the mechanism of Nrf2 induction by falcarindiol. NMR analysis revealed that the conjugated diacetylene carbons of falcarindiol acted as electrophilic moieties to form adducts with a cysteine (Cys) thiol. In addition, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and circular dichroism spectroscopy, it was demonstrated that falcarindiol alkylated Cys residues in Keap1 and altered the Keap1 secondary structure. Transfection studies using the purified Keap1 protein, a luciferase reporter construct, and an Nrf2-expressing plasmid indicated that the intact Keap1 protein suppressed Nrf2-mediated ARE-luciferase activity. On the other hand, the falcarindiol-alkylated Keap1 protein did not suppress such activity. Treatment of HEK293 cells overexpressing Keap1 with falcarindiol generated a high molecular weight (HMW) form of Keap1. Furthermore, the Cys151 residue in Keap1 was found to be uniquely required for not only the formation of HMW Keap1 but also an increase in ARE-luciferase activity by falcarindiol. Our results demonstrate that falcarindiol having conjugated diacetylene carbons covalently modifies the Cys151 residue in Keap1 and that the

Identification of 6-octadecynoic acid from a methanol extract of Marrubium vulgare L. as a peroxisome proliferator-activated receptor γ agonist

Energy Technology Data Exchange (ETDEWEB)

Ohtera, Anna; Miyamae, Yusaku; Nakai, Naomi [Graduate School of Biostudies, Kyoto University, Kyoto 606-8502 (Japan); Kawachi, Atsushi; Kawada, Kiyokazu; Han, Junkyu; Isoda, Hiroko [Alliance for Research on North Africa (ARENA), University of Tsukuba, Ibaraki 305-8572 (Japan); Faculty of Life and Environment, University of Tsukuba, Ibaraki 305-8572 (Japan); Neffati, Mohamed [Arid Zone Research Institute (IRA), Médenine 4119 (Tunisia); Akita, Toru; Maejima, Kazuhiro [Nippon Shinyaku CO., LTD., Kyoto 601-8550 (Japan); Masuda, Seiji; Kambe, Taiho [Graduate School of Biostudies, Kyoto University, Kyoto 606-8502 (Japan); Mori, Naoki; Irie, Kazuhiro [Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 (Japan); Nagao, Masaya, E-mail: mnagao@kais.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Kyoto 606-8502 (Japan)

2013-10-18

Highlights: •6-ODA, a rare fatty acid with a triple bond, was identified from Marrubium vulgare. •6-ODA was synthesized from petroselinic acid as a starting material. •6-ODA stimulated lipid accumulation in HSC-T6 and 3T3-L1 cells. •The first report of a fatty acid with a triple bond functioning as a PPARγ agonist. •This study sheds light on novel functions of a fatty acid with a triple bond. -- Abstract: 6-Octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified in the methanol extract of Marrubium vulgare L. as an agonist of peroxisome proliferator-activated receptor γ (PPARγ). Fibrogenesis caused by hepatic stellate cells is inhibited by PPARγ whose ligands are clinically used for the treatment of diabetes. Plant extracts of Marrubium vulgare L., were screened for activity to inhibit fibrosis in the hepatic stellate cell line HSC-T6 using Oil Red-O staining, which detects lipids that typically accumulate in quiescent hepatic stellate cells. A methanol extract with activity to stimulate accumulation of lipids was obtained. This extract was found to have PPARγ agonist activity using a luciferase reporter assay. After purification using several chromatographic methods, 6-ODA, a fatty acid with a triple bond, was identified as a candidate of PPARγ agonist. Synthesized 6-ODA and its derivative 9-octadecynoic acid (9-ODA), which both have a triple bond but in different positions, activated PPARγ in a luciferase reporter assay and increased lipid accumulation in 3T3-L1 adipocytes in a PPARγ-dependent manner. There is little information about the biological activity of fatty acids with a triple bond, and to our knowledge, this is the first report that 6-ODA and 9-ODA function as PPARγ agonists.

The Nrf1 CNC-bZIP protein is regulated by the proteasome and activated by hypoxia.

Science.gov (United States)

Chepelev, Nikolai L; Bennitz, Joshua D; Huang, Ting; McBride, Skye; Willmore, William G

2011-01-01

Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. We found that proteasomal inhibitors MG-132 and clasto-lactacystin-β-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status. © 2011 Chepelev et al.

Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

Directory of Open Access Journals (Sweden)

Huarong Guo

2012-09-01

Full Text Available p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase and pRL-CMV-luc (CMV promoter linked to Renilla luciferase into marine flatfish flounder gill (FG cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation, but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl phthalate (DEHP, a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner.

Xenoestrogenic and dioxin-like activity in blood of East Greenland polar bears (Ursus maritimus)

DEFF Research Database (Denmark)

Erdmann, Simon Erik; Dietz, Rune; Sonne, Christian

2013-01-01

chromatography (HPLC). ER mediated transactivity was determined using the ER luciferase reporter MVLN cell assay. The extracts were tested alone (XER) and together with 17β-estradiol (E2) as a physiological mimic (XERcomp). Dioxins and dioxin-like (DL) compounds were extracted by a combination of SPE...... and the Supelco Dioxin Prep System®. AhR mediated dioxin-like transactivity was determined using the AhR luciferase reporter Hepa 1.12cR cell assay. Agonistic ER transactivity was elicited by 19% of the samples, and a further increased E2 induced ER response was found for 52%, whereas 17% antagonized the E2...

The inhibition effect of 2,3,7,8-tetrachlorinated dibenzo-p-dioxin-induced aryl hydrocarbon receptor activation in human hepatoma cells with the treatment of cadmium chloride

International Nuclear Information System (INIS)

Chao, How-Ran; Tsou, Tsui-Chun; Chen, Hung-Ta; Chang, Eddy Essen; Tsai, Feng-Yuan; Lin, Ding-Yan; Chen, Fu-An; Wang, Ya-Fen

2009-01-01

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), considered as endocrine disruptors, tend to accumulate in fatty tissues. Dioxin-responsive element chemical activated luciferase gene expression assay (DRE-luciferase assay) has been recognized as a semi-quantitative method for screening dioxins for its fast and low-cost as compared with HRGC/HRMS. However, some problems with the bioassay, including specificity, detection variation resulted from different cleanup strategies, and uncertainty of false-negative or false-positive results, remain to be overcome. Cadmium is a prevalent environmental contaminant around the world. This study was aimed to examine the effects of cadmium on the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced activation of aryl hydrocarbon receptor (AhR)-mediated gene expression in human hepatoma cells (Huh7-DRE-Luc cells and Huh7 cells). Ethoxyresorufin-O-deethylase (EROD) and DRE-luciferase assay were employed to determine the enzyme activity of cytochrome P450 1A1 (CYP1A1) and activation of AhR, respectively. The results showed that Cd 2+ levels significantly inhibited the induction of TCDD-induced CYP1A1 and DRE luciferase activation in hepatoma cells. The 50% inhibited concentrations (IC 50 ) of CdCl 2 were 0.414 μM (95% confidence interval (C.I.): 0.230-0.602 μM) in Huh7-DRE-Luc cells and 23.2 μM (95% C.I.: 21.7-25.4 μM) in Huh7 cells. Accordingly, prevention of interference with non-dioxin-like compounds in a DRE-luciferase assay is of great importance in an extensive cleanup procedure.

Role of hypoxia-inducible factor-α in hepatitis-B-virus X protein-mediated MDR1 activation

International Nuclear Information System (INIS)

Han, Hyo-Kyung; Han, Chang Yeob; Cheon, Eun-Pa; Lee, Jaewon; Kang, Keon Wook

2007-01-01

The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1α (HIF-1α) and induced the nuclear translocation of C/EBPβ. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1α siRNA but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1α activation, and suggest HIF-1α for the therapeutic target of HBV-mediated chemoresistance

Activity report 1977

International Nuclear Information System (INIS)

Krishnan, L.V.; Lee, S.M.; Sahoo, D.; Sreedharan, O.M.; Srinivasan, G.

1979-05-01

The Research and Development (R and D) activities of the Reactor Research Centre, Kalpakkam, during the year 1977 are reported. The report is presented in the form of individual summaries of R and D activities most of which are connected with the FBTR project. These R and D activities deal with various aspects of the FBTR project such as reactor physics, reactor design, reactor engineering, fabrication, testing and quality assurance of reactor materials and components, reactor chemistry, reactor safety, reprocessing, systems analysis and instrumentation. (M.G.B.)

Molecular Imaging of the ATM Kinase Activity

Energy Technology Data Exchange (ETDEWEB)

Williams, Terence M. [Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States); Nyati, Shyam [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Ross, Brian D. [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Rehemtulla, Alnawaz, E-mail: alnawaz@umich.edu [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

2013-08-01

Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

Estrogen induction of telomerase activity through regulation of the mitogen-activated protein kinase (MAPK dependent pathway in human endometrial cancer cells.

Directory of Open Access Journals (Sweden)

Chunxiao Zhou

Full Text Available Given that prolonged exposure to estrogen and increased telomerase activity are associated with endometrial carcinogenesis, our objective was to evaluate the interaction between the MAPK pathway and estrogen induction of telomerase activity in endometrial cancer cells. Estradiol (E2 induced telomerase activity and hTERT mRNA expression in the estrogen receptor (ER-α positive, Ishikawa endometrial cancer cell line. UO126, a highly selective inhibitor of MEK1/MEK2, inhibited telomerase activity and hTERT mRNA expression induced by E2. Similar results were also found after transfection with ERK 1/2-specific siRNA. Treatment with E2 resulted in rapid phosphorylation of p44/42 MAPK and increased MAPK activity which was abolished by UO126. The hTERT promoter contains two estrogen response elements (EREs, and luciferase assays demonstrate that these EREs are activated by E2. Exposure to UO126 or ERK 1/2-specific siRNA in combination with E2 counteracted the stimulatory effect of E2 on luciferase activity from these EREs. These findings suggest that E2-induction of telomerase activity is mediated via the MAPK pathway in human endometrial cancer cells.

miR-130a activates apoptotic signaling through activation of caspase-8 in taxane-resistant prostate cancer cells.

Science.gov (United States)

Fujita, Yasunori; Kojima, Toshio; Kawakami, Kyojiro; Mizutani, Kosuke; Kato, Taku; Deguchi, Takashi; Ito, Masafumi

2015-10-01

The acquisition of drug resistance is one of the most malignant phenotypes of cancer and identification of its therapeutic target is a prerequisite for the development of novel therapy. MicroRNAs (miRNAs) have been implicated in various types of cancer and proposed as potential therapeutic targets for patients. In the present study, we aimed to identify miRNA that could serve as a therapeutic target for taxane-resistant prostate cancer. In order to identify miRNAs related to taxane-resistance, miRNA profiling was performed using prostate cancer PC-3 cells and paclitaxel-resistant PC-3 cell lines established from PC-3 cells. Microarray analysis of mRNA expression was also conducted to search for potential target genes of miRNA. Luciferase reporter assay was performed to examine miRNA binding to the 3'-UTR of target genes. The effects of ectopic expression of miRNA on cell growth, tubulin polymerization, drug sensitivity, and apoptotic signaling pathway were investigated in a paclitaxel-resistant PC-3 cell line. The expression of miR-130a was down-regulated in all paclitaxel-resistant cell lines compared with parental PC-3 cells. Based on mRNA microarray analysis and luciferase reporter assay, we identified SLAIN1 as a direct target gene for miR-130a. Transfection of a miR-130a precursor into a paclitaxel-resistant cell line suppressed cell growth and increased the sensitivity to paclitaxel. Lastly, ectopic expression of miR-130a did not affect the polymerized tubulin level, but activated apoptotic signaling through activation of caspase-8. Our results suggested that reduced expression of miR-130a may be involved in the paclitaxel-resistance and that miR-130a could be a therapeutic target for taxane-resistant prostate cancer patients. © 2015 Wiley Periodicals, Inc.

Antiosteoporotic compounds from seeds of Cuscuta chinensis.

Science.gov (United States)

Yang, Lijuan; Chen, Qianfeng; Wang, Fei; Zhang, Guolin

2011-05-17

The seeds of Cuscuta chinensis (Tu-Si-Zi, TSZ) have long been used for the treatment of osteoporosis in China and some Asian countries. The compounds in TSZ responsible for the antiosteoporotic activity are still poorly understood. The present study was designed to investigate the osteogenic compounds in TSZ, and to evaluate their antiosteoporotic effects in osteoblastic cells. Osteoblast-like UMR-106 cells were used for bioactivity-guided isolation of the active compounds. The activity of alkaline phosphatase (ALP) in UMR-106 cells was measured by p-nitrophenyl sodium phosphate assay. The proliferation of UMR-106 cells was assayed by Alamar-Blue method. Estrogenic activity of the extracts and isolated compounds was evaluated by activation of estrogen response element (ERE) luciferase reporter expression in HeLa cells co-transfected with human estrogen receptor subtypes (ERα or ERβ) expression vectors and 5×ERE luciferase reporter plasmid. Antiestrogenic activity of the extracts and isolated compounds were evaluated by activation of activator protein-1 (AP-1) luciferase reporter expression in HeLa cells co-transfected with human estrogen receptor subtypes (ERα or ERβ) expression vectors and 6×AP-1 luciferase reporter plasmid. ALP-guided fractionation led to the isolation of five known flavonoids, quercetin, kaempferol, isorhamnetin, hyperoside and astragalin from the crude ethanolic extract of TSZ. Further study showed that kaempferol and hyperoside significantly increased the ALP activity in UMR-106 cells. Astragalin promoted the proliferation of UMR-106 cells whereas other compounds had no such effect. The isolated compounds showed estrogenic activity but quercetin, kaempferol and isorhamnetin showed more potent ERβ agonist activity. However, compared with their ER agonist activity, only quercetin and kaempferol showed potent ER antagonist activity by activating ERα/β-mediated AP-1 reporter expression. Our findings validated the clinical use of TSZ in

Validation of the use of an artificial mitochondrial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial transgene expression.

Science.gov (United States)

Yamada, Yuma; Ishikawa, Takuya; Harashima, Hideyoshi

2017-08-01

Mitochondria have their own gene expression system that is independent of the nuclear system, and control cellular functions in cooperation with the nucleus. While a number of useful technologies for achieving nuclear transgene expression have been reported, only a few have focused on mitochondria. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. We designed and constructed pCMV-mtLuc (CGG) that contains a CMV promotor derived from Cytomegalovirus and an artificial mitochondrial genome with a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. Nluc luciferase activity measurements showed that the pCMV-mtLuc (CGG) efficiently produced the Nluc luciferase protein in human HeLa cells. Moreover, we optimized the mitochondrial transfection of pCMV-mtLuc (CGG) using a MITO-Porter system, a liposome-based carrier for mitochondrial delivery via membrane fusion. As a result, we found that transfection of pCMV-mtLuc (CGG) by MITO-Porter modified with the KALA peptide (cationic amphipathic cell-penetrating peptide) showed a high mitochondrial transgene expression. The developed mitochondrial transgene expression system represents a potentially useful tool for the fields of nanoscience and nanotechnology for controlling the intracellular microenvironment via the regulation of mitochondrial function and promises to open additional innovative research fields of study. Copyright © 2017 Elsevier Ltd. All rights reserved.

Activity report 1975

International Nuclear Information System (INIS)

Ananthakrishna, G.; Ramanathan, N.; Rodriguez, P.; Shankar Singh, R.; Venkataraman, S.

1977-08-01

The report covers a wide range of R and D activities in the Reactor Research Centre, Kalpakkam (India). The scientific and technical activities in various fields such as: reactor physics, reactor design, reactor engineering, design of sodium circuits, reactor fuel handling, electrical and instrumentation engineering for FBTR, data processing system, reactor operation studies, reactor construction, materials science, metallurgy, reprocessing, instrumentation, safety research and engineering services, have been reported in brief. (A.K.)

The regulation of transactivator of transcription on the activity of DNA-PKcs promoter

International Nuclear Information System (INIS)

Yang Tianyi; Zhang Shimeng; Qin Xia; Li Bing; Liu Xiaodan; Zhou Pingkun

2012-01-01

Objective: To explore the influence of human immunodeficiency virus transactivator of transcription (TAT) on the promoter activity of DNA dependent protein kinase catalytic subunit (DNA-PKcs). Methods: The truncated promoters of DNA-PKcs were cloned by PCR from the template DNA from HeLa genomic DNA, and the pGL3-basic-DNA-PKcs promoter reporter plasmids were constructed. The activity of DNA-PKcs promoters was detected by dual-luciferase reporter assay system. A Lac-repressor and Lacoperator based green fluorescent protein imaging system was used to assay the chromatin remodeling activity. Results: A series of reporter plasmids harboring the truncated promoters of DNA-PKcs from -939 bp to -1 bp were constructed. The sequence of -64 bp to-1 bp was identified as a critical element for the activity of DNA-PKes promoter. TAT can suppress the activity of DNA-PKcs promoter. TAT participates in the regulation of the large scale chromatin relaxation. Ionizing radiation attenuates the activity of TAT played in the chromatin remodeling. Conclusion: TAT represses the promoter activity of DNA repair protein DNA-PKcs, and also play a role of large scale chromatin remodeling which can te attenuated by ionizing radiation. (authors)

Optical Imaging of Tumor Hypoxia and Evaluation of Efficacy of a Hypoxia-Targeting Drug in Living Animals

Directory of Open Access Journals (Sweden)

Hiroshi Harada

2005-07-01

Full Text Available Solid tumors containing more hypoxic regions show a more malignant phenotype by increasing the expression of genes encoding angiogenic and metastatic factors. Hypoxia-inducible factor-1 (HIF-1 is a master transcriptional activator of such genes, and thus, imaging and targeting hypoxic tumor cells where HIF-1 is active are important in cancer therapy. In the present study, HIF-1 activity was monitored via an optical in vivo imaging system by using a luciferase reporter gene under the regulation of an artificial HIF-1-dependent promoter, 5HRE. To monitor tumor hypoxia, we isolated a stable reporter-transfectant, HeLa/5HRE-Luc, which expressed more than 100-fold luciferase in response to hypoxic stress, and observed bioluminescence from its xenografts. Immunohistochemical analysis of the xenografts with a hypoxia marker, pimonidazole, confirmed that the luciferase-expressing cells were hypoxic. Evaluation of the efficacy of a hypoxia-targeting prodrug, TOP3, using this optical imaging system revealed that hypoxic cells were significantly diminished by TOP3 treatment. Immunohistochemical analysis of the TOP3-treated xenografts confirmed that hypoxic cells underwent apoptosis and were removed after TOP3 treatment. These results demonstrate that this model system using the 5HRE-luciferase reporter construct provides qualitative information (hypoxic status of solid tumors and enables one to conveniently evaluate the efficacy of cancer therapy on hypoxia in malignant solid tumors.

Home Activities Reports

Data.gov (United States)

Department of Housing and Urban Development — This monthly report is an Excel spreadsheet, broken up by state. PJs can use this report to view activities with the 2012 or 2013 program year in IDIS, including;...

Homebuyer Activities Report

Data.gov (United States)

Department of Housing and Urban Development — This monthly report is an Excel spreadsheet. PJs can use this report to view homebuyer activities with the 2012 or 2013 program year in IDIS that are in final draw,...

Activation of Nrf2 is required for up-regulation of the π class of glutathione S-transferase in rat primary hepatocytes with L-methionine starvation.

Science.gov (United States)

Lin, Ai-Hsuan; Chen, Haw-Wen; Liu, Cheng-Tze; Tsai, Chia-Wen; Lii, Chong-Kuei

2012-07-04

Numerous genes expression is regulated in response to amino acid shortage, which helps organisms adapt to amino acid limitation. The expression of the π class of glutathione (GSH) S-transferase (GSTP), a highly inducible phase II detoxification enzyme, is regulated mainly by activates activating protein 1 (AP-1) binding to the enhancer I of GSTP (GPEI). Here we show the critical role of nuclear factor erythroid-2-related factor 2 (Nrf2) in up-regulating GSTP gene transcription. Primary rat hepatocytes were cultured in a methionine-restricted medium, and immunoblotting and RT-PCR analyses showed that methionine restriction time-dependently increased GSTP protein and mRNA expression over a 48 h period. Nrf2 translocation to the nucleus, nuclear proteins binding to GPEI, and antioxidant response element (ARE) luciferase reporter activity were increased by methionine restriction as well as by l-buthionine sulfoximine (BSO), a GSH synthesis inhibitor. Transfection with Nrf2 siRNA knocked down Nrf2 expression and reversed the methionine-induced GSTP expression and GPEI binding activity. Chromatin immunoprecipitation assay confirmed the binding of Nrf2 to the GPEI. Phosphorylation of extracellular signal-regulated kinase 2 (ERK2) was increased in methionine-restricted and BSO-treated cells. ERK2 siRNA abolished methionine restriction-induced Nrf2 nuclear translocation, GPEI binding activity, ARE-luciferase reporter activity, and GSTP expression. Our results suggest that the up-regulation of GSTP gene transcription in response to methionine restriction likely occurs via the ERK-Nrf2-GPEI signaling pathway.

Inhibition of Nuclear Transcription Factor-κB and Activation of Peroxisome Proliferator-Activated Receptors in HepG2 Cells by Cucurbitane-Type Triterpene Glycosides from Momordica charantia

Science.gov (United States)

Nhiem, Nguyen Xuan; Yen, Pham Hai; Ngan, Nguyen Thi Thanh; Quang, Tran Hong; Kiem, Phan Van; Minh, Chau Van; Tai, Bui Huu; Cuong, Nguyen Xuan; Song, Seok Bean

2012-01-01

Abstract Momordica charantia: is used to treat various diseases, including inflammatory conditions. Previous reports indicated that the extract of this plant inhibits activation of nuclear transcription factor-κB (NF-κB) but activates peroxisome proliferator-activated receptor (PPAR). Additionally, cucurbitane-type triterpene glycosides are the main bioactive components of the fruit of M. charantia. Therefore, we investigated the anti-inflammatory activity of 17 cucurbitane-type triterpene glycosides (1–17) isolated from this plant. Their inhibition of NF-κB and activation of PPAR activities in HepG2 cells were measured using luciferase reporter and PPAR subtype transactivation assays. Compounds 6 and 8 were found to inhibit NF-κB activation stimulated by tumor necrosis factor-α (TNFα) in a dose-dependent manner. With 50% inhibition concentration (IC50) values of 0.4 μM, compounds 6 and 8 were more potent inhibitors than the positive control, sulfasalazine (IC50=0.9 μM). Compounds 4, 6, and 8 also inhibited TNFα-induced expressions of inducible nitric oxide synthase and cyclooxygenase-2 mRNA. However, only compound 13 significantly increased PPARγ transactivation. PMID:22248180

Potent PPARα activator derived from tomato juice, 13-oxo-9,11-octadecadienoic acid, decreases plasma and hepatic triglyceride in obese diabetic mice.

Directory of Open Access Journals (Sweden)

Young-il Kim

Full Text Available Dyslipidemia is a major risk factor for development of several obesity-related diseases. The peroxisome proliferator-activated receptor α (PPARα is a ligand-activated transcription factor that regulates energy metabolism. Previously, we reported that 9-oxo-10,12-octadecadienoic acid (9-oxo-ODA is presented in fresh tomato fruits and acts as a PPARα agonist. In addition to 9-oxo-ODA, we developed that 13-oxo-9,11-octadecadienoic acid (13-oxo-ODA, which is an isomer of 9-oxo-ODA, is present only in tomato juice. In this study, we explored the possibility that 13-oxo-ODA acts as a PPARα agonist in vitro and whether its effect ameliorates dyslipidemia and hepatic steatosis in vivo. In vitro luciferase assay experiments revealed that 13-oxo-ODA significantly induced PPARα activation; moreover, the luciferase activity of 13-oxo-ODA was stronger than that of 9-oxo-ODA and conjugated linoleic acid (CLA, which is a precursor of 13-oxo-ODA and is well-known as a potent PPARα activator. In addition to in vitro experiment, treatment with 13-oxo-ODA decreased the levels of plasma and hepatic triglycerides in obese KK-Ay mice fed a high-fat diet. In conclusion, our findings indicate that 13-oxo-ODA act as a potent PPARα agonist, suggesting a possibility to improve obesity-induced dyslipidemia and hepatic steatosis.

Comparison of in vitro hormone activities of novel flame retardants TBB, TBPH and their metabolites TBBA and TBMEPH using reporter gene assays.

Science.gov (United States)

Klopčič, Ivana; Skledar, Darja Gramec; Mašič, Lucija Peterlin; Dolenc, Marija Sollner

2016-10-01

The anti-androgenic and anti-thyroid hormonal activities of the two novel brominated flame retardants, TBB and TBPH and of their metabolites TBBA and TBMEPH have been compared using the luciferase reporter gene assays. Only the parent compounds TBB and TBPH exhibited anti-glucocorticoid activity with IC50 values of 1.9 μM and 0.3 μM. Furthermore, mode of action for these two compounds is by direct competing to the glucocorticoid receptor (GR) with IC50 values of 0.03 μM and 0.002 μM. All four tested compounds possess anti-androgenic and anti-thyroid hormonal activities, without agonist activities on the respective receptors. Anti-androgenic activities with IC50 values of 43.5 μM, 0.1 μM, 47.5 μM and 1.3 μM were found for TBB, TBPH, TBBA and TBMEPH. The anti-thyroid hormonal IC50 values of 37.5 μM, 0.1 μM, 22.8 μM and 32.3 μM for TBB, TBPH, TBBA and TBMEPH, together with the above quoted results, indicate that metabolism can modify anti-androgenic, anti-glucocorticoid and anti-thyroid hormonal effects of these novel brominated flame retardants. Furthermore, the parent flame retardants are shown to be able to disrupt the function of the GR as antagonists by direct competition to the receptor. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development of an ultralow-light-level luminescence image analysis system for dynamic measurements of transcriptional activity in living and migrating cells.

Science.gov (United States)

Maire, E; Lelièvre, E; Brau, D; Lyons, A; Woodward, M; Fafeur, V; Vandenbunder, B

2000-04-10

We have developed an approach to study in single living epithelial cells both cell migration and transcriptional activation, which was evidenced by the detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescence inverted microscope, connected to an ultralow-light-level photon-counting camera and an image-acquisition card associated to specialized image analysis software running on a PC computer. Using a simple method based on a thin calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photon-counting cameras designed to observe luminescence. This setup allows us to measure by image analysis the luminescent light emitted by individual cells stably expressing a luciferase reporter vector. The sensitivity of the camera was adjusted to a high value, which required the use of a segmentation algorithm to eliminate the background noise. Following mathematical morphology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highlight the usefulness of our image acquisition chain and mathematical morphology software to quantify the kinetics of luminescence changes in migrating cells.

SPE-HPLC purification of endocrine disrupting compounds from human serum for assessment of xenoestrogenic activity

DEFF Research Database (Denmark)

Hjelmborg, P.S.; Ghisari, Mandana; Bonefeld-Jørgensen, Eva

2006-01-01

Assessment of xenoestrogenic activity in human serum samples requires the removal of endogenous sex hormones to assure that the activity measured originates from xenobiotic compounds only. Serum samples representing high, medium and lower accumulation of persistent organic pollutants (POPs) were...... measured by ERE-CALUX was validated and considered to be a valuable tool to assess the combined ER effect of lipophilic serum POPs where additive/synergistic and agonistic/antagonistic effects are integrated giving an overall estimate of exposure and bioactivity....... for the study. MVLN cells, stably transfected with an estrogen receptor (ER) luciferase reporter vector (ERE-CALUX), were exposed to the reconstituted SPE-HPLC extracts for determination of the integrated estrogenic activity. The effects of PCBs were analyzed by direct in vitro exposure of PCBs (138, 153...

E-Divisionn activities report

International Nuclear Information System (INIS)

Barschall, H.H.

1980-07-01

This report describes some of the activities in E (Experimental Physics) Division during the past year. E-Division carries out research and development in areas related to the missions of the Laboratory. Many of the activities are in pure and applied atomic and nuclear physics. In addition this report describes work on accelerators, radiation damage, microwaves, and plasma diagnostics

E-Division activities report

International Nuclear Information System (INIS)

Barschall, H.H.

1979-07-01

This report describes some of the activities in E (Experimental Physics) Division during the past year. E-Division carries out research and development in areas related to the missions of the Laboratory. Many of the activities are in pure and applied atomic and nuclear physics. In addition, this report describes work on accelerators, radiation damage, microwaves, and plasma diagnostics

Cytocidal activities of topoisomerase 1 inhibitors and 5-azacytidine against pheochromocytoma/paraganglioma cells in primary human tumor cultures and mouse cell lines.

Directory of Open Access Journals (Sweden)

James F Powers

Full Text Available There is currently no effective treatment for metastatic pheochromocytomas and paragangliomas. A deficiency in current chemotherapy regimens is that the metastases usually grow very slowly. Drugs that target dividing tumor cells have therefore had limited success. To improve treatment, new strategies and valid experimental models are required for pre-clinical testing. However, development of models has itself been hampered by the absence of human pheochromocytoma/paraganglioma cell lines for cultures or xenografts. Topoisomerase 1 (TOP1 inhibitors are drugs that interfere with mechanisms that maintain DNA integrity during transcription in both quiescent and dividing cells. We used primary cultures of representative human tumors to establish the cytotoxicity of camptothecin, a prototypical TOP1 inhibitor, against non-dividing pheochromocytoma/paraganglioma cells, and then employed a mouse pheochromocytoma model (MPC to show that efficacy of low concentrations of camptothecin and other TOP1 inhibitors is increased by intermittent coadministration of sub-toxic concentrations of 5-azacytidine, a DNA methylation inhibitor that modulates transcription. We then tested the same drugs against a clonal MPC derivative that expresses CMV reporter-driven luciferase and GFP, intended for in vivo drug testing. Unexpectedly, luciferase expression, bioluminescence and GFP expression were paradoxically increased by both camptothecin and SN38, the active metabolite of irinotecan, thereby masking cell death. Expression of chromogranin A, a marker for neuroendocrine secretory granules, was not increased, indicating that the drug effects on levels of luciferase and GFP are specific to the GFP-luciferase construct rather than generalized cellular responses. Our findings provide proof of principle for use of TOP1 inhibitors against pheochromocytoma/paraganglioma and suggest novel strategies for enhancing efficacy and reducing toxicity by optimizing the combination and

Disruption of bbe02 by Insertion of a Luciferase Gene Increases Transformation Efficiency of Borrelia burgdorferi and Allows Live Imaging in Lyme Disease Susceptible C3H Mice.

Directory of Open Access Journals (Sweden)

Kamfai Chan

Full Text Available Lyme disease is the most prevalent tick-borne disease in North America and Europe. The causative agent, Borrelia burgdorferi persists in the white-footed mouse. Infection with B. burgdorferi can cause acute to persistent multisystemic Lyme disease in humans. Some disease manifestations are also exhibited in the mouse model of Lyme disease. Genetic manipulation of B. burgdorferi remains difficult. First, B. burgdorferi contains a large number of endogenous plasmids with unique sequences encoding unknown functions. The presence of these plasmids needs to be confirmed after each genetic manipulation. Second, the restriction modification defense systems, including that encoded by bbe02 gene lead to low transformation efficiency in B. burgdorferi. Therefore, studying the molecular basis of Lyme pathogenesis is a challenge. Furthermore, investigation of the role of a specific B. burgdorferi protein throughout infection requires a large number of mice, making it labor intensive and expensive. To overcome the problems associated with low transformation efficiency and to reduce the number of mice needed for experiments, we disrupted the bbe02 gene of a highly infectious and pathogenic B. burgdorferi strain, N40 D10/E9 through insertion of a firefly luciferase gene. The bbe02 mutant shows higher transformation efficiency and maintains luciferase activity throughout infection as detected by live imaging of mice. Infectivity and pathogenesis of this mutant were comparable to the wild-type N40 strain. This mutant will serve as an ideal parental strain to examine the roles of various B. burgdorferi proteins in Lyme pathogenesis in the mouse model in the future.

RTE activity report 2005

International Nuclear Information System (INIS)

2006-01-01

The RTE (electric power transport network) is the french manger of the electric power transport. This activity report provides information on the company results for the year 2005: panorama of the year, management and organization, the place of RTE in the european market, the customers, the industrial tool, the environment the human resources, the international activity and the management report. (A.L.B.)

pLIVE-EGFP: A liver specific vector carrying the EGFP reporter for ...

African Journals Online (AJOL)

STORAGESEVER

2009-10-19

Oct 19, 2009 ... system for monitoring gene expression, intracellular protein trafficking or localization. Several types of reporter systems, such as secretory alkaline phosphatase (SEAP), luciferase, β-galactosidase, or fluorescent proteins (FPs), have been widely applied in these researches (Naylor,. 1999; Branchini, et al., ...

Cucurbitane Triterpenoid from Momordica charantia Induces Apoptosis and Autophagy in Breast Cancer Cells, in Part, through Peroxisome Proliferator-Activated Receptor γ Activation

Directory of Open Access Journals (Sweden)

Jing-Ru Weng

2013-01-01

Full Text Available Although the antitumor activity of the crude extract of wild bitter gourd (Momordica charantia L. has been reported, its bioactive constituents and the underlying mechanism remain undefined. Here, we report that 3β,7β-dihydroxy-25-methoxycucurbita-5,23-diene-19-al (DMC, a cucurbitane-type triterpene isolated from wild bitter gourd, induced apoptotic death in breast cancer cells through peroxisome proliferator-activated receptor (PPAR γ activation. Luciferase reporter assays indicated the ability of DMC to activate PPARγ, and pharmacological inhibition of PPARγ protected cells from DMC's antiproliferative effect. Western blot analysis indicated that DMC suppressed the expression of many PPARγ-targeted signaling effectors, including cyclin D1, CDK6, Bcl-2, XIAP, cyclooxygenase-2, NF-κB, and estrogen receptor α, and induced endoplasmic reticulum stress, as manifested by the induction of GADD153 and GRP78 expression. Moreover, DMC inhibited mTOR-p70S6K signaling through Akt downregulation and AMPK activation. The ability of DMC to activate AMPK in liver kinase (LK B1-deficient MDA-MB-231 cells suggests that this activation was independent of LKB1-regulated cellular metabolic status. However, DMC induced a cytoprotective autophagy presumably through mTOR inhibition, which could be overcome by the cotreatment with the autophagy inhibitor chloroquine. Together, the ability of DMC to modulate multiple PPARγ-targeted signaling pathways provides a mechanistic basis to account for the antitumor activity of wild bitter gourd.

DNA repair in human fibroblasts, as reflected by host-cell reactivation of a transfected UV-irradiated luciferase gene, is not related to donor age

International Nuclear Information System (INIS)

Merkle, Thomas J.; O'Brien, Katherine; Brooks, Philip J.; Tarone, Robert E.; Robbins, Jay H.

2004-01-01

The effect of donor age on the ability of mammalian cells to repair ultraviolet (UV)-induced DNA damage has been studied using several approaches, most recently via assays that measure the host-cell reactivation (HCR) of UV-irradiated reporter gene-containing plasmid vectors following their transfection into cells. Plasmid HCR assays indirectly quantify a cell line's ability to perform nucleotide excision repair (NER) by measuring the enzyme activity of the repaired reporter gene, e.g., chloramphenical acetyltransferase (cat) or luciferase (luc), and are useful in studies investigating whether increasing age may be a risk factor for the deficient repair of potentially cancer-causing, sunlight-induced, DNA lesions in skin cells. In our study, we quantified the DNA repair ability of cultured, nontransformed, human skin fibroblast lines through their HCR of a transfected UV-C-irradiated plasmid containing luc. HCR was measured at various times after transfection in five lines from normal donors of ages 21-96 years, and from one donor who had xeroderma pigmentosum (XP). The normal lines displayed increasing HCR at successive post-transfection time points and showed no significant correlation between HCR and donor age. The XP-A line, known to be markedly deficient in NER of UV-induced DNA damage, showed minimal evidence of HCR compared to the normal lines. To further assess potential variation in HCR with donor age, fibroblast lines from five old donors, ages 84-94 years, were compared with lines from five young donors, ages 17-26 years. While significant differences in HCR were found between some lines, no significant difference was found between the young and old age groups (P=0.44). Our study provides no indication that the higher incidence of skin cancer observed with increasing age is due to an age-related decrease in the ability to repair UV-induced DNA damage

DNA repair in human fibroblasts, as reflected by host-cell reactivation of a transfected UV-irradiated luciferase gene, is not related to donor age

Energy Technology Data Exchange (ETDEWEB)

Merkle, Thomas J.; O' Brien, Katherine; Brooks, Philip J.; Tarone, Robert E.; Robbins, Jay H

2004-10-04

The effect of donor age on the ability of mammalian cells to repair ultraviolet (UV)-induced DNA damage has been studied using several approaches, most recently via assays that measure the host-cell reactivation (HCR) of UV-irradiated reporter gene-containing plasmid vectors following their transfection into cells. Plasmid HCR assays indirectly quantify a cell line's ability to perform nucleotide excision repair (NER) by measuring the enzyme activity of the repaired reporter gene, e.g., chloramphenical acetyltransferase (cat) or luciferase (luc), and are useful in studies investigating whether increasing age may be a risk factor for the deficient repair of potentially cancer-causing, sunlight-induced, DNA lesions in skin cells. In our study, we quantified the DNA repair ability of cultured, nontransformed, human skin fibroblast lines through their HCR of a transfected UV-C-irradiated plasmid containing luc. HCR was measured at various times after transfection in five lines from normal donors of ages 21-96 years, and from one donor who had xeroderma pigmentosum (XP). The normal lines displayed increasing HCR at successive post-transfection time points and showed no significant correlation between HCR and donor age. The XP-A line, known to be markedly deficient in NER of UV-induced DNA damage, showed minimal evidence of HCR compared to the normal lines. To further assess potential variation in HCR with donor age, fibroblast lines from five old donors, ages 84-94 years, were compared with lines from five young donors, ages 17-26 years. While significant differences in HCR were found between some lines, no significant difference was found between the young and old age groups (P=0.44). Our study provides no indication that the higher incidence of skin cancer observed with increasing age is due to an age-related decrease in the ability to repair UV-induced DNA damage.

Activity report 2002

International Nuclear Information System (INIS)

2002-01-01

This annual report deals with the activities, the program management and the financial aspects of the ANDRA (National Agency for the radioactive wastes management) during 2002. After a presentation of the activities during the year 2002, it provides the financial accounting, data on company cash, the ANDRA missions and publications. (A.L.B.)

Active Components with Inhibitory Activities on IFN-γ/STAT1 and IL-6/STAT3 Signaling Pathways from Caulis Trachelospermi

Directory of Open Access Journals (Sweden)

Xiao-Ting Liu

2014-08-01

Full Text Available Initial investigation for new active herbal extract with inhibiting activity on JAK/STAT signaling pathway revealed that the extract of Caulis Trachelospermi, which was separated by 80% alcohol extraction and subsequent HP-20 macroporous resin column chromatography, was founded to strongly inhibit IFN-γ-induced STAT1-responsive luciferase activity (IFN-γ/STAT1 with IC50 value of 2.43 μg/mL as well as inhibiting IL-6-induced STAT3-responsive luciferase activity (IL-6/STAT3 with IC50 value of 1.38 μg/mL. Subsequent study on its active components led to the isolation and identification of two new dibenzylbutyrolactone lignans named 4-demethyltraxillaside (1 and nortrachelogenin 4-O-β-d-glucopyranoside (2, together with six known compounds. The lignan compounds 1–4 together with other lignan compounds isolated in previous study were tested the activities on IFN-γ/STAT1 and IL-6/STAT3 pathways. The following result showed that the main components trachelogenin and arctigenin had corresponding activities on IFN-γ/STAT1 pathway with IC50 values of 3.14 μM and 9.46 μM as well as trachelogenin, arctigenin and matairesinol strongly inhibiting IL-6/STAT3 pathway with IC50 values of 3.63 μM, 6.47 μM and 2.92 μM, respectively.

E-Division activities report

International Nuclear Information System (INIS)

Barschall, H.H.

1981-07-01

This report describes some of the activities in E (Experimental Physics) Division during the past year. E-Division carries out research and development in areas related to the missions of the Laboratory. Many of the activities are in pure and applied atomic and nuclear physics and in material science. In addition this report describes work on accelerators, microwaves, plasma diagnostics, determination of atmospheric oxygen and of nitrogen in tissue

Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A

DEFF Research Database (Denmark)

Gottwein, Judith M; Jensen, Tanja B; Mathiesen, Christian K

2011-01-01

to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log(10) focus-forming units (FFU)/ml. 2a(J6) with ¿40 or ¿25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFP¿40 acquired various...... deletions in EGFP, while 2a(J6)¿40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5A¿40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those...

A high-throughput cellular assay to quantify the p53-degradation activity of E6 from different human papillomavirus types.

Science.gov (United States)

Gagnon, David; Archambault, Jacques

2015-01-01

A subset of human papillomaviruses (HPVs), known as the high-risk types, are the causative agents of cervical cancer and other malignancies of the anogenital region and oral mucosa. The capacity of these viruses to induce cancer and to immortalize cells in culture relies in part on a critical function of their E6 oncoprotein, that of promoting the poly-ubiquitination of the cellular tumor suppressor protein p53 and its subsequent degradation by the proteasome. Here, we describe a cellular assay to measure the p53-degradation activity of E6 from different HPV types. This assay is based on a translational fusion of p53 to Renilla luciferase (Rluc-p53) that remains sensitive to degradation by high-risk E6 and whose steady-state levels can be accurately measured in standard luciferase assays. The p53-degradation activity of any E6 protein can be tested and quantified in transiently transfected cells by determining the amount of E6-expression vector required to reduce by half the levels of RLuc-p53 luciferase activity (50 % effective concentration [EC50]). The high-throughput and quantitative nature of this assay makes it particularly useful to compare the p53-degradation activities of E6 from several HPV types in parallel.

Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

Science.gov (United States)

Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

1977-01-01

The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

Structure-activity relationship study of Aib-containing amphipathic helical peptide-cyclic RGD conjugates as carriers for siRNA delivery.

Science.gov (United States)

Wada, Shun-Ichi; Takesada, Anna; Nagamura, Yurie; Sogabe, Eri; Ohki, Rieko; Hayashi, Junsuke; Urata, Hidehito

2017-12-15

The conjugation of Aib-containing amphipathic helical peptide with cyclo(-Arg-Gly-Asp-d-Phe-Cys-) (cRGDfC) at the C-terminus of the helix peptide (PI) has been reported to be useful for constructing a carrier for targeted siRNA delivery into cells. In order to explore structure-activity relationships for the development of potential carriers for siRNA delivery, we synthesized conjugates of Aib-containing amphipathic helical peptide with cRGDfC at the N-terminus (PII) and both the N- and C-termini (PIII) of the helical peptide. Furthermore, to examine the influence of PI helical chain length on siRNA delivery, truncated peptides containing 16 (PIV), 12 (PV), and 8 (PVI) amino acid residues at the N-terminus of the helical chain were synthesized. PII and PIII, as well as PI, could deliver anti-luciferase siRNA into cells to induce the knockdown of luciferase stably expressed in cells. In contrast, all of the truncated peptides were unlikely to transport siRNA into cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

Science.gov (United States)

Bongiorni, Silvia; Tilesi, Francesca; Bicorgna, Silvia; Iacoponi, Francesca; Willems, Daniela; Gargani, Maria; D'Andrea, MariaSilvia; Pilla, Fabio; Valentini, Alessio

2014-11-07

Success of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes. We tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and

Quantification of PRL/Stat5 signaling with a novel pGL4-CISH reporter.

Science.gov (United States)

Fang, Feng; Antico, Giovanni; Zheng, Jiamao; Clevenger, Charles V

2008-02-06

Elevations of serum prolactin (PRL) are associated with an increased risk for breast cancer. PRL signaling through its prolactin receptor (PRLr) involves the Jak2/Stat5 pathway. Luciferase-based reporter assays have been widely used to evaluate the activity of this pathway. However, the existing reporters are often not sensitive enough to monitor the effect of PRL in this pathway. In this study, a new biologically relevant reporter, pGL4-CISH, was generated to study the PRL/Jak2/Stat5 signaling pathway. The sensitivity of pGL4-CISH to detect PRL was superior to that of several other commonly utilized Stat5-responsive reporters. Interestingly, the enhanced function pGL4-CISH was restricted to the estrogen receptor positive (ER+) human breast cancer cell lines T47D and MCF7, but not in the ER-MDA-231, BT-474, or MCF10A cell lines. Overexpression of Stat5 further enhanced the effect of PRL on pGL4-CISH. These studies demonstrate that pGL4-CISH is a novel and sensitive reporter for assessing the activity of the PRL/Stat5 signaling pathway in the ER+ human breast cancer cells.

Comparison of intra-organellar chaperone capacity for dealing with stress-induced protein unfolding.

Science.gov (United States)

Hageman, Jurre; Vos, Michel J; van Waarde, Maria A W H; Kampinga, Harm H

2007-11-23

Molecular chaperones are essential for cells to prevent that partially unfolded proteins form non-functional, toxic aggregates. This requirement is increased when cells experience protein unfolding stresses and such could affect all compartments in the eukaryotic cell. Whether all organelles are equipped with comparable chaperone capacities is largely unknown, mainly due to the lack of suitable reporters that allow such a comparison. Here we describe the development of fluorescent luciferase reporters that are sorted to various cellular locations (nucleus, cytoplasm, endoplasmic reticulum, and peroxisomes) and that differ minimally in their intrinsic thermal stability properties. When heating living cells, the rate of inactivation was most rapid for the nuclear-targeted luciferase, indicating that the nucleus is the most sensitive organelle toward heat-induced denaturing stress. Post-heat re-activation, however, occurred at equal kinetics irrespective of luciferase localization. Also, induction of thermotolerance by a priming heat treatment, that coordinately up-regulates all heat-inducible chaperones, resulted in a transient heat resistance of the luciferase in all organelles in a comparable manner. Overexpression of the main heat-inducible Hsp70 family member, HspA1A, protected only the cytosolic and nuclear, but not the other luciferases. Together, our data suggest that in each compartment investigated, including the peroxisome in which so far no chaperones could be detected, chaperone machines are present and can be induced with activities similar to those present in the cytosolic/nuclear compartment.

LANSCE Activity Report 1995-1998

International Nuclear Information System (INIS)

Amy Robinson; Audrey Archuleta; Barbara Maes; Dan Strottman; Earl Hoffman; Garth Tietjen; Gene Farnum; Geoff Greene; Joyce Roberts; Ken Johnson; Paul Lewis; Roger Pynn; Stan Schriber; Steve Sterbenz; Steve Wender; Sue Harper

1999-01-01

The Los Alamos Neutron Science Center Activity Report describes scientific and technological progress and achievements in LANSCE Division during the period of 1995 to 1998. This report includes a message from the Division Director, an overview of LANSCE, sponsor overviews, research highlights, advanced projects and facility upgrades achievements, experimental and user program accomplishments, news and events, and a list of publications. The research highlights cover the areas of condensed-matter science and engineering, accelerator science, nuclear science, and radiography. This report also contains a compact disk that includes an overview, the Activity Report itself, LANSCE operations progress reports for 1996 and 1997, experiment reports from LANSCE users, as well as a search capability

2001 activity report

International Nuclear Information System (INIS)

2002-01-01

Managing radioactive waste for a better control in the future. The Quadrennial State/Andra contract, the double ISO 9001 (quality) and ISO 140001 (environment) certification and three diversified missions: industry, research and information. Those are the highlights of the activity report where the reader will be able to learn more about the Agency's major achievements in 2001. An appendix contains the management report of the Agency

Activity report 1976

International Nuclear Information System (INIS)

Ananthakrishna, G.; Ramanathan, N.; Rodriguez, P.; Shankar Singh, R.; Venkataraman, S.

1978-11-01

The research and development activities of the Reactor Research Centre, Kalpakkam, during 1976 are reported. The main thrust of the R and D activities is directed towards the FBTR Project and they deal with the various aspects of the project such as reactor physics, reactor design, fuel handling, reactor instrumentation, reactor operation, reactor construction, reactor safety, reactor materials, reactor chemistry and fuel reprocessing. (M.G.B.)

Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Qiao Yawei; Spitz, Margaret R.; Guo Zhaozheng; Hadeyati, Mohammad; Grossman, Lawrence; Kraemer, Kenneth H.; Wei Qingyi

2002-11-30

As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.

ANDRA, 2006 activity report

International Nuclear Information System (INIS)

2006-01-01

The French national agency of radioactive waste management (ANDRA) was marked in 2006 by two outstanding events: the publication of the national inventory of radioactive wastes and valorisable materials, and the vote of the law from June 28, 2006 relative to the sustainable management of radioactive wastes and materials. This road-map law has an impact on ANDRA's activities for the coming years. This activity report presents several 2006 highlights of ANDRA's missions as well: the public service mission, the by-law about the effluents of the Aube plant for the storage of low-medium activity wastes, the building of the first 'double-cell' at the very-low activity waste storage plant of Aube, the research studies about the project of deep underground disposal of high-medium activity, long-living wastes, and the public information about ANDRA's technical and scientific know-how. The management and financial reports are attached in appendix. (J.S.)

The E-screen test and the MELN gene-reporter assay used for determination of estrogenic activity in fruits and vegetables in relation to pesticide residues.

Science.gov (United States)

Schilirò, Tiziana; Porfido, Arianna; Longo, Annalisa; Coluccia, Sara; Gilli, Giorgio

2013-12-01

Endocrine-disrupting chemicals (EDCs) may lead to adverse systemic effects by interfering with normal hormone homeostasis, and diet is considered to be among the main routes of EDC exposure. The present study investigated the total estrogenic activity of fruits and vegetables by calculating the 17-β-estradiol equivalent quantity (EEQ) using two in vitro tests: the human breast cancer cell line (MCF-7 BUS) proliferation assay (E-screen test) and the luciferase-transfected human breast cancer cell line (MELN) gene-reporter assay. Of the 24 analyzed fruits and vegetables, 14 contained from 1 to 4 pesticide residues in concentrations ranging from 0.02 to 1.19 ppm, whereas the other 10 did not contain any pesticide residues. The EEQ values for all positive samples ranged from 0.010 to 0.616 μg/100g for the above in vitro tests. Our study demonstrates that estrogenic activity was present in fruits and vegetables and that the concentration of allowable pesticide residues and EEQ values were positively correlated; however, no correlation was found by comparing the estrogenic activity and the intrinsic content of phytoestrogens obtained from the available literature. A theoretical adult dietary intake of 0.7-0.9 ng EEQ/L/day from fruits and vegetables was calculated. Copyright © 2013 Elsevier Ltd. All rights reserved.

pedometer-measured physical activity, self-reported physical activity

African Journals Online (AJOL)

between self-reported and pedometer-measured physical activity was also determined. Results. Average ... Methods. This was a cross-sectional study among employed South African adults. Participant ... acquired information on physical activity habits. Questions ..... How many days of monitoring predict physical activity and ...

Activity report 1980

International Nuclear Information System (INIS)

Drent, W.; Delande, E.

1981-07-01

The EUROCHEMIC Company's activity report for fiscal year 1980 covers the following topics: administrative and commercial matters; personnel; cleaning and decontamination works in the reprocessing plant; operation of the bituminization facility; active treatment of the spent solvent in the Eurowatt facility; operation of the waste sorting and shredding unit, preparing the plutonium contaminated solid wastes for conditioning and recovery of plutonium; radiation protection and library and documentation. (AF)

Bmi-1 promotes the aggressiveness of glioma via activating the NF-kappaB/MMP-9 signaling pathway

International Nuclear Information System (INIS)

Jiang, Lili; Wu, Jueheng; Yang, Yi; Liu, Liping; Song, Libing; Li, Jun; Li, Mengfeng

2012-01-01

The prognosis of human glioma is poor, and the highly invasive nature of the disease represents a major impediment to current therapeutic modalities. The oncoprotein B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) has been linked to the development and progression of glioma; however, the biological role of Bmi-1 in the invasion of glioma remains unclear. A172 and LN229 glioma cells were engineered to overexpress Bmi-1 via stable transfection or to be silenced for Bmi-1 expression using RNA interfering method. Migration and invasiveness of the engineered cells were assessed using wound healing assay, Transwell migration assay, Transwell matrix penetration assay and 3-D spheroid invasion assay. MMP-9 expression and activity were measured using real-time PCR, ELISA and the gelatin zymography methods. Expression of NF-kappaB target genes was quantified using real-time PCR. NF-kappaB transcriptional activity was assessed using an NF-kappaB luciferase reporter system. Expression of Bmi-1 and MMP-9 in clinical specimens was analyzed using immunohistochemical assay. Ectopic overexpression of Bmi-1 dramatically increased, whereas knockdown of endogenous Bmi-1 reduced, the invasiveness and migration of glioma cells. NF-kappaB transcriptional activity and MMP-9 expression and activity were significantly increased in Bmi-1-overexpressing but reduced in Bmi-1-silenced cells. The reporter luciferase activity driven by MMP-9 promoter in Bmi-1-overexpressing cells was dependent on the presence of a functional NF-kappaB binding site, and blockade of NF-kappaB signaling inhibited the upregulation of MMP-9 in Bmi-1 overexpressing cells. Furthermore, expression of Bmi-1 correlated with NF-kappaB nuclear translocation as well as MMP-9 expression in clinical glioma samples. Bmi-1 may play an important role in the development of aggressive phenotype of glioma via activating the NF-kappaB/MMP-9 pathway and therefore might represent a novel therapeutic

Bmi-1 promotes the aggressiveness of glioma via activating the NF-kappaB/MMP-9 signaling pathway

Directory of Open Access Journals (Sweden)

Jiang Lili

2012-09-01

Full Text Available Abstract Background The prognosis of human glioma is poor, and the highly invasive nature of the disease represents a major impediment to current therapeutic modalities. The oncoprotein B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1 has been linked to the development and progression of glioma; however, the biological role of Bmi-1 in the invasion of glioma remains unclear. Methods A172 and LN229 glioma cells were engineered to overexpress Bmi-1 via stable transfection or to be silenced for Bmi-1 expression using RNA interfering method. Migration and invasiveness of the engineered cells were assessed using wound healing assay, Transwell migration assay, Transwell matrix penetration assay and 3-D spheroid invasion assay. MMP-9 expression and activity were measured using real-time PCR, ELISA and the gelatin zymography methods. Expression of NF-kappaB target genes was quantified using real-time PCR. NF-kappaB transcriptional activity was assessed using an NF-kappaB luciferase reporter system. Expression of Bmi-1 and MMP-9 in clinical specimens was analyzed using immunohistochemical assay. Results Ectopic overexpression of Bmi-1 dramatically increased, whereas knockdown of endogenous Bmi-1 reduced, the invasiveness and migration of glioma cells. NF-kappaB transcriptional activity and MMP-9 expression and activity were significantly increased in Bmi-1-overexpressing but reduced in Bmi-1-silenced cells. The reporter luciferase activity driven by MMP-9 promoter in Bmi-1-overexpressing cells was dependent on the presence of a functional NF-kappaB binding site, and blockade of NF-kappaB signaling inhibited the upregulation of MMP-9 in Bmi-1 overexpressing cells. Furthermore, expression of Bmi-1 correlated with NF-kappaB nuclear translocation as well as MMP-9 expression in clinical glioma samples. Conclusions Bmi-1 may play an important role in the development of aggressive phenotype of glioma via activating the NF

Activity report 1998

International Nuclear Information System (INIS)

1998-01-01

The ''Institut de Protection et de Surete Nucleaire'' (IPSN), carries out researches and expert studies needed to manage nuclear risks and their consequences on people and environment. These activities cover the installation safety, the radioactive matter transport, the public health and environmental control, the nuclear matters safety and control and the crisis situation management. The IPSN have got also a mission of public information and participates to international actions in the domain of scientific researches or expert's report. This annual report presents the highlights of the year 1998, information on staff, budgets and geographical situation. Then technical and scientific papers gathers the activities of the year covered by the IPSN: the ''Free Tribune'', installations and radioactive wastes safety, crisis and nuclear matter management, public health and environment control, international cooperation. (A.L.B.)

Activity report 1995-1998

International Nuclear Information System (INIS)

1998-01-01

The aim of this report is to outline the scientific activity of the ''Service des Photons Atomes et Molecules'' (DRECAM), during the period 1995-1998: activity in terms of structure, scientific research programs and results, publications, training and contracts. DRECAM is one of the service of the ''Direction des Sciences de la Matiere'' (DSM), itself depending of the CEA Institution. The scientific domains covered by this report are: the excited atoms, multicharged ions - atoms collisions, molecules and aggregates in extreme conditions, covalent structures, new light sources. (A.L.B.)

Marine Natural Product Honaucin A Attenuates Inflammation by Activating the Nrf2-ARE Pathway.

Science.gov (United States)

Mascuch, Samantha J; Boudreau, Paul D; Carland, Tristan M; Pierce, N Tessa; Olson, Joshua; Hensler, Mary E; Choi, Hyukjae; Campanale, Joseph; Hamdoun, Amro; Nizet, Victor; Gerwick, William H; Gaasterland, Teresa; Gerwick, Lena

2018-03-23

The cyanobacterial marine natural product honaucin A inhibits mammalian innate inflammation in vitro and in vivo. To decipher its mechanism of action, RNA sequencing was used to evaluate differences in gene expression of cultured macrophages following honaucin A treatment. This analysis led to the hypothesis that honaucin A exerts its anti-inflammatory activity through activation of the cytoprotective nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile response element (ARE/EpRE) signaling pathway. Activation of this pathway by honaucin A in cultured human MCF7 cells was confirmed using an Nrf2 luciferase reporter assay. In vitro alkylation experiments with the natural product and N-acetyl-l-cysteine suggest that honaucin A activates this pathway through covalent interaction with the sulfhydryl residues of the cytosolic repressor protein Keap1. Honaucin A presents a potential therapeutic lead for diseases with an inflammatory component modulated by Nrf2-ARE.

The cis decoy against the estrogen response element suppresses breast cancer cells via target disrupting c-fos not mitogen-activated protein kinase activity.

Science.gov (United States)

Wang, Li Hua; Yang, Xiao Yi; Zhang, Xiaohu; Mihalic, Kelly; Xiao, Weihua; Farrar, William L

2003-05-01

Breast cancer, the most common malignancy in women, has been demonstrated to be associated with the steroid hormone estrogen and its receptor (ER), a ligand-activated transcription factor. Therefore, we developed a phosphorothiolate cis-element decoy against the estrogen response element (ERE decoy) to target disruption of ER DNA binding and transcriptional activity. Here, we showed that the ERE decoy potently ablated the 17beta-estrogen-inducible cell proliferation and induced apoptosis of human breast carcinoma cells by functionally affecting expression of c-fos gene and AP-1 luciferase gene reporter activity. Specificity of the decoy was demonstrated by its ability to directly block ER binding to a cis-element probe and transactivation. Moreover, the decoy failed to inhibit ER-mediated mitogen-activated protein kinase signaling pathways and cell growth of ER-negative breast cancer cells. Taken together, these data suggest that estrogen-mediated cell growth of breast cancer cells can be preferentially restricted via targeted disruption of ER at the level of DNA binding by a novel and specific decoy strategy applied to steroid nuclear receptors.

Development of DNA vaccines for fish

DEFF Research Database (Denmark)

Heppell, Joël; Lorenzen, Niels; Armstrong, Neil K.

1998-01-01

Disease control is one of the major concerns in the aquaculture industry. However, there are no vaccines available for the prevention of many piscine infectious diseases, especially those of viral and parasitic origin. DNA-based vaccination could circumvent several problems associated with tradit......Disease control is one of the major concerns in the aquaculture industry. However, there are no vaccines available for the prevention of many piscine infectious diseases, especially those of viral and parasitic origin. DNA-based vaccination could circumvent several problems associated...... with traditional methods of immunization, but little is known on its efficacy in fish. The luciferase and lacZ reporter genes were used to characterize expression of plasmid-encoded genes in rainbow trout and zebra fish injected intramuscularly. For a given dose of DNA, the luciferase activity was higher in fish...... than in mouse muscle. The enzyme activity in fish peaked with 1 μg of DNA and remained constant for over 12 weeks, but it was not limited to the injected muscle since luciferase activity was also detected in the gills. Thin sections of rainbow trout muscle injected with the lacZ reporter gene showed...

HOME Participating Jurisdictions Open Activities Reports

Data.gov (United States)

Department of Housing and Urban Development — This report is an MS Excel spreadsheet broken up by state. Participating Jurisdictions can use this report to view open activities in IDIS including activities with...

Oleamide activates peroxisome proliferator-activated receptor gamma (PPARγ in vitro

Directory of Open Access Journals (Sweden)

Dionisi Mauro

2012-05-01

Full Text Available Abstract Background Oleamide (ODA is a fatty acid primary amide first identified in the cerebrospinal fluid of sleep-deprived cats, which exerts effects on vascular and neuronal tissues, with a variety of molecular targets including cannabinoid receptors and gap junctions. It has recently been reported to exert a hypolipidemic effect in hamsters. Here, we have investigated the nuclear receptor family of peroxisome proliferator-activated receptors (PPARs as potential targets for ODA action. Results Activation of PPARα, PPARβ and PPARγ was assessed using recombinant expression in Chinese hamster ovary cells with a luciferase reporter gene assay. Direct binding of ODA to the ligand binding domain of each of the three PPARs was monitored in a cell-free fluorescent ligand competition assay. A well-established assay of PPARγ activity, the differentiation of 3T3-L1 murine fibroblasts into adipocytes, was assessed using an Oil Red O uptake-based assay. ODA, at 10 and 50 μM, was able to transactivate PPARα, PPARβ and PPARγ receptors. ODA bound to the ligand binding domain of all three PPARs, although complete displacement of fluorescent ligand was only evident for PPARγ, at which an IC50 value of 38 μM was estimated. In 3T3-L1 cells, ODA, at 10 and 20 μM, induced adipogenesis. Conclusions We have, therefore, identified a novel site of action of ODA through PPAR nuclear receptors and shown how ODA should be considered as a weak PPARγ ligand in vitro.

Performance of a novel keratinocyte-based reporter cell line to screen skin sensitizers in vitro

International Nuclear Information System (INIS)

Emter, Roger; Ellis, Graham; Natsch, Andreas

2010-01-01

In vitro tests are needed to replace animal tests to screen for the skin sensitization potential of chemicals. Skin sensitizers are electrophilic molecules and the Nrf2-electrophile-sensing pathway comprising the repressor protein Keap1, the transcription factor Nrf2 and the antioxidant response element (ARE) is emerging as a toxicity pathway induced by skin sensitizers. Previously, we screened a large set of chemicals in the reporter cell line AREc32, which contains an eight-fold repeat of the rat GSTA2 ARE-sequence upstream of a luciferase reporter gene in the human breast cancer cell line MCF7. This approach was now further developed to bring it closer to the conditions in the human skin and to propose a fully standardized assay. To this end, a luciferase reporter gene under control of a single copy of the ARE-element of the human AKR1C2 gene was stably inserted into HaCaT keratinocytes. A standard operating procedure was developed whereby chemicals are routinely tested at 12 concentrations in triplicate for significant induction of gene activity. We report results from this novel assay on (i) a list of reference chemicals published by ECVAM, (ii) the ICCVAM list of chemicals for validation of alternative endpoints in the LLNA and (iii) on a more general list of 67 chemicals derived from the ICCVAM database. For comparison, peptide reactivity data are presented for the same chemicals. The results indicate a good predictive value of this approach for hazard identification. Its technical simplicity, the high-throughput format and the good predictivity may make this assay a candidate for rapid validation to meet the tight deadline to replace animal tests for skin sensitization by 2013 set by the European authorities.

Activated AMPK inhibits PPAR-{alpha} and PPAR-{gamma} transcriptional activity in hepatoma cells.

Science.gov (United States)

Sozio, Margaret S; Lu, Changyue; Zeng, Yan; Liangpunsakul, Suthat; Crabb, David W

2011-10-01

AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-α (PPAR-α) are critical regulators of short-term and long-term fatty acid oxidation, respectively. We examined whether the activities of these molecules were coordinately regulated. H4IIEC3 cells were transfected with PPAR-α and PPAR-γ expression plasmids and a peroxisome-proliferator-response element (PPRE) luciferase reporter plasmid. The cells were treated with PPAR agonists (WY-14,643 and rosiglitazone), AMPK activators 5-aminoimidazole-4-carboxamide riboside (AICAR) and metformin, and the AMPK inhibitor compound C. Both AICAR and metformin decreased basal and WY-14,643-stimulated PPAR-α activity; compound C increased agonist-stimulated reporter activity and partially reversed the effect of the AMPK activators. Similar effects on PPAR-γ were seen, with both AICAR and metformin inhibiting PPRE reporter activity. Compound C increased basal PPAR-γ activity and rosiglitazone-stimulated activity. In contrast, retinoic acid receptor-α (RAR-α), another nuclear receptor that dimerizes with retinoid X receptor (RXR), was largely unaffected by the AMPK activators. Compound C modestly increased AM580 (an RAR agonist)-stimulated activity. The AMPK activators did not affect PPAR-α binding to DNA, and there was no consistent correlation between effects of the AMPK activators and inhibitor on PPAR and the nuclear localization of AMPK-α subunits. Expression of either a constitutively active or dominant negative AMPK-α inhibited basal and WY-14,643-stimulated PPAR-α activity and basal and rosiglitazone-stimulated PPAR-γ activity. We concluded that the AMPK activators AICAR and metformin inhibited transcriptional activities of PPAR-α and PPAR-γ, whereas inhibition of AMPK with compound C activated both PPARs. The effects of AMPK do not appear to be mediated through effects on RXR or on PPAR/RXR binding to DNA. These effects are independent of kinase activity and instead appear to

Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor

NARCIS (Netherlands)

Blankvoort, B.M.G.; Groene, E.M. de; Meeteren-Kreikamp, A.P. van; Witkamp, R.F.; Rodenburg, R.J.T.; Aarts, J.M.M.J.G.

2001-01-01

The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2

NMDA modulates oligodendrocyte differentiation of subventricular zone cells through PKC activation

Directory of Open Access Journals (Sweden)

Fabio eCavaliere

2013-12-01

Full Text Available Multipotent cells from the juvenile subventricular zone (SVZ possess the ability to differentiate into new neural cells. Depending on local signals, SVZ can generate new neurons, astrocytes or oligodendrocytes. We previously demonstrated that activation of NMDA receptors in SVZ progenitors increases the rate of oligodendrocyte differentiation. Here we investigated the mechanisms involved in NMDA receptor-dependent differentiation. Using functional studies performed with the reporter gene luciferase we found that activation of NMDA receptor stimulates PKC. In turn, stimulation of PKC precedes the activation of NADPH oxidase (NOX as demonstrated by translocation of the p67phox subunit to the cellular membrane. We propose that NOX2 is involved in the transduction of the signal from NMDA receptors through PKC activation as the inhibitor gp91 reduced their pro-differentiation effect. In addition, our data and that from other groups suggest that signaling through the NMDA receptor/PKC/NOX2 cascade generates ROS that activate the PI3/mTOR pathway and finally leads to the generation of new oligodendrocytes.

[miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

Science.gov (United States)

Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

2016-12-23

Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples ( n =5) and cervical intraepithelial neoplasia tissue samples ( n =5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group ( P cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group ( P HeLa cells through targeted regulating the expression of K-ras gene. In human cervical cancer tissues of a small sample set, the expression of miR-143 is downregulated, and the expression of K-ras is upregulated.

ITER activities status report: April 1990

International Nuclear Information System (INIS)

1990-01-01

Beginning in 1988 and continuing through 1990, the four Parties involved, under the auspices of the IAEA, have been cooperating in the ITER Conceptual Design Activities. This activity resulted in a single conceptual design for a facility that could achieve the objectives established for ITER. This report is a second interim report which updates the previous report of December 1989, and in particular addresses considerations relevant to a possible next phase of cooperation, discussions with a view toward negotiations on an instrument to allow Engineering Design Activities. 5 refs, 3 figs, 1 tab

A rapid method for the determination of microbial susceptibility using the firefly luciferase assay for adenosine triphosphate (ATP)

Science.gov (United States)

Vellend, H.; Tuttle, S. A.; Barza, M.; Weinstein, L.; Picciolo, G. L.; Chappelle, E. W.

1975-01-01

Luciferase assay for adenosine triphosphate (ATP) was optimized for pure bacteria in broth in order to evaluate if changes in bacterial ATP content could be used as a rapid measure of antibiotic effect on microorganisms. Broth cultures of log phase bacteria were incubated at 310 K (37 C) for 2.5 hours at antimicrobial concentrations which resulted in the best discrimination between sensitive and resistant strains. Eighty-seven strains of 11 bacterial species were studied for their susceptibility to 12 commonly used antimicrobial agents: ampicillin, Penicillin G, nafcillin, carbenicillin, cephalothin, tetracycline, erythromycin, clindamycin, gentamicin, nitrofurantoin, colistin, and chloramplenicol. The major advantage of the ATP system over existing methods of rapid microbial susceptibility testing is that the assay can be made specific for bacterial ATP.

[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

Science.gov (United States)

Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

2004-01-01

To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P ras4B can enhance the expression of ERalpha and

In vitro nuclear receptor inhibition and cytotoxicity of hydraulic fracturing chemicals and their binary mixtures.

Science.gov (United States)

Bain, Peter A; Kumar, Anu

2018-05-01

The widespread use of hydraulic fracturing (HF) in oil and gas extraction operations has led to concern over environmental risks posed by chemicals used in HF fluids. Here we employed a suite of stable luciferase reporter gene assays to investigate the potential for selected HF chemicals or geogenics to activate or antagonise nuclear receptor signalling. We screened three biocides (bronopol [BP], glutaraldehyde [GA], and tetrakis(hydroxymethyl)phosphonium sulfate [THPS]), a surfactant (2-butoxyethanol), a friction reducer (polyacrylamide), and a coal seam geogenic (o-cresol) for their potential to act as agonists or antagonists of the estrogen receptor, androgen receptor, progesterone receptor (PR), glucocorticoid receptor or peroxisome proliferator-activated receptor gamma (PPARγ). None of the chemicals induced luciferase activity in any of assays used in the study. In antagonistic mode, BP, GA and THPS caused reductions in luciferase activity in the reporter assays at higher concentrations (50-100 μM), while at low concentrations (2-10 μM) GA and THPS enhanced luciferase activity in some assays relative to controls. None of the other tested chemicals exhibited antagonism in the selected assays. In most cases, altered receptor signalling only occurred at concentrations exhibiting cytotoxicity. However, PPARγ activity, and to a lesser extent PR activity, were inhibited by THPS at sub-cytotoxic concentrations. The majority of binary combinations tested exhibited significantly less-than-additive cytotoxicity, and none of the combinations exhibited synergistic cytotoxicity. In summary, the results of the present study indicate that the selected chemicals are not likely to function as direct agonists of the nuclear receptors tested, and only one chemical, THPS was an apparent partial antagonist of two nuclear receptors. Copyright © 2017. Published by Elsevier Ltd.

Evidence for a relief of repression mechanism for activation of the human telomerase reverse transcriptase promoter.

Science.gov (United States)

Wang, Shuwen; Zhu, Jiyue

2003-05-23

The transcriptional activation of human telomerase reverse transcriptase (hTERT) is an important step during cellular immortalization and tumorigenesis. To study how this activation occurs during immortalization, we have established a set of genetically related pre-crisis cells and their immortal progeny. As expected, hTERT mRNA was detected in our telomerase-positive immortal cells but not in pre-crisis cells or telomerase-negative immortal cells. However, transiently transfected luciferase reporters controlled by hTERT promoter sequences exhibited similar levels of luciferase activity in both telomerase-positive and -negative cells, suggesting that the endogenous chromatin context is likely required for hTERT regulation. Analysis of chromatin susceptibility to DNase I digestion consistently identified a DNase I hypersensitivity site (DHS) near the hTERT transcription initiation site in telomerase-positive cells. In addition, the histone deacetylase inhibitor trichostatin A (TSA) induced hTERT transcription and also a general increase in chromatin sensitivity to DNase treatment in telomerase-negative cells. The TSA-induced hTERT transcription in pre-crisis cells was accompanied by the formation of a DHS at the hTERT promoter. Furthermore, the TSA-induced hTERT transcription and chromatin alterations were not blocked by cycloheximide, suggesting that this induction does not require de novo protein synthesis and that TSA induces hTERT expression through the inhibition of histone deacetylation at the hTERT promoter. Taken together, our results suggest that the endogenous chromatin environment plays a critical role in the regulation of hTERT expression during cellular immortalization.

Regulation of NF-κB activity in astrocytes: effects of flavonoids at dietary-relevant concentrations

International Nuclear Information System (INIS)

Spilsbury, Alison; Vauzour, David; Spencer, Jeremy P.E.; Rattray, Marcus

2012-01-01

Highlights: ► We tested the hypothesis that low concentrations of flavonoids inhibit NF-κB in astrocytes. ► Primary cultured astrocytes possess a functional κB-system, measured using luciferase assays. ► Seven flavonoids (100 nM–1 μM) failed to reduce NF-κB activity in astrocytes. ► Four flavonoids (100 nM–1 μM) failed to reduce TNFa-stimulated NF-κB activity in astrocytes. ► (−)-Epicatechin did not regulate nuclear translocation of the NF-κB subunit, p65. -- Abstract: Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Sustained activation of nuclear transcription factor κB (NF-κB) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-κB signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNFα, 150 ng/ml) increased NF-κB-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative IκBα construct. In addition, TNFα increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-κB activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((−)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1–1 μM) for 18 h. None of the flavonoids modulated constitutive or TNFα-induced NF-κB activity. Therefore, we conclude that NF-κB signalling in astrocytes is not a major target for flavonoids.

Quantification of PRL/Stat5 signaling with a novel pGL4-CISH reporter

Directory of Open Access Journals (Sweden)

Zheng Jiamao

2008-02-01

Full Text Available Abstract Background Elevations of serum prolactin (PRL are associated with an increased risk for breast cancer. PRL signaling through its prolactin receptor (PRLr involves the Jak2/Stat5 pathway. Luciferase-based reporter assays have been widely used to evaluate the activity of this pathway. However, the existing reporters are often not sensitive enough to monitor the effect of PRL in this pathway. Results In this study, a new biologically relevant reporter, pGL4-CISH, was generated to study the PRL/Jak2/Stat5 signaling pathway. The sensitivity of pGL4-CISH to detect PRL was superior to that of several other commonly utilized Stat5-responsive reporters. Interestingly, the enhanced function pGL4-CISH was restricted to the estrogen receptor positive (ER+ human breast cancer cell lines T47D and MCF7, but not in the ER-MDA-231, BT-474, or MCF10A cell lines. Overexpression of Stat5 further enhanced the effect of PRL on pGL4-CISH. Conclusion These studies demonstrate that pGL4-CISH is a novel and sensitive reporter for assessing the activity of the PRL/Stat5 signaling pathway in the ER+ human breast cancer cells.

CDB-4124 and its putative monodemethylated metabolite, CDB-4453, are potent antiprogestins with reduced antiglucocorticoid activity: in vitro comparison to mifepristone and CDB-2914.

Science.gov (United States)

Attardi, Barbara J; Burgenson, Janet; Hild, Sheri A; Reel, Jerry R; Blye, Richard P

2002-02-25

To obtain selective antiprogestins, we have examined the in vitro antiprogestational/antiglucocorticoid properties of two novel compounds, CDB-4124 and the putative monodemethylated metabolite, CDB-4453, in transcription and receptor binding assays and compared them to CDB-2914 and mifepristone. All four antiprogestins bound with high affinity to rabbit uterine progestin receptors (PR) and recombinant human PR-A and PR-B (rhPR-A, rhPR-B) and were potent inhibitors of R5020-induced transactivation of the PRE2-tk-luciferase (PRE2-tk-LUC) reporter plasmid and endogenous alkaline phosphatase production in T47D-CO human breast cancer cells. None of these compounds exhibited agonist activity in these cells. Induction of luciferase activity was potentiated about five-fold by 8-Br-cAMP under basal conditions and to the same extent in the presence of the PR antagonists. Mifepristone bound to rabbit thymic glucocorticoid receptors (GR) with approximately twice the avidity of the CDB antiprogestins. Inhibition of GR-mediated transcription of PRE2-tk-LUC was assessed in HepG2 human hepatoblastoma cells. Mifepristone exhibited greater antiglucocorticoid activity than CDB-2914, 4124, and 4453, about 12-, 22-, and 185-fold, respectively. Thus, while there was a good correlation between binding to PR and functional activity of these antiprogestins, GR binding was not predictive of their glucocorticoid antagonist activity. In agreement with our in vivo results, CDB-4124 and CDB-4453, as well as CDB-2914, are potent antiprogestins in vitro, but show considerably less antiglucocorticoid activity than mifepristone.

Activity report. July 1993 - June 1994

International Nuclear Information System (INIS)

1994-09-01

The Department of Physics and Measurement Technology, Biology and Chemistry (IFM) presents its 24th consecutive annual progress report. It contains a brief description of activities in research and education within the department. The report is intended as an information for colleagues and institutions. The present report contains activities for the academic year July 1993 to June 1994. IFM has been in operation for slightly more than two decades, and has during that time expanded quite substantially. The number of employees is around 270 including 110 graduate students. The number of papers accepted for publication or published in highly reputed international scientific journals is on a high level, which gives a good indication of the quality of research performed at IFM. IFM is also very active in education for undergraduates. Examples of such activities are given in our report. Almost 30% of the total budget is allocated to undergraduate educational programmes

1996 Activity report

International Nuclear Information System (INIS)

1996-01-01

The 1996 activity report of the Direction for Waste Management of the CEA, is presented. Its activities have been developed in the framework of the general CEA cleaning plan, and include the management of wastes generated before 1992 in R and D CEA facilities, the disposal of spent fuels generated by research reactors (Orphee and Siloe) or prototype reactors (EL4 at Brennilis), and the dismantling and cleaning of obsolete facilities such as the plutonium radiochemistry building at Fontenay-aux-Roses and the pilot workshop at Marcoule

miR-582-5p is upregulated in patients with active tuberculosis and inhibits apoptosis of monocytes by targeting FOXO1.

Directory of Open Access Journals (Sweden)

Yanhua Liu

Full Text Available Macrophage apoptosis is a host innate defense mechanism against tuberculosis (TB. In this study, we found that percentage of apoptotic cells in peripheral blood monocytes from patients with active TB was lower than that from healthy controls (p<0.001. To understand whether microRNAs can modulate apoptosis of monocytes, we investigated differentially expressed microRNAs in patients with active TB. miR-582-5p was mainly expressed in monocytes and was upregulated in patients with active TB. The apoptotic percentage of THP-1 cells transfected with miR-582-5p mimics was significantly lower than those transfected with negative control of microRNA mimics (p<0.001, suggesting that miR-582-5p could inhibit apoptosis of monocytes. To our knowledge, the role of miR-582-5p in regulating apoptosis of monocytes has not been reported so far. Systematic bioinformatics analysis indicated that FOXO1 might be a target gene for miR-582-5p and its 3'UTR contains potential binding sites for miR-582-5p. To determine whether miR-582-5p could influence FOXO1 expression, miR-582-5p mimics or negative control of microRNA mimics were transfected into THP-1 cells. RT-PCR and western blot analysis showed that the miR-582-5p could suppress both FOXO1 mRNA and protein expression. Co-transfection of miR-582-5p and FOXO1 3'UTR-luciferase reporter vector into cells demonstrated that significant decrease in luciferase activity was only found in reporter vector that contained a wild type sequence of FOXO1 3'UTR, suggesting that miR-582-5p could directly target FOXO1. In conclusion, miR-582-5p inhibited apoptosis of monocytes by down-regulating FOXO1 expression and might play an important role in regulating anti-M. tuberculosis directed immune responses.

Development of Androgen- and Estrogen-Responsive bio-assays, members of a panel of human cell line-based highly selective steroid-responsive bioassays

NARCIS (Netherlands)

Sonneveld, E.; Jansen, H.J.

2004-01-01

We have established highly sensitive and specific androgen and estrogen reporter cell lines which we have named AR (androgen receptor) and ERα (estrogen receptor alpha) CALUX® (Chemically Activated LUciferase eXpression), respectively. Both bioassays are member of a panel of CALUX reporter cell

Development of androgen-and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid-responsive bioassays

NARCIS (Netherlands)

Sonneveld, E.; Jansen, H.J..; Riteco, J.A.C.; Brouwer, A.

2005-01-01

We have established highly sensitive and specific androgen and estrogen reporter cell lines which we have named AR (androgen receptor) and ERα (estrogen receptor alpha) CALUX® (Chemically Activated LUciferase eXpression), respectively. Both bioassays are member of a panel of CALUX reporter cell

CEREM activity report

International Nuclear Information System (INIS)

1997-01-01

This 1997 issue of the activity report of the CEREM (CEA) gives a general overview of the CEREM organization, activities and human resources with some budgetary information. The main activities described concern the materials fabrication for the nuclear and automotive industries, the powders metallurgy (molding by injection, cold pressing modelling), the simulation of the hot and cold working processes, the surface treatments, the joining (brazed joints, sintering, welding), the electrochemical storage of the energy (fuel cells, lithium accumulators), the corrosion and the physico-chemistry of fluids, the mechanical properties and the cracks, the robots for nuclear units dismantlement, the alteration mechanisms of properties under different solicitations (thermal, mechanical, chemical, irradiation) and the solidification-crystallogenesis. (O.M.)

Warm Water Bath Stimulates Phase-Shifts of the Peripheral Circadian Clocks in PER2::LUCIFERASE Mouse

Science.gov (United States)

Kuriki, Daisuke; Haraguchi, Atsushi; Shibata, Shigenobu

2014-01-01

Circadian clocks in the peripheral tissues of mice are known to be entrained by pulse stimuli such as restricted feeding, novel wheel running, and several other agents. However, there are no reports on high temperature pulse-mediated entrainment on the phase-shift of peripheral clocks in vivo. Here we show that temperature treatment of mice for two days at 41°C, instead of 37°C, for 1–2 h during the inactive period, using a temperature controlled water bath stimulated phase-advance of peripheral clocks in the kidney, liver, and submandibular gland of PER2::LUCIFERASE mice. On the other hand, treatment for 2 days at 35°C ambient room temperature for 2 h did not cause a phase-advance. Maintenance of mice at 41°C in a water bath, sustained the core body temperature at 40–41°C. However, the use of 37°C water bath or the 35°C ambient room temperature elevated the core body temperature to 38.5°C, suggesting that at least a core body temperature of 40–41°C is necessary to cause phase-advance under light-dark cycle conditions. The temperature pulse stimulation at 41°C, instead of 37°C water bath for 2 h led to the elevated expression of Per1 and Hsp70 in the peripheral tissue of mice. In summary, the present study demonstrates that transient high temperature pulse using water bath during daytime causes phase-advance in mouse peripheral clocks in vivo. The present results suggest that hot water bath may affect the phase of peripheral clocks. PMID:24933288

A small scale cell culture system to analyze mechanobiology using reporter gene constructs and polyurethane dishes

DEFF Research Database (Denmark)

Seefried, Lothar; Mueller-Deubert, Sigrid; Wentzer, Thomas Schwarz

2010-01-01

of mechanotransduction and its crosstalk with biochemically induced signal transduction, AP1 and SP1 luciferase reporter gene constructs were cloned and transfected into various cell lines and primary cells. A newly developed bioreactor and small-scale 24-well polyurethane dishes were used to apply cyclic stretching...

ITER activities status report: December 1989

International Nuclear Information System (INIS)

1990-01-01

Beginning in 1988 and continuing through 1990, the four Parties involved, under the auspices of the IAEA, have been cooperating in the ITER Conceptual Design Activities. This activity resulted in a single conceptual design for a facility that could achieve the objectives established for ITER. This report is an interim report which gives a brief summary of the Conceptual Design Activities through December, 1989. 4 figs, 1 tab

Activity report 1984

International Nuclear Information System (INIS)

Drent, W.

1985-07-01

For the fiscal year 1984, the technical activities of Eurochemic were: a) the cleaning of the vessels having contained medium level liquid wastes and the incorporation into bitumen of the resulting solution, b) the conditioning of plutonium contaminated solid waste, c) the civil engineering works of Building 29 were completed and the mounting of equipment was begun. The installation will assure the surface storage of the blocks resulting from vitrification, by means of Pamela installation of the high-level liquid wastes. The report includes administrative matters as well as health and safety activity. (AF)

Induction of human adiponectin gene transcription by telmisartan, angiotensin receptor blocker, independently on PPAR-γ activation

International Nuclear Information System (INIS)

Moriuchi, Akie; Yamasaki, Hironori; Shimamura, Mika; Kita, Atsushi; Kuwahara, Hironaga; Fujishima, Keiichiro; Satoh, Tsuyoshi; Fukushima, Keiko; Fukushima, Tetsuya; Hayakawa, Takao; Mizuguchi, Hiroyuki; Nagayama, Yuji; Abiru, Norio; Kawasaki, Eiji; Eguchi, Katsumi

2007-01-01

Adiponectin, an adipose tissue-specific plasma protein, has been shown to ameliorate insulin resistance and inhibit the process of atherosclerosis. Recently, several reports have stated that angiotensin type 1 receptor blockers (ARBs), increase adiponectin plasma level, and ameliorate insulin resistance. Telmisartan, a subclass of ARBs, has been shown to be a partial agonist of the peroxisome proliferator-activated receptor (PPAR)-γ, and to increase the plasma adiponectin level. However, the transcriptional regulation of the human adiponectin gene by telmisartan has not been determined yet. To elucidate the effect of telmisartan on adiponectin, the stimulatory regulation of human adiponectin gene by telmisartan was investigated in 3T3-L1 adipocytes, utilizing adenovirus-mediated luciferase reporter gene-transferring technique. This study indicates that telmisartan may stimulate adiponectin transcription independent of PPAR-γ

Activity report 1983

International Nuclear Information System (INIS)

Delande, E.; Drent, W.

1984-07-01

The reports presents an account of the activities of the Eurochemic Company for the chemical processing of irradiated fuels, in the fiscal year l982. It deals with legal and administrative matters, industrial development, plant operation, new works, health and safety, library and documentation, and conferences and symposia. (AF)

Activity report 1992

International Nuclear Information System (INIS)

1993-01-01

This activity report of the DSIN ( Direction of Nuclear Installations Safety) is divided into 4 parts bearing on: (1) Organisation, Safety principles, Regulations (2) Nuclear basis installations following (3) Surveillance by the regional directions of the Ministry of Industry, Research and Environment (4) Other missions of the DSIN

Assessment report on research and development activities. Activity: 'Advanced science research' (Interim report)

Energy Technology Data Exchange (ETDEWEB)

NONE

2012-11-15

Japan Atomic Energy Agency (hereinafter referred to as 'JAEA') consulted an assessment committee, 'Evaluation Committee of Research Activities for Advanced Science Research' (hereinafter referred to as 'Committee') for interim assessment of 'Advanced Science Research,' in accordance with 'General Guideline for the Evaluation of Government Research and Development (R and D) Activities' by Cabinet Office, Government of Japan, 'Guideline for Evaluation of R and D in Ministry of Education, Culture, Sports, Science and Technology' and 'Regulation on Conduct for Evaluation of R and D Activities' by JAEA. In response to the JAEA's request, the Committee assessed the research programs and activities of the Advanced Science Research Center (hereinafter referred to as 'ASRC') for the period of two years from April 2010. The Committee evaluated the management and the research programs of the ASRC based on the explanatory documents prepared by the ASRC and the oral presentations with questions-and-answers by the Director and the research group leaders. This report summarizes the result of the assessment by the Committee with the Committee report attached from page 7. (author)

Methylated polycyclic aromatic hydrocarbons and/or their metabolites are important contributors to the overall estrogenic activity of polycyclic aromatic hydrocarbon-contaminated soils.

Science.gov (United States)

Lam, Monika M; Engwall, Magnus; Denison, Michael S; Larsson, Maria

2018-02-01

In the present study 42 polycyclic aromatic compounds (PACs) were investigated for their estrogenic potential using the VM7Luc4E2 transactivation assay. Relative potencies were determined for mass-balance analysis. In addition, compounds were tested in combination with the estrogen receptor (ER) antagonist ICI182,780 (ICI) and the aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor α-naphthoflavone. Luciferase induction and CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity were measured to assess whether the estrogenic activity was elicited by the compound itself and/or by its metabolites. Relative potencies ranged between 10 -7 and 10 -4 . The ability of ICI to decrease luciferase activity stimulated by all compounds indicated that the induction responses were ER-dependent. The aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor α-naphthoflavone decreased luciferase induction and EROD activity by several compounds, including the methylated chrysenes, suggesting that metabolites of these chemicals contributed to ER activation. Several PACs, such as acridine and its derivatives, appear to directly activate the ER. Furthermore, extracts of soils from industrial areas were examined using this bioassay, and estrogenic activity was detected in all soil samples. Mass-balance analysis using a combination of relative potencies and chemical analysis of the samples suggested that polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs, such as 1- and 3-methylchrysene, are important contributors to the overall estrogenic activity. However, these results revealed that a considerable proportion of the estrogenic activity in the soil remained unexplained, indicating the presence of other significant estrogenic compounds. Environ Toxicol Chem 2018;37:385-397. © 2017 SETAC. © 2017 SETAC.

PPARγ induces growth inhibition and apoptosis through upregulation of insulin-like growth factor-binding protein-3 in gastric cancer cells

Energy Technology Data Exchange (ETDEWEB)

Kim, S.Y. [Department of Pediatrics, Chonbuk National University Hospital, Jeonju (Korea, Republic of); Biomedical Research Institute, School of Medicine, Chonbuk National University Hospital, Jeonju (Korea, Republic of); Kim, M.S.; Lee, M.K. [Department of Pediatrics, Chonbuk National University Hospital, Jeonju (Korea, Republic of); Kim, J.S.; Yi, H.K. [Department of Biochemistry, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Nam, S.Y. [Department of Alternative Therapy, Jeonju University, Jeonju (Korea, Republic of); Lee, D.Y.; Hwang, P.H. [Department of Pediatrics, Chonbuk National University Hospital, Jeonju (Korea, Republic of); Biomedical Research Institute, School of Medicine, Chonbuk National University Hospital, Jeonju (Korea, Republic of)

2015-01-13

Peroxisome proliferator activator receptor-gamma (PPARγ) is a ligand-activated transcriptional factor involved in the carcinogenesis of various cancers. Insulin-like growth factor-binding protein-3 (IGFBP-3) is a tumor suppressor gene that has anti-apoptotic activity. The purpose of this study was to investigate the anticancer mechanism of PPARγ with respect to IGFBP-3. PPARγ was overexpressed in SNU-668 gastric cancer cells using an adenovirus gene transfer system. The cells in which PPARγ was overexpressed exhibited growth inhibition, induction of apoptosis, and a significant increase in IGFBP-3 expression. We investigated the underlying molecular mechanisms of PPARγ in SNU-668 cells using an IGFBP-3 promoter/luciferase reporter system. Luciferase activity was increased up to 15-fold in PPARγ transfected cells, suggesting that PPARγ may directly interact with IGFBP-3 promoter to induce its expression. Deletion analysis of the IGFBP-3 promoter showed that luciferase activity was markedly reduced in cells without putative p53-binding sites (-Δ1755, -Δ1795). This suggests that the critical PPARγ-response region is located within the p53-binding region of the IGFBP-3 promoter. We further demonstrated an increase in PPARγ-induced luciferase activity even in cells treated with siRNA to silence p53 expression. Taken together, these data suggest that PPARγ exhibits its anticancer effect by increasing IGFBP-3 expression, and that IGFBP-3 is a significant tumor suppressor.

Activity report 1977

International Nuclear Information System (INIS)

Delande, E.; Drent, W.

1978-07-01

The principal activities of Eurochemic plant in the fields of reprocessing and the solidification and conditioning of liquid wastes are presented in this report that covers the period 1 January to 31 December 1977. Works in the plant are closely related to the handling of fuel element and the treatment of solid wastes as well as the management of liquid wastes. Industrial development activities have been primarily devoted to the solidification of HEWC (highly enriched waste concentrate) by means of the Lotes process; the treatment of spent solvent by means of the Eurowatt process; the treatment of the plutonium-bearing solid wastes by the Eurowetcomb process and the conditioning of highly activity non-combustible solid wastes by incorporation into polymer-concrete matrix . (AF)

Activity report 1978

International Nuclear Information System (INIS)

Delande, E.; Drent, W.

1979-07-01

The principal activities of Eurochemic plant in the fields of reprocessing and the solidification and conditioning of liquid and solid wastes are presented in this report that covers the period 1 January to 31 December 1978. Works in the plant are closely related to the decontamination in cooperation with specialized firms. Active operation of bituminization facility was started in June, and a total of 66 m 3 of medium level waste was incorporated into bitumen by the end of 1978. The associated storage facility was in active operation since June and preliminary studies for its extension were completed. A facility was completed for the conditioning of solid wastes arising from the decontamination operation of solid wastes whch were stored under water during plant operation. (AF)

VGB activity report 1989/1990

International Nuclear Information System (INIS)

1990-01-01

This annual report of the VGB Association is intended to contribute to an exchange of experience and results obtained by activities on the technical and scientific levels, regarding safety, availability, and economics of power plant operation. The report further presents availability and damage statistics compiled by power plant operators. The material of the report is arranged by sections, the first, more general part reporting on organisational aspects and activities of the VGB such as conferences, publications, lectures, etc. The following parts deal with the activities carried out by the various main sections of the VGB, presenting the latest developments and knowledge gathered, related to such main subjects as thermal power plants, fuelling and furnaces, emissions, power plant chemistry, materials and stresses, building construction, and research work. A summary of power plant data is followed by facts and data of power plant engineering from all VGB member countries: Argentina, Australia, Brazil, Peoples Republic of China, Denmark, Federal Republic of Germany, Finland, France, Greece, Great Britain, India, Iran, Ireland, Israel, Yugoslavia, Korea, Luxembourg, New Zealand, Netherlands, Norway, Austria, Portugal, Sweden, Switzerland, Zimbabwe, Spain, South Africa, USA. (HS) [de

VGB activity report 1988/1989

International Nuclear Information System (INIS)

1989-01-01

This annual report of the VGB Association is intended to contribute to an exchange of experience and results obtained by activities on the technical and scientific levels, regarding safety, availability, and economics of power plant operation. The report further presents availability and damage statistics compiled by power plant operators. The material of the report is arranged by sections, the first, more general part reporting on organisational aspects and activities of the VGB such as conferences, publications, lectures, etc. The following parts deal with the activities carried out by the various main sections of the VGB, presenting the latest developments and knowledge gathered, related to such main subjects as thermal power plants, fuelling and furnaces, emissions, power plant chemistry, materials and stresses, building construction, and research work. A summary of power plant data is followed by facts and data of power plant engineering from all VGB member countries: Argentina, Australia, Brazil, Peoples Republic of China, Denmark, Federal Republic of Germany, Finland, France, Greece, Great Britain, India, Iran, Ireland, Israel, Yugoslavia, Korea, Luxembourg, New Zealand, Netherlands, Norway, Austria, Portugal, Sweden, Switzerland, Zimbabwe, Spain, South Africa, USA. (HS) [de

TRANSCRIPTIONAL INHIBITION OF INTERLEUKIN-12 PROMOTER ACTIVITY IN LEISHMANIA SPP.-INFECTED MACROPHAGES

Science.gov (United States)

Jayakumar, Asha; Widenmaier, Robyn; Ma, Xiaojing; McDowell, Mary Ann

2009-01-01

To establish and persist within a host, Leishmania spp. parasites delay the onset of cell-mediated immunity by suppressing interleukin-12 (IL-12) production from host macrophages. Although it is established that Leishmania spp.-infected macrophages have impaired IL-12 production, the mechanisms that account for this suppression remain to be completely elucidated. Using a luciferase reporter assay assessing IL-12 transcription, we report here that Leishmania major, Leishmania donovani, and Leishmania chagasi inhibit IL-12 transcription in response to interferon-gamma, lipopolysaccharide, and CD40 ligand and that Leishmania spp. lipophosphoglycan, phosphoglycans, and major surface protein are not necessary for inhibition. In addition, all the Leishmania spp. strains and life-cycle stages tested inhibited IL-12 promoter activity. Our data further reveal that autocrine-acting host factors play no role in the inhibitory response and that phagocytosis signaling is necessary for inhibition of IL-12. PMID:18372625

MiR-155 is upregulated in patients with active tuberculosis and inhibits apoptosis of monocytes by targeting FOXO3.

Science.gov (United States)

Huang, Jian; Jiao, Junhua; Xu, Weihua; Zhao, Huayang; Zhang, Chunxiao; Shi, Yan; Xiao, Zhijian

2015-11-01

The aim of the present study was to investigate the association between microRNA (miR)-155 and apoptosis of monocytes infected by Mycobacterium tuberculosis, to examine the effect of forkhead box O3 (FOXO3) on miR‑155. The present study analysed the apoptosis of CD14+ in the peripheral blood of patients with active tuberculosis, disposed the THP‑1 human monocytic cell line by BCG and examined the expression of miR‑155. Furthermore, the expression of FOXO3 in THP‑1 cells was determined, and wild- and mutant-type luciferase reporter plasmids containing FOXO3 3'‑untranslated regions (UTRs) were constructed to analyse the expression of luciferase. Finally, an over‑expression plasmid was constructed, and THP-1 cells were transfected with control miRNA, miR‑155 and the plasmid, which revealed that miR‑155 inhibited the apoptosis of THP‑1 cells. miR‑155 in the THP‑1 cells infected by BCG was upregulated and apoptosis also increased. However, the apoptosis declined when the cells were transfected with the control miRNA and miR‑155. Folllowing transfection with miR‑155, the expression of FOXO3 decreased. Transfection with miR‑155 and the FOXO3 3'-UTRs significantly reduced luciferase, and overexpression of FOXO3 reversed the inhibitory role of miR‑155. From these results, it was concluded that mycobacteria can improve the level of miR‑155, while BCG can induce apoptosis in THP‑1 cells. The results suggested FOXO3 is a downstream target gene of miR‑155, which combines 3'-UTRs to inhibit the expression of FOXO3.

Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin

International Nuclear Information System (INIS)

Paeshuyse, Jan; Coelmont, Lotte; Vliegen, Inge; Hemel, Johan van; Vandenkerckhove, Jan; Peys, Eric; Sas, Benedikt; Clercq, Erik De; Neyts, Johan

2006-01-01

We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC 5 ) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78 ± 21 μM. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 μM) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin

Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin

Energy Technology Data Exchange (ETDEWEB)

Paeshuyse, Jan [Rega Institute for Medical Research, Minderbroedersstraat 10, KULeuven, B-3000 Leuven (Belgium); Coelmont, Lotte [Rega Institute for Medical Research, Minderbroedersstraat 10, KULeuven, B-3000 Leuven (Belgium); Vliegen, Inge [Rega Institute for Medical Research, Minderbroedersstraat 10, KULeuven, B-3000 Leuven (Belgium); Hemel, Johan van [Kemin Pharma, Atealaan 4H, B-2200 Herentals (Belgium); Vandenkerckhove, Jan [Kemin Pharma, Atealaan 4H, B-2200 Herentals (Belgium); Peys, Eric [Kemin Pharma, Atealaan 4H, B-2200 Herentals (Belgium); Sas, Benedikt [Kemin Pharma, Atealaan 4H, B-2200 Herentals (Belgium); Clercq, Erik De [Rega Institute for Medical Research, Minderbroedersstraat 10, KULeuven, B-3000 Leuven (Belgium); Neyts, Johan [Rega Institute for Medical Research, Minderbroedersstraat 10, KULeuven, B-3000 Leuven (Belgium)

2006-09-15

We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC{sub 5}) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78 {+-} 21 {mu}M. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 {mu}M) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin.

Activity report 1998

International Nuclear Information System (INIS)

1998-01-01

The aim of this report is to outline the main developments of the ''Departement des Reacteurs Experimentaux'', (DRE) during the year 1998. DRE is one of the Department of the ''Direction des Reacteurs Nucleaires'', itself depending of the CEA Institution. After a presentation of the year highlights, this report gathers the main research and development programs. The second part concerns the production of radioisotopes, the silicon doping, the neutron radiography, the Orphee experiments and the activation analysis. The installations management, the closed reactors improvement program and the effluents and wastes processing of Grenoble, are presented in the other parts. Data on staff, budget and safety are also provided. (A.L.B.)

Activity report 1999

International Nuclear Information System (INIS)

1999-01-01

The aim of this report is to outline the main developments of the ''Departement des Reacteurs Experimentaux'', (DRE) during the year 1999. DRE is one of the Department of the ''Direction des Reacteurs Nucleaires'', itself depending of the CEA Institution. After a presentation of the year highlights, this report gathers the main research and development programs. The second part concerns the production of radioisotopes, the silicon doping, the neutron radiography, the Orphee experiments and the activation analysis. The installations management, the closed reactors improvement program and the effluents and wastes processing of Grenoble, are presented in the other parts. Data on staff, budget and safety are also provided. (A.L.B.)

Reporters to monitor cellular MMP12 activity

Science.gov (United States)

Cobos-Correa, Amanda; Mall, Marcus A.; Schultz, Carsten

2010-02-01

Macrophage elastase, also called MMP12, belongs to a family of proteolytic enzymes whose best known physiological function is the remodeling of the extracellular matrix. Under certain pathological conditions, including inflammation, chronic overexpression of MMP12 has been observed and its elevated proteolytic activity has been suggested to be the cause of pulmonary emphysema. However, it was until recently impossible to monitor the activity of MMP12 under disease conditions, mainly due to a lack of detection methods. Recent development of new reporters for monitoring MMP12 activity in living cells, such as LaRee1, provided novel insights into the pathobiology of MMP12 in pulmonary inflammation.1 In the future, these reporters might contribute to improved diagnosis and in finding better treatments for chronic inflammatory lung diseases and emphysema. Our approach for visualizing MMP12 activity is based on peptidic, membrane-targeted FRET (Foerster Resonance Energy Transfer) reporters. Here we describe a set of new reporters containing different fluorophore pairs as well as modifications in the membrane-targeting lipid moiety. We studied the influence of these modifications on reporter performance and the reporter mobility on live cell membranes by FRAP (fluorescence recovery after photobleaching). Finally, we generated several new fluorescently labeled MMP inhibitors based on the peptidic reporter structures as prototypes for future tools to inhibit and monitor MMP activity at the same time.

IPSN activity report 1999

International Nuclear Information System (INIS)

1999-01-01

The ''Institut de Protection et de Surete Nucleaire'' (IPSN), carries out researches and expert studies needed to manage nuclear risks and their consequences on people and environment. These activities cover the installation safety, the radioactive matter transport, the public health and environmental control, the nuclear matters safety and control and the crisis situation management. The IPSN have got also a mission of public information and participates to international actions in the domain of scientific researches or expert's report. This annual report presents the highlights of the year 1999, information on staff, budgets and geographical situation. Then technical and scientific papers gathers the activities of the year covered by the IPSN: the ''Free Tribune'', installations and radioactive wastes safety, crisis and nuclear matter management, public health and environment control, international cooperation. (A.L.B.)

Microwave-assisted synthesis, molecular docking and antitubercular activity of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives

Czech Academy of Sciences Publication Activity Database

Mohan, S. B.; Kumar, B. V. V. R.; Dinda, S. C.; Naik, D.; Seenivasan, S. P.; Kumar, V.; Rana, D. N.; Brahmkshatriya, Pathik

2012-01-01

Roč. 22, č. 24 (2012), s. 7539-7542 ISSN 0960-894X Institutional research plan: CEZ:AV0Z40550506 Keywords : antitubercular * binding interactions * luciferase reporter phage (LRP) assay * microwave-assisted * molecular docking Subject RIV: CC - Organic Chemistry Impact factor: 2.338, year: 2012

Considerations for potency equivalent calculations in the Ah receptor-based CALUX bioassay: normalization of superinduction results for improved sample potency estimation.

Science.gov (United States)

Baston, David S; Denison, Michael S

2011-02-15

The chemically activated luciferase expression (CALUX) system is a mechanistically based recombinant luciferase reporter gene cell bioassay used in combination with chemical extraction and clean-up methods for the detection and relative quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like halogenated aromatic hydrocarbons in a wide variety of sample matrices. While sample extracts containing complex mixtures of chemicals can produce a variety of distinct concentration-dependent luciferase induction responses in CALUX cells, these effects are produced through a common mechanism of action (i.e. the Ah receptor (AhR)) allowing normalization of results and sample potency determination. Here we describe the diversity in CALUX response to PCDD/Fs from sediment and soil extracts and not only report the occurrence of superinduction of the CALUX bioassay, but we describe a mechanistically based approach for normalization of superinduction data that results in a more accurate estimation of the relative potency of such sample extracts. Copyright © 2010 Elsevier B.V. All rights reserved.

Activity report 1981

International Nuclear Information System (INIS)

Delande, E.; Drent, W.

1982-07-01

This report reviews the main activities of the Eurochemic company for the fiscal year 1981. These activities are: the plant operation including the safeguards for special nuclear materials, the management of radioactive wastes and the analytical services, new works such as vitrification project, Pamela building and the design for the storage of vitrified wastes. The industrial development concerns the Eurowatt process for solvent treatment, the wet combustion for plutonium bearing solid wastes, the separation of mercury, the incorporation of solid wastes into a polymer concrete matrix and the Pamela process. The health and safety division, the library and documentation service and the administrative matters are also presented. (AF)

Report of Activity - 1997

International Nuclear Information System (INIS)

Hammer, Philippe

1999-01-01

The Report of Activity on 1997 published by Waste Management Division (Direction chargee de la Gestion des Dechets) of French Atomic Energy Authority (Commissariat a l'Energie Atomique) presents the current activity in the framework of CEA Recovery Plan for Civil Centers initiated in 1991, aiming at managing all the radioactive problems issued from its Research and Development Activities. This plan covers three principal directions: management of radioactive wastes resulting from CEA research activities, reprocessing or storing the spent or un-used fuel resulting from research or prototype reactors, recovery measures and dismantling of nuclear facilities. In the field of radioactive waste management important projects, assuring the waste processing equipment, were developed in 1997 at Saclay (STELLA Installation), Cadarache (AGATE Installation) and Marcoule (ATENA Installation). The Radioactive Waste Storage TFA was put into operation at Cadarache. In the field of out-of-use fuel, in 1997 the fuel reprocessing units CARAMEL and UNGG were finished while as temporary storage for fuels waiting to be processed the CASCAD facility at Cadarache was assigned. Finally, concerning the recovery/dismantling activities, priority was granted to recover: the Radiochemical Lab. at Fontenay, the pilot workshop and UP1 plant of COGEMA at Marcoule, the EL4 reactor (in a joint action with EDF), and the AT1 pilot workshop at Cherbourg. The report is structured on five chapters with the following content: 1. The radioactive wastes; 2. The out-of-use fuels; 3. The dismantling of the definitively stopped installations; 4. The waste management at DAM (Direction des Applications Militaires); 5. Recovery activities at Marcoule

Assessment report of research and development activities. Activity: 'Advanced science research' (Pre-review report)

International Nuclear Information System (INIS)

2010-11-01

Japan Atomic Energy Agency (hereinafter referred to as 'JAEA') consulted an assessment committee, 'Evaluation Committee of Research Activities for Advanced Science Research' (hereinafter referred to as 'Committee') for prior assessment of 'Advanced Science Research,' in accordance with 'General Guideline for the Evaluation of Government Research and Development (R and D) Activities' by Cabinet Office, Government of Japan, 'Guideline for Evaluation of R and D in Ministry of Education, Culture, Sports, Science and Technology' and 'Regulation on Conduct for Evaluation of R and D Activities' by JAEA. In response to the JAEA's request, the Committee assessed the research program and activities of the Advanced Science Research Center (hereinafter referred to as 'ASRC') for the period of five years from April 2010. The Committee evaluated the management and the research program of the ASRC based on the explanatory documents prepared by the ASRC and the oral presentations with questions-and-answers by the Director and the research group leaders. This report summarizes the result of the assessment by the Committee with the Committee report attached from page 7. (author)

IPSN's 2001 activity report

International Nuclear Information System (INIS)

2001-01-01

This report summarizes the 2001 activities and operation of the French institute of nuclear protection and safety (IPSN) which has become the institute of radioprotection and nuclear safety (IRSN) after its merging with the office of protection against ionizing radiations (OPRI). IPSN's activities cover: the safety of nuclear facilities, the safety of nuclear wastes, the management of emergencies, the management of sensible materials, the protection of the environment and of the public health, some research activities with foreign partners (central and eastern Europe, Asia..), and a partnership with the GRS, the German homologue of IPSN. (J.S.)

Development and Characterisation of a Novel NF-κB Reporter Cell Line for Investigation of Neuroinflammation

Directory of Open Access Journals (Sweden)

Marie-Theres Zeuner

2017-01-01

Full Text Available Aberrant activation of the transcription factor NF-κB, as well as uncontrolled inflammation, has been linked to autoimmune diseases, development and progression of cancer, and neurological disorders like Alzheimer’s disease. Reporter cell lines are a valuable state-of-the art tool for comparative analysis of in vitro drug screening. However, a reporter cell line for the investigation of NF-κB-driven neuroinflammation has not been available. Thus, we developed a stable neural NF-κB-reporter cell line to assess the potency of proinflammatory molecules and peptides, as well as anti-inflammatory pharmaceuticals. We used lentivirus to transduce the glioma cell line U251-MG with a tandem NF-κB reporter construct containing GFP and firefly luciferase allowing an assessment of NF-κB activity via fluorescence microscopy, flow cytometry, and luminometry. We observed a robust activation of NF-κB after exposure of the reporter cell line to tumour necrosis factor alpha (TNFα and amyloid-β peptide [1-42] as well as to LPS derived from Salmonella minnesota and Escherichia coli. Finally, we demonstrate that the U251-NF-κB-GFP-Luc reporter cells can be used for assessing the anti-inflammatory potential of pharmaceutical compounds using Bay11-7082 and IMD0354. In summary, our newly generated cell line is a robust and cost-efficient tool to study pro- and anti-inflammatory potential of drugs and biologics in neural cells.

Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression.

Science.gov (United States)

DeSmet, Marsha L; Fleet, James C

2017-10-01

High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH) 2 D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH) 2 D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH) 2 D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH) 2 D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention. Copyright © 2017 Elsevier Ltd. All rights reserved.

Space-time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the IAA19 promoter fusion with a destabilized luciferase reporter.

Science.gov (United States)

Yamamoto, Kotaro T; Watahiki, Masaaki K; Matsuzaki, Jun; Satoh, Soichirou; Shimizu, Hisayo

2017-07-01

Imaging analysis was carried out during the gravitropic response of etiolated Arabidopsis hypocotyls, using an IAA19 promoter fusion of destabilized luciferase as a probe. From the bright-field images we obtained the local deflection angle to the vertical, A, local curvature, C, and the partial derivative of C with respect to time, [Formula: see text]. These were determined every 19.9 µm along the curvilinear length of the hypocotyl, at ~10 min intervals over a period of ~6 h after turning hypocotyls through 90° to the horizontal. Similarly from the luminescence images we measured the luminescence intensity of the convex and concave flanks of the hypocotyl as well as along the median of the hypocotyl, to determine differential expression of auxin-inducible IAA19. Comparison of these parameters as a function of time and curvilinear length shows that the gravitropic response is composed of three successive elements: the first and second curving responses and a decurving response (autostraightening). The maximum of the first curving response occurs when A is 76° along the entire length of the hypocotyl, suggesting that A is the sole determinant in this response; in contrast, the decurving response is a function of both A and C, as predicted by the newly-proposed graviproprioception model (Bastien et al., Proc Natl Acad Sci USA 110:755-760, 2013). Further, differential expression of IAA19, with higher expression in the convex flank, is observed at A = 44°, and follows the Sachs' sine law. This also suggests that IAA19 is not involved in the first curving response. In summary, the gravitropic response of Arabidopsis hypocotyls consists of multiple elements that are each determined by separate principles.

miR-200c targets nuclear factor IA to suppress HBV replication and gene expression via repressing HBV Enhancer I activity.

Science.gov (United States)

Tian, Hui; He, Zhenkun

2018-03-01

Hepatitis B virus (HBV) chronic infection is a health problem in the worldwide, with a underlying higher risk of liver cirrhosis and hepaticocellular carcinoma. A number of studies indicate that microRNAs (miRNAs) play vital roles in HBV replication. This study was designed to explore the potential molecular mechanism of miR-200c in HBV replication. The expression of miR-200c, nuclear factor IA (NFIA) mRNA, HBV DNA, and HBV RNA (pregenomic RNA (pgRNA), and total RNA) were measured by qRCR. The levels of HBsAg and HBeAg were detected by ELISA. NFIA expression at protein level was measured by western blot. The direct interaction between miR-200c and NFIA were identified by Targetscan software and Dual-Luciferase reporter analysis. Enhance I activity were detected by Dual-Luciferase reporter assay. miR-200c expression was prominently reduced in pHBV1.3-tranfected Huh7 and in stable HBV-producing cell line (HepG2.2.15). The enforced expression of miR-200c significantly suppressed HBV replication, as demonstrated by the reduced levels of HBV protein (HBsAg and HBeAg) and, DNA and RNA (pgRNA and total RNA) levels. NFIA was proved to be a target of miR-200c and NFIA overexpression notably stimulated HBV replication. In addition, the inhibitory effect of miR-200c on HBV Enhance I activity was abolished following restoration of NFIA. miR-200c repressed HBV replication by directly targeting NFIA, which might provide a novel therapeutic target for HBV infection. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Nuclear factor-kappaB activation correlates with better prognosis and Akt activation in human gastric cancer.

Science.gov (United States)

Lee, Byung Lan; Lee, Hye Seung; Jung, Jieun; Cho, Sung Jin; Chung, Hee-Yong; Kim, Woo Ho; Jin, Young-Woo; Kim, Chong Soon; Nam, Seon Young

2005-04-01

Because the biological significance of constitutive nuclear factor-kappaB (NF-kappaB) activation in human gastric cancer is unclear, we undertook this study to clarify the regulatory mechanism of NF-kappaB activation and its clinical significance. Immunohistochemistry for NF-kappaB/RelA was done on 290 human gastric carcinoma specimens placed on tissue array slides. The correlations between NF-kappaB activation and clinicopathologic features, prognosis, Akt activation, tumor suppressor gene expression, or Bcl-2 expression were analyzed. We also did luciferase reporter assay, Western blot analysis, and reverse transcription-PCR using the SNU-216 human gastric cancer cell line transduced with retroviral vectors containing constitutively active Akt or the NF-kappaB repressor mutant of IkappaBalpha. Nuclear expression of RelA was found in 18% of the gastric carcinomas and was higher in early-stage pathologic tumor-node-metastasis (P = 0.019). A negative correlation was observed between NF-kappaB activation and lymphatic invasion (P = 0.034) and a positive correlation between NF-kappaB activation and overall survival rate of gastric cancer patients (P = 0.0228). In addition, NF-kappaB activation was positively correlated with pAkt (P = 0.047), p16 (P = 0.004), adenomatous polyposis coli (P Smad4 (P = 0.002), and kangai 1 (P Akt. NF-kappaB activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis and Akt activation. These findings suggest that NF-kappaB activation is a valuable prognostic variable in gastric carcinoma.

Activity report 1993

International Nuclear Information System (INIS)

1994-01-01

This activity report of the DSIN (Direction of Nuclear Installations Safety) is divided into 13 chapters bearing on: (1)Nuclear safety (2) Nuclear safety organisation (3) Regulations (4) Nuclear installations inspections (5) Crisis organisation (6) Information and communication (7) International relations (8) PWR (9)Other reactors (10) Experimental reactors, laboratories and other installations (11) Fuel cycle centers (12) Radioactive wastes storages (13) Nuclear installations definitively stopped

Fisetin inhibits epidermal growth factor-induced migration of ARPE-19 cells by suppression of AKT activation and Sp1-dependent MMP-9 expression.

Science.gov (United States)

Lin, Hung-Yu; Chen, Yong-Syuan; Wang, Kai; Chien, Hsiang-Wen; Hsieh, Yi-Hsien; Yang, Shun-Fa

2017-01-01

Proliferative vitreoretinopathy (PVR) can result in abnormal migration of RPE cells. Fisetin is a naturally occurring compound that has been reported to have antitumor effects, but its effects on epidermal growth factor (EGF)-induced cell migration and the underlying mechanisms remain unclear. Effects of fisetin on EGF-induced cell viability and migration were examined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and in vitro migration assays. Reverse transcription-PCR (RT-PCR) and immunoblotting were performed to evaluate matrix metallopeptidase-9 (MMP-9) expression and activation of specificity protein-1 (Sp1) and protein kinase B (AKT) in ARPE-19 cells treated with EGF and with or without fisetin. Luciferase and chromatin immunoprecipitation (ChIP) assays were performed to examine Sp1 transcription activity and MMP-9 binding activity. Fisetin did not affect ARPE-19 cell viability and significantly inhibited the EGF-induced migration capacity of ARPE-19 cells. Furthermore, fisetin exerted an antimigratory effect and suppressed MMP-9 mRNA and protein expression. Treatment with EGF induced phosphorylation of AKT and expression of MMP-9 and Sp1. Fisetin combined with LY294002 (an inhibitor of AKT) prevented the EGF-induced migration involved in downregulation of Sp1 and MMP-9 expression. Luciferase and ChIP assays suggested that fisetin remarkably decreased the EGF-induced transcription activity of MMP-9 and Sp1 and inhibited EGF-mediated Sp1 from directly binding to the MMP-9 promoter in ARPE-19 cells. Fisetin inhibited EGF-induced cell migration via modulation of AKT/Sp1-dependent MMP-9 transcriptional activity. Therefore, fisetin may be a potential agent in the treatment of migratory PVR diseases.

Responsible Development of AREVA's Mining Activities - Report 2012

International Nuclear Information System (INIS)

2013-09-01

By defining AREVA's strategy and policies, this report aims to demonstrate the company's performance in the key areas of mining activity responsibility: ethics and governance, social report, the environment, occupational health and safety, community involvement, commitments to stakeholders. The data given cover the assets for which AREVA acts as operator in uranium mining activities: exploration, project development, production and rehabilitation. The consolidated data target activities in France, Canada, Niger, Kazakhstan, Mongolia, Gabon and Namibia. Activities in the Central African Republic and those linked to La Mancha no longer fall within the scope of this report (sale of assets in 2012). This report is the third edition of this annual exercise

Development and characterization of a green fluorescent protein-based rat cell bioassay system for detection of AH receptor ligands

Energy Technology Data Exchange (ETDEWEB)

Zhao Bin; Denison, M. [California Univ., Davis, CA (United States). Dept. of Environmental Toxicology

2004-09-15

Proper epidemiological, risk assessment and exposure analysis of TCDD and related HAHs requires accurate measurements of these chemicals both in the species of interest and in various exposure matrices (i.e. biological, environmental, food and feed). While high-resolution instrumental analysis techniques are established for these chemicals, these procedures are very costly, time-consuming and are impractical for large scale sampling studies. Accordingly, numerous bioanalytical methods have been developed for the detection of these chemicals in extracts from a variety of matrices, the majority of which take the advantage of the ability of these chemicals to activate one or more aspects of the AhR-dependent mechanism of action. One of the most sensitive bioassay systems developed to date is the so-called CALUX (Chemically Activated Luciferase Expression) assay, which is based on novel recombinant cell lines that contain a stably transfected dioxin (AhR)-responsive firefly luciferase gene. Treatment of these cells with TCDD and related HAHs and polycyclic aromatic hydrocarbons (PAHs), as well as other AhR ligands, results in induction of reporter gene expression in a time-, dose-, AhR-, and chemical-specific manner. The level of reporter gene expression correlates with the total concentration of the TCDD-like AhR inducers (agonists) present in the sample. Although the firefly luciferase reporter gene contributes to the high degree of sensitivity of the assay, it also has limitations with respect to our need for a rapid and inexpensive bioassay for high-throughput screening analysis. Accordingly, we previously developed a stably transfected murine cell line containing an AhRresponsive enhanced green fluorescent protein (EGFP) reporter gene. This cell line provided us with a high-throughput cell bioassay system for identification and characterization of AhR agonists and antagonists. Here we have extended these studies and describe the development, optimization, and

Screening of molecular cell targets for carcinogenic heterocyclic aromatic amines by using CALUX® reporter gene assays.

Science.gov (United States)

Steinberg, Pablo; Behnisch, Peter A; Besselink, Harrie; Brouwer, Abraham A

2017-06-01

Heterocyclic aromatic amines (HCAs) are compounds formed when meat or fish are cooked at high temperatures for a long time or over an open fire. To determine which pathways of toxicity are activated by HCAs, nine out of the ten HCAs known to be carcinogenic in rodents (2-amino-9H-pyrido[2,3-b]indole (AαC), 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)) were tested in the estrogen receptor α (ERα), androgen receptor (AR), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor γ2 (PPARγ2), polycyclic aromatic hydrocarbons (PAH), Nrf2, and p53 CALUX® reporter gene assays. Trp-P-1 was the only HCA that led to a positive response in the ERα, PPARγ2, and Nrf2 CALUX® assays. In the PAH CALUX® assay, Trp-P-2, MeAαC, and AαC induced luciferase activity to a greater extent than MeIQ and PhIP. In the p53 CALUX® assay without a coupled metabolic activation, only Trp-P-1 and Trp-P-2 enhanced luciferase expression; when a metabolic activation step was coupled to the p53 CALUX® assay, Trp-P-1, Glu-P-2, MeIQ, MeIQx, and PhIP induced a positive response. No HCA was positive in the AR and GR CALUX® assays. Taken together, the results obtained show that the battery of CALUX® assays performed in the present study can successfully be used to screen for molecular cell targets of carcinogenic compounds such as HCAs.

Activity report 1993-1994

International Nuclear Information System (INIS)

1995-05-01

This reports presents the Leon Brillouin Laboratory (LLB) activity during 1993-1994. Concerning the physico-chemical and biological systems, polymers are still a main field of the LLB. New ideas and experiments appeared also in the field water, ionic solids, polyelectrolyte and biologicals systems. Concerning structures and phase transitions, activity in the field of crystalline structures has been reinforced. Disordered systems, metallurgy and material fields were also developed. Concerning magnetism and superconductivity, important results were obtained. (TEC). 159 refs., 241 figs., 29 tabs

Nitric Oxide Synthase Type III Overexpression By Gene Therapy Exerts Antitumoral Activity In Mouse Hepatocellular Carcinoma

Directory of Open Access Journals (Sweden)

Raúl González

2015-08-01

Full Text Available Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO synthase type III (NOS-3 overexpression induces cell death in hepatoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. Hepa 1-6 cells were used for in vitro and in vivo experiments. The first generation adenovirus was designed to overexpress NOS-3 (or GFP and luciferase cDNA under the regulation of murine alpha-fetoprotein (AFP and Rous Sarcoma Virus (RSV promoters, respectively. Both adenoviruses were administered through the tail vein two weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-Luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8 activity in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by l-NAME or p53 siRNA. The tail vein infusion of AFP-NOS- 3/RSV-Luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

RTE - Activity report 2011

International Nuclear Information System (INIS)

2012-01-01

RTE, an independent subsidiary of EDF, is the French electricity transmission system operator. It is a public service company responsible for operating, maintaining and developing the high and extra high voltage network. It guarantees the reliability and proper operation of the power network. RTE transports electricity between electricity suppliers (French and European) and consumers, whether they are electricity distributors or industrial consumers directly connected to the transmission system. The mission of RTE is to balance the electricity supply and demand in real time. This publication gathers in a single document the 2011 Activity and Sustainable development report of RTE, the Management report with the consolidated financial statements, and the Report from the chairman of the supervisory board

RTE - Activity report 2010

International Nuclear Information System (INIS)

2011-01-01

RTE, an independent subsidiary of EDF, is the French electricity transmission system operator. It is a public service company responsible for operating, maintaining and developing the high and extra high voltage network. It guarantees the reliability and proper operation of the power network. RTE transports electricity between electricity suppliers (French and European) and consumers, whether they are electricity distributors or industrial consumers directly connected to the transmission system. The mission of RTE is to balance the electricity supply and demand in real time. This publication gathers in a single document the 2010 Activity and Sustainable development report of RTE, the Management report with the consolidated financial statements, and the Report from the chairman of the supervisory board

Transcriptional activity of Pax3 is co-activated by TAZ

International Nuclear Information System (INIS)

Murakami, Masao; Tominaga, Junji; Makita, Ryosuke; Uchijima, Yasunobu; Kurihara, Yukiko; Nakagawa, Osamu; Asano, Tomoichiro; Kurihara, Hiroki

2006-01-01

Pax3 is a transcription factor which functions in embryonic development and human diseases. In a yeast two-hybrid screen with full-length Pax3 as bait, we isolated a clone encoding transcriptional co-activator with PDZ-binding motif (TAZ) from an E10.5 mouse embryo cDNA library. Co-immunoprecipitation and nuclear co-localization of TAZ with Pax3 suggest that their association is functionally relevant. In situ hybridization revealed TAZ and Pax3 expression to partially overlap in the paraxial mesoderm, limb buds, and the neural tube. In C2C12 myoblast cells and NIH3T3 cells, TAZ enhanced the transcriptional activity of Pax3 on artificial and microphthalmia-associated transcription factor promoter-luciferase constructs, suggesting that TAZ can function as a co-activator of Pax3. Functional interaction between Pax3 and TAZ may provide a clue to clarifying the mechanism by which Pax3 serves as a transcriptional activator during embryogenesis

Peroxisome Proliferator-Activated Receptor γ Regulates the Expression of Lipid Phosphate Phosphohydrolase 1 in Human Vascular Endothelial Cells

Directory of Open Access Journals (Sweden)

Yazi Huang

2014-01-01

Full Text Available Lipid phosphate phosphohydrolase 1 (LPP1, a membrane ectophosphohydrolase regulating the availability of bioactive lipid phosphates, plays important roles in cellular signaling and physiological processes such as angiogenesis and endothelial migration. However, the regulated expression of LPP1 remains largely unknown. Here, we aimed to examine a role of peroxisome proliferator-activated receptor γ (PPARγ in the transcriptional control of LPP1 gene expression. In human umbilical vein endothelial cells (HUVECs, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR demonstrated that activation of PPARγ increased the mRNA level of LPP1. Chromatin immunoprecipitation assay showed that PPARγ binds to the putative PPAR-responsive elements (PPREs within the 5′-flanking region of the human LPP1 gene. Genomic fragment containing 1.7-kilobase of the promoter region was cloned by using PCR. The luciferase reporter assays demonstrated that overexpression of PPARγ and rosiglitazone, a specific ligand for PPARγ, could significantly upregulate the reporter activity. However, site-directed mutagenesis of the PPRE motif abolished the induction. In conclusion, our results demonstrated that PPARγ transcriptionally activated the expression of LPP1 gene in ECs, suggesting a potential role of PPARγ in the metabolism of phospholipids.

Andra 1997 activity report

International Nuclear Information System (INIS)

1998-07-01

This activity report comprises two parts. The first part describes the missions and activities of the ANDRA (the French agency for the management of radioactive wastes): quality control of wastes produced by EdF, CEA, Cogema and the 1100 other small producers, design, construction and management of storage centres, anticipation and development of new management solutions, research and evaluation studies on deep storage facilities, inventory and control of all radioactive materials present in the French territory. The second part is a presentation of financial data. (J.S.)

100 Areas technical activities report, engineers - March 1951

Energy Technology Data Exchange (ETDEWEB)

1951-04-01

This is the monthly 100 areas technical activities report from the engineering division for the month of March 1951. It reports on engineering activities related directly to the different production reactors, and gives progress reports on various engineering projects which are in development by the engineering group.

A Requirement for ZAK Kinase Activity in Canonical TGF-β Signaling

Directory of Open Access Journals (Sweden)

Shyam Nyati

2016-12-01

Full Text Available The sterile alpha motif and leucine zipper containing kinase ZAK (AZK, MLT, MLK7, is a MAPK-kinase kinase (MKKK. Like most MAPKKKs which are known to activate the c-Jun. amino-terminal kinase (JNK pathway, ZAK has been shown to participate in the transduction of Transforming growth factor-β (TGF-β-mediated non-canonical signaling. A role for ZAK in SMAD-dependent, canonical TGF-β signaling has not been previously appreciated. Using a combination of functional genomics and biochemical techniques, we demonstrate that ZAK regulates canonical TGFβRI/II signaling in lung and breast cancer cell lines and may serve as a key node in the regulation of TGFBR kinase activity. Remarkably, we demonstrate that siRNA mediated depletion of ZAK strongly inhibited TGF-β dependent SMAD2/3 activation and subsequent promoter activation (SMAD binding element driven luciferase expression; SBE4-Luc. A ZAK specific inhibitor (DHP-2, dose-dependently activated the bioluminescent TGFBR-kinase activity reporter (BTR, blocked TGF-β induced SMAD2/3 phosphorylation and SBE4-Luc activation and cancer cell-invasion. In aggregate, these findings identify a novel role for the ZAK kinase in canonical TGF-β signaling and an invasive cancer cell phenotype thus providing a novel target for TGF-β inhibition.

Recovery Efficiency Test Project: Phase 1, Activity report

Energy Technology Data Exchange (ETDEWEB)

Overbey, W.K. Jr.; Wilkins, D.W.; Keltch, B.; Saradji, B.; Salamy, S.P.

1988-04-01

This report is the second volume of the Recovery Efficiency Test Phase I Report of Activities. Volume 1 covered selection, well planning, drilling, coring, logging and completion operations. This volume reports on well testing activities, reclamation activities on the drilling site and access roads, and the results of physical and mechanical properties tests on the oriented core material obtained from a horizontal section of the well. 3 refs., 21 figs., 10 tabs.

1980/1981 activities report

International Nuclear Information System (INIS)

1982-03-01

The National Uranium Resource Evaluation (NURE) had as its major goal the development of reliable, timely, and comprehensive estimates of the uranium resources of the United States. Geologic, geochemical, geophysical, and other data were acquired which contributed to assessment of the distribution and magnitude of these uranium resources, as well as to determination of areas favorable for the occurrence of uranium and to establishment of potential resource estimates. The data-collection phase of the NURE program ended September 30, 1980. The NURE program was organized around six major activity elements: Quadrangle Evaluation, Data Acquisition, Resource Assessment, Geologic Studies, Technology Applications, and Mineral Economics. This fifth activities report presents summary results on work performed during the period 1980/1981 in each of these element areas, together with appendices that cite individual project data and resulting reports. By the end of 1981, evaluation of an additional 28 quadrangles had been completed, bringing to 163 the total number covered by the NURE program

75 FR 75586 - Confidentiality of Suspicious Activity Reports

Science.gov (United States)

2010-12-03

...] RIN 1550-AC26 Confidentiality of Suspicious Activity Reports AGENCY: The Office of Thrift Supervision... implementing the Bank Secrecy Act (BSA) governing the confidentiality of a suspicious activity report (SAR) to... corporations regulated by the OTS, to keep certain records and make certain reports that have been determined...

75 FR 75576 - Confidentiality of Suspicious Activity Reports

Science.gov (United States)

2010-12-03

...-2010-0019] RIN 1557-AD17 Confidentiality of Suspicious Activity Reports AGENCY: The Office of the... suspicious activity report (SAR) to: clarify the scope of the statutory prohibition on the disclosure by a... institutions, including national banks regulated by the OCC, to keep certain records and make certain reports...

Regulation of NF-{kappa}B activity in astrocytes: effects of flavonoids at dietary-relevant concentrations

Energy Technology Data Exchange (ETDEWEB)

Spilsbury, Alison [Reading School of Pharmacy, University of Reading, Reading RG6 6UB (United Kingdom); Vauzour, David; Spencer, Jeremy P.E. [Molecular Nutrition Group, Centre for Integrative Neuroscience and Neurodynamics, School of Chemistry, Food and Pharmacy, University of Reading, Reading RG6 6AP (United Kingdom); Rattray, Marcus, E-mail: m.a.n.rattray@reading.ac.uk [Reading School of Pharmacy, University of Reading, Reading RG6 6UB (United Kingdom)

2012-02-17

Highlights: Black-Right-Pointing-Pointer We tested the hypothesis that low concentrations of flavonoids inhibit NF-{kappa}B in astrocytes. Black-Right-Pointing-Pointer Primary cultured astrocytes possess a functional {kappa}B-system, measured using luciferase assays. Black-Right-Pointing-Pointer Seven flavonoids (100 nM-1 {mu}M) failed to reduce NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer Four flavonoids (100 nM-1 {mu}M) failed to reduce TNFa-stimulated NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer (-)-Epicatechin did not regulate nuclear translocation of the NF-{kappa}B subunit, p65. -- Abstract: Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Sustained activation of nuclear transcription factor {kappa}B (NF-{kappa}B) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-{kappa}B signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNF{alpha}, 150 ng/ml) increased NF-{kappa}B-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative I{kappa}B{alpha} construct. In addition, TNF{alpha} increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-{kappa}B activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((-)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1-1 {mu}M) for 18 h. None of the flavonoids modulated constitutive or

Biological validation of a sample preparation method for ER-CALUX bioanalysis of estrogenic activity in sediments using mixtures of xeno-estrogens

NARCIS (Netherlands)

Houtman, C.J.; Houten, Y.K.; Leonards, P.E.G.; Brouwer, A.; Lamoree, M.H.; Legler, J.

2006-01-01

The combined estrogenic effects of mixtures of environmental pollutants in the in vitro ER-CALUX (chemical activated luciferase gene expression) bioassay were examined to biologically validate a sample preparation method for the analysis of estrogenic compounds in sediment. The method used

Biolonical validation of a sample preparation method for ER-CALUX bioanalysis of estrogenic activity in sediment using mixtures of xeno-estrogens

NARCIS (Netherlands)

Houtman, C.J.; Houten, Van Y.K.; Leonards, P.E.G.; Brouwer, A.; Lamoree, M.H.; Legler, J.

2006-01-01

The combined estrogenic effects of mixtures of environmental pollutants in the in vitro ER-CALUX (chemical activated luciferase gene expression) bioassay were examined to biologically validate a sample preparation method for the analysis of estrogenic compounds in sediment. The method used

Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases

Directory of Open Access Journals (Sweden)

Xinxia Zhao

2016-03-01

Full Text Available Myostatin (MSTN is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs in tandem with single-stranded DNA oligonucleotides (ssODNs. We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

Activation of farnesoid X receptor downregulates monocyte chemoattractant protein-1 in murine macrophage

Energy Technology Data Exchange (ETDEWEB)

Li, Liangpeng; Zhang, Qian; Peng, Jiahe; Jiang, Chanjui; Zhang, Yan; Shen, Lili; Dong, Jinyu; Wang, Yongchao; Jiang, Yu, E-mail: yujiang0207@163.com

2015-11-27

Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily, which plays important roles in bile acids/lipid homeostasis and inflammation. Monocyte chemoattractant protein-1 (MCP-1) contributes to macrophage infiltration into body tissues during inflammation. Here we investigated whether FXR can regulate MCP-1 expression in murine macrophage. FXR activation down regulate MCP-1 mRNA and protein levels in ANA-1 and Raw264.7 cells. Luciferase reporter assay, Gel shift and Chromatin immunoprecipitation assays have revealed that the activated FXR bind to the FXR element located in −738 bp ∼  −723 bp in MCP-1 promoter. These results suggested that FXR may serve as a novel target for regulating MCP-1 levels for the inflammation related diseases therapies. - Highlights: • FXR is expressed in murine macrophage cell line. • FXR down regulates MCP-1 expression. • FXR binds to the DR4 in MCP-1 promoter.

Activity report. July 1992 - June 1993

International Nuclear Information System (INIS)

1993-09-01

The Department of Physics and Measurement Technology, Biology and Chemistry (IFM) presents its 23nd consecutive annual progress report. It contains a brief description of activities in research and education within the department. The report is intended as an information for colleagues and institutions. The present report contains activities for the academic year July 1992 to June 1993. According to the present plans the erection of the new building will start in the beginning of 1994 and the construction time will be less than two years. The picture on the cover is taken from an ongoing research project at IFM. It shows a polymer light-emitting diode. A red polymer is sandwiched between a transparent conducting glass electrode and two narrow Aluminium electrodes. When a voltage of approximately 10 V is applied between the conducting glass electrode and one of the two Aluminium electrodes yellow-orange light is emitted from the polymer. Studies of these diodes are part of the activities in Applied Physics

A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma.

Science.gov (United States)

Söling, Ariane; Theiss, Christian; Jungmichel, Stephanie; Rainov, Nikolai G

2004-08-04

BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.

HUD Initiated Activity Cancellation Reports

Data.gov (United States)

Department of Housing and Urban Development — This monthly report displays all HOME activities automatically cancelled by IDIS. Effective January 1, 2011, and the beginning of every month thereafter, committed...

Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

Directory of Open Access Journals (Sweden)

Mamaghani Shadi

2009-04-01

Full Text Available Abstract Background Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. Methods GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. Results GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-XL, and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. Conclusion GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard

Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

International Nuclear Information System (INIS)

Mamaghani, Shadi; Patel, Satish; Hedley, David W

2009-01-01

Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-X L , and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard chemotherapy agent gemcitabine. This lack of synergy might be

Activity report 1998-1999

International Nuclear Information System (INIS)

1999-01-01

The aim of this report is to outline the main developments of the ''Departement d'Ecophysiologie Vegetale et de Microbiologie'', (DEVM) during the period 1998-1999. DEVM is one of the Department of the CEA Institution. This report presents the activities of the laboratories of the department. Among these laboratories, two laboratories have applications of radiation: the laboratory of vegetal radiobiology and the laboratory of research on food quality. The research programs on radiation effects on living organisms and regulation of food or drug radiation process, respectively for the two laboratories, are discussed. (A.L.B.)

Quantitative and Functional Requirements for Bioluminescent Cancer Models.

Science.gov (United States)

Feys, Lynn; Descamps, Benedicte; Vanhove, Christian; Vermeulen, Stefan; Vandesompele, J O; Vanderheyden, Katrien; Messens, Kathy; Bracke, Marc; De Wever, Olivier

2016-01-01

Bioluminescent cancer models are widely used but detailed quantification of the luciferase signal and functional comparison with a non-transfected control cell line are generally lacking. In the present study, we provide quantitative and functional tests for luciferase-transfected cells. We quantified the luciferase expression in BLM and HCT8/E11 transfected cancer cells, and examined the effect of long-term luciferin exposure. The present study also investigated functional differences between parental and transfected cancer cells. Our results showed that quantification of different single-cell-derived populations are superior with droplet digital polymerase chain reaction. Quantification of luciferase protein level and luciferase bioluminescent activity is only useful when there is a significant difference in copy number. Continuous exposure of cell cultures to luciferin leads to inhibitory effects on mitochondrial activity, cell growth and bioluminescence. These inhibitory effects correlate with luciferase copy number. Cell culture and mouse xenograft assays showed no significant functional differences between luciferase-transfected and parental cells. Luciferase-transfected cells should be validated by quantitative and functional assays before starting large-scale experiments. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity.

NARCIS (Netherlands)

Boerboom, A.M.A.; Vermeulen, M.; Woude, H. van der; Bremer, B.I.; Lee-Hilz, Y.Y.; Kampman, E.; Bladeren, P.J. van; Rietjens, I.M.C.M.; Aarts, J.

2006-01-01

The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity

NARCIS (Netherlands)

Boerboom, A.M.J.F.; Vermeulen, M.; Woude, H. van der; Bremer, B.I.; Lee-Hilz, Y.Y.; Kampman, E.; Bladeren, P.J. van; Rietjens, I.M.C.M.; Aarts, J.M.M.J.G.

2006-01-01

The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

ILL experimental reports and theory college activities

International Nuclear Information System (INIS)

1987-01-01

This volume is the collection of reports describing experimental work performed at the I.L.L. They have been arranged by subject (from the I.L.L. classification scheme) and are published under their proposal number. (Only those reports received up to Jan. 19, 1987 have been included). In order to have connection between the theory group and the experimental activities of the I.L.L., scientific activity of theoreticians has been included in the form of a personal review of activity. For any further details, readers should contact directly individual scientists

Fisetin inhibits epidermal growth factor–induced migration of ARPE-19 cells by suppression of AKT activation and Sp1-dependent MMP-9 expression

Science.gov (United States)

Lin, Hung-Yu; Chen, Yong-Syuan; Wang, Kai; Chien, Hsiang-Wen

2017-01-01

Purpose Proliferative vitreoretinopathy (PVR) can result in abnormal migration of RPE cells. Fisetin is a naturally occurring compound that has been reported to have antitumor effects, but its effects on epidermal growth factor (EGF)–induced cell migration and the underlying mechanisms remain unclear. Methods Effects of fisetin on EGF-induced cell viability and migration were examined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and in vitro migration assays. Reverse transcription–PCR (RT–PCR) and immunoblotting were performed to evaluate matrix metallopeptidase-9 (MMP-9) expression and activation of specificity protein-1 (Sp1) and protein kinase B (AKT) in ARPE-19 cells treated with EGF and with or without fisetin. Luciferase and chromatin immunoprecipitation (ChIP) assays were performed to examine Sp1 transcription activity and MMP-9 binding activity. Results Fisetin did not affect ARPE-19 cell viability and significantly inhibited the EGF-induced migration capacity of ARPE-19 cells. Furthermore, fisetin exerted an antimigratory effect and suppressed MMP-9 mRNA and protein expression. Treatment with EGF induced phosphorylation of AKT and expression of MMP-9 and Sp1. Fisetin combined with LY294002 (an inhibitor of AKT) prevented the EGF-induced migration involved in downregulation of Sp1 and MMP-9 expression. Luciferase and ChIP assays suggested that fisetin remarkably decreased the EGF-induced transcription activity of MMP-9 and Sp1 and inhibited EGF-mediated Sp1 from directly binding to the MMP-9 promoter in ARPE-19 cells. Conclusions Fisetin inhibited EGF-induced cell migration via modulation of AKT/Sp1–dependent MMP-9 transcriptional activity. Therefore, fisetin may be a potential agent in the treatment of migratory PVR diseases. PMID:29296070

NEA activities in 1980. 9. Activity Report

International Nuclear Information System (INIS)

1981-01-01

This report describes the main features of the Agency's work during 1980 and discusses the state and prospects of the nuclear industry in OECD countries. Trends in nuclear power, radiological and environmental impacts of nuclear fuel cycle activities, nuclear safety research and licensing, nuclear law, nuclear development and fuel cycle studies technical co-operation, nuclear science organisation and administration are reviewed

Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux in a mammalian cell line.

Directory of Open Access Journals (Sweden)

Dan M Close

Full Text Available The bacterial luciferase (lux gene cassette consists of five genes (luxCDABE whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2 was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp from Vibrio harveyi. FMNH(2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

Report of Laboratory Activity, 1996 - 1997

International Nuclear Information System (INIS)

1997-01-01

This report presents the activity of the Laboratory of Particle Physics and Cosmology of College de France on the years 1996-1997 in the fields of Cosmic Physics, Observational Cosmology, Neutrino Experiments, HELLAZ Project, Instrumentation, DELPHI Experiment, Research of Quark-Gluon Plasma, Research on Dark Matter, Theory, Parallel Processing. Also, are mentioned the activities in computer software, electronics, mechanics, general service, publications, external relations, seminars and collaborations. In the field of Cosmic Physics there are described the current experiments on cosmic gamma rays, the work with AUGER observatory and simulations. In the field of observational cosmology there are mentioned the search for baryonic dark matter and studies on type Ia supernovae. In the field of neutrino studies there are described the searches on neutrino oscillations on a 1 km base, while in the framework of HELLAZ project there is reported the work on solar neutrinos. In the field of instrumentation there are mentioned the work on Hybrid Photon Detector and the contribution of the laboratory to the LHC-B Experiment at CERN and on long-base RICH experiment. In the framework of DELPHI experiment at LEP there are reported investigations on beauty particles, new particles and detector performances. There are given results obtained in the field of Quark-Gluon Plasma studies. There are described the research and development works with the dark matter detectors. In the field of theory there are reported studies on the proton structure, photon-photon collisions, the physics of the excited leptons and studies on neutron stars. Also, in this field there is reported the studies in Quantum Chromodynamics and physics of top quark. In the section devoted to parallel processing there are mentioned the research activities related to actinide burning by accelerators and simulations in nuclear medicine issues, electron channelling in crystals and beam-beam effect in colliders. The

1990-1991 activity report

International Nuclear Information System (INIS)

1992-01-01

The 1990-1991 activity report of LYON Nuclear Physics Institute is presented and includes the following topics: scientific and technical works in theoretical physics, high and intermediate energy nuclear physics, low and mean energy nuclear physics and multi-field physics (by means of AMPHORA multidetector, EUROGAM high performance gamma multidetector); theses, congress contributions and published papers

Report of Activity, 1996 - 1997

International Nuclear Information System (INIS)

Augustin, Jean-Eudes

1998-01-01

This report covers the activity developed in the Lyon Institute of Nuclear Physics in the years 1996-1997. The INP-Lyon is a mixed research unit, dependent on Lyon 1 Claude Bernard University, on one side, and on CNRS Institute of Nuclear and Particle Physics, on the other side. Its personnel of some 200 fellows is formed up of a half of researchers and undergraduate researchers, the other half being engineers, technicians and administration personnel. The principal activities in INP Lyon are the experimental research in sub-atomic physics as well as the training by research of the master and enrolled towards Ph D students. The main research problems concern the particle physics nuclear matter and the particle astrophysics as well as the works in theoretical physics, radioactive product management and the interactions ions/clusters - matter. The report contains the following chapters: Physics at LEP, Experiment preparation for LHC, Hadronic and Nuclear Matter, Astro-particles, Theoretical Physics, Ions/clusters - matter and physico-chemistry of the ion-matter interaction, Technical activities, Scientific life at INP -Lyon

Activities Report 2008 PATTEC-Mali

International Nuclear Information System (INIS)

2008-01-01

This annual report is a summary of main activities undertaken in 2008 under the Pan African Tsetse and Trypanosomiasis project in continuation of 2007. Further results about integrated actitivities of combating the vector are presented

ANCCLI. 2012 activity report

International Nuclear Information System (INIS)

2012-01-01

This report first proposes a synthesis of the ANCCLI (the national association of local information committees) progress report, and then a presentation of the context (relationships between CLIs and the ANCCLI, situation at the local, European and international levels). It presents the various ANCCLI's bodies (scientific committee, permanent groups, task officers' club) and their activities. It proposes an overview of partnership with different agencies and bodies (ASN, IRSN, ministries, and HCTISN or High committee for transparency and information on nuclear safety). It presents the ANCCLI's European strategy, and the national and international participations of the ANCCLI to various actions and bodies

ANDRA. 2003 activity report

International Nuclear Information System (INIS)

2004-05-01

This document includes both the activity report and the management and financial situation report of the French national agency for the management of radioactive wastes (ANDRA). Content: ANDRA's missions: 1 - the industrial mission (short-lived low- and medium-level wastes, a new disposal facility for the very low level wastes, wastes from diffuse nuclear origins); 2 - the research mission (methodology and approach, knowledge gained in 2003, the 2003 experimental program of the Meuse-Haute Marne underground laboratory, the three approaches applied to research, projects for radium- and graphite-bearing wastes, international relations); 3 - information mission (public expectations and specific answers, inventory mission). (J.S.)

Activity report june 1996 - may 1998

International Nuclear Information System (INIS)

1998-01-01

The aim of this report is to outline the scientific activity of the ''Service de Physique Theorique'' (SPhT), during the period june 1996-may 1998: activity in terms of structure, scientific research programs and results, publications, training and contracts. SPhT is one of the service of the ''Direction des Sciences de la Matiere'' (DSM), itself depending of the CEA Institution. The scientific domains covered by this report are: the statistical physics (phase transition, biophysics, soft matter, quantum systems); mathematical physics and fields theory (strings theory, random matrix, quantum theory); astrophysics, nuclear physics and particles physics. (A.L.B.)

Useful Bicistronic Reporter System for Studying Poly(A Site-Defining cis Elements and Regulation of Alternative Polyadenylation

Directory of Open Access Journals (Sweden)

Zhongyuan Deng

2018-01-01

Full Text Available The link between polyadenylation (pA and various biological, behavioral, and pathological events of eukaryotes underlines the need to develop in vivo polyadenylation assay methods for characterization of the cis-acting elements, trans-acting factors and environmental stimuli that affect polyadenylation efficiency and/or relative usage of two alternative polyadenylation (APA sites. The current protein-based CAT or luciferase reporter systems can measure the polyadenylation efficiency of a single pA site or candidate cis element but not the choice of two APA sites. To address this issue, we developed a set of four new bicistronic reporter vectors that harbor either two luciferase or fluorescence protein open reading frames connected with one Internal Ribosome Entry Site (IRES. Transfection of single or dual insertion constructs of these vectors into mammalian cells demonstrated that they could be utilized not only to quantify the strength of a single candidate pA site or cis element, but also to accurately measure the relative usage of two APA sites at both the mRNA (qRT-PCR and protein levels. This represents the first reporter system that can study polyadenylation efficiency of a single pA site or element and regulation of two APA sites at both the mRNA and protein levels.

Agrice 2002. Activity report

International Nuclear Information System (INIS)

2002-01-01

Renewable raw materials derived from agricultural crops are used to manufacture bio-products in energy, chemicals and materials industries. Renewable feedstocks address a double need. On the one hand, they offer alternatives to fossil resources, and on the other hand, they are a response to today's environmental challenges: mitigation of the greenhouse affect, reduction of air, soil and water pollution, innocuousness and biodegradability of products. A new chemistry must be created. Large-scale and ongoing research work must be committed to this goal. The research effort implies active coordination of all the various actors involved - multidisciplinary research teams, agro-industrial enterprises, user industries (petrochemicals, chemicals, materials), and specific agricultural branches. With these ends in mind the scientific interest group AGRICE - Agriculture for Chemicals and Energy - was established by public authorities and eight partners in France in 1994. The AGRICE consortium was renewed for six years in 2001, with a broader membership base. AGRICE now includes the following partners: - the research arms of the Commissariat a l'Energie Atomique (CEA), the Institut Francais du Petrole (IFP), and the Institut National de Recherche Agronomique (INRA), - professional organisations representing producers of oilseeds (ONIDOL), grains (AGPB) and beets (CGB), - corporate members ATOFINA, AUTOBAR PACKAGING France, BAYER CROP SCIENCE, CERESTAR France, LIMAGRAIN Agro-Industrie, RHODIA, TOTAL FINA ELF, - the French ministries of Agriculture, Industry, Research, and the Environment, - the French Agency for the Environment and Energy Management (ADEME), - the French Agency for Innovation (ANVAR). ADEME is responsible for managing AGRICE. The consortium is involved in major international undertakings, notably through the European Renewable Resources and Materials Association (ERRMA). This report presents: 1 - the AGRICE profile, scope of activity, structure and Operations

Activity Report of Reactor Physics Division - 1997

International Nuclear Information System (INIS)

Singh, Om Pal

1998-01-01

The research and development activities of the Reactor Physics Division of the Indira Gandhi Centre for Atomic Research (IGCAR), Kalpakkam during 1997 are reported. The activities are arranged under the headings: nuclear data processing and validation, PFBR and KAMINI core physics, FBTR core physics, radioactivity and shielding and safety analysis. A list of publications of the Division and seminars delivered are included at the end of the report

Momordica charantia Inhibits Inflammatory Responses in Murine Macrophages via Suppression of TAK1.

Science.gov (United States)

Yang, Woo Seok; Yang, Eunju; Kim, Min-Jeong; Jeong, Deok; Yoon, Deok Hyo; Sung, Gi-Ho; Lee, Seungihm; Yoo, Byong Chul; Yeo, Seung-Gu; Cho, Jae Youl

2018-01-01

Momordica charantia known as bitter melon is a representative medicinal plant reported to exhibit numerous pharmacological activities such as antibacterial, antidiabetic, anti-inflammatory, anti-oxidant, antitumor, and hypoglycemic actions. Although this plant has high ethnopharmacological value for treating inflammatory diseases, the molecular mechanisms by which it inhibits the inflammatory response are not fully understood. In this study, we aim to identify the anti-inflammatory mechanism of this plant. To this end, we studied the effects of its methanol extract (Mc-ME) on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Specifically, we evaluated nitric oxide (NO) production, mRNA expression of inflammatory genes, luciferase reporter gene activity, and putative molecular targets. Mc-ME blocked NO production in a dose-dependent manner in RAW264.7 cells; importantly, no cytotoxicity was observed. Moreover, the mRNA expression levels of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were decreased by Mc-ME treatment in a dose-dependent manner. Luciferase assays and nuclear lysate immunoblotting analyses strongly indicated that Mc-ME decreases the levels of p65 [a nuclear factor (NF)-[Formula: see text]B subunit] and c-Fos [an activator protein (AP)-1 subunit]. Whole lysate immunoblotting assays, luciferase assays, and overexpression experiments suggested that transforming growth factor [Formula: see text]-activated kinase 1 (TAK1) is targeted by Mc-ME, thereby suppressing NF-[Formula: see text]B and AP-1 activity via downregulation of extracellular signal-regulated kinases (ERKs) and AKT. These results strongly suggest that Mc-ME exerts its anti-inflammatory activity by reducing the action of TAK1, which also affects the activation of NF-[Formula: see text]B and AP-1.

Tributyltin or triphenyltin inhibits aromatase activity in the human granulosa-like tumor cell line KGN.

Science.gov (United States)

Saitoh, M; Yanase, T; Morinaga, H; Tanabe, M; Mu, Y M; Nishi, Y; Nomura, M; Okabe, T; Goto, K; Takayanagi, R; Nawata, H

2001-11-23

The superimposition of male sex organs (penis and vas deferens) in a female gastropod, called imposex, is widely attributed to the exposure to tributyltin (TBT) compounds, used world-wide in antifouling paints for ships. It has been hypothesized that the TBT-induced imposex is mediated by an increasing androgen level relative to the estrogen level, namely a decreased conversion of androgens to estrogens (i.e., aromatization). In the present study, we tested this hypothesis by examining the effects of TBT or triphenyltin (TPT) on the aromatase activity in a cultured human granulosa-like tumor cell line, KGN, which was recently established by our group. Treatment with more than 1000 ng/ml TBT compounds was very toxic to the cells and caused immediate cell death within 24 h, while 200 ng/ml was found to cause apoptosis of the cells. Treatment of the KGN cells for more than 48 h with 20 ng/ml TBT or TPT, which is a concentration level reported to cause imposex in marine species, did not affect cell proliferation but significantly suppressed the aromatase activity determined by a [(3)H]H(2)O release assay. Treatment with 20 ng/ml TBT compounds for 7 days also resulted in a reduction of the E2 production from Delta 4-androstenedione stimulated by db-cAMP. The changes in the aromatase activity by TBT compounds were associated with comparable changes in P450arom mRNA assessed by RT-PCR. The luciferase activity of the P450arom promoter II (1 kb) decreased after the addition of 20 ng/ml TBT compounds in transfected KGN cells either in a basic state or in states stimulated by db-cAMP. The Ad4BP-dependent increase in the luciferase activity of P450arom promoter II was also downregulated by such treatments. These results indicate that TBT compounds inhibited the aromatase activity and also decreased the P450arom mRNA level at the transcriptional level in KGN cells. The direct inhibitory effect of TBT compounds on the aromatase activity may therefore partly explain the induction

Positive radionuclide imaging of miRNA expression using RILES and the human sodium iodide symporter as reporter gene is feasible and supports a protective role of miRNA-23a in response to muscular atrophy.

Directory of Open Access Journals (Sweden)

Viorel Simion

Full Text Available MicroRNAs (miRNAs are key players in many biological processes and are considered as an emerging class of pharmacology drugs for diagnosis and therapy. However to fully exploit the therapeutic potential of miRNAs, it is becoming crucial to monitor their expression pattern using medical imaging modalities. Recently, we developed a method called RILES, for RNAi-Inducible Luciferase Expression System that relies on an engineered regulatable expression system to switch-ON the expression of the luciferase gene when a miRNA of interest is expressed in cells. Here we investigated whether replacing the luciferase reporter gene with the human sodium iodide symporter (hNIS reporter gene will be also suited to monitor the expression of miRNAs in a clinical setting context. We provide evidence that radionuclide imaging of miRNA expression using hNIS is feasible although it is not as robust as when the luciferase reporter gene is used. However, under appropriate conditions, we monitored the expression of several miRNAs in cells, in the liver and in the tibialis anterior muscle of mice undergoing muscular atrophy. We demonstrated that radiotracer accumulation in transfected cells correlated with the induction of hNIS and with the expression of miRNAs detected by real time PCR. We established the kinetic of miRNA-23a expression in mice and demonstrated that this miRNA follows a biphasic expression pattern characterized by a loss of expression at a late time point of muscular atrophy. At autopsy, we found an opposite expression pattern between miRNA-23a and one of the main transcriptional target of this miRNA, APAF-1, and as downstream target, Caspase 9. Our results report the first positive monitoring of endogenously expressed miRNAs in a nuclear medicine imaging context and support the development of additional work to establish the potential therapeutic value of miRNA-23 to prevent the damaging effects of muscular atrophy.

Positive radionuclide imaging of miRNA expression using RILES and the human sodium iodide symporter as reporter gene is feasible and supports a protective role of miRNA-23a in response to muscular atrophy.

Science.gov (United States)

Simion, Viorel; Sobilo, Julien; Clemoncon, Rudy; Natkunarajah, Sharuja; Ezzine, Safia; Abdallah, Florence; Lerondel, Stephanie; Pichon, Chantal; Baril, Patrick

2017-01-01

MicroRNAs (miRNAs) are key players in many biological processes and are considered as an emerging class of pharmacology drugs for diagnosis and therapy. However to fully exploit the therapeutic potential of miRNAs, it is becoming crucial to monitor their expression pattern using medical imaging modalities. Recently, we developed a method called RILES, for RNAi-Inducible Luciferase Expression System that relies on an engineered regulatable expression system to switch-ON the expression of the luciferase gene when a miRNA of interest is expressed in cells. Here we investigated whether replacing the luciferase reporter gene with the human sodium iodide symporter (hNIS) reporter gene will be also suited to monitor the expression of miRNAs in a clinical setting context. We provide evidence that radionuclide imaging of miRNA expression using hNIS is feasible although it is not as robust as when the luciferase reporter gene is used. However, under appropriate conditions, we monitored the expression of several miRNAs in cells, in the liver and in the tibialis anterior muscle of mice undergoing muscular atrophy. We demonstrated that radiotracer accumulation in transfected cells correlated with the induction of hNIS and with the expression of miRNAs detected by real time PCR. We established the kinetic of miRNA-23a expression in mice and demonstrated that this miRNA follows a biphasic expression pattern characterized by a loss of expression at a late time point of muscular atrophy. At autopsy, we found an opposite expression pattern between miRNA-23a and one of the main transcriptional target of this miRNA, APAF-1, and as downstream target, Caspase 9. Our results report the first positive monitoring of endogenously expressed miRNAs in a nuclear medicine imaging context and support the development of additional work to establish the potential therapeutic value of miRNA-23 to prevent the damaging effects of muscular atrophy.

NEA activities in 1983. 12. Activity report

International Nuclear Information System (INIS)

1984-01-01

This report describes the main features of the Agency's work during 1983 and discusses the state and prospects of the nuclear industry in OECD countries. Trends in nuclear power, nuclear development and the fuel cycles nuclear safety technology and licensing, radiological and environmental impacts of nuclear fuel cycle activities, legal affairs, nuclear science, joint undertakings and other NEA joint projects, organisation and administration are reviewed

SIRT3 is a Mitochondrial Tumor Suppressor and Genetic Loss Results in a Murine Model for ER/PR-Positive Mammary Tumors Connecting Metabolism and Carcinogenesis Mitochondrial Tumor Suppressor. Revision

Science.gov (United States)

2013-11-01

dependent gene expression, as shown by co-transfection assays using an HRE luciferase reporter (Fig. 4b, bar 1 vs. 2). In addition, exposure to NAC...transfected with p3x- HRE -luciferase with or without NAC or stigmatellin and 40 hours afterwards luciferase levels were determined. (c) MTCLT3

RTE - Activity and sustainable development report 2016 + Business report - Consolidated accounts

International Nuclear Information System (INIS)

2017-01-01

This document gathers several reports. The first one is an activity report which proposes an overview of RTE activities by outlining the contribution to the improvement of territory, customer, and electric power system performance, the contribution to innovation in terms of energy and digital transition (for example, development of smart grids), and development perspectives in terms of use of open data, of development of partnerships, and of work organisation. The next report is a business report which gives and comments financial and legal information (discussion of various accounting aspects and elements), social information (employment, work organisation, social relationships, safety, health, life quality, training, equal opportunities), environmental information (general policy, circular economy, struggle against climate change and adaptation, protection and development of biodiversity), and societal information. The last part contains a table of consolidated accounts followed by a detailed discussion and comment of its content

Agrice 2001. Activity report

International Nuclear Information System (INIS)

2002-01-01

AGRICE was created in 1994 to support research on new renewable plant-based products other than foodstuffs, to open up new markets for agriculture, and to take action on today's environmental challenges: fighting the greenhouse effect, curbing pollution (VOCs, effluents, etc.), eco-toxics, preservation of health, and depletion of fossil energy supplies. The scope of AGRICE's activity covers primarily the industrial conversion of crop production to chemicals (lubricants, surfactants, solvents, etc.), energy (liquid and solid biofuels) and materials (agro-materials, biopolymers). AGRICE's brief is to stimulate applied technological research. The consortium first tackled work aimed at substituting plant-based products for products derived from fossil resources. This strategy has been progressively widened to take into account the inherent characteristics of plant-based products in order to meet new needs. In addition to research focusing on industrial products and processes, this action spans the study of agronomic improvements, project economics, markets, and energy and environmental assessments. This report presents: 1 - the AGRICE profile, scope of activity (Biofuels (vehicles and non-vehicles), Biomolecules, Biomaterials), structure and Operations, and sectors of focus; 2 - the year 2001: Toward a new golden age of renewable plant based resources?, Financial Report and Applications, Noteworthy Achievements in 2001, Events in 2001, European Joint Undertakings; 3 - appendixes: Breakdown of AGRICE grants by sector 1994-2001, Financial report 1994-2001, Partners, The ADEME / AGRICE team, Projects initiated and completed in 2001

Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases.

Science.gov (United States)

Takahashi, Yuki; Nishikawa, Makiya; Kobayashi, Naoki; Takakura, Yoshinobu

2005-07-20

Silencing of oncogenes or other genes contributing to tumor malignancy or progression by RNA interference (RNAi) offers a promising approach to treating tumor patients. To achieve RNAi-based tumor therapy, a small interfering RNA (siRNA) or siRNA-expressing vector needs to be delivered to tumor cells, but little information about its in vivo delivery has been reported. In this study, we examined whether the expression of the target gene in tumor cells can be suppressed by the delivery of RNAi effectors to primary and metastatic tumor cells. To quantitatively evaluate the RNAi effects in tumor cells, mouse melanoma B16-BL6 cells were stably transfected with both firefly (a model target gene) and sea pansy (an internal standard gene) luciferase genes to obtain B16-BL6/dual Luc cells. The target gene expression in subcutaneous primary tumors of B16-BL6/dual Luc cells was significantly suppressed by direct injection of the RNAi effectors followed by electroporation. The expression in metastatic hepatic tumors was also significantly reduced by an intravenous injection of either RNAi effector by the hydrodynamics-based procedure. These results indicate that the both RNAi effectors have a potential to silence target gene in tumor cells in vivo when successfully delivered to tumor cells.

Report of Activity, 1996 - 1997. Vol. 3. General activities - Technical Research

International Nuclear Information System (INIS)

Pouthas, Joel; Hutin, Christiane; Niogret Mathias

1999-01-01

This is the third tome of the Report of Activity of IPN - Orsay on 1996-1997. It deals with general activities and technical research. Summary reports and short communications are grouped in the following sections: 1 - Accelerators with the sub-divisions 1.1 - R and D on superconducting RF cavities; 1.2 - SPIRAL Project; 1.3 - Contribution to the LHC Project; 1.4 - Tandem; 2 - Targets, Sources and Injectors; 3 - Detectors and related instrumentation; 4 - Electronics; 5 - Data processing; 6 - Radioprotection and medical applications

Cell-Penetrating Bispecific Antibodies for Targeting Oncogenic Transcription Factors in Advanced Prostate Cancer

Science.gov (United States)

2015-10-01

observations with DHT -stimulated luciferase activity assays, enzalutamide had no effect on the constitutive induction of luciferase activity by the AR...to expess firefly luciferase under the control of a synthetic ARE. Similar studies were also performed with intact LNCaP cells stimulated with DHT ...enzalutamide. B. Demonstration that 3E10-AR441 can inhibit non-genomic, AR-dependent signaling. These studies used LNCaP cells stimulated with DHT , with a

Oleocanthal Modulates Estradiol-Induced Gene Expression Involving Estrogen Receptor α.

Science.gov (United States)

Keiler, Annekathrin Martina; Djiogue, Sefirin; Ehrhardt, Tino; Zierau, Oliver; Skaltsounis, Leandros; Halabalaki, Maria; Vollmer, Günter

2015-09-01

Oleocanthal is a bioactive compound from olive oil. It has attracted considerable attention as it is anti-inflammatory, antiproliferative, and has been shown to possess neuroprotective properties in vitro and in vivo. Delineated from its polyphenolic structure, the aim of this study was to characterize oleocanthal towards estrogenic properties. This might contribute to partly explain the beneficial effects described for the Mediterranean diet. Estrogenic properties of oleocanthal were assessed by different methods: a) stimulation of reporter gene activity in MVLN or RNDA cells either expressing estrogen receptor α or β, b) stimulation of luciferase reporter gene activity in U2OS osteosarcoma cells expressing estrogen receptor α or β, and c) elucidation of the impact on estradiol-induced gene expression in U2OS cells transduced with both estrogen receptors. Depending on the cell line origin, oleocanthal inhibited luciferase activity (MVLN, U2OS-estrogen receptor β) or weakly induced reporter gene activity at 10 µM in U2OS-estrogen receptor α cells. However, oleocanthal inhibited stimulation of luciferase activity by estradiol from both estrogen receptors. Oleocanthal, if given alone, did not stimulate gene expression in U2OS cells, but it significantly modulated the response of estradiol. Oleocanthal enhanced the effect of estradiol on the regulation of those genes, which are believed to be regulated through heterodimeric estrogen receptors. As the estrogenic response pattern of oleocanthal is rather unique, we compared the results obtained with oleacein. Oleocanthal binds to both estrogen receptors inducing estradiol-agonistic or antiagonistic effects depending on the cell line. Regarding regulation of gene expression in U2OS-estrogen receptor α/β cells, oleocanthal and oleacein enhanced estradiol-mediated regulation of heterodimer-regulated genes. Georg Thieme Verlag KG Stuttgart · New York.

SIRT3 is a Mitochondrial Tumor Suppressor and Genetic Loss Results in a Murine Model for ER/PR-Positive Mammary Tumors Connecting Metabolism and Carcinogenesis SIRT3 is a Mitochondrial Tumor Suppressor

Science.gov (United States)

2012-09-01

well as HIF-1α dependent gene expression, as shown by co-transfection assays using an HRE luciferase reporter (Fig. 4b, bar 1 vs. 2). In addition...MEFs were co-transfected with p3x- HRE -luciferase with or without NAC or stigmatellin and 40 hours afterwards luciferase levels were determined. (c

Screening to Identify Commonly Used Chinese Herbs That Affect ERBB2 and ESR1 Gene Expression Using the Human Breast Cancer MCF-7 Cell Line

Directory of Open Access Journals (Sweden)

Jen-Hwey Chiu

2014-01-01

Full Text Available Aim. Our aim the was to screen the commonly used Chinese herbs in order to detect changes in ERBB2 and ESR1 gene expression using MCF-7 cells. Methods. Using the MCF-7 human breast cancer cell line, cell cytotoxicity and proliferation were evaluated by MTT and trypan blue exclusion assays, respectively. A luciferase reporter assay was established by transient transfecting MCF-7 cells with plasmids containing either the ERBB2 or the ESR1 promoter region linked to the luciferase gene. Chinese herbal extracts were used to treat the cells at 24 h after transfection, followed by measurement of their luciferase activity. The screening results were verified by Western blotting to measure HER2 and ERα protein expression. Results. At concentrations that induced little cytotoxicity, thirteen single herbal extracts and five compound recipes were found to increase either ERBB2 or ESR1 luciferase activity. By Western blotting, Si-Wu-Tang, Kuan-Shin-Yin, and Suan-Tsao-Ren-Tang were found to increase either HER2 or ERα protein expression. In addition, Ligusticum chuanxiong was shown to have a great effect on ERBB2 gene expression and synergistically with estrogen to stimulate MCF-7 cell growth. Conclusion. Our results provide important information that should affect clinical treatment strategies among breast cancer patients who are receiving hormonal or targeted therapies.

Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

Science.gov (United States)

Ngo, Tony; Coleman, James L J; Smith, Nicola J

2015-01-01

Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.

Validation of reported physical activity for cholesterol control using two different physical activity instruments

Directory of Open Access Journals (Sweden)

Amy Z Fan

2009-08-01

Full Text Available Amy Z Fan1, Sandra A Ham2, Shravani Reddy Muppidi3, Ali H Mokdad41Behavioral Surveillance Branch, Division of Adult and Community Health, National Center for Chronic Disease Prevention and Health Promotion; 2Physical Activity and Health Branch, Division of Nutrition, Physical Activity, and Obesity, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, GA, USA; 3College of Public Health, University of Georgia, Athens, GA, USA; 4Institute for Health Metrics and Evaluation, University of Washington, Seattle, WA, USAAbstract: The National Cholesterol Education Program recommends increasing physical activity to improve cholesterol levels and overall cardiovascular health. We examined whether US adults who reported increasing their physical activity to control or lower blood cholesterol following physician’s advice or on their own efforts had higher levels of physical activity than those who reported that they did not. We used data from the National Health and Nutrition Examination Survey 2003–2004, which implemented two physical activity assessment instruments. The physical activity questionnaire (PAQ assessed self-reported frequency, intensity, and duration of leisure-time, household, and transportation-related physical activity in the past month. Physical movement was objectively monitored using a waist accelerometer that assessed minute-by-minute intensity (counts of movement/minute during waking time over a 7-day period. We adjusted our analysis for age, gender, race/ethnicity, educational attainment, and body mass index. Participants who reported increasing physical activity to control blood cholesterol had more PAQ-assessed physical activity and more accelerometer-assessed active days per week compared to those who did not. However, there were no significant differences in cholesterol levels between comparison groups. These findings suggest that self-report of exercising

Activity report 1988-1989

International Nuclear Information System (INIS)

Dagnegaard, E.; Johansson, Rolf.

1989-12-01

This report covers the activities of the department during the period 1 July 1988 - 30 June 1989, which is the academic year 1988/89. Research has continued in established areas such as adaptive control, computer aided control engineering, robotics, and information technology. The program package Simnon, that has been developed at the Department, is now available for mainframes, IBM-PC compatibles, and Sun workstations

Activity report July 1990 - June 1991

International Nuclear Information System (INIS)

1991-11-01

The Department of Physics and Measurement Technology, Biology and Chemistry (IFM) presents its 21st consecutive annual progress report. It contains a brief description of activities in research and education within the department. The present report contains activities for the academic year July 1990 to June 1991. IFM has been in operation for slightly more than 20 years, and has during that time expanded quite substantially. The number of employees is presently around 250 (90 with a PhD). The number of graduate students amounts to around 90, most of them in different areas of physics. Most of the expansion has taken place in the area of research. IFM is also very active in undergraduate education. About 30% of the total budget is allocated to undergraduate educational activities. Twelve graduate students have defended their PhD-thesis during the elapsed academic year. IFM have added several new professorships to the staff since last year. These are in Computational Physics, Experimental Semi-conductor Physics, Interface Biology and Thin Film Physics

PPARd IS A LIPID SENSOR AND A REGULATOR OF FATTY ACID OXIDATION IN PANCREATIC β-CELLS

DEFF Research Database (Denmark)

Ravnskjær, Kim; Nielsen, Tina; Børgesen, Michael

to stimulate fatty acid oxidation when activated by agonists in myocytes, cardiomycytes and adipo-cytes. A role for PPARd as regulator of basal fatty acid catabolism and energy expenditure was therefore suggested. Here we show that PPARd is the most abundantly expressed PPAR subtype in both primary pancreatic...... islets and in the insulinoma cell line INS-1E. This is reflected at the functional level in activity assays using a PPRE-driven luciferase reporter construct. The fatty acids oleic, arachidonic and linolenic acid are able to acivate this construct synergistically with the synthetic RXR agonist LG100268....... Selective activation of PPARd in INS-1E cells with the PPARd agonist L165041 in the presence or absence of the RXRa agonist LG100268 induces luciferase activity 3- and 7-fold respectively and mimics the effect of the fatty acids. The same subset genes involved in fatty acid uptake and oxidation...

Report on the risk communication activities

International Nuclear Information System (INIS)

Takashita, Hirofumi; Yonezawa, Rika; Shobu, Nobuhiro; Ayame, Junko; Gunji, Ikuko; Asanuma, Misuzu

2008-10-01

This report summarizes the risk communication activities of Nuclear Fuel Cycle Engineering Laboratories conducted by mainly Risk Communication Study Office. To gain trust and understanding of the local community for nuclear energy, a 'Risk Communication Study Team' was set up at former Tokai Works, Japan Nuclear Cycle Development Institute (JNC) in January, 2001. When former JNC and former Japan Atomic Energy Research Institute (JAERI) were merged in October, 2005, the Team was changed to 'Risk Communication Study Office' in Nuclear Fuel Cycle Engineering Laboratories. This report shows the risk communication activities implemented from January, 2001 to March 2007. The activities are classified into three categories: research and study on risk communication, message design and development of information transmission tools, and implementation of risk communication. As for the research and study, we investigated the risk communication about nuclear energy in Western countries. As for the message design, we made messages under our basic policy of showing information clearly that residents want to know. We have prepared about 300 messages in slide format (Microsoft Power Point) and compiled them in a report. More easy-to understand messages were created from the viewpoint of the residents by working in collaboration with local residents, not making messages by ourselves. As for the development of information transmission tools, we have been developing a portal website, 'Risk Information Navigator', which provides information about risks that exist in daily life such as traffic accidents and natural disasters, as well as risk information on nuclear technology. We produced videos and environmental panels as information transmission tools, and made poster panels, clear holder, Karuta' (Japanese card game), and so on in the collaboration work with residents. As a practice of risk communication, we have been carrying out Cycle Friendly Talk', which is direct dialogue within

Assessment report on research and development activities. Activity: 'Advanced science research' (Interim report)

International Nuclear Information System (INIS)

2012-11-01

Japan Atomic Energy Agency (hereinafter referred to as “JAEA”) consulted an assessment committee, “Evaluation Committee of Research Activities for Advanced Science Research” (hereinafter referred to as “Committee”) for interim assessment of “Advanced Science Research,” in accordance with “General Guideline for the Evaluation of Government Research and Development (R and D) Activities” by Cabinet Office, Government of Japan, “Guideline for Evaluation of R and D in Ministry of Education, Culture, Sports, Science and Technology” and “Regulation on Conduct for Evaluation of R and D Activities” by JAEA. In response to the JAEA's request, the Committee assessed the research programs and activities of the Advanced Science Research Center (hereinafter referred to as “ASRC”) for the period of two years from April 2010. The Committee evaluated the management and the research programs of the ASRC based on the explanatory documents prepared by the ASRC and the oral presentations with questions-and-answers by the Director and the research group leaders. This report summarizes the result of the assessment by the Committee with the Committee report attached from page 7. (author)

SIRT3 is a Mitochondrial Tumor Suppressor and Genetic Loss Results in a Murine Model for ER/PR Positive Mammary Tumors Connecting Metabolism and Carcinogenesis

Science.gov (United States)

2011-09-01

as well as HIF-1 dependent gene expression, as shown by co-transfection assays using an HRE luciferase reporter (Fig. 4b, bar 1 vs. 2). In...antibody. (b) MEFs were co-transfected with p3x- HRE -luciferase with or without NAC or stigmatellin and 40 hours afterwards luciferase levels were

Chemical Characterization and Antioxidant, Antimicrobial, and Anti-Inflammatory Activities of South Brazilian Organic Propolis

Science.gov (United States)

Tiveron, Ana Paula; Rosalen, Pedro Luiz; Franchin, Marcelo; Lacerda, Risia Cristina Coelho; Bueno-Silva, Bruno; Benso, Bruna; Denny, Carina; Ikegaki, Masaharu; de Alencar, Severino Matias

2016-01-01

South Brazilian organic propolis (OP), which has never been studied before, was assessed and its chemical composition, scavenging potential of reactive oxygen species, antimicrobial and anti-inflammatory activities are herein presented. Based on the chemical profile obtained using HPLC, OP was grouped into seven variants (OP1–OP7) and all of them exhibited high scavenging activity, mainly against superoxide and hypochlorous acid species. OP1, OP2, and OP3 had the smallest minimal inhibitory concentration (MIC) against Gram-positive bacteria Streptococcus mutans, Streptococcus oralis, and Streptococcus aureus (12.5–100 μg/mL). OP1, OP2, OP3, and OP4 were more effective against Pseudomonas aeruginosa (Gram-negative), with MIC values ranging from 100 to 200 μg/mL. OP6 showed anti-inflammatory activity by decreasing NF-kB activation and TNF-α release in RAW 264.7 macrophages, and expressing the NF-κB-luciferase reporter stable gene. Therefore, south Brazilian OP can be considered an excellent source of bioactive compounds with great potential of application in the pharmaceutical and food industry. PMID:27802316

Report of Activity, 1997

International Nuclear Information System (INIS)

Lacoste, Andre-Claude; Livolant, Michel

1998-01-01

The Institute of Nuclear Protection and Safety (Institut de Protection et de Surete Nucleaire) - IPSN carries out research and expert studies needed in managing nuclear risk and consequences of nuclear events on population and environment, i.e. supervises the safety of installations, the prevention and study of accidents, the human health and environmental conditions, the safety of transport and has also the task of organizing the management of the crisis situations. IPSN has the mission of informing the public and participates in numerous international actions. Thus, since 1989 IPSN and its German counterpart, GRS, co-operate towards improving of the nuclear safety in Europe and developing a common approach of international issues. As acting for public authorities (government departments in charge with industry, environment, health or labor), IPSN grants technical authorizations on request of DSIN exploiters. The IPSN personnel amounts up to 1300 employees of whom around two thirds are engineers, researchers, physicians, agronomists, and veterinarians. This IPSN annual report contains in the 'Free Tribune' section two general papers entitled: 'The federalist role of research at IPSN' by Catherine Lecompte and 'The dose, a central issue in radioprotection debates' by Annie Sugier. Then five sub-reports follow: 1.The safety of nuclear facilities and radioactive wastes; 2.Crisis management; 3. The management of sensitive materials (protection and control of sensitive materials and safety analysis of the radioactive materials transport); 4.Protection of human health; 5.Environment protection. The report also presents the international activities, the cooperation IPSN/GRS, the budget, the staff and the human resource policy and the communication activity

Modeling Ebola Virus Genome Replication and Transcription with Minigenome Systems.

Science.gov (United States)

Cressey, Tessa; Brauburger, Kristina; Mühlberger, Elke

2017-01-01

In this chapter, we describe the minigenome system for Ebola virus (EBOV), which reconstitutes EBOV polymerase activity in cells and can be used to model viral genome replication and transcription. This protocol comprises all steps including cell culture, plasmid preparation, transfection, and luciferase reporter assay readout.

Activity report 1976

International Nuclear Information System (INIS)

Drent, W.; Delande, E.

1977-07-01

The principal activities of Eurochemic plant in the fields of reprocessing and the closing-down program are presented in this report that covers the period 1 January to 31 December 1976. Works in the plant are closely related to the decontamination of the process cells and the completation of the bituminization plant and storage facility. The solidification processes for the high-level liquid waste have been definied and the negotiations in view of setting up an international R and D program for the embedding of solidified high-level waste into metal patrices have been pursued. (A.F.)

Structural Features and Transcriptional Activity of Chicken PPARs (α, β, and γ

Directory of Open Access Journals (Sweden)

Ichiro Takada

2013-01-01

Full Text Available While an understanding of lipid metabolism in chickens is critical for a further improvement of food production, there are few studies concerning differences in lipid metabolism mechanisms between chickens and other species at a molecular level. Chickens have three PPAR gene subtypes (α, β, and γ that function differently from those present in humans and mice. The chicken PPAR-gamma (cPPARγ gene is shorter than that in humans and lacks a γ2 isoform. Moreover, in serum-free media, cPPARγ shows high transcriptional activity without exogenous ligands. Luciferase reporter assays were used to examine the effect of sera on cPPAR transcriptional activities and showed that adult bovine serum and chicken serum highly activate cPPARα and β functions. Moreover, we found that bezafibrate induces the transactivation function of cPPARβ, but not human PPARδ (human PPARβ ortholog. This ligand selectivity relies on one amino acid residue (chicken: Val419, human: Met444. These results show the possibilities for unique functions of cPPARs on chicken-specific lipid glucose metabolism. As such, a better understanding of the molecular mechanisms of lipid metabolism in chickens could result in higher productivity for the poultry industry.

The flavonoid fisetin promotes osteoblasts differentiation through Runx2 transcriptional activity.

Science.gov (United States)

Léotoing, Laurent; Davicco, Marie-Jeanne; Lebecque, Patrice; Wittrant, Yohann; Coxam, Véronique

2014-06-01

Flavonoids represent a group of polyphenolic compounds commonly found in daily nutrition with proven health benefits. Among this group, the flavonol fisetin has been previously shown to protect bone by repressing osteoclast differentiation. In the present study, we investigated the role of fisetin in regulating osteoblasts physiology. In vivo mice treated with LPSs exhibited osteoporosis features associated with a dramatic repression of osteoblast marker expression. In this model, inhibition of osteocalcin and type I collagen alpha 1 transcription was partially countered by a daily consumption of fisetin. Interestingly, in vitro, fisetin promoted both osteoblast alkaline phosphatase activity and mineralization process. To decipher how fisetin may exert its positive effect on osteoblastogenesis, we analyzed its ability to control the runt-related transcription factor 2 (Runx2), a key organizer in developing and maturing osteoblasts. While fisetin did not impact Runx2 mRNA and protein levels, it upregulated its transcriptional activity. Actually, fisetin stimulated the luciferase activity of a reporter plasmid driven by the osteocalcin gene promoter that contains Runx2 binding sites and promoted the mRNA expression of osteocalcin and type I collagen alpha 1 targets. Bone sparing properties of fisetin also rely on its positive influence on osteoblast differentiation and activity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Induction Effect of Am80 and TSA on ESC Differentiation via Regulation of Stra8 in Chicken.

Science.gov (United States)

Zhang, Yani; Zuo, Qisheng; Liu, Zhiyong; Li, Dong; Tang, Beibei; Xiao, Tian-Rong; Lian, Chao; Wang, Yingjie; Jin, Kai; Wang, Yilin; Zhang, Wenhui; Li, Bichun

2015-01-01

Stra8 encodes stimulated by retinoic acid gene 8, a protein that is important for initiation of meiosis in mammals and birds. This study was aimed at identifying the active control area of chicken STRA8 gene core promoter, to screen optimum inducers of the STRA8 gene, thus to enhance the differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells. Fragments of chicken STRA8 gene promoter were cloned into fluorescent reporter plasmids and transfected into DF-1 cells. Then Dual-Luciferase® Reporter Assay System was used to identify the activity of the STRA8 gene under different inducers. Our studies showed that the promoter fragment -1055 bp to +54 bp of Suqin chicken Stra8 revealed the strongest activity. The dual-luciferase® reporter showed that Tamibarotene (Am80) and TrichostatinA (TSA) could significantly enhance STRA8 transcription. The in vitro inductive culture of chicken ESCs demonstrated that spermatogonial stem cells (SSC)-like cells appeared and Integrinβ1 protein was expressed on day 10, indicating that Am80 and TSA can promote ESCs differentiation into SSCs via regulation of Stra8.

TIGF Activity Report 2007

International Nuclear Information System (INIS)

2007-01-01

Total Infrastructure Gaz France (TIGF) provides and develops natural gas transport and storage services on a European scale to meet its customers' needs. It achieves a turnover of almost 300 million Euros and employs some 370 people. Backed by 50 years of expertise, TIGF is a major player in the natural gas market in 15 departements in South-West France. TIGF in figures: 5,000 km of pipelines, 13% of the large scale French transport gas pipeline network, 15% of the national gas volume transit operations, 22% of France's gas storage capacity. This document is the 2007 activity report of TIGF. Content: Message from the Chairman, TIGF, the birth of an ambition, Governance, Management report - overview of the 2007 Financial Year, Market deregulation and development prospects

Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

Energy Technology Data Exchange (ETDEWEB)

Zhang, Lianying [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); College of Life Science, Dezhou University, Dezhou 253023 (China); Ren, Xiao-Min; Wan, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); Guo, Liang-Hong, E-mail: LHGuo@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China)

2014-09-15

Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group. For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.

[Establishment of an iRFP and luciferase dual-color fluorescence-traced hepatocellular carcinoma transplantation model in nude mice].

Science.gov (United States)

Li, Hongjun; Yang, Tianhua; Huang, Yanping; Liu, Mingzhu; Qin, Zhongqiang; Chu, Fei; Li, Zhenghong; Li, Yonghai

2017-11-01

Objective To establish a hepatocellular carcinoma xenograft model in nude mice which could stably express gene and be monitored dynamically. Methods We first constructed the lentiviral particles containing luciferase (Luc) and near-infrared fluorescent protein (iRFP) and puromycin resistance gene, and then transduced them into the HepG2 hepatoma cells. The cell line stably expressing Luc and iRFP genes were screened and inoculated into nude mice to establish xenograft tumor model. Tumor growth was monitored using in vivo imaging system. HE staining and immunohistochemistry were used to evaluate the pathological features and tumorigenic ability. Results HepG2 cells stably expressing iRFP and Luc were obtained; with the engineered cell line, xenograft model was successfully established with the features of proper tumor developing time and high rate of tumor formation as well as typical pathological features as showed by HE staining and immunohistochemistry. Conclusion Hepatocellular carcinoma model in nude mice with the features of stable gene expression and dynamical monitoring has been established successfully with the HepG2-iRFP-Luc cell line.

Evaluation of estrogen receptor alpha activation by glyphosate-based herbicide constituents

OpenAIRE

Mesnage, Robin; Phedonos, Alexia; Biserni, Martina; Arno, Matthew; Balu, Sucharitha; Corton, J. Christopher; Ugarte, Ricardo; Antoniou, Michael N.

2017-01-01

The safety, including endocrine disruptive capability, of glyphosate-based herbicides (GBHs) is a matter of intense debate. We evaluated the estrogenic potential of glyphosate, commercial GBHs and polyethoxylated tallowamine adjuvants present as co-formulants in GBHs. Glyphosate (≥10,000 μg/L or 59 μM) promoted proliferation of estrogen-dependent MCF-7 human breast cancer cells. Glyphosate also increased expression of an estrogen response element-luciferase reporter gene (ERE-luc) in T47D-KBl...

Active sites environmental monitoring program FY 1997 annual report

International Nuclear Information System (INIS)

Morrissey, C.M.; Marshall, D.S.; Cunningham, G.R.

1998-03-01

This report summarizes the activities conducted by the Active Sites Environmental Monitoring Program (ASEMP) from October 1996 through September 1997. The purpose of the program is to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 North. This report continues a series of annual and semiannual reports that present the results of ASEMP monitoring activities. This report details monitoring results for fiscal year (FY) 1997 from SWSA 6, including the Interim Waste Management Facility (IWMF) and the Hillcut Disposal Test Facility (HDTF), and (2) TRU-waste storage areas in SWSA 5 N. This report presents a summary of the methodology used to gather data for each major area along with the FY 1997 results. Figures referenced in the text are found in Appendix A and data tables are presented in Appendix B

Hepatitis C virus NS2 protein activates cellular cyclic AMP-dependent pathways

International Nuclear Information System (INIS)

Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki

2007-01-01

Chronic infection of the hepatitis C virus (HCV) leads to liver cirrhosis and cancer. The mechanism leading to viral persistence and hepatocellular carcinoma, however, has not been fully understood. In this study, we show that the HCV infection activates cellular cAMP-dependent pathways. Expression of a luciferase reporter gene controlled by a basic promoter with the cAMP response element (CRE) was significantly elevated in human hepatoma Huh-7 cells infected with the HCV JFH1. Analysis with viral subgenomic replicons indicated that the HCV NS2 protein is responsible for the effect. Furthermore, the level of cellular transcripts whose stability is known to be regulated by cAMP was specifically reduced in cells harboring NS2-expressing replicons. These results allude to the HCV NS2 protein having a novel function of regulating cellular gene expression and proliferation through the cAMP-dependent pathway

Requirement of ERα and basal activities of EGFR and Src kinase in Cd-induced activation of MAPK/ERK pathway in human breast cancer MCF-7 cells

Energy Technology Data Exchange (ETDEWEB)

Song, Xiulong, E-mail: songxiulong@hotmail.com; Wei, Zhengxi; Shaikh, Zahir A., E-mail: zshaikh@uri.edu

2015-08-15

Cadmium (Cd) is a common environmental toxicant and an established carcinogen. Epidemiological studies implicate Cd with human breast cancer. Low micromolar concentrations of Cd promote proliferation of human breast cancer cells in vitro. The growth promotion of breast cancer cells is associated with the activation of MAPK/ERK pathway. This study explores the mechanism of Cd-induced activation of MAPK/ERK pathway. Specifically, the role of cell surface receptors ERα, EGFR, and Src kinase was evaluated in human breast cancer MCF-7 cells treated with 1–3 μM Cd. The activation of ERK was studied using a serum response element (SRE) luciferase reporter assay. Receptor phosphorylation was detected by Western blot analyses. Cd treatment increased both the SRE reporter activity and ERK1/2 phosphorylation in a concentration-dependent manner. Cd treatment had no effect on reactive oxygen species (ROS) generation. Also, blocking the entry of Cd into the cells with manganese did not diminish Cd-induced activation of MAPK/ERK. These results suggest that the effect of Cd was likely not caused by intracellular ROS generation, but through interaction with the membrane receptors. While Cd did not appear to activate either EGFR or Src kinase, their inhibition completely blocked the Cd-induced activation of ERK as well as cell proliferation. Similarly, silencing ERα with siRNA or use of ERα antagonist blocked the effects of Cd. Based on these results, it is concluded that not only ERα, but also basal activities of EGFR and Src kinase are essential for Cd-induced signal transduction and activation of MAPK/ERK pathway for breast cancer cell proliferation. - Highlights: • Low micromolar concentrations of Cd rapidly activate ERK1/2 in MCF-7 cells. • Signal transduction and resulting cell proliferation require EGFR, ERα, and Src. • These findings implicate Cd in promotion of breast cancer.

Specific detection of peste des petits ruminants virus antibodies in sheep and goat sera by the luciferase

International Nuclear Information System (INIS)

Berguido, F.J.; Bodjo, S.C.; Loitsch, A.; Diallo, A.

2016-01-01

Full text: Peste des petits ruminants (PPR) is a contagious and often fatal transboundary animal disease affecting mostly sheep, goats and wild small ruminants. This disease is endemic in most of Africa, the Middle, Near East, and large parts of Asia. The casual agent is peste des petits ruminants virus (PPRV), which belongs to the genus Morbilivirus in the family Paramyxoviridae. This genus also includes measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). All are closely related viruses with serological cross reactivity. In this study, we have developed a Luciferase Immunoprecipitation System (LIPS) for the rapid detection of antibodies against PPRV in serum samples and for specific differentiation from antibodies against RPV. PPR and rinderpest (RP) serum samples were assayed by PPR-LIPS and two commercially available PPR cELISA tests. The PPR-LIPS showed high sensitivity and specificity for the samples tested and showed no cross reactivity with RPV unlike the commercial PPR cELISA tests which did not cross react with RPV. Based on the results shown in this study, PPR-LIPS is presented as a good candidate for the specific serosurveillance of PPR. (author)

Activity report for 1992

International Nuclear Information System (INIS)

1993-01-01

ADEME means agency for defence of environment and for mastery of energy, these two aims are ADEME's mission; to serve its mission, this agency has to mobilize managers able to reduce tensions on natural resources and to limit impacts on environment; the concerned areas are transport, building, industry because of their weight in energy consumption, in pollutants emission and waste recovery. Here is the report of its activity for the year 1992

Advanced Light Source Activity Report 1997/1998

International Nuclear Information System (INIS)

Greiner, Annette

1999-01-01

This Lawrence Berkeley National Laboratory, Advanced Light Source (ALS) activity report for 1997/98 discusses the following topics: Introduction and Overview; Science Highlights; Facility Report; Special Events; ALS Advisory Panels 1997/98; ALS Staff 1997/98 and Facts and Figures for the year

Advanced Light Source Activity Report 1997/1998

Energy Technology Data Exchange (ETDEWEB)

Greiner, Annette (ed.)

1999-03-01

This Lawrence Berkeley National Laboratory, Advanced Light Source (ALS) activity report for 1997/98 discusses the following topics: Introduction and Overview; Science Highlights; Facility Report; Special Events; ALS Advisory Panels 1997/98; ALS Staff 1997/98 and Facts and Figures for the year.

Nuclear Energy Division. 2009 Activity report

International Nuclear Information System (INIS)

2009-01-01

After a presentation of the future investment programme of the nuclear energy department at the French national Nuclear Research Center (CEA), this report proposes a description of tomorrow's industrial nuclear systems (back-end of future fuel cycle, fourth generation systems, basic scientific and technological research), describes how current nuclear industrial systems are optimized (front-end and back-end of fuel cycle, second and third generation reactors). It presents the main tools for nuclear development: simulation programme, the Jules Horowitz reactor project, maintenance of specific facilities, research valorisation. It reports the activities related to the clean-up and dismantling in different nuclear sites, presents the activities of CEA's nuclear research centres (Saclay, Cadarache, Marcoule), briefly presents the transverse material programme, recalls some events, and gives some key figures

MicroRNA-137 dysregulation predisposes to osteoporotic fracture by impeding ALP activity and expression via suppression of leucine-rich repeat-containing G-protein-coupled receptor 4 expression.

Science.gov (United States)

Liu, Xiangjun; Xu, Xiaohui

2018-08-01

Osteoporosis is defined as a loss of bone mass and deterioration of its architecture resulting in bone weakness, which becomes prone to fracture. The objective of this study was to investigate the molecular mechanism by which miR-137 can reduce the risk of fracture in patients with osteoporosis. An online miRNA database and a luciferase reporter assay system were used to confirm that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) was the target of miR-137. Real-time PCR and western blot analysis were used to study miR-137 mRNA, the expression of LGR4 mRNA and protein among different groups or cells transfected with a scrambled miRNA control, miR-137 mimic, LGR4 siRNA and miR-137 inhibitor. Expression of miR-137 was upregulated to higher levels in cells isolated from osteoporosis patients with fracture than in those without fracture. The 'seed sequence' was found to be located within the 3' untranslated region (3'-UTR) of LGR4 mRNA by searching an online miRNA database. Luciferase reporter assay was performed to confirm that LGR4 is a direct target gene of miR-137 with a potential binding site in the 3'UTR of LGR4. Luciferase activity of cells transfected with wild-type LGR4 3'UTR was much lower than that of the cells transfected with mutant LGR4 3'UTR. The results of real-time PCR and immunohistochemistry experiments demonstrated that the expression levels of LGR4 mRNA and protein were much higher in osteoporosis patients with fracture than osteoporosis patients without fracture. We found that the expression levels of LGR4 mRNA and protein were clearly upregulated following transfection with miR-137 inhibitor, while noticeably downregulated following transfection with miR-137 mimic when compared with the scramble control. Furthermore, the expression of ALP mRNA and ALP activity in bone tissue were much higher in osteoporosis patients with fracture than those without fracture. In conclusion, these data prove that the overexpression of

Anti-inflammatory effects of Zea mays L. husk extracts.

Science.gov (United States)

Roh, Kyung-Baeg; Kim, Hyoyoung; Shin, Seungwoo; Kim, Young-Soo; Lee, Jung-A; Kim, Mi Ok; Jung, Eunsun; Lee, Jongsung; Park, Deokhoon

2016-08-19

Zea mays L. (Z. mays) has been used for human consumption in the various forms of meal, cooking oil, thickener in sauces and puddings, sweetener in processed food and beverage products, bio-disel. However, especially, in case of husk extract of Z. mays, little is known about its anti-inflammatory effects. Therefore, in this study, the anti-inflammatory effects of Z. mays husk extract (ZMHE) and its mechanisms of action were investigated. The husks of Z. Mays were harvested in kangwondo, Korea. To assess the anti-inflammatory activities of ZMHE, we examined effects of ZMHE on nitric oxide (NO) production, and release of soluble intercellular adhesion molecule-1 (sICAM-1) and eotaxin-1. The expression level of inducible nitric oxide synthase (iNOS) gene was also determined by Western blot and luciferase reporter assays. To determine its mechanisms of action, a luciferase reporter assay for nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) was introduced. ZMHE inhibited lipopolysaccharide (LPS)-induced production of NO in RAW264.7 cells. In addition, expression of iNOS gene was reduced, as confirmed by Western blot and luciferase reporter assays. Effects of ZMHE on the AP-1 and NF-kB promoters were examined to elucidate the mechanism of its anti-inflammatory activity. Activation of AP-1 and NF-kB promoters induced by LPS was significantly reduced by ZMHE treatment. In addition, LPS-induced production of sICAM-1 and IL-4-induced production of eotaxin-1 were all reduced by ZMHE. Our results indicate that ZMHE has anti-inflammatory effects by downregulating the expression of iNOS gene and its downregulation is mediated by inhibiting NF-kB and AP-1 signaling.

Environmental contaminants activate human and polar bear (Ursus maritimus) pregnane X receptors (PXR, NR1I2) differently.

Science.gov (United States)

Lille-Langøy, Roger; Goldstone, Jared V; Rusten, Marte; Milnes, Matthew R; Male, Rune; Stegeman, John J; Blumberg, Bruce; Goksøyr, Anders

2015-04-01

Many persistent organic pollutants (POPs) accumulate readily in polar bears because of their position as apex predators in Arctic food webs. The pregnane X receptor (PXR, formally NR1I2, here proposed to be named promiscuous xenobiotic receptor) is a xenobiotic sensor that is directly involved in metabolizing pathways of a wide range of environmental contaminants. In the present study, we comparably assess the ability of 51 selected pharmaceuticals, pesticides and emerging contaminants to activate PXRs from polar bears and humans using an in vitro luciferase reporter gene assay. We found that polar bear PXR is activated by a wide range of our test compounds (68%) but has a slightly more narrow ligand specificity than human PXR that was activated by 86% of the 51 test compounds. The majority of the agonists identified (70%) produces a stronger induction of the reporter gene via human PXR than via polar bear PXR, however with some notable and environmentally relevant exceptions. Due to the observed differences in activation of polar bear and human PXRs, exposure of each species to environmental agents is likely to induce biotransformation differently in the two species. Bioinformatics analyses and structural modeling studies suggest that amino acids that are not part of the ligand-binding domain and do not interact with the ligand can modulate receptor activation. Copyright © 2015. Published by Elsevier Inc.

TOTAL 2003 activities report in brief

International Nuclear Information System (INIS)

2004-01-01

This activities report presents the activities of the petroleum industry Group Total for the year 2003. The following topics are detailed: the corporate social responsibility with the environment stewardship, the energy future management, the safety enhancing, the human resources and the ethics and local development; the shareholder information with the Total share and the share-holding structure; the activities with informations on the Group, the main events, the upstream exploration and production,, the downstream refining, marketing, trading and shipping, the chemicals with overview 2003, base chemical and polymers, performance and specialities. (A.L.B.)

Annual report scientific activities 1992

Energy Technology Data Exchange (ETDEWEB)

NONE

1993-04-01

This report summarizes the activities at TRIUMF for the year 1992. Three major achievements cited in this report are experiments by a group of Columbia University scientists who elucidated the superconducting properties of 60C (''Buckyballs''), a second work led by the University of Toronto that used the radioactive nitrogen to solve some long-standing puzzles about how all the heavy elements of our universe are generated from protons and alpha particles in the dying moments of a collapsing star, and the TRIUMF PET program in which new strides have been taken through brain imaging, in understanding Parkinson's disease.

Development of a cell-based reporter assay for screening of inhibitors of hypoxia-inducible factor 2-induced gene expression.

Science.gov (United States)

Woldemichael, Girma M; Vasselli, James R; Gardella, Roberta S; McKee, Tawnya C; Linehan, W Marston; McMahon, James B

2006-09-01

Reporter cell lines have been developed for the identification of inhibitors of gene expression enhanced by hypoxia-inducible factor 2, which has been implicated as a transcription factor involved in the tumorigenesis of clear cell renal carcinoma. Stably transformed reporter clones of the human renal clear cell carcinoma cell line 786-O were generated by transfection or retroviral infection. Luciferase reporter expression in the vectors used was driven by either the natural human vascular endothelial growth factor (VEGF) promoter-enhancer or by the VEGF and the human endothelial nitric oxide synthase enhancers modulating minimal human cytomegalovirus promoter. Utility of the generated reporter cell lines was validated by introducing the von Hippel-Lindau protein complex and testing for reporter inducibility by hypoxia. The dynamic range in reporter activity under hypoxic stress was found to be at least 30- to 40-fold, with a signal-to-noise ratio of 60:1. Properties of the cell lines such as tolerance to up to 3% DMSO, signal stability with multiple in vitro passages, and utility in both 96- and 384-well plate formats indicated their suitability for use in a high-throughput screen. In addition, the potential use of these reporter lines in the evaluation of high-throughput screening hits in vivo in various mice models has been demonstrated.

ACRO -Activity report for 2008

International Nuclear Information System (INIS)

2008-01-01

After having recalled the history, organisation, missions, and personnel of the ACRO (Association for the Control of Radioactivity in the West of France) this report presents the activities performed by this association in 2008. The relative importance of the different missions is assessed and the analysis laboratory is presented as well as the measurements it performs. The activities are then presented. They concern the radio-ecological assessment, education and training, participation to work groups and institutional commissions, radio-ecological surveys and assessments, international actions in some countries (Belarus), regulatory controls (of industrial wastes and medical effluents, of radon), and publications

ACRO -Activity report for 2007

International Nuclear Information System (INIS)

2007-01-01

After having recalled the history, organisation, missions, and personnel of the ACRO (Association for the Control of Radioactivity in the West of France) this report presents the activities performed by this association in 2007. The relative importance of the different missions is assessed and the analysis laboratory is presented as well as the measurements it performs. The activities are then presented. They concern the radio-ecological assessment, education and training, participation to work groups and institutional commissions, radio-ecological surveys and assessments, international actions in some countries (Belarus, Japan, Korea), regulatory controls (of industrial wastes and medical effluents, of radon), and publications

20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol negatively regulates activation of STAT3 and ERK pathways and exhibits anti-cancer effects in HepG2 cells.

Science.gov (United States)

Ai, Hui-Han; Zhou, Zi-Long; Sun, Lu-Guo; Yang, Mei-Ting; Li, Wei; Yu, Chun-Lei; Song, Zhen-Bo; Huang, Yan-Xin; Wu, Yin; Liu, Lei; Yang, Xiao-Guang; Zhao, Yu-Qing; Bao, Yong-Li; Li, Yu-Xin

2017-11-01

The pro-inflammatory cytokine interleukin 6 (IL-6), via activating its downstream JAK/STAT3 and Ras/ERK signaling pathways, is involved in cell growth, proliferation and anti-apoptotic activities in various malignancies. To screen inhibitors of IL-6 signaling, we constructed a STAT3 and ERK dual-pathway responsive luciferase reporter vector (Co.RE). Among several candidates, the natural compound 20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol (25-OCH 3 -PPD, GS25) was identified to clearly inhibit the luciferase activity of Co.RE. GS25 was confirmed to indeed inhibit activation of both STAT3 and ERK pathways and expression of downstream target genes of IL-6, and to predominantly decrease the viability of HepG2 cells via induction of cell cycle arrest and apoptosis. Interestingly, GS25 showed preferential inhibition of HepG2 cell viability relative to normal liver L02 cells. Further investigation showed that GS25 could not induce apoptosis and block activation of STAT3 and ERK pathways in L02 cells as efficiently as in HepG2 cells, which may result in differential effects of GS25 on malignant and normal liver cells. In addition, GS25 was found to potently suppress the expression of endogenous STAT3 at a higher concentration and dramatically induce p38 phosphorylation in HepG2 cells, which could mediate its anti-cancer effects. Finally, we demonstrated that GS25 also inhibited tumor growth in HepG2 xenograft mice. Taken together, these findings indicate that GS25 elicits its anti-cancer effects on HepG2 cells through multiple mechanisms and has the potential to be used as an inhibitor of IL-6 signaling. Thus, GS25 may be developed as a treatment for hepatocarcinoma with low toxicity on normal liver tissues as well as other inflammation-associated diseases.

A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging

International Nuclear Information System (INIS)

Ponomarev, Vladimir; Vider, Jelena; Shavrin, Aleksander; Ageyeva, Ludmila; Tourkova, Vilia; Doubrovin, Michael; Serganova, Inna; Beresten, Tatiana; Ivanova, Anna; Blasberg, Ronald; Balatoni, Julius; Bornmann, William; Gelovani Tjuvajev, Juri

2004-01-01

Two genetic reporter systems were developed for multimodality reporter gene imaging of different molecular-genetic processes using fluorescence, bioluminescence (BLI), and nuclear imaging techniques. The eGFP cDNA was fused at the N-terminus with HSV1-tk cDNA bearing a nuclear export signal from MAPKK (NES-HSV1-tk) or with truncation at the N-terminus of the first 45 amino acids (Δ45HSV1-tk) and with firefly luciferase at the C-terminus. A single fusion protein with three functional subunits is formed following transcription and translation from a single open reading frame. The NES-TGL (NES-TGL) or Δ45HSV1-tk/GFP/luciferase (Δ45-TGL) triple-fusion gene cDNAs were cloned into a MoMLV-based retrovirus, which was used for transduction of U87 human glioma cells. The integrity, fluorescence, bioluminescence, and enzymatic activity of the TGL reporter proteins were assessed in vitro. The predicted molecular weight of the fusion proteins (130 kDa) was confirmed by western blot. The U87-NES-TGL and U87-Δ45-TGL cells had cytoplasmic green fluorescence. The in vitro BLI was 7- and 13-fold higher in U87-NES-TGL and U87-Δ45-TGL cells compared to nontransduced control cells. The Ki of 14 C-FIAU was 0.49±0.02, 0.51±0.03, and 0.003±0.001 ml/min/g in U87-NES-TGL, U87-Δ45-TGL, and wild-type U87 cells, respectively. Multimodality in vivo imaging studies were performed in nu/nu mice bearing multiple s.c. xenografts established from U87-NES-TGL, U87-Δ45-TGL, and wild-type U87 cells. BLI was performed after administration of d-luciferin (150 mg/kg i.v.). Gamma camera or PET imaging was conducted at 2 h after i.v. administration of [ 131 I]FIAU (7.4 MBq/animal) or [ 124 I]FIAU (7.4 MBq/animal), respectively. Whole-body fluorescence imaging was performed in parallel with the BLI and radiotracer imaging studies. In vivo BLI and gamma camera imaging showed specific localization of luminescence and radioactivity to the TGL transduced xenografts with background levels of activity

A high-resolution peak fractionation approach for streamlined screening of nuclear-factor-E2-related factor-2 activators in Salvia miltiorrhiza.

Science.gov (United States)

Zhang, Hui; Luo, Li-Ping; Song, Hui-Peng; Hao, Hai-Ping; Zhou, Ping; Qi, Lian-Wen; Li, Ping; Chen, Jun

2014-01-24

Generation of a high-purity fraction library for efficiently screening active compounds from natural products is challenging because of their chemical diversity and complex matrices. In this work, a strategy combining high-resolution peak fractionation (HRPF) with a cell-based assay was proposed for target screening of bioactive constituents from natural products. In this approach, peak fractionation was conducted under chromatographic conditions optimized for high-resolution separation of the natural product extract. The HRPF approach was automatically performed according to the predefinition of certain peaks based on their retention times from a reference chromatographic profile. The corresponding HRPF database was collected with a parallel mass spectrometer to ensure purity and characterize the structures of compounds in the various fractions. Using this approach, a set of 75 peak fractions on the microgram scale was generated from 4mg of the extract of Salvia miltiorrhiza. After screening by an ARE-luciferase reporter gene assay, 20 diterpene quinones were selected and identified, and 16 of these compounds were reported to possess novel Nrf2 activation activity. Compared with conventional fixed-time interval fractionation, the HRPF approach could significantly improve the efficiency of bioactive compound discovery and facilitate the uncovering of minor active components. Copyright © 2013 Elsevier B.V. All rights reserved.

Technical activities report: Mechanical development studies

Energy Technology Data Exchange (ETDEWEB)

Alexander, W.K.

1952-05-01

This monthly progress report covers activities in the following areas: pile control; shielding studies; process tube assembly; pile operating equipment; power recovery; pile piping; and test laboratory. A detailed discussion is given for each topic.

Report on activities 2009

OpenAIRE

Universitat Oberta de Catalunya. eLearn Center (eLC)

2010-01-01

2009 Report on activities of the eLearn Center, the e-learning research, innovation and training center of the UOC. Memòria d'activitat de l'eLearn Center, el centre de recerca, innovació i formació en e-learning de la UOC, corresponent a l'any 2009. Memoria de actividad de la eLearn Center, el centro de investigación, innovación y formación en e-learning de la UOC, correspondiente al año 2009.

Report on activities 2010

OpenAIRE

Universitat Oberta de Catalunya. eLearn Center (eLC)

2011-01-01

2010 Report on activities of the eLearn Center, the e-learning research, innovation and training center of the UOC. Memòria d'activitat de l'eLearn Center, el centre de recerca, innovació i formació en e-learning de la UOC, corresponent a l'any 2010. Memoria de actividad del eLearn Center, el centro de investigación, innovación y formación en e-learning de la UOC, correspondiente al año 2010.

Report on activities 2011

OpenAIRE

Universitat Oberta de Catalunya. eLearn Center (eLC)

2012-01-01

2011 Report on activities of the eLearn Center, the e-learning research, innovation and training centre of the UOC. Memòria d'activitat de l'eLearn Center, el centre de recerca, innovació i formació en e-learning de la UOC, corresponent a l'any 2011. Memoria de actividad del eLearn Center, el centro de investigación, innovación y formación en e-learning de la UOC, correspondiente al año 2011.

Activity report 2004

International Nuclear Information System (INIS)

2005-04-01

Constituted by a public and commercial establishment, the French National Radioactive Waste Management Agency (ANDRA) is responsible for the long term management of radioactive waste. This activity report presents the three missions of the ANDRA during the year 2004. The industrial mission deals with the Aube center disposal and the Manche center disposal facilities and the collection effort concerning the diffuse nuclear waste. The research mission focuses on the presentation of several topics: the repository design by studying various potential architectures, the reversibility and the safety assessment under operating conditions and the description of the behavior of repository at various space and time scales. The last mission concerns the information and the communication program. (A.L.B.)

Activity Progress report 1982-1985

International Nuclear Information System (INIS)

1986-01-01

This reports gives a summary of all activities of the Elementary Particle Physics Department at Saclay between the beginning of 1982 and the end of 1985. The experiments in progress or in preparation are presented by subject. The main technical studies and achievements are also described. Finally lists of publications and information concerning Department organization are given [fr

Annual activity report of Atomic Energy Organization of Iran

International Nuclear Information System (INIS)

1976-07-01

The second year activities of Atomic Energy Organization of Iran (AEOI) was reported as follows: Activites of two R and D centres at Tehran, and one in Isfahan which is concentrated on technical and industrial problems of nuclear power plant; site selection from geographical, geological and environmental points of view; nuclear power construction in the vicinity of Bushehr; supply and quantity survey of nuclear fuel for the next 20 years and the activity program of AEOI on nuclear safety. This report also included nuclear information centre activities plus administrative organization and international relations activities

Assessment report of research and development activities FY2014. Activity: 'Advanced science research' (Final report)

International Nuclear Information System (INIS)

2015-09-01

Japan Atomic Energy Agency (hereinafter referred to as 'JAEA') consulted an assessment committee, 'Evaluation Committee of Research Activities for Advanced Science Research' (hereinafter referred to as 'Committee') for final evaluation and prior assessment of 'Advanced Science Research,' in accordance with 'General Guideline for the Evaluation of Government Research and Development (R and D) Activities' by Cabinet Office, Government of Japan, 'Guideline for Evaluation of R and D in Ministry of Education, Culture, Sports, Science and Technology' and 'Regulation on Conduct for Evaluation of R and D Activities' by JAEA. In response to the JAEA's request, the Committee assessed the research programs and activities of the Advanced Science Research Center (hereinafter referred to as 'ASRC') for the period of five years from April 2010 and the research programs from April 2015. The Committee evaluated the management and the research programs of the ASRC based on the explanatory documents prepared by the ASRC and the oral presentations with questions-and-answers by the Director and the research group leaders. This report summarizes the results of the assessment by the Committee with the Committee report attached. (author)

Report of Activity 1997

International Nuclear Information System (INIS)

Menjon, Gerard

1998-04-01

The document is a report of activity of the Direction of Studies and Research of the France's Electricity Authority (Electricite de France) on 1997. It contains the following six main sections: 1. development; 2. environment; 3. development and managing of production-transport system; 4. production; 5. international relations; 6. future developments. The second section deals with the air and water quality, reduction of energy network impact on environment and the case of renewable energies. The following section is devoted to the research and development problems raised by the management of production-transport system. The section number four reports about the experience obtained in the operation of N4 stage, particularly, in the conception of REP 2000. Also mentioned are progress in the field of virtual reality used in professional formation, robot intervention in case of leaks in reactor vessel, multi-recycling of plutonium in PWR, etc. The fifth section reports about the international collaboration in the field of superconductivity, superconductor cables, combined cycles with integrated gasification of carbon and biomass and energy programs in Morocco and China. Finally, it is mentioned the launching of the Galaxie-server in charge with the documentation management of EDF

KFK annual report on research and development activities in 1988

International Nuclear Information System (INIS)

1989-01-01

This annual report on R and D activities in the year 1988 is published by the Karlsruhe Nuclear Research Centre in compliance with section 13.4 of the corporate statutes. The presentation adopts the arrangement of the subjects and items as given in the R and D programme planning of the centre. The reports given by the various institutes and main departments have been collected in their relevant subject groups, with reference being given to the reporting source. Summary reports of activities are obtainable direct from the relevant institutes or main departments. The annual report reviews the progress achieved in every of the R and D projects listed in the KfK programms, and the material is arranged so as to facilitate a comparison between planned and achieved purposes, and to show activities within their entire context, as the R and D programm frequently distributes research tasks for a common project to various institutes. Detailed accounts can be found in the numerous scientific-technical publications listed, or in the KfK status reports. In compliance with the underlying programme budgeting report, the annual report, upon decision by the competent bodies, also presents a summary report of R and D activities in 1988. (orig.) [de

Advanced Light Source Activity Report 2000

International Nuclear Information System (INIS)

Greiner, A.; Moxon, L.; Robinson, A.; Tamura, L.

2001-01-01

This is an annual report, detailing activities at the Advanced Light Source for the year 2000. It includes highlights of scientific research by users of the facility as well as information about the development of the facility itself

Advanced Light Source Activity Report 2000

Energy Technology Data Exchange (ETDEWEB)

Greiner, A.; Moxon, L.; Robinson, A.; Tamura, L.

2001-04-01

This is an annual report, detailing activities at the Advanced Light Source for the year 2000. It includes highlights of scientific research by users of the facility as well as information about the development of the facility itself.

Activity report june 1998 - may 2000

International Nuclear Information System (INIS)

2000-01-01

The aim of this report is to outline the scientific activity of the ''Service de Physique Theorique'' (SPhT), during the period june 1998-may 2000: activity in terms of structure, scientific research programs and results, publications, training and contracts. SPhT is one of the service of the ''Direction des Sciences de la Matiere'' (DSM), itself depending of the CEA Institution. The scientific domains covered by this report are: the quantum field theory and mathematical physics (phase transitions, integrable systems, random matrix, gravity and strings, quantum chaos and dynamical systems, correlation and super-correlation); nuclear physics, particle physics and astrophysics (quantum chromodynamics, physic beyond the standard model, the early Universe, from cosmic microwave background anisotropies to galaxies); statistical physics and condensed matter (statistical physics and models, spin glasses, quantum systems, soft condensed matter and biological physics). (A.L.B.)

Materials Science Division activity report 1991-1993

International Nuclear Information System (INIS)

Amarendra, G.; Tiwari, A.M.; Subramanian, N.; Venugopal Rao, G.

1995-01-01

This progress report gives an account of the various research and developmental activities carried out at the Materials Science Division of the Indira Gandhi Centre for Atomic Research, Kalpakkam during 1991-93. It also gives a summary of the results of the research activities, describes the experimental facilities and also list the publications

Responsible Development of Areva's Mining Activities. 2010 report

International Nuclear Information System (INIS)

2010-01-01

After a map indicating the location of the main Areva's mining sites, this report provides several key figures and data and discusses the evolution of this activity which is part of the business core of the AREVA company. It gives a statement of values and principles, governance and commitments related to this activity. In order to report this activity, it addresses several topics: reduction of industrial risks, protection of workers and populations, rational consumption of water and energy resources, biodiversity preservation, management over time of waste rock and mining tailings, sustainable integration into territories, and contribution to social development. This document is proposed in French and in English

Activity report of Reactor Physics Division : 1990

International Nuclear Information System (INIS)

Mohanakrishnan, P.

1991-01-01

The major Research and Development and Project activities carried out during the year 1990 in Reactor Physics Division are presented in the form of summaries in this report. The various activities are organised under the following areas : (1) Nuclear Data Evaluation, Processing and Validation, (2) Core Physics and Analysis, (3) Reactor Kinetics and Safety Analysis, (4) Noise Analysis, and (5) Radiation Transport and Shielding. FBTR was restarted in July 1990 and the power was raised upto 500 kW. A number of low power physics experiments on reactivity coefficients, kinetics and noise, neutron flux and gamma dose in B cells, were performed, which are discussed in this report. (author). figs., tabs

Nuclear energy Division - 2011 Activity report

International Nuclear Information System (INIS)

2012-01-01

This document reports the activity of the Nuclear Energy Department (DEN) within the CEA. It evokes its international relationship (participation to international initiatives, cooperation with different countries), describes the scientific activity within the DEN, presents the Advanced Material Program, and the activities undertaken in different fields: future nuclear industrial systems (fourth generation reactors, downstream part of the future fuel cycle, fundamental scientific and technological research), optimization of the present nuclear industrial activity (second and third generation reactors, nuclear security, upstream and downstream part of the present fuel cycle), tools for nuclear development (numerical simulation, Jules Horowitz reactor), cleaning up and nuclear dismantling (dismantling strategy, the Passage project in Grenoble, works in Marcoule, the Aladin project in Fontenay, waste and material flow management, nuclear support installations, transports). It finally addresses the specific activities of the Marcoule, Cadarache and Saclay centres

Experimental evidence for the physiological role of bacterial luciferase in the protection of cells against oxidative stress.

Science.gov (United States)

Szpilewska, Hanna; Czyz, Agata; Wegrzyn, Grzegorz

2003-11-01

The origin and function of bioluminescence was considered a problematic question of the Charles Darwin theory. Early evolution of bacterial luminescence and its current physiological importance seem to be especially mysterious. Recently, it was proposed that stimulation of DNA repair may be a physiological role for production of light by bacterial cells. On the other hand, it was also proposed that primary role of luminescent systems could be detoxification of the deleterious oxygen derivatives. Although some previous results might suggest that this hypothesis can be correct, until now experimental evidence for such a mechanism operating in bacterial cells and having physiological importance was generally lacking. Here we demonstrate that in the presence of various oxidants (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, and ferrous ions) at certain concentrations in the culture medium, growth of Vibrio harveyi mutants luxA and luxB, but not of the mutant luxD, is severely impaired relative to wild-type bacteria. This deleterious effect of oxidants on the mutants luxA and luxB could be significantly reduced by addition of the antioxidants A-TEMPO or 40H-TEMPO. We conclude that bacterial luciferase may indeed play a physiological role in the protection of cells against oxidative stress.

105-DR Large Sodium Fire Facility closure activities evaluation report

International Nuclear Information System (INIS)

Adler, J.G.

1996-01-01

This report evaluates the closure activities at the 105-DR Large Sodium Fire Facility. The closure activities discussed include: the closure activities for the structures, equipment, soil, and gravel scrubber; decontamination methods; materials made available for recycling or reuse; and waste management. The evaluation compares these activities to the regulatory requirements and closure plan requirements. The report concludes that the areas identified in the closure plan can be clean closed

Applied nuclear physics group - activities report. 1977-1997

International Nuclear Information System (INIS)

Appoloni, Carlos Roberto

1998-06-01

This report presents the activities conducted by the Applied Nuclear Physics group of the Londrina State University - Applied Nuclear Physics Laboratory - Brazil, from the activities beginning (1977) up to the end of the year 1997

Physics Laboratory technical activities, 1991. Final report

International Nuclear Information System (INIS)

Gebbie, K.B.

1992-02-01

The report summarizes research projects, measurement method development, calibration and testing, and data evaluation activities that were carried out during calendar year 1991 in the NIST Physics Laboratory. These activities fall in the areas of electron and optical physics, atomic physics, molecular physics, radiometric physics, quantum metrology, ionizing radiation, time and frequency, quantum physics, and fundamental constants

Discovery of peroxisome proliferator-activated receptor α (PPARα) activators with a ligand-screening system using a human PPARα-expressing cell line.

Science.gov (United States)

Tachibana, Keisuke; Yuzuriha, Tomohiro; Tabata, Ryotaro; Fukuda, Syohei; Maegawa, Takashi; Takahashi, Rika; Tanimoto, Keiichi; Tsujino, Hirofumi; Nunomura, Kazuto; Lin, Bangzhong; Matsuura, Yoshiharu; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro Js; Kodama, Tatsuhiko; Kobayashi, Tadayuki; Ishimoto, Kenji; Miyachi, Hiroyuki; Doi, Takefumi

2018-05-15

Peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor that belongs to the superfamily of nuclear hormone receptors. PPARα is mainly expressed in the liver, where it activates fatty acid oxidation and lipoprotein metabolism and improves plasma lipid profiles. Therefore, PPARα activators are often used to treat patients with dyslipidemia. To discover additional PPARα activators as potential compounds for use in hypolipidemic drugs, here we established human hepatoblastoma cell lines with luciferase reporter expression from the promoters containing peroxisome proliferator responsive elements (PPRE) and tetracycline-regulated expression of full-length human PPARα to quantify the effects of chemical ligands on PPARα activity. Using the established cell-based PPARα-activator screening system to screen a library of > 12,000 chemical compounds, we identified several hit compounds with basic chemical skeletons different from those of known PPARα agonists. One of the hit compounds, a 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivative we termed compound 3, selectively up-regulated PPARα transcriptional activity, leading to PPARα target gene expression both in vitro and in vivo. Of note, the half-maximal effective concentrations of the hit compounds were lower than that of the known PPARα ligand fenofibrate. Finally, fenofibrate or compound 3 treatment of high fructose-fed rats having elevated plasma triglyceride levels for 14 days indicated that compound 3 reduces plasma triglyceride levels with similar efficiency as fenofibrate. These observations raise the possibility that 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives might be effective drug candidates for selective targeting of PPARα to manage dyslipidemia. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Summary reports of activities under visiting research program

International Nuclear Information System (INIS)

1984-01-01

Technical Report of the Research Reactor Institute, Kyoto University, is published any time as a prompt report as occasion requires. In this report, the title, names of reporters and summary of each of 53 researches which were carried out under the visiting research program in the first half of 1983 are given. The titles are, for example, a mechanism for the anodic oxidation of GaAs, gamma-gamma perturbed angular correlation study of relaxation phenomena, the studies on the genesis of volcanic rocks by partition of trace elements, neutron activation analysis of geochemical standard rock samples, studies on the changes of elements in the fur of rats with various disease, neutron activation analysis on glacier ice strata and glaciegla lake sediments in Nepal Himalayas, low temperature irradiation effects on bcc metals, organ and its subcellular distribution of manganese ion in animals, structure analysis of orthorhombic BaTiO 3 by profile analysis method, regional distribution of Mn, Zn and Cu in human brain, neutron diffraction study of Mn-Ni alloy, structural change of KCoF 3 in the vicinity of the critical region, activation analysis of trace elements in tumor tissues (thyroid) and so on. (Kako, I.)

Long noncoding RNA HOTAIR, a hypoxia-inducible factor-1α activated driver of malignancy, enhances hypoxic cancer cell proliferation, migration, and invasion in non-small cell lung cancer.

Science.gov (United States)

Zhou, Chunxia; Ye, Lincai; Jiang, Chuan; Bai, Jie; Chi, Yongbin; Zhang, Haibo

2015-12-01

Despite the fact that great advances have been made in the management of non-small cell lung cancer (NSCLC), the prognosis of advanced NSCLC remains very poor. HOX transcript antisense intergenic RNA (HOTAIR) has been identified as an oncogenic long noncoding RNA (lncRNA) that is involved in the progression of a variety of carcinomas and acts as a negative prognostic biomarker. Yet, little is known about the effect of HOTAIR in the hypoxic microenvironment of NSCLC. The expression and promoter activity of HOTAIR were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and luciferase reporter assay. The function of the hypoxia-inducible factor-1α (HIF-1α) binding site to hypoxia-responsive elements (HREs) in the HOTAIR promoter region was tested by luciferase reporter assay with nucleotide substitutions. The binding of HIF-1α to the HOTAIR promoter in vivo was confirmed by chromatin immunoprecipitation assay (CHIP) and electrophoretic mobility shift assay (EMSA). The effect of HIF-1α suppression by small interference RNA or YC-1 on HOTAIR expression was also determined. In the present study, we demonstrated that HOTAIR was upregulated by hypoxia in NSCLC cells. HOTAIR is a direct target of HIF-1α through interaction with putative HREs in the upstream region of HOTAIR in NSCLC cells. Furthermore, HIF-1α knockdown or inhibition could prevent HOTAIR upregulation under hypoxic conditions. Under hypoxic conditions, HOTAIR enhanced cancer cell proliferation, migration, and invasion. These data suggested that suppression of HOTAIR upon hypoxia of NSCLC could be a novel therapeutic strategy.

The Evolution of the Bacterial Luciferase Gene Cassette (lux as a Real-Time Bioreporter

Directory of Open Access Journals (Sweden)

Gary Sayler

2012-01-01

Full Text Available The bacterial luciferase gene cassette (lux is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted.

Epigenetic Control of Prolyl and Asparaginyl Hydroxylases in Prostate Cancer

Science.gov (United States)

2011-07-01

melanoma, renal carcinoma and breast cancer cell lines. Furthermore, we show that neither HIF-1a protein levels nor hypoxic response through an HRE ...constitutively expressed gene to control for equivalent DNase digestion between the cell lines examined. HRE -Luciferase assay Cell lines ,85% confluent in 60...mm dishes were transfected with an HRE -luciferase reporter vector [19] (2.5 mg) and Renilla luciferase (1.5 mg) according to Lipofectamine 2000

Active Sites Environmental Monitoring Program. FY 1993: Annual report

International Nuclear Information System (INIS)

Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.; Marsh, J.D.

1994-08-01

This report continues a series of annual and semiannual reports that present the results of the Active Sites Environmental Monitoring Program (ASEMP) monitoring activities. The report details monitoring data for fiscal year (FY) 1993 and is divided into three major areas: SWSA 6 [including tumulus pads, Interim Waste Management Facility (IWMF), and other sites], the low-level Liquid-Waste Solidification Project (LWSP), and TRU-waste storage facilities in SWSA 5 N. The detailed monitoring methodology is described in the second revision of the ASEMP program plan. This report also presents a summary of the methodology used to gather data for each major area along with the results obtained during FY 1993

Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo

International Nuclear Information System (INIS)

Rivera, Angel A.; Wang Minghui; Suzuki, Kaori; Uil, Taco G.; Krasnykh, Victor; Curiel, David T.; Nettelbeck, Dirk M.

2004-01-01

The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdΔE1AIL), the early gene E2B (AdΔE2BIL), or the late fiber gene (AdΔfiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdΔE2BIL, compared with nonreplicating AdTL, which contained the luciferase gene expressed from the strong CMV promoter. However, 6 days after infection, luciferase activities were approximately four (AdΔE1AIL) to six (AdΔfiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdΔE2BIL, AdΔfiberIL, and AdTL. AdΔfiberIL and the parental virus, Ad5-Δ24, resulted in similar cytotoxicity, but AdΔE2BIL and AdΔE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdΔE2BIL and AdΔfiberIL, but substantial for AdΔE1AIL. Our observations suggest that insertion of IRES-transgene cassettes into viral transcription units is an attractive strategy for the development of armed oncolytic adenoviruses with defined kinetics and strength of transgene expression

Reported frequency of physical activity in a large epidemiological study: relationship to specific activities and repeatability over time

Directory of Open Access Journals (Sweden)

Reeves Gillian K

2011-06-01

Full Text Available Abstract Background How overall physical activity relates to specific activities and how reported activity changes over time may influence interpretation of observed associations between physical activity and health. We examine the relationships between various physical activities self-reported at different times in a large cohort study of middle-aged UK women. Methods At recruitment, Million Women Study participants completed a baseline questionnaire including questions on frequency of strenuous and of any physical activity. About 3 years later 589,896 women also completed a follow-up questionnaire reporting the hours they spent on a range of specific activities. Time spent on each activity was used to estimate the associated excess metabolic equivalent hours (MET-hours and this value was compared across categories of physical activity reported at recruitment. Additionally, 18,655 women completed the baseline questionnaire twice, at intervals of up to 4 years; repeatability over time was assessed using the weighted kappa coefficient (κweighted and absolute percentage agreement. Results The average number of hours per week women reported doing specific activities was 14.0 for housework, 4.5 for walking, 3.0 for gardening, 0.2 for cycling, and 1.4 for all strenuous activity. Time spent and the estimated excess MET-hours associated with each activity increased with increasing frequency of any or strenuous physical activity reported at baseline (tests for trend, P weighted = 0.71 for questionnaires administered less than 6 months apart, and 52% (κweighted = 0.51 for questionnaires more than 2 years apart. Corresponding values for any physical activity were 57% (κweighted = 0.67 and 47% (κweighted = 0.58. Conclusions In this cohort, responses to simple questions on the frequency of any physical activity and of strenuous activity asked at baseline were associated with hours spent on specific activities and the associated estimated excess MET

Responsible Development of Areva's Mining Activities - Report 2011

International Nuclear Information System (INIS)

2012-09-01

The results consolidated at Mining Business Group level and presented in this document mainly relate to the uranium production sites for which Areva is the majority operator. For each stage of the mining life cycle (exploration, project development, operation, closure, redevelopment) much of the data is also reported on a national and international reporting level, and is presented in this document when deemed necessary for the understanding of Areva's activities. This document reports the extra-financial performance of Areva's mining activities for the period from 1 January to 31 December 2011. The extra-financial performance of mining activities presented in this report is underpinned by the Areva group reporting process called STAR (for Sustainability Tools Advanced Reporting). It is supplemented by information collected from in-house experts or from other reporting documents deemed to be valid sources of reference for the subjects discussed. Content: 1 - Approach to responsibility (Being a Responsible Mining Stakeholder, The Fundamentals of Areva's Approach); 2 - Activities (Activities Experiencing Strong Growth, Uranium as a Core Business); 3 - Teams (A Proactive Employment Policy, Employee Health and Safety, Focus: 'I am committed to maintaining safety' campaign yields exemplary results in Namibia); 4 - Environmental policy (Management of Challenges, The Environment Throughout the Entire Mining Lifecycle, Focus: hybrid electricity generation system for prospecting camps in Australia); 5 - Social commitment (Transparency and Openness to Dialogue, Community Involvement, Focus: working group with communities of the province of Saskatchewan in Canada); 6 - Performance (The Main Sustainable Development Indicators, Scope of this Report); 7 - Appendices (Glossary, Communications associated with this report)

RTE activity report 2005; RTE rapport d'activite 2005

Energy Technology Data Exchange (ETDEWEB)

NONE

2006-07-01

The RTE (electric power transport network) is the french manger of the electric power transport. This activity report provides information on the company results for the year 2005: panorama of the year, management and organization, the place of RTE in the european market, the customers, the industrial tool, the environment the human resources, the international activity and the management report. (A.L.B.)

Influence of some inhibiting and activating substances on the light reaction in vitro of photobacterium phosphoreum

NARCIS (Netherlands)

Terpstra, Willemke; Steenbergen, C.L.M.

1964-01-01

Addition of H2O2 to a bacterial luciferase preparation resulted in the formation of a compound with a fluoresence maximum at about 470 mμ. This compond was probably the same as the one that is formed as a result of ultraviolet irradiation of luciferase preparations after addition of FMNH2. It is

Battelle-Northwest monthly activities report, February 1965

Energy Technology Data Exchange (ETDEWEB)

1964-03-15

Activities for each of the following departments are discussed in this report: Reactor and Materials Technology Dept.; Physics and Instruments Dept.; Chemistry Dept.; Biology Dept.; Applied Mathematics Dept.; Radiation Protection Dept.; and the Test Reactor and Engineering Services Dept.. Activities are in support of Hanford reactors (production reactors, N-reactor, PRTR reactor, etc) and reprocessing and radioactive waste management efforts at Hanford.

Effect of ATRX and G-Quadruplex Formation by the VNTR Sequence on α-Globin Gene Expression.

Science.gov (United States)

Li, Yue; Syed, Junetha; Suzuki, Yuki; Asamitsu, Sefan; Shioda, Norifumi; Wada, Takahito; Sugiyama, Hiroshi

2016-05-17

ATR-X (α-thalassemia/mental retardation X-linked) syndrome is caused by mutations in chromatin remodeler ATRX. ATRX can bind the variable number of tandem repeats (VNTR) sequence in the promoter region of the α-globin gene cluster. The VNTR sequence, which contains the potential G-quadruplex-forming sequence CGC(GGGGCGGGG)n , is involved in the downregulation of α-globin expression. We investigated G-quadruplex and i-motif formation in single-stranded DNA and long double-stranded DNA. The promoter region without the VNTR sequence showed approximately twofold higher luciferase activity than the promoter region harboring the VNTR sequence. G-quadruplex stabilizers hemin and TMPyP4 reduced the luciferase activity, whereas expression of ATRX led to a recovery in reporter activity. Our results demonstrate that stable G-quadruplex formation by the VNTR sequence downregulates the expression of α-globin genes and that ATRX might bind to and resolve the G-quadruplex. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Andrographolide suppresses high glucose-induced fibronectin expression in mesangial cells via inhibiting the AP-1 pathway.

Science.gov (United States)

Lan, Tian; Wu, Teng; Gou, Hongju; Zhang, Qianqian; Li, Jiangchao; Qi, Cuiling; He, Xiaodong; Wu, Pingxiang; Wang, Lijing

2013-11-01

Mesangial cells (MCs) proliferation and accumulation of glomerular matrix proteins such as fibronectin (FN) are the early features of diabetic nephropathy, with MCs known to upregulate matrix protein synthesis in response to high glucose. Recently, it has been found that andrographolide has renoprotective effects on diabetic nephropathy. However, the molecular mechanism underlying these effects remains unclear. Cell viability and proliferation was evaluated by MTT. FN expression was examined by immunofluorescence and immunoblotting. Activator protein-1 (AP-1) activation was assessed by immunoblotting, luciferase reporter and electrophoretic mobility shift assays. Andrographolide significantly decreased high glucose-induced cell proliferation and FN expression in MCs. Exposure of MCs to high glucose markedly stimulated the expression of phosphorylated c-jun, whereas the stimulation was inhibited by andrographolide. Plasmid pAP-1-Luc luciferase reporter assay showed that andrographolide blocked high glucose-induced AP-1 transcriptional activity. EMSA assay demonstrated that increased AP-1 binding to an AP-1 binding site at -1,029 in the FN gene promoter upon high glucose stimulation, and the binding were disrupted by andrographolide treatment. These data indicate that andrographolide suppresses high glucose-induced FN expression by inhibiting AP-1-mediated pathway. © 2013 Wiley Periodicals, Inc.

National energy ombudsman - 2010 activity report

International Nuclear Information System (INIS)

2010-01-01

This report first gives an overview of the evolutions noticed on the energy market (natural gas and electric power) from the mediator's point of view for the consumer protection: improvement of transparency, struggle against energy precariousness, improvement of the protection of European consumers. Some figures and a description of a typical week of work are given to illustrate the mediator's activity. Solutions are proposed to improve practices: excess payment, index correction, set prices, first necessity tariff, and bill readability. Some social indicators are given and a financial report is provided

Imaging and Targeting of Hypoxic Tumor Cells with Use of HIF-1-2

International Nuclear Information System (INIS)

Kizaka-Kondoh, Shinae; Harada, Hiroshi; Tanaka, Shotaro; Hiraoka, Masahiro

2006-01-01

This paper describes imaging (visualization) of transplanted tumor cells under hypoxia in vivo and molecular targeting to kill those cells by inducing their apoptosis. HIF (hypoxia inducible factor) concerned with angiogenesis is induced specifically in hypoxic tumor cells and its activity can be visualized by transfection of reporter vector construct of fluorescent protein GFP or luciferase. Authors established the transfected tumor cells with the plasmid p5HRE-luciferase and when transplanted in the nude mouse, those cells emitted light dependently to their hypoxic conditions, which could be visualized by in vivo imaging system (IVIS) with CCD camera. Authors prepared the oxygen-dependent degradation-procaspase 3-fusion protein (TOP3) to target the hypoxic tumor cells for enhancing their apoptotic signaling, whose apoptosis was actually observed by the IVIS. Reportedly, radiation transiently activates HIF-1 and combination treatment of radiation and TOP3 resulted in the enhanced death of tumor cells. Interestingly, the suppression of tumor growth lasted longer than expected, probably due to inhibition of angiogenesis. Authors called this anti-tumor strategy as the micro-environmental targeting. (T.I.)

β-Sitosterol and flavonoids isolated from Bauhinia malabarica found during screening for Wnt signaling inhibitory activity.

Science.gov (United States)

Park, Hyun Young; Toume, Kazufumi; Arai, Midori A; Koyano, Takashi; Kowithayakorn, Thaworn; Ishibashi, Masami

2014-01-01

Screening with a cell-based luciferase assay was conducted to identify bioactive natural products which inhibit Wnt signaling activity-guided separation of an MeOH extract of Bauhinia malabarica (Caesalpiniaceae) leaves yielded five compounds, which were identified as β-sitosterol (1), quercetin (2), 6,8-C-dimethyl kaempferol-3-O-rhamnopyranoside (3), hyperin (4), and 6,8-C-dimethyl kaempferol-3-methyl ether (5). The tested compounds 1, 3, and 5 exhibited Wnt signaling inhibitory activity, with IC50 values of 0.77, 0.74, and 16.6 μM, respectively.

The examination of urine samples for pathogenic microbes by the luciferase assay for ATP. 1: The effect of the presence of fungi, fungal like bacteria and kidney cells in urine samples

Science.gov (United States)

Bush, V. N.

1973-01-01

A method for accurately determining urinary tract infections in man is introduced. The method is based on adenosine triphosphate (ATP) concentration in urine samples after removing nonbacterial ATP. Adenosine triphosphate concentration is measured from the bioluminescent reaction of luciferase when mixed with ATP. An examination was also made of the effectiveness of rupturing agents on monkey kidney cells Candia albicans, a Rhodotorula species, and a Streptomyces species in determining whether these cells could contribute ATP to the bacterial ATP value of a urine sample.

Hanford Laboratories monthly activities report, November 1964

Energy Technology Data Exchange (ETDEWEB)

1964-12-15

This is the monthly report for the Hanford Laboratories Operation, November 1964. Reactor fuels, chemistry, dosimetry, separation processes, reactor technology, financial activities, biology operation, and physics and instrumentation research.

Status report on ILDG activities

Energy Technology Data Exchange (ETDEWEB)

Jansen, K. [Deutsches Elektronen-Synchrotron (DESY), Zeuthen (Germany). John von Neumann-Inst. fuer Computing NIC

2006-09-15

A status report about the International Lattice Data Grid (ILDG) is given. Different countries participating in the ILDG have created regional lattice data grid solutions that are implemented, working and used. The remaining task and the focus of present activities is the development of the interoperability of these regional grids. A first, successful step in this direction is a metadata catalogue service which is already working interoperable. (orig.)

Status report on ILDG activities

International Nuclear Information System (INIS)

Jansen, K.

2006-09-01

A status report about the International Lattice Data Grid (ILDG) is given. Different countries participating in the ILDG have created regional lattice data grid solutions that are implemented, working and used. The remaining task and the focus of present activities is the development of the interoperability of these regional grids. A first, successful step in this direction is a metadata catalogue service which is already working interoperable. (orig.)

Thrombin-induced, TNFR-dependent miR-181c downregulation promotes MLL1 and NF-κB target gene expression in human microglia.

Science.gov (United States)

Yin, Min; Chen, Zhiying; Ouyang, Yetong; Zhang, Huiyan; Wan, Zhigang; Wang, Han; Wu, Wei; Yin, Xiaoping

2017-06-29

Controlling thrombin-driven microglial activation may serve as a therapeutic target for intracerebral hemorrhage (ICH). Here, we investigated microRNA (miRNA)-based regulation of thrombin-driven microglial activation using an in vitro thrombin toxicity model applied to primary human microglia. A miRNA array identified 22 differential miRNA candidates. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) identified miR-181c as the most significantly downregulated miRNA. TargetScan analysis identified mixed lineage leukemia-1 (MLL1) as a putative gene target for miR-181c. qRT-PCR was applied to assess tumor necrosis factor-alpha (TNF-α), miR-181c, and MLL1 levels following thrombin or proteinase-activated receptor-4-specific activating peptide (PAR4AP) exposure. Anti-TNF-α antibodies and tumor necrosis factor receptor (TNFR) silencing were employed to test TNF-α/TNFR dependence. A dual-luciferase reporter system and miR-181c mimic transfection assessed whether mir-181c directly binds to and negatively regulates MLL1. Nuclear factor kappa-B (NF-κB)-dependent luciferase reporter assays and NF-κB target gene expression were assessed in wild-type (MLL1+) and MLL1-silenced cells. Thrombin or PAR4AP-induced miR-181c downregulation (p < 0.05) and MLL1 upregulation (p < 0.05) that were dependent upon TNF-α/TNFR. miR-181c decreased wild-type MLL1 3'-UTR luciferase reporter activity (p < 0.05), and a miR-181c mimic suppressed MLL1 expression (p < 0.05). Thrombin treatment increased, while miR-181c reduced, NF-κB activity and NF-κB target gene expression in both wild-type (MLL1+) and MLL1-silenced cells (p < 0.05). Thrombin-induced, TNF-α/TNFR-dependent miR-181c downregulation promotes MLL1 expression, increases NF-κB activity, and upregulates NF-κB target gene expression. As miR-181c opposes thrombin's stimulation of pro-inflammatory NF-κB activity, miR-181c mimic therapy may show promise in controlling thrombin

Western Gas Sands Project. Quarterly Basin Activities Report

Energy Technology Data Exchange (ETDEWEB)

Atkinson, C H

1979-01-31

This report is a summation of 3 months' drilling and testing activities in the four primary WGSP study areas: Greater Green River Basin, Northern Great Plains Province, Piceance Basin, and Uinta Basin. The monitoring of basin activities is part of resource assessment. (DLC)

Robust heat-inducible gene expression by two endogenous hsp70-derived promoters in transgenic Aedes aegypti

Science.gov (United States)

Carpenetti, Tiffany L. G.; Aryan, Azadeh; Myles, Kevin M.; Adelman, Zach N.

2011-01-01

Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. Reverse genetic approaches to the study of gene function in this mosquito have been limited by the lack of a robust inducible promoter to allow precise temporal control over a protein-encoding or hairpin RNA transgene. Likewise, investigations into the molecular and biochemical basis of vector competence would benefit from the ability to activate an anti-pathogen molecule at specific times during infection. We have characterized the ability of genomic sequences derived from two Ae. aegypti hsp70 genes to drive heat-inducible expression of a reporter in both transient and germline transformation contexts. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Luciferase expression in transgenic Ae. aegypti increased by ∼25-50 fold in whole adults by four hours after heat-shock, with significant activity (∼20 fold) remaining at 24 hr. Heat-induced expression was even more dramatic in midgut tissues, with one strain showing a ∼2500-fold increase in luciferase activity. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules. PMID:22142225

Activity Report 1990-1991

International Nuclear Information System (INIS)

Dagnegaard, E.; Aastroem, K.J.

1991-10-01

This report covers the activities at the Department of Automatic Control at Lund University during the period 1 July 1990 - 30 June 1991, which is the academic year 1990/91. During this period Bo Eliasson and Lars Rundqwist completed their PhD theses. This brings the total number of PhDs graduating from our department to 33. Twenty-one students completed their MSc degree at the department. One book, seventeen journal papers and 48 conference papers were published by staff members. Research has continued in established areas such as adaptive control, expert control, computer aided control engineering, robotics, and information technology

Hanford Laboratories operation monthly activities report, November 1957

Energy Technology Data Exchange (ETDEWEB)

1957-12-15

This is the monthly report for the Hanford Laboratories Operation. Metallurgy, reactor fuels, physics and instrumentation, reactor technology, chemistry, separation processes, biology, financial activities, employee relations, laboratories auxiliaries, radiation protection, operation research, inventions, visits, and personnel status are discussed. This report is for November 1957.

Hanford Laboratories operation monthly activities report, November 1956

Energy Technology Data Exchange (ETDEWEB)

1956-12-21

This is the monthly report for the Hanford Laboratories Operation. Metallurgy, reactor fuels, physics and instrumentation, reactor technology, chemistry, separation processes, biology, financial activities, employee relations, laboratories auxiliaries, radiation protection, operations research, inventions, visits, and personnel status are discussed. This report is for November, 1956.

Hanford Laboratories Operation monthly activities report, October 1958

Energy Technology Data Exchange (ETDEWEB)

1958-11-15

This is the monthly report for the Hanford Laboratories Operation. Metallurgy, reactor fuels, physics and instrumentation, reactor technology, chemistry, separation processes, biology, financial activities, employee relations, laboratories auxiliaries, radiation protection, operation research, inventions, visits, and personnel status are discussed. This report is for October 1958.

Hanford Laboratories operation monthly activities report, January 1957

Energy Technology Data Exchange (ETDEWEB)

1957-02-15

This is the monthly report for the Hanford Laboratories Operation. Metallurgy, reactor fuels, physics and instrumentation, reactor technology, chemistry, separation processes, biology, financial activities, employee relations, laboratories auxiliaries, radiation protection, operation research, inventions, visits, and personnel status are discussed. This report is for January 1957.

Hanford Laboratories Operation monthly activities report, October 1957

Energy Technology Data Exchange (ETDEWEB)

1957-11-15

This is the monthly report for the Hanford Laboratories Operation. Metallurgy, reactor fuels, physics and instrumentation, reactor technology, chemistry, separation processes, biology, financial activities, employee relations, laboratories auxiliaries, radiation protection, operation research, inventions, visits, and personnel status are discussed. This report is for October 1957.

Hanford Laboratories Operation monthly activities report, September 1956

Energy Technology Data Exchange (ETDEWEB)

1956-10-19

This is the monthly report for the Hanford Laboratories Operation. Metallurgy, reactor fuels, physics and instrumentation, reactor technology, chemistry, separation processes, biology, financial activities, employee relations, laboratories auxiliaries, radiation protection, operation research, inventions, visits, and personnel status are discussed. This report is for September 1956.

Hanford Laboratories Operation monthly activities report, March 1960

Energy Technology Data Exchange (ETDEWEB)

1960-04-15

This is the monthly report for the Hanford Laboratories Operation. Metallurgy, reactor fuels, physics and instrumentation, reactor technology, chemistry, separation processes, biology, financial activities, employee relations, laboratories auxiliaries, radiation protection, operation research, inventions, visits, and personnel status are discussed. This report is for March 1960.

Hanford Laboratories operation monthly activities report, February 1958

Energy Technology Data Exchange (ETDEWEB)

1958-03-15

This is the monthly report for the Hanford Laboratories Operation. Metallurgy, reactor fuels, physics and instrumentation, reactor technology, chemistry, separation processes, biology, financial activities, employee relations, laboratories auxiliaries, radiation protection, operation research, inventions, visits, and personnel status are discussed. This report is for February 1958.

Hanford Laboratories Operation monthly activities report, December 1957

Energy Technology Data Exchange (ETDEWEB)

1958-01-15

This is the monthly report for the Hanford Laboratories Operation. Metallurgy, reactor fuels, physics and instrumentation, reactor technology, chemistry, separation processes, biology, financial activities, employee relations, laboratories auxiliaries, radiation protection, operation research, inventions, visits, and personnel status are discussed. This report is for December 1957.

Educational differences in the validity of self-reported physical activity

NARCIS (Netherlands)

Winckers, Annemarie N. E.; Mackenbach, Joreintje D.; Compernolle, Sofie; Nicolaou, Mary; van der Ploeg, Hidde P.; de Bourdeaudhuij, Ilse; Brug, Johannes; Lakerveld, Jeroen

2015-01-01

The assessment of physical activity for surveillance or population based studies is usually done with self-report questionnaires. However, bias in self-reported physical activity may be greater in lower educated than in higher educated populations. The aim of the present study is to describe

Educational differences in the validity of self-reported physical activity

NARCIS (Netherlands)

Winckers, A.N.; Mackenbach, J.D.; Compernolle, S.; Nicolaou, M.; van der Ploeg, H.P.; de Bourdeaudhuij, I.; Brug, J.; Lakerveld, J.

2015-01-01

Background: The assessment of physical activity for surveillance or population based studies is usually done with self-report questionnaires. However, bias in self-reported physical activity may be greater in lower educated than in higher educated populations. The aim of the present study is to

Calibration of self-report tools for physical activity research: the Physical Activity Questionnaire (PAQ).

Science.gov (United States)

Saint-Maurice, Pedro F; Welk, Gregory J; Beyler, Nicholas K; Bartee, Roderick T; Heelan, Kate A

2014-05-16

The utility of self-report measures of physical activity (PA) in youth can be greatly enhanced by calibrating self-report output against objectively measured PA data.This study demonstrates the potential of calibrating self-report output against objectively measured physical activity (PA) in youth by using a commonly used self-report tool called the Physical Activity Questionnaire (PAQ). A total of 148 participants (grades 4 through 12) from 9 schools (during the 2009-2010 school year) wore an Actigraph accelerometer for 7 days and then completed the PAQ. Multiple linear regression modeling was used on 70% of the available sample to develop a calibration equation and this was cross validated on an independent sample of participants (30% of sample). A calibration model with age, gender, and PAQ scores explained 40% of the variance in values for the percentage of time in moderate-to-vigorous PA (%MVPA) measured from the accelerometers (%MVPA = 14.56 - (sex*0.98) - (0.84*age) + (1.01*PAQ)). When tested on an independent, hold-out sample, the model estimated %MVPA values that were highly correlated with the recorded accelerometer values (r = .63) and there was no significant difference between the estimated and recorded activity values (mean diff. = 25.3 ± 18.1 min; p = .17). These results suggest that the calibrated PAQ may be a valid alternative tool to activity monitoring instruments for estimating %MVPA in groups of youth.

Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment.

Science.gov (United States)

Eyre, Nicholas S; Aloia, Amanda L; Joyce, Michael A; Chulanetra, Monrat; Tyrrell, D Lorne; Beard, Michael R

2017-07-01

The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Selective Estrogen Receptor Modulator (SERM)-like Activities of Diarylheptanoid, a Phytoestrogen from Curcuma comosa, in Breast Cancer Cells, Pre-osteoblast Cells, and Rat Uterine Tissues.

Science.gov (United States)

Thongon, Natthakan; Boonmuen, Nittaya; Suksen, Kanoknetr; Wichit, Patsorn; Chairoungdua, Arthit; Tuchinda, Patoomratana; Suksamrarn, Apichart; Winuthayanon, Wipawee; Piyachaturawat, Pawinee

2017-05-03

Diarylheptanoids from Curcuma comosa, of the Zingiberaceae family, exhibit diverse estrogenic activities. In this study we investigated the estrogenic activity of a major hydroxyl diarylheptanoid, 7-(3,4 -dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (compound 092) isolated from C. comosa. The compound elicited different transcriptional activities of estrogen agonist at low concentrations (0.1-1 μM) and antagonist at high concentrations (10-50 μM) using luciferase reporter gene assay in HEK-293T cells. In human breast cancer (MCF-7) cells, compound 092 showed an anti-estrogenic activity by down-regulating ERα-signaling and suppressing estrogen-responsive genes, whereas it attenuated the uterotrophic effect of estrogen in immature ovariectomized rats. Of note, compound 092 promoted mouse pre-osteoblastic (MC3T3-E1) cell differentiation and the related bone markers, indicating its positive osteogenic effect. Our findings highlight a new, nonsteroidal, estrogen agonist/antagonist of catechol diarylheptanoid from C. comosa, which is scientific evidence supporting its potential as a dietary supplement to prevent bone loss with low risk of breast and uterine cancers in postmenopausal women.

Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data

DEFF Research Database (Denmark)

Kannan, Soumya; Sams, Thomas; Maury, Jérôme

2018-01-01

activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process. In fact, some promoters that are often thought of as constitutive can show changes in activity when growth conditions change. For these reasons, we have developed a system......Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds......, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter...

Lung Cancer Chemopreventive Activity of Patulin Isolated from Penicillium vulpinum

Directory of Open Access Journals (Sweden)

Aymeric Monteillier

2018-03-01

Full Text Available Lung cancer is the most lethal form of cancer in the world. Its development often involves an overactivation of the nuclear factor kappa B (NF-κB pathway, leading to increased cell proliferation, survival, mobility, and a decrease in apoptosis. Therefore, NF-κB inhibitors are actively sought after for both cancer chemoprevention and therapy, and fungi represent an interesting unexplored reservoir for such molecules. The aim of the present work was to find naturally occurring lung cancer chemopreventive compounds by investigating the metabolites of Penicillium vulpinum, a fungus that grows naturally on dung. Penicillium vulpinum was cultivated in Potato Dextrose Broth and extracted with ethyl acetate. Bioassay-guided fractionation of this extract was performed by measuring NF-κB activity using a HEK293 cell line transfected with an NF-κB-driven luciferase reporter gene. The mycotoxin patulin was identified as a nanomolar inhibitor of TNF-α-induced NF-κB activity. Immunocytochemistry and Western blot analyses revealed that its mechanism of action involved an inhibition of p65 nuclear translocation and was independent from the NF-κB inhibitor α (IκBα degradation process. Enhancing its interest in lung cancer chemoprevention, patulin also exhibited antiproliferative, proapoptotic, and antimigration effects on human lung adenocarcinoma cells through inhibition of the Wnt pathway.

ANDRA - 2012 activity and sustainable development report

International Nuclear Information System (INIS)

Renauld, Valerie; Levert, Anne-Sophie; Muzerelle, Sophie; Cassoli, Emmanuel

2013-07-01

After a brief presentation of the main events during 2012, a recall of the ANDRA's mission (national inventory of radioactive materials and wastes), and a brief presentation of staffing data, this report presents and comments the activities of the ANDRA in the industrial domain (the Cigeo project for deep geological disposal of radioactive wastes, activities of the Aube centres and of the Manche storage centre, perspectives for low-activity long-life wastes, decontamination of polluted sites), in the scientific field (European projects, support to education and training, participation to a colloquium on clays, activities in the field of numerical simulation, activities of the 'Observatoire de l'Environnement'). The next part address the international activities, local commitments (in relationship with the different storage and industrial centres, or in the field of education and training), and activities related to communication and information

Physical activity in police beyond self-report.

Science.gov (United States)

Ramey, Sandra L; Perkhounkova, Yelena; Moon, Mikyung; Tseng, Hui-Chen; Wilson, Annerose; Hein, Maria; Hood, Kristin; Franke, Warren D

2014-03-01

Police officers have a higher risk for cardiovascular disease. Reductions in occupational physical activity may contribute to the risk, yet there have been few efforts to characterize the physical demands of police work beyond self-report. To compare measured physical activity between work and off-duty hours and assess the effects of stress on physical activity. Officers (n = 119) from six departments wore a pattern recognition monitor for 96 hours to measure total energy expenditure (kilocalorie per hour) (1k/cal = 4184 joules), activity intensity, and step count per hour. Participants were more active on their off-duty days than at work; the effects of stress on physical activity seemed moderated by sex. Police work is primarily a sedentary occupation, and officers tend to be more active on their off-duty days than during their work hours.

Data describing the effect of DRD4 promoter polymorphisms on promoter activity

Directory of Open Access Journals (Sweden)

Shoin Tei

2016-06-01

Full Text Available This data article tested whether polymorphisms within the dopamine D4 receptor (DRD4 gene promoter can lead to differences in the promoter activity. The variants, a 120-bp variable number tandem repeat (VNTR, −906 T/C, −809 G/A, −616G/C, and −521C/T, were introduced into the DRD4 promoter and the promoter activity was measured in a neural cell line using the luciferase assay. However, no differences were detected among the haplotypes investigated, and the in vitro data obtained from our protocol could not support the involvement of DRD4 promoter polymorphisms in heritable human traits.

Evaluation of estrogen receptor alpha activation by glyphosate-based herbicide constituents.

Science.gov (United States)

Mesnage, Robin; Phedonos, Alexia; Biserni, Martina; Arno, Matthew; Balu, Sucharitha; Corton, J Christopher; Ugarte, Ricardo; Antoniou, Michael N

2017-10-01

The safety, including the endocrine disruptive capability, of glyphosate-based herbicides (GBHs) is a matter of intense debate. We evaluated the estrogenic potential of glyphosate, commercial GBHs and polyethoxylated tallowamine adjuvants present as co-formulants in GBHs. Glyphosate (≥10,000 μg/L or 59 μM) promoted proliferation of estrogen-dependent MCF-7 human breast cancer cells. Glyphosate also increased the expression of an estrogen response element-luciferase reporter gene (ERE-luc) in T47D-KBluc cells, which was blocked by the estrogen antagonist ICI 182,780. Commercial GBH formulations or their adjuvants alone did not exhibit estrogenic effects in either assay. Transcriptomics analysis of MCF-7 cells treated with glyphosate revealed changes in gene expression reflective of hormone-induced cell proliferation but did not overlap with an ERα gene expression biomarker. Calculation of glyphosate binding energy to ERα predicts a weak and unstable interaction (-4.10 kcal mol -1 ) compared to estradiol (-25.79 kcal mol -1 ), which suggests that activation of this receptor by glyphosate is via a ligand-independent mechanism. Induction of ERE-luc expression by the PKA signalling activator IBMX shows that ERE-luc is responsive to ligand-independent activation, suggesting a possible mechanism of glyphosate-mediated activation. Our study reveals that glyphosate, but not other components present in GBHs, can activate ERα in vitro, albeit at relatively high concentrations. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Minimum Data Set Active Resident Information Report

Data.gov (United States)

U.S. Department of Health & Human Services — The MDS Active Resident Report summarizes information for residents currently in nursing homes. The source of these counts is the residents MDS assessment record....

Identification of a functional element in the promoter of the silkworm (Bombyx mori) fat body-specific gene Bmlp3.

Science.gov (United States)

Xu, Hanfu; Deng, Dangjun; Yuan, Lin; Wang, Yuancheng; Wang, Feng; Xia, Qingyou

2014-08-01

30K proteins are a group of structurally related proteins that play important roles in the life cycle of the silkworm Bombyx mori and are largely synthesized and regulated in a time-dependent manner in the fat body. Little is known about the upstream regulatory elements associated with the genes encoding these proteins. In the present study, the promoter of Bmlp3, a fat body-specific gene encoding a 30K protein family member, was characterized by joining sequences containing the Bmlp3 promoter with various amounts of 5' upstream sequences to a luciferase reporter gene. The results indicated that the sequences from -150 to -250bp and -597 to -675bp upstream of the Bmlp3 transcription start site were necessary for high levels of luciferase activity. Further analysis showed that a 21-bp sequence located between -230 and -250 was specifically recognized by nuclear factors from silkworm fat bodies and BmE cells, and could enhance luciferase reporter-gene expression 2.8-fold in BmE cells. This study provides new insights into the Bmlp3 promoter and contributes to the further clarification of the function and developmental regulation of Bmlp3. Copyright © 2014. Published by Elsevier B.V.

Annual report on nuclear physics activities

International Nuclear Information System (INIS)

Beck, R.; Bueche, G.; Fluegge, G.

1982-02-01

This report surveys the activities in fundamental research from July 1, 1980 to June 30, 1981 at the three institutes of the KfK which are concerned with nuclear physics. The research program comprises laser spectroscopy, nuclear reactions with light ions and physics at medium and higher energies. (orig.) [de

Aryl hydrocarbon receptor activation and CYP1A induction by cooked food-derived carcinogenic heterocyclic amines in human HepG2 cell lines.

Science.gov (United States)

Sekimoto, Masashi; Sumi, Haruna; Hosaka, Takuomi; Umemura, Takashi; Nishikawa, Akiyoshi; Degawa, Masakuni

2016-11-01

The ability of nine cooked food-derived heterocyclic aromatic amines (HCAs), such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methylpyrido[12-a:3',2'-d]imidazole (Glu-P-1), 2-amino-pyrido[12-a:3',2'-d]imidazole hydrochloride (Glu-P-2), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP), to activate human aryl hydrocarbon receptor (hAhR) was examined using a HepG2-A10 cell line, which has previously established from human hepatocarcinoma-derived HepG2 cells for use in hAhR-based luciferase reporter gene assays. Trp-P-1, Trp-P-2, AαC, MeAαC, IQ and MeIQx showed a definite ability to induce not only luciferase (hAhR activation) in HepG2-A10 cells but also cytochrome P450 (CYP)1A1/1A2 mRNAs in HepG2 cells, while such the ability of Glu-P-1, Glu-P-2, and PhIP was very low. In addition, all the HCAs examined, especially MeAαC and MeIQx, had a definite capacity for inhibiting the activity of ethoxyresorfin O-deethylase (CYP1As, especially CYP1A1). The present findings demonstrate that all the HCAs examined have the ability to activate hAhR and its target genes, and further confirm that these HCAs become good substrates for human CYP1A subfamily enzyme(s). Copyright © 2016 Elsevier Ltd. All rights reserved.

Hanford Laboratories monthly activities report, October 1964

Energy Technology Data Exchange (ETDEWEB)

1964-11-16

The monthly report for the Hanford Laboratories Operation, October 1964. Reactor fuels, chemistry, dosimetry, separation processes, reactor technology, financial activities, biology operation, and physics and instrumentation research, and applied mathematics operations are discussed.

Receptor for activated protein kinase C 1 suppresses gastric tumor progression through nuclear factor-kB pathway.

Science.gov (United States)

Yong-Zheng, X; Wan-Li, M; Ji-Ming, M; Xue-Qun, R

2015-12-01

Nuclear factor-kB (NF-kB) activity is crucial for survival and proliferation of many kinds of malignancies, including gastric cancer (GC). The receptor for activated protein kinase C 1 (RACK1) is known to regulate tumor development, whereas the underlined mechanism has not been described clearly. We analyzed expression of RACK1 in paired human GC samples by both real-time polymerase chain reaction (PCR) and western blot. Effects of RACK inhibition with small interfering RNA or its overexpression in cultured GC cell lines were evaluated in cell viabilities. NF-kB signaling was investigated using luciferase reporter assay and real-time PCR. RACK1 was significantly decreased in GC samples. Knockdown of RACK elevated GC cell viabilities, whereas overexpression of RACK1 suppressed tumorigenesis of GC cells. Importantly, NF-kB signaling was enhanced after RACK1 expression was inhibited, suggesting the negative regulation of the pro-oncogenic NF-kB activity by RACK1 might contribute to its tumor suppressor role in GC cells. Our results support that RACK1 suppresses gastric tumor progression through the NF-kB signaling pathway.

Progress report on scientific and technological activities: 1996

International Nuclear Information System (INIS)

1997-01-01

An overview of research and development activities of the Constituyentes Atomic Center, National Atomic Energy Commission (Argentine), during the period January-December 1996 is presented. The research and development activities are reported under the following headings: Fuel Elements; Non Destructive and Structural Test; Physics; Materials; Chemistry; Radiobiology Nuclear Reactors; Institute of Technology; Quality Management; Technical Assistance to Industry (SATI); Computer and Communication Services Center; Nuclear Power Stations Support Group. List of publications by the scientific staff of the Atomic Center is provided. The list includes paper published in journals, papers presented in conferences, symposia, etc., and technical reports

Progress report on scientific and technological activities: 1995

International Nuclear Information System (INIS)

1996-01-01

An overview of research and development activities of the Constituyentes Atomic Center, National Atomic Energy Commission (Argentine), during the period January-December 1995 is presented. The research and development activities are reported under the following headings: Fuel Elements; Non Destructive and Structural Test; Physics; Materials; Chemistry; Radiobiology Nuclear Reactors; Institute of Technology; Quality Management; Technical Assistance to Industry (SATI); Computer and Communication Services Center; Nuclear Power Stations Support Group. List of publications by the scientific staff of the Atomic Center is provided. The list includes paper published in journals, papers presented in conferences, symposia, etc., and technical reports

Activity report of Reactor Physics Division - 1993

International Nuclear Information System (INIS)

Indira, R.

1994-01-01

The research and development (R and D) activities of the Reactor Physics Division of Indira Gandhi Centre for Atomic Research (IGCAR), Kalpakkam during 1993 are reported. The activities are arranged under the headings: Nuclear Data Processing and validation, Core Physics and Operation Studies, Reactor Kinetics and Safety analysis, Reactor Noise Analysis and Radiation Transport and Shielding Studies. List of publication is given at the end. (author). refs., figs., tabs

Activity report of Reactor Physics Division-1995

International Nuclear Information System (INIS)

Gopalakrishnan, V.

1996-01-01

The research and development (R and D) activities of the Reactor Physics Division of Indira Gandhi Centre for Atomic Research (IGCAR), Kalpakkam during 1995 are reported. The activity are arranged under the headings: Nuclear Data Processing and Validation, Core Physics and Operation Studies, Reactor Kinetics and Safety analysis, Reactor Noise Analysis and Radiation Transport and Shielding Studies. List of publication is given at the end. refs., figs., tabs

Activity report of Reactor Physics Division - 1993

Energy Technology Data Exchange (ETDEWEB)

Indira, R [ed.; Indira Gandhi Centre for Atomic Research, Kalpakkam (India)

1994-12-31

The research and development (R and D) activities of the Reactor Physics Division of Indira Gandhi Centre for Atomic Research (IGCAR), Kalpakkam during 1993 are reported. The activities are arranged under the headings: Nuclear Data Processing and validation, Core Physics and Operation Studies, Reactor Kinetics and Safety analysis, Reactor Noise Analysis and Radiation Transport and Shielding Studies. List of publication is given at the end. (author). refs., figs., tabs.

Characterization of retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope glycoproteins of four serotypes of dengue viruses

International Nuclear Information System (INIS)

Hu, H.-P.; Hsieh, S.-C.; King, C.-C.; Wang, W.-K.

2007-01-01

In this study, we successfully established retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope (PrM/E) proteins of each of the four serotypes of dengue viruses, which caused the most important arboviral diseases in this century. Co-sedimentation of the dengue E protein and HIV-1 core proteins by sucrose gradient analysis of the pseudotype reporter virus of dengue virus type 2, D2(HIVluc), and detection of HIV-1 core proteins by immunoprecipitation with anti-E monoclonal antibody suggested that dengue viral proteins were incorporated into the pseudotype viral particles. The infectivity in target cells, as assessed by the luciferase activity, can be inhibited by the lysosomotropic agents, suggesting a pH-dependent mechanism of entry. Amino acid substitutions of the leucine at position 107, a critical residue at the fusion loop of E protein, with lysine resulted in severe impairment in infectivity, suggesting that entry of the pseudotype reporter virus is mediated through the fusogenic properties of E protein. With more and more dengue viral sequences available from different outbreaks worldwide, this sensitive and convenient tool has the potential to facilitate molecular characterization of the PrM/E proteins of dengue field isolates

Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction.

Science.gov (United States)

Lum, H; Jaffe, H A; Schulz, I T; Masood, A; RayChaudhury, A; Green, R D

1999-09-01

We investigated the hypothesis that cAMP-dependent protein kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An E1-, E3-, replication-deficient adenovirus (Ad) vector was constructed containing the complete sequence of PKA inhibitor (PKI) gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 microM thrombin increased transendothelial albumin clearance rate (0.012 +/- 0.003 and 0.035 +/- 0.005 microl/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKI resulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC infected with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was present. Also, F + I treatment of HMEC transfected with reporter plasmid containing the cAMP response element-directed transcription of the luciferase gene resulted in an almost threefold increase in luciferase activity. Overexpression of PKI inhibited this induction of luciferase activity. The results show that Ad-mediated overexpression of PKI in endothelial cells abrogated the cAMP-mediated protection against increased endothelial permeability, providing direct evidence that cAMP-dependent protein kinase promotes endothelial barrier function.

The activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 to the distal CCAAT box of the RhoB promoter

International Nuclear Information System (INIS)

Ahn, Jiwon; Choi, Jeong-Hae; Won, Misun; Kang, Chang-Mo; Gyun, Mi-Rang; Park, Hee-Moon; Kim, Chun-Ho; Chung, Kyung-Sook

2011-01-01

Highlights: → Regulation of transcriptional activation of RhoB is still unclear. → We examine the effect of p38 MAPK inhibition, and c-Jun and RhoB depletion on UV-induced RhoB expression and apoptosis. → We identify the regions of RhoB promoter necessary to confer UV responsiveness using pRhoB-luciferase reporter assays. → c-Jun, ATF2 and p300 are dominantly associated with NF-Y on the distal CCAAT box. → The activation of p38 MAPK primarily contribute to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins on distal CCAAT box of RhoB promoter. -- Abstract: The Ras-related small GTP-binding protein RhoB is rapidly induced in response to genotoxic stresses caused by ionizing radiation. It is known that UV-induced RhoB expression results from the binding of activating transcription factor 2 (ATF2) via NF-Y to the inverted CCAAT box (-23) of the RhoB promoter. Here, we show that the association of c-Jun with the distal CCAAT box (-72) is primarily involved in UV-induced RhoB expression and p38 MAPK regulated RhoB induction through the distal CCAAT box. UV-induced RhoB expression and apoptosis were markedly attenuated by pretreatment with the p38 MAPK inhibitor. siRNA knockdown of RhoB, ATF2 and c-Jun resulted in decreased RhoB expression and eventually restored the growth of UV-irradiated Jurkat cells. In the reporter assay using luciferase under the RhoB promoter, inhibition of RhoB promoter activity by the p38 inhibitor and knockdown of c-Jun using siRNA occurred through the distal CCAAT box. Immunoprecipitation and DNA affinity protein binding assays revealed the association of c-Jun and p300 via NF-YA and the dissociation of histone deacetylase 1 (HDAC1) via c-Jun recruitment to the CCAAT boxes of the RhoB promoter. These results suggest that the activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins to the distal CCAAT box of the RhoB promoter in Jurkat cells.

Energy National Mediator activity report 2009

International Nuclear Information System (INIS)

2009-01-01

After some data illustrating the activity of the Energy National Mediator in 2009, and an interview of a representative of this institution who comments its practice, this report proposes the opinions of the different involved actors (communities, consumer associations, providers, and so on) about the mediator. It puts the adopted strategy in perspective from the past year to the coming one. It describes the missions: information, advice, protection. It reports actions, recommendations and facts for 2009 in terms of consumer information, group mediation, poverty management, samples of analysed disputes. It presents the social organisation and gives a financial assessment of the institution

RTE - activity report 2008

International Nuclear Information System (INIS)

2008-01-01

RTE is a public company that operates, maintains and develops the largest high and very high-voltage electricity network in Europe. With its 100,000 km of lines, its 45 cross-border connections and its central geographical location, at the crossroads of European exchange, RTE has decisive assets for responding to the new economic and environmental challenges related to its activity, and for taking part actively in the construction of the internal electricity market. Since 2000, in the context of the public-service duties entrusted to it by the law, RTE has been the guarantor of the correct functioning and reliability of the French electricity system. It ensures free and equitable access to its network by all users, while respecting the code of good conduct that is checked annually by the Energy Regulatory Commission. Its income (public transmission network usage prices) and its expenditure (annual investment programme) are fixed by this regulatory authority. In accordance with the law, RTE is fully independent within the EDF group. Its status as a limited company with board of directors and supervisory board ensures its neutrality in relation to all players in the electricity market. In 2008: RTE's Turnover: euros 4,221 million, investments: euros 835 million, Employees: 8,536. This activity report presents: 1 - an interview with Dominique Maillard Chairman of the RTE Executive Board; 2 - 2008, a transitional year for RTE: overview and key figures, governance and organisation, significant events; 3 - Investments and clients, the fundamentals of RTE's assets: investing, adapting the network, ensuring the security of the system, dialogue with clients; 4 - RTE's men and women, more effective and higher added value: recruiting, preserving diversity, strengthening team spirit, supporting human resources; 5 - RTE, player and vehicle for sustainable development: a proactive commitment by RTE, participating in regional development, RTE, a socially-responsible company