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Sample records for ahr-dependent luciferase activity

  1. Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases.

    PubMed

    Viviani, V R; Arnoldi, F G C; Venkatesh, B; Neto, A J S; Ogawa, F G T; Oehlmeyer, A T L; Ohmiya, Y

    2006-10-01

    The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR) and red-emitting (PxRE) luciferases of railroad worms Phrixotrix viviani and P. hirtus[OLE1]. These luciferases were expressed and purified, and their active-site properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics. The substrate affinities and catalytic efficiency of PxRE luciferase are also higher than those of PxGR luciferase. Fluorescence studies with 8-anilino-1-naphthalene sulfonic acid and 6-p-toluidino-2-naphthalene sulfonic acid showed that the PxRE luciferase luciferin-binding site is more polar than that of PxGR luciferase, and it is sensitive to guanidine. Mutagenesis and modelling studies suggest that several invariant residues in the putative luciferin-binding site of PxRE luciferase cannot interact with excited oxyluciferin. These results suggest that one portion of the luciferin-binding site of the red-emitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.

  2. Improvement of thermostability and activity of firefly luciferase through [TMG][Ac] ionic liquid mediator.

    PubMed

    Ebrahimi, Mehdi; Hosseinkhani, Saman; Heydari, Akbar; Khavari-Nejad, Ramazan Ali; Akbari, Jafar

    2012-10-01

    Firefly luciferase catalyzes production of light from luciferin in the presence of Mg(2+)-ATP and oxygen. This enzyme has wide range of applications in biotechnology and development of biosensors. The low thermal stability of wild-type firefly luciferase is a limiting factor in most applications. Improvements in activity and stability of few enzymes in the presence of ionic liquids were shown in many reports. In this study, kinetic and thermal stability of firefly luciferase from Photinus pyralis in the presence of three tetramethylguanidine-based ionic liquids was investigated. The enzyme has shown improved activity in the presence of [1, 1, 3, 3-tetramethylguanidine][acetate], but in the presence of [TMG][trichloroacetate] and [TMG][triflouroacetate] activity, it decreased or unchanged significantly. Among these ionic liquids, only [TMG][Ac] has increased the thermal stability of luciferase. Incubation of [TMG][Ac] with firefly luciferase brought about with decrease of K(m) for ATP.

  3. Fe65 does not stabilize AICD during activation of transcription in a luciferase assay

    SciTech Connect

    Huysseune, Sandra; Kienlen-Campard, Pascal; Octave, Jean-Noel . E-mail: octave@nchm.ucl.ac.be

    2007-09-21

    The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount of AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment.

  4. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters

    PubMed Central

    Buckley, Suzanne M. K.; Delhove, Juliette M. K. M.; Perocheau, Dany P.; Karda, Rajvinder; Rahim, Ahad A.; Howe, Steven J.; Ward, Natalie J.; Birrell, Mark A.; Belvisi, Maria G.; Arbuthnot, Patrick; Johnson, Mark R.; Waddington, Simon N.; McKay, Tristan R.

    2015-01-01

    The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruses to neonatal rodents as an alternative to the existing technology of generating germline transgenic light producing rodents. At this age, neonates acquire immune tolerance to the conditionally responsive luciferase reporter. This simple and transferrable procedure permits surrogate quantitation of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4−/− mice or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter constructs in somatotransgenic mice, respectively. PMID:26138224

  5. Effects of iodide on the fluorescence and activity of the hydroperoxyflavin intermediate of Vibrio harveyi luciferase.

    PubMed

    Huang, Shouqin; Tu, Shiao-Chun

    2005-01-01

    The 4a-hydroperoxy-4a,5-dihydroFMN intermediate (II or HFOOH) of Vibrio harveyi luciferase is known to transform from a low quantum yield IIx to a high quantum yield (lambdamax 485 nm, uncorrected) IIy fluorescent species on exposure to excitation light. Similar results were observed with II prepared from the alphaH44A luciferase mutant, which is very weak in bioluminescence activity. Because of the rapid decay of the alphaH44A II, its true fluorescence was obscured by the more intense 520 nm fluorescence (uncorrected) from its decay product oxidized flavin mononucleotide (FMN). Potassium iodide (KI) at 0.2 M was effective in quenching the FMN fluorescence, leaving the 485 nm fluorescence of II from both the wild-type (WT) and alphaH44A luciferase readily detectable. For both II species, the luciferase-bound peroxyflavin was well shielded from KI quenching. KI also enhanced the decay rates of both the WT and alphaH44A II. For alphaH44A, the transformation of IIx to IIy can be induced by KI in the dark, and it is proposed to be a consequence of a luciferase conformational change. The WT II formed a bioluminescence-inactive complex with KI, resulting in two distinct decay time courses based on absorption changes and decreases of bioluminescence activity of II.

  6. Systemic activation of NF-κB driven luciferase activity in transgenic mice fed advanced glycation end products modified albumin.

    PubMed

    Nass, Norbert; Bayreuther, Kristina; Simm, Andreas

    2017-04-01

    Advanced glycation end products (AGEs) are stable end products of the Maillard reaction and accumulate with progressing ageing and degenerative diseases. Significant amounts of AGE-modified peptides are also consumed with processed food. AGEs bind to specific receptors, especially the receptor of AGEs (RAGE). Activation of RAGE then evokes intracellular signalling, finally resulting in the activation of the NF-κB transcription factor and therefore a proinflammatory state. We here analysed, whether NF-κB is activated in short term upon feeding an AGE-modified protein in-vivo. Transgenic mice expressing firefly luciferase under the control of an NF-κB responsive promoter were intraperitoneally injected or fed with AGE-modified- or control albumin and luciferase expression was analysed by in-vivo imaging and by in-vitro by determination of luciferase enzyme activity in heart, lung, gut, spleen, liver and kidney. In all organs, an activation of the luciferase reporter gene was observed in response to AGE-BSA feeding, however with different intensity and timing. The gut exhibited highest luciferase activity and this activity peaked 6-8 h post AGE-feeding. In heart and kidney, luciferase activity increased for up to 12 h post feeding. All other organs tested, exhibited highest activity at 10 h after AGE-consumption. Altogether, these data demonstrate that feeding AGE-modified protein resulted in a transient and systemic activation of the NF-κB reporter.

  7. Formation of active bacterial luciferase between interspecific subunits in vivo.

    PubMed

    Almashanu, S; Tuby, A; Hadar, R; Einy, R; Kuhn, J

    1995-01-01

    Interspecific complementation between luxAs and luxBs from Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi and Xenorhabdus luminescens was examined in vivo. The individual genes from these species were cloned on different compatible plasmids or amplified by PCR and brought together to yield cis combinations without extraneous DNA. The beta subunits from V. harveyi and X. luminescens form active enzyme only with alpha subunits from one of these species. All other combinations yield active enzymes. The lack of activity of the V. harveyi and X. luminescens beta subunits with the alpha subunits from V. fischeri and P. leiognathi results from a lack of association. This was shown by in vivo competition in which these beta subunits were overproduced in comparison with the beta and alpha of V. fischeri. No reduction in light was found. Overall, the in vivo results parallel those found in vitro using isolated denatured subunits and renaturation by removal of the denaturant.

  8. High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

    PubMed Central

    Trimmer, Casey; Snyder, Lindsey L.; Mainland, Joel D.

    2014-01-01

    Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system. PMID:24961834

  9. Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

    PubMed Central

    Mofford, David M.; Reddy, Gadarla Randheer; Miller, Stephen C.

    2014-01-01

    Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation. PMID:24616520

  10. In vivo characterization of an AHR-dependent long non-coding RNA required for proper Sox9b expression.

    PubMed

    Garcia, Gloria R; Goodale, Britton C; Wiley, Michelle W; La Du, Jane K; Hendrix, David A; Tanguay, Robert L

    2017-04-06

    Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long non-coding RNA (slincR) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2-dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests a relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurological and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway.

  11. Analysis of structural changes in active site of luciferase adsorbed on nanofabricated hydrophilic Si surface by molecular-dynamics simulations

    SciTech Connect

    Nishiyama, Katsuhiko; Hoshino, Tadatsugu

    2007-05-21

    Interactions between luciferase and a nanofabricated hydrophilic Si surface were explored by molecular-dynamics simulations. The structural changes in the active-site residues, the residues affecting the luciferin binding, and the residues affecting the bioluminescence color were smaller on the nanofabricated hydrophilic Si surface than on both a hydrophobic Si surface and a hydrophilic Si surface. The nanofabrication and wet-treatment techniques are expected to prevent the decrease in activity of luciferase on the Si surface.

  12. Localization of the active site of an enzyme, bacterial luciferase, using two-quantum affinity modification

    NASA Astrophysics Data System (ADS)

    Benimetskaya, L. Z.; Gitelzon, I. I.; Kozionov, Andrew L.; Novozhilov, S. Y.; Petushkov, V. N.; Rodionova, N. S.; Stockman, Mark I.

    1991-11-01

    For the first time the method of two-quantum affinity modification has been employed to probe the structure of an enzyme, bacterial luciferase. Position of the flavin-binding site of this enzyme, which was previously unknown, has been established. The obtained data indicate that the flavin site is positioned on the (alpha) -subunit. The closest contact of the protein chain of the enzyme with the chromophoric group of the flavin takes place near 80 +/- 10 and 120 +/- 10 amino acid residues; the regions 50 +/- 10 and 215 +/- 10 are also close to the flavin. The established localization does not contradict suggestions on positions of the flavin and phosphate sites of the bacterial luciferase, which had earlier been made from the data on evolutionary stability of various luciferases. The present method can, in principle, be applied to a great number of enzymes, including all flavin-dependent enzymes. Enzymatic catalysis has high speed and specificity. Creation of a method of determination of the elements of the primary structure of a protein, making up the active site (in which substratum conversion occurs), could be a significant advance in clearing up mechanisms of enzymatic catalysis. It was proposed to localize active sites of the enzymes, whose substrata are chromophores, using this method of two-quantum affinity modification. An enzyme- substratum complex is irradiated with laser light of sufficiently long wavelength ((lambda) 300 nm) which is not directly absorbed by the enzyme. Two-quantum quasiresonant excitation of the substratum activates it to the state with energy 5-7 eV, which is then radiativelessly transferred to neighboring protein groups. This energy exceeds the energy of activation of peptide bond breakage. Therefore, the enzyme will be disrupted in the vicinity of its active site. In the present paper the above approach has been implemented for the first time. Information has been obtained about the position of the flavin-binding site of bacterial

  13. Fimbrolide Natural Products Disrupt Bioluminescence of Vibrio By Targeting Autoinducer Biosynthesis and Luciferase Activity.

    PubMed

    Zhao, Weining; Lorenz, Nicola; Jung, Kirsten; Sieber, Stephan A

    2016-01-18

    Vibrio is a model organism for the study of quorum sensing (QS) signaling and is used to identify QS-interfering drugs. Naturally occurring fimbrolides are important tool compounds known to affect QS in various organisms; however, their cellular targets have so far remained elusive. Here we identify the irreversible fimbrolide targets in the proteome of living V. harveyi and V. campbellii via quantitative mass spectrometry utilizing customized probes. Among the major hits are two protein targets with essential roles in Vibrio QS and bioluminescence. LuxS, responsible for autoinducer 2 biosynthesis, and LuxE, a subunit of the luciferase complex, were both covalently modified at their active-site cysteines leading to inhibition of activity. The identification of LuxE unifies previous reports suggesting inhibition of bioluminescence downstream of the signaling cascade and thus contributes to a better mechanistic understanding of these QS tool compounds.

  14. Bioluminescent Reaction by Immobilized Luciferase

    NASA Astrophysics Data System (ADS)

    Tanaka, Ryuta; Takahama, Eriko; Iinuma, Masataka; Ikeda, Takeshi; Kadoya, Yutaka; Kuroda, Akio

    We have investigated an effect of immobilization of luciferase molecules at the optical fiber end on a bioluminescent reaction. The time dependence of measured count rates of emitted photons has been analyzed by fitting with numerical solution of differential equations including the effect of the product-inhibitor and the deactivation of the luciferase. Through the analysis, we have successfully extracted kinetic constants such as, reaction rate, number of active luciferase molecules, etc. Ratio of active molecules to total luciferase molecules in immobilization was one order of magnitude lower than that in solution. The reaction rate of the bioluminescent process was also different from the one of free luciferase in solution.

  15. Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

    PubMed

    Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

    2011-07-01

    The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

  16. Construction and evaluation of luciferase reporter phages for the detection of active and non-replicating tubercle bacilli

    PubMed Central

    Dusthackeer, Azger; Kumar, Vanaja; Subbian, Selvakumar; Sivaramakrishnan, Gomathi; Zhu, Guofang; Subramanyam, Balaji; Hassan, Sameer; Nagamaiah, Selvakumar; Chan, John; Rama, Narayanan Paranji

    2008-01-01

    The luciferase reporter phages (LRP) show great promise for diagnostic mycobacteriology. Though conventional constructs developed from lytic phages such as D29 and TM4 are highly specific, they lack sensitivity. We have isolated and characterized Che12, the first true temperate phage infecting M. tuberculosis. Since the tuberculosis (TB) cases among HIV infected population result from the reactivation of latent bacilli, it would be useful to develop LRP that can detect dormant bacteria. During dormancy, pathogenic mycobacteria switch their metabolism involving divergent genes than during normal, active growth phase. Since the promoters of these genes can potentially function during dormancy, they were exploited for the construction of novel mycobacterial luciferase reporter phages. The promoters of hsp60, isocitrate lyase (icl), and alpha-crystallin (acr) genes from M. tuberculosis were used for expressing firefly luciferase gene (FFlux) in both Che12 and TM4 phages and their efficiency was evaluated in detecting dormant bacteria from clinical isolates of M. tuberculosis. These LRP constructs exhibited detectable luciferase activity in dormant as well as in actively growing M. tuberculosis. The TM4 ts mutant based constructs showed about one log increase in light output in three of the five tested clinical isolates and in M. tuberculosis H37Rv compared to conventional lytic reporter phage, phAE129. By refining the LRP assay format further, an ideal rapid assay can be designed not only to diagnose active and dormant TB but also to differentiate the species and to find their drug susceptibility pattern. PMID:18272245

  17. Dioxin induces Ahr-dependent robust DNA demethylation of the Cyp1a1 promoter via Tdg in the mouse liver

    NASA Astrophysics Data System (ADS)

    Amenya, Hesbon Z.; Tohyama, Chiharu; Ohsako, Seiichiroh

    2016-10-01

    The aryl hydrocarbon receptor (Ahr) is a highly conserved nuclear receptor that plays an important role in the manifestation of toxicity induced by polycyclic aromatic hydrocarbons. As a xenobiotic sensor, Ahr is involved in chemical biotransformation through activation of drug metabolizing enzymes. The activated Ahr cooperates with coactivator complexes to induce epigenetic modifications at target genes. Thus, it is conceivable that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent Ahr ligand, may elicit robust epigenetic changes in vivo at the Ahr target gene cytochrome P450 1a1 (Cyp1a1). A single dose of TCDD administered to adult mice induced Ahr-dependent CpG hypomethylation, changes in histone modifications, and thymine DNA glycosylase (Tdg) recruitment at the Cyp1a1 promoter in the liver within 24 hrs. These epigenetic changes persisted until 40 days post-TCDD treatment and there was Cyp1a1 mRNA hyperinduction upon repeat administration of TCDD at this time-point. Our demethylation assay using siRNA knockdown and an in vitro methylated plasmid showed that Ahr, Tdg, and the ten-eleven translocation methyldioxygenases Tet2 and Tet3 are required for the TCDD-induced DNA demethylation. These results provide novel evidence of Ahr-driven active DNA demethylation and epigenetic memory. The epigenetic alterations influence response to subsequent chemical exposure and imply an adaptive mechanism to xenobiotic stress.

  18. Dioxin induces Ahr-dependent robust DNA demethylation of the Cyp1a1 promoter via Tdg in the mouse liver

    PubMed Central

    Amenya, Hesbon Z.; Tohyama, Chiharu; Ohsako, Seiichiroh

    2016-01-01

    The aryl hydrocarbon receptor (Ahr) is a highly conserved nuclear receptor that plays an important role in the manifestation of toxicity induced by polycyclic aromatic hydrocarbons. As a xenobiotic sensor, Ahr is involved in chemical biotransformation through activation of drug metabolizing enzymes. The activated Ahr cooperates with coactivator complexes to induce epigenetic modifications at target genes. Thus, it is conceivable that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent Ahr ligand, may elicit robust epigenetic changes in vivo at the Ahr target gene cytochrome P450 1a1 (Cyp1a1). A single dose of TCDD administered to adult mice induced Ahr-dependent CpG hypomethylation, changes in histone modifications, and thymine DNA glycosylase (Tdg) recruitment at the Cyp1a1 promoter in the liver within 24 hrs. These epigenetic changes persisted until 40 days post-TCDD treatment and there was Cyp1a1 mRNA hyperinduction upon repeat administration of TCDD at this time-point. Our demethylation assay using siRNA knockdown and an in vitro methylated plasmid showed that Ahr, Tdg, and the ten-eleven translocation methyldioxygenases Tet2 and Tet3 are required for the TCDD-induced DNA demethylation. These results provide novel evidence of Ahr-driven active DNA demethylation and epigenetic memory. The epigenetic alterations influence response to subsequent chemical exposure and imply an adaptive mechanism to xenobiotic stress. PMID:27713569

  19. Assessment of shikonin for potential estrogenic activity by dual-luciferase reporter based bioluminescent measurements in vitro.

    PubMed

    Zhang, Yi; Li, Ping-Ping

    2011-02-01

    Shikonin, an active component of Lithospermum erythrorhizon Sieb. et Zucc., shows multiple pharmacological properties. However, the estrogenic activity of shikonin is remaining unclear. We assessed the potential estrogenic activity of shikonin with dual-luciferase reporter assay and bioluminescent measurements, by using transient cotransfection with estrogen dependent plasmid pERE-TK-Luc and internal control plasmid pRL-TK in MCF-7 cells. Estrogenic activity of shikonin, even at high concentration did not alter significantly compared to negative control (p > 0.05) and were significantly lower than those with E2 (p < 0.01). Concluding, shikonin demonstrates no estrogenic activity in vitro.

  20. Effect of antiangiogenic therapy on luciferase activity in a cytomegalovirus- or HSP70-promoter-transfected M21 tumor model

    NASA Astrophysics Data System (ADS)

    Hundt, Walter; Schink, Christian; Steinbach, Silke; O'Connell-Rodwell, Caitlin E.; Kiessling, Andreas; Librizzi, Damiano; Burbelko, Mykhaylo; Guccione, Samira

    2012-06-01

    We investigated the effect of targeted gene therapy on heat shock protein 70 expression (Hsp70) and protein production (HSP70) in a melanoma tumor model (M21; M21-L). M21 and M21-L cells transfected with a plasmid containing the Hsp70 (Hspa1b) or the cytomegalovirus (CMV) promoter and the luciferase reporter gene were injected into mice; the resulting tumors grew to a size of 650 mm3. Mice (five per group) were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein complex [RGD-NP/RAF(-)] or with a nanoparticle control. Bioluminescence imaging (IVIS®, Xenogen, USA) was performed at 12, 24, 48, and 72 h after the treatment cycle. Western blot analysis of HSP70 protein was performed to monitor protein expression. The size of the treated M21 tumors remained fairly constant (647.8+/-103.4 mm2 at the beginning versus 704.8+/-94.4 mm3 at the end of the experiment). The size of the M21-L tumors increased, similar to the untreated control tumors. Bioluminescent imaging demonstrated that when transcription was controlled by the CMV promoter, luciferase activity decreased to 17.9%+/-4.3% of baseline values in the treated M21 tumors. When transcription was controlled by the Hsp70 promoter, the highest luciferase activity (4.5+/-0.7-fold increase over base-line values) was seen 24 h after injection in the M21 tumors; however, no luciferase activity was seen in the M21-L tumors. In accordance with bioluminescent imaging, western blot analysis showed a peak in HSP70 production at 24 h after the injection of the RGD-NP/RAF(-) complex in the M21 tumors; however, no HSP70 protein induction was seen in the M21-L tumors. Thus, targeted antiangiogenic therapy can induce Hsp70 expression and HSP70 protein in melanoma tumors.

  1. Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis

    PubMed Central

    van den Bogaard, Ellen H.; Bergboer, Judith G.M.; Vonk-Bergers, Mieke; van Vlijmen-Willems, Ivonne M.J.J.; Hato, Stanleyson V.; van der Valk, Pieter G.M.; Schröder, Jens Michael; Joosten, Irma; Zeeuwen, Patrick L.J.M.; Schalkwijk, Joost

    2013-01-01

    Topical application of coal tar is one of the oldest therapies for atopic dermatitis (AD), a T helper 2 (Th2) lymphocyte–mediated skin disease associated with loss-of-function mutations in the skin barrier gene, filaggrin (FLG). Despite its longstanding clinical use and efficacy, the molecular mechanism of coal tar therapy is unknown. Using organotypic skin models with primary keratinocytes from AD patients and controls, we found that coal tar activated the aryl hydrocarbon receptor (AHR), resulting in induction of epidermal differentiation. AHR knockdown by siRNA completely abrogated this effect. Coal tar restored filaggrin expression in FLG-haploinsufficient keratinocytes to wild-type levels, and counteracted Th2 cytokine–mediated downregulation of skin barrier proteins. In AD patients, coal tar completely restored expression of major skin barrier proteins, including filaggrin. Using organotypic skin models stimulated with Th2 cytokines IL-4 and IL-13, we found coal tar to diminish spongiosis, apoptosis, and CCL26 expression, all AD hallmarks. Coal tar interfered with Th2 cytokine signaling via dephosphorylation of STAT6, most likely due to AHR-regulated activation of the NRF2 antioxidative stress pathway. The therapeutic effect of AHR activation herein described opens a new avenue to reconsider AHR as a pharmacological target and could lead to the development of mechanism-based drugs for AD. PMID:23348739

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

    SciTech Connect

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

    2008-08-29

    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.

  3. Firefly luciferase inhibition.

    PubMed

    Leitão, João M M; Esteves da Silva, Joaquim C G

    2010-10-05

    Firefly luciferase (Luc) is the most studied of the luciferase enzymes and the mechanism and kinetics of the reactions catalyzed by this enzyme have been relatively well characterized. Luc catalyzes the bioluminescent reaction involving firefly luciferin (D-LH(2)), adenosine triphosphate (ATP), magnesium ion and molecular oxygen with the formation of an electronically excited species (oxyluciferin), inorganic pyrophosphate (PPi), carbon dioxide and adenosine monophosphate (AMP). Luc also catalyzes other non-luminescent reactions, which can interfere with the light production mechanism. Following electronic relaxation, the excited oxyluciferin emits radiation in the visible region of the electromagnetic spectrum (550-570 nm). Among the various possible compounds, several classes of inhibitory substances interfere with the activity of this enzyme: here, we consider substrate-related compounds, intermediates or products of the Luc catalyzed reactions, in addition to anesthetics and, fatty acids. This review summarizes the main inhibitors of Luc and the corresponding inhibition kinetic parameters.

  4. Development of Neh2-Luciferase Reporter and Its Application for High Throughput Screening and Real-Time Monitoring of Nrf2 Activators

    PubMed Central

    Smirnova, Natalya A.; Haskew-Layton, Renee E.; Basso, Manuela; Hushpulian, Dmitry M.; Payappilly, Jimmy B.; Speer, Rachel E.; Ahn, Young-Hoon; Rakhman, Ilay; Cole, Philip A.; Pinto, John T.; Ratan, Rajiv R.; Gazaryan, Irina G.

    2011-01-01

    SUMMARY The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degradation of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biologically active compounds to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found—nordihydroguaiaretic acid, fisetin, and gedunin—induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism. PMID:21700211

  5. An enzymatic assay based on luciferase Ebola virus-like particles for evaluation of virolytic activity of antimicrobial peptides.

    PubMed

    Peskova, Marie; Heger, Zbynek; Janda, Petr; Adam, Vojtech; Pekarik, Vladimir

    2017-02-01

    Antimicrobial peptides are currently considered as promising antiviral compounds. Current assays to evaluate the effectivity of peptides against enveloped viruses based on liposomes or hemolysis are encumbered by the artificial nature of liposomes or distinctive membrane composition of used erythrocytes. We propose a novel assay system based on enzymatic Ebola virus-like particles containing sensitive luciferase reporter. The assay was validated with several cationic and anionic peptides and compared with lentivirus inactivation and hemolytic assays. The assay is sensitive and easy to perform in standard biosafety level laboratory with potential for high-throughput screens. The use of virus-like particles in the assay provides a system as closely related to the native viruses as possible eliminating some issues associated with other more artificial set ups. We have identified CAM-W (KWKLWKKIEKWGQGIGAVLKWLTTWL) as a peptide with the greatest antiviral activity against infectious lentiviral vectors and filoviral virus-like particles.

  6. Use Of Low Light Image Microscopy To Monitor Genetically Engineered Bacterial Luciferase Gene Expression In Living Cells And Gene Activation Throughout The Development Of A Transgenic Organism

    NASA Astrophysics Data System (ADS)

    Langridge, W. H.; Escher, Alan P.; Baga, M.; O'Kane, Dennis J.; Wampler, John E.; Koncz, C.; Schell, John D.; Szalay, A. A.

    1989-12-01

    Procaryotic and eucaryotic expression vectors which contain a marker gene for selection of transformants linked to genes encoding bacterial luciferase for detection of promoter activated gene expression in vivo were used to transform the appropriate host organisms and drug resistant colonies, cells, or calli were obtained. Bacterial luciferase expression was measured by a luminescence assay for quantitative determination of promoter activation. The cellular localization of bacteria inside the host plant cell cytoplasm was achieved in a single infected plant cell based on the light emitting ability of the genetically engineered bacteria. In addition, the bacterial luciferase marker gene fusions were used to monitor cell type, tissue, and organ specific gene expression in transgenic plants in vivo. To monitor physiological changes during ontogeny of a transformed plant, low light video microscopy, aided by real time image processing techniques developed specifically to enhance extreme low light images, was successfully applied.

  7. Coupling ex vivo electroporation of mouse retinas and luciferase reporter assays to assess rod-specific promoter activity.

    PubMed

    Boulling, Arnaud; Escher, Pascal

    2016-07-01

    Ex vivo electroporation of mouse retinas is an established tool to modulate gene expression and to study cell type-specific gene expression. Here we coupled ex vivo electroporation to luciferase reporter assays to facilitate the study of rod-photoreceptor-specific gene promoters. The activity of the rod-specific proximal bovine rhodopsin promoter was significantly increased in C57BL/6J wild-type retinas at postnatal days 1 and 7 by 3.4-fold and 8.7-fold respectively. In C57BL/6J Nr2e3(rd7/rd7) retinas, where the rod photoreceptor-specific nuclear receptor Nr2e3 is not expressed, a significant increase by 2.5-fold was only observed at postnatal day 7. Cone-specific S-opsin promoter activity was not modulated in C57BL/6J wild-type and Nr2e3(rd7/rd7) retinas. Taken together, we describe an easily implementable protocol to assess rod-specific promoter activity in a physiological context resembling that of the developing postnatal mouse retina.

  8. [Comparison of the relative luciferase activity in secondary CEF by different heterogenous strong promoters, MDV gB promoter and the composed promoters].

    PubMed

    Qiu, Ya-Feng; Ge, Fei-Fei; Xu, Xue-Qing; Chen, Pu-Yan

    2006-04-01

    To improve the protection efficiency of the recombinant Marek' s disease viruses (MDV) in chickens with or without maternal antibodies,the work of selecting the optimal promoters for the construction of recombinant MDV was carried out. Combined with the efficient genetic manipulation, the composed promoters was constructed by use of the MDV gB core promoter with the regulatory elements from the early immediately promoter and enhancer of hCMV, the promoter and enhancer of SV40 or the partial enhancer of hCMV. And these composed promoters were ligased to the luciferase to construct the eukaryotic expressing vectors and named PhCMV-gB, Psv-gB and Pen-gB, respectively. In vitro, these vectors and internal standard plasmid (pSV-beta-LacZ) were transiently co-transfected into secondary CEF by FuGene 6 Transfection Reagent. Furthermore, cells were harvested 48 hours after transfection. Then the luciferase activity was detected by a luciferase assay kit, at the same time, the beta-galactosidase enzyme activity was detected by a beta-galactosidase enzyme assay kit, and the luciferase activity was corrected by the beta-galactosidase enzyme activity to get the relative luciferase activity. The relative luciferase activity was used as the transcriptional activity. By comparison of the relative luciferase activity of every promoter, it was found that these composed promoters could more effectively drive the reporter gene expression than the full legth of gB promoter did. Among them, PhCMV-gB robustly drove the reporter gene expression. On the other hand, PSV-gB and Pen-gB appeared to have the same strength; But compared with the commercial strong promoters, the transcriptional activity of the composed promoter were less than as or the same as that of the strong promoters. Therefore, at a sense, it can be proposed that these composed promoters have not only the characteristic of MDV gB promoter, but also that of the commercial strong promoters. These provide the choices for

  9. A novel luciferase based reporter system to monitor activation of the ErbB2/Her2/neu pathway non-invasively during radiotherapy

    PubMed Central

    Wolf, Frank; Li, Wenrong; Li, Fang; Li, Chuan-Yuan

    2010-01-01

    Purpose To develop a split-luciferase based reporter system that allows for non-invasive monitoring of activation of the Her2/neu pathway in vivo in a quantitative and sensitive manner. Methods and Materials Fusion proteins of the ErbB2/Her2/neu receptor to the N-terminal fragment of luciferase as well as of its downstream binding partner Shc to the C-terminal fragment of luciferase have been engineered based on the rationale that upon activation and binding of the Her2 receptor molecule to Shc, luciferase function will be reconstituted. Thus the resulting bioluminescence signals can serve as a surrogate measure of receptor activation. Results We show that our reporter systems functions well in vitro in breast cancer cells and in vivo in xenograft tumors. In particular, the activities of Her2/neu in xenograft tumors could be monitored serially for an extended period of time after radiotherapy. Conclusions We believe that the novel ErbB2/Her2/neu reporter presented here is a powerful tool to study the biology of the Her2-neu pathway in vitro as well as in vivo. It should also facilitate the development and rapid evaluation of new Her2/neu targeted therapeutics. PMID:20934271

  10. Novel Luciferase-Based Reporter System to Monitor Activation of ErbB2/Her2/neu Pathway Noninvasively During Radiotherapy

    SciTech Connect

    Wolf, Frank; Li Wenrong; Li Fang; Li Chuanyuan

    2011-01-01

    Purpose: To develop a split-luciferase-based reporter system that allows for noninvasive monitoring of activation of the Her2/neu pathway in vivo in a quantitative and sensitive manner. Methods and Materials: Fusion proteins of the ErbB2/Her2/neu receptor to the N-terminal fragment of luciferase and of its downstream binding partner Shc to the C-terminal fragment of luciferase have been engineered owing to the rationale that on activation and binding of the Her2 receptor molecule to Shc, luciferase function will be reconstituted. Thus, the resulting bioluminescence signals can serve as a surrogate measure of receptor activation. Results: We have shown that our reporter systems functions well in vitro in breast cancer cells and in vivo in xenograft tumors. In particular, the activities of Her2/neu in xenograft tumors could be monitored serially for an extended period after radiotherapy. Conclusions: We believe that the novel ErbB2/Her2/neu reporter we have presented is a powerful tool to study the biology of the Her2-neu pathway in vitro and in vivo. It should also facilitate the development and rapid evaluation of new Her2/neu-targeted therapeutic agents.

  11. Exploiting NanoLuc luciferase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity.

    PubMed

    Cevenini, Luca; Calabretta, Maria Maddalena; Lopreside, Antonia; Tarantino, Giuseppe; Tassoni, Annalisa; Ferri, Maura; Roda, Aldo; Michelini, Elisa

    2016-12-01

    The availability of smartphones with high-performance digital image sensors and processing power has completely reshaped the landscape of point-of-need analysis. Thanks to the high maturity level of reporter gene technology and the availability of several bioluminescent proteins with improved features, we were able to develop a bioluminescence smartphone-based biosensing platform exploiting the highly sensitive NanoLuc luciferase as reporter. A 3D-printed smartphone-integrated cell biosensor based on genetically engineered Hek293T cells was developed. Quantitative assessment of (anti)-inflammatory activity and toxicity of liquid samples was performed with a simple and rapid add-and-measure procedure. White grape pomace extracts, known to contain several bioactive compounds, were analyzed, confirming the suitability of the smartphone biosensing platform for analysis of untreated complex biological matrices. Such approach could meet the needs of small medium enterprises lacking fully equipped laboratories for first-level safety tests and rapid screening of new bioactive products. Graphical abstract Smartphone-based bioluminescence cell biosensor.

  12. Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane

    PubMed Central

    2014-01-01

    Background Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene. Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. Results The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences. Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures. Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes. In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. Conclusions We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred

  13. Computational analysis and functional expression of ancestral copepod luciferase.

    PubMed

    Takenaka, Yasuhiro; Noda-Ogura, Akiko; Imanishi, Tadashi; Yamaguchi, Atsushi; Gojobori, Takashi; Shigeri, Yasushi

    2013-10-10

    We recently reported the cDNA sequences of 11 copepod luciferases from the superfamily Augaptiloidea in the order Calanoida. They were classified into two groups, Metridinidae and Heterorhabdidae/Lucicutiidae families, by phylogenetic analyses. To elucidate the evolutionary processes, we have now further isolated 12 copepod luciferases from Augaptiloidea species (Metridia asymmetrica, Metridia curticauda, Pleuromamma scutullata, Pleuromamma xiphias, Lucicutia ovaliformis and Heterorhabdus tanneri). Codon-based synonymous/nonsynonymous tests of positive selection for 25 identified copepod luciferases suggested that positive Darwinian selection operated in the evolution of Heterorhabdidae luciferases, whereas two types of Metridinidae luciferases had diversified via neutral mechanism. By in silico analysis of the decoded amino acid sequences of 25 copepod luciferases, we inferred two protein sequences as ancestral copepod luciferases. They were expressed in HEK293 cells where they exhibited notable luciferase activity both in intracellular lysates and cultured media, indicating that the luciferase activity was established before evolutionary diversification of these copepod species.

  14. Oestrogenic activity of a textile industrial wastewater treatment plant effluent evaluated by the E-screen test and MELN gene-reporter luciferase assay.

    PubMed

    Schilirò, Tiziana; Porfido, Arianna; Spina, Federica; Varese, Giovanna Cristina; Gilli, Giorgio

    2012-08-15

    This study quantified the biological oestrogenic activity in the effluent of a textile industrial wastewater treatment plant (IWWTP) in northwestern Italy. Samples of the IWWTP effluent were collected monthly, both before and after tertiary treatment (ozonation). After solid phase extraction, all samples were subjected to two in vitro tests of total estrogenic activity, the human breast cancer cell line (MCF-7 BUS) proliferation assay, or E-screen test, and the luciferase-transfected human breast cancer cell line (MELN) gene-reporter assay, to measure the 17β-oestradiol equivalent quantity (EEQ). In the E-screen test, the mean EEQ values were 2.35±1.68 ng/L pre-ozonation and 0.72±0.58 ng/L post-ozonation; in the MELN gene-reporter luciferase assay, the mean EEQ values were 4.18±3.54 ng/L pre-ozonation and 2.53±2.48 ng/L post-ozonation. These results suggest that the post-ozonation IWWTP effluent had a lower oestrogenic activity (simple paired t-tests, p<0.05). The average reduction of estrogenic activity of IWWTP effluent after ozonation was 67±26% and 52±27% as measured by E-screen test and MELN gene-reporter luciferase assay, respectively. There was a positive and significant correlation between the two tests (Rho S=0.650, p=0.022). This study indicates that the environmental risk is low because oestrogenic substances are deposited into the river via IWWTP at concentrations lower than those at which chronic exposure has been reported to affect the endocrine system of living organisms.

  15. Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors

    PubMed Central

    Kilianski, Andy; Mielech, Anna M.; Deng, Xufang

    2013-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors. PMID:23986593

  16. Discovery, adaptation and transcriptional activity of two tick promoters: Construction of a dual luciferase reporter system for optimization of RNA interference in Rhipicephalus (Boophilus) microplus cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dual luciferase reporter systems are valuable tools for functional genomic studies, but have not previously been developed for use in tick cell culture. We evaluated expression of available luciferase constructs in tick cell cultures derived from Rhipicephalus (Boophilus) microplus, an important vec...

  17. Relationship between stability and bioluminescence color of firefly luciferase.

    PubMed

    Maghami, Parvaneh; Ranjbar, Bijan; Hosseinkhani, Saman; Ghasemi, Atiyeh; Moradi, Ali; Gill, Pooria

    2010-03-01

    Firefly luciferase catalyzes the oxidation of luciferin in the presence of ATP, Mg(2+) and molecular oxygen. The bioluminescence color of firefly luciferases is identified by the luciferase structure and assay conditions. Amongst different types of beetles, luciferase from Phrixotrix railroad worm (PhRE) with a unique additional residue (Arg353) naturally emits red bioluminescence color. By insertion of Arg356 in luciferase of Lampyris turkestanicus, corresponding to Arg353 in Phrixotrix hirtus, the color of the emitted light was changed to red. To understand the effect of this position on the bioluminescence color shift, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus pyralis luciferase. Comparison of mutants with native luciferase shows that mutation brought an increase in the content of secondary structure and globular compactness of (P. pylalis) luciferase. Comparative study of chemical denaturation of native and mutant luciferases by activity measurement, intrinsic and extrinsic fluorescence, circular dichroism, and DSC techniques revealed that insertion of positively charged residues (Arg, Lys) in the flexible loop (352-358) plays a significant role on the stability of (P. pyralis) luciferase and changes the light color to red.

  18. Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.

    PubMed

    Viviani, V R; Simões, A; Bevilaqua, V R; Gabriel, G V M; Arnoldi, F G C; Hirano, T

    2016-08-30

    Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also

  19. Isolation and properties of the luciferase stored in the ovary of the scyphozoan medusa Periphylla periphylla.

    PubMed

    Shimomura, O; Flood, P R; Inouye, S; Bryan, B; Shimomura, A

    2001-12-01

    Bioluminescence of the medusa Periphylla is based on the oxidation of coelenterazine catalyzed by luciferase. Periphylla has two types of luciferase: the soluble form luciferase L, which causes the exumbrellar bioluminescence display of the medusa, and the insoluble aggregated form, which is stored as particulate material in the ovary, in an amount over 100 times that of luciferase L. The eggs are especially rich in the insoluble luciferase, which drastically decreases upon fertilization. The insoluble form could be solubilized by 2-mercaptoethanol, yielding a mixture of luciferase oligomers with molecular masses in multiples of approximately 20 kDa. Those having the molecular masses of 20 kDa, 40 kDa, and 80 kDa were isolated and designated, respectively, as luciferase A, luciferase B, and luciferase C. The luminescence activities of Periphylla luciferases A, B, and C were 1.2 approximately 4.1 x 10(16) photon/mg. s, significantly higher than any coelenterazine luciferase known, and the quantum yields of coelenterazine catalyzed by these luciferases (about 0.30 at 24 degrees C) are comparable to that catalyzed by Oplophorus luciferase (0.34 at 22 degrees C), which has been considered the most efficient coelenterazine luciferase until now. Luciferase L (32 kDa) could also be split by 2-mercaptoethanol into luciferase A and an accessory protein (approx. 12 kDa), as yet uncharacterized. Luciferases A, B, and C are highly resistant to inactivation: their luminescence activities are only slightly diminished at pH 1 and pH 11 and are enhanced in the presence of 1 approximately 2 M guanidine hydrochloride; but they are less stable to heating than luciferase L, which is practically unaffected by boiling.

  20. Chaperones rescue luciferase folding by separating its domains.

    PubMed

    Scholl, Zackary N; Yang, Weitao; Marszalek, Piotr E

    2014-10-10

    Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process.

  1. Chaperones Rescue Luciferase Folding by Separating Its Domains*

    PubMed Central

    Scholl, Zackary N.; Yang, Weitao; Marszalek, Piotr E.

    2014-01-01

    Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process. PMID:25160632

  2. Design of disulfide bridge as an alternative mechanism for color shift in firefly luciferase and development of secreted luciferase.

    PubMed

    Nazari, Mahboobeh; Hosseinkhani, Saman

    2011-07-01

    The bioluminescence reaction, which uses luciferin, Mg(2+)-ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and emits visible light. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. 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 three separate mutant enzymes with a single bridge. Moreover, C(81)-A(105)C mutant luciferase was modified and successfully secreted to the extracellular medium. By introduction of disulfide bridges using site-directed mutagenesis in Photinus pyralis luciferase the color of emitted light was changed to red and the optimum temperature of activity was also increased (up to 10 °C more than wild type). Amongst mutants with a disulfide bridge, P(451)C-V(469)C and L(306)C-L(309)C mutants exhibit a single peak in the red region of the spectrum at pH 7.8. It is worthwhile to note that with the design of a secreted luciferase, the increased optimum temperature, thermostability and emission of red light might make mutant luciferase suitable reporters for the study of gene expression in high through-put screening.

  3. Firefly luciferase gene contains a cryptic promoter

    PubMed Central

    Vopálenský, Václav; Mašek, Tomáš; Horváth, Ondřej; Vicenová, Blanka; Mokrejš, Martin; Pospíšek, Martin

    2008-01-01

    A firefly luciferase (FLuc) counts among the most popular reporters of present-day molecular and cellular biology. In this study, we report a cryptic promoter activity in the luc+ gene, which is the most frequently used version of the firefly luciferase. The FLuc coding region displays cryptic promoter activity both in mammalian and yeast cells. In human CCL13 and Huh7 cells, cryptic transcription from the luc+ gene is 10–16 times weaker in comparison to the strong immediate-early cytomegalovirus promoter. Additionally, we discuss a possible impact of the FLuc gene cryptic promoter on experimental results especially in some fields of the RNA-oriented research, for example, in analysis of translation initiation or analysis of miRNA/siRNA function. Specifically, we propose how this newly described cryptic promoter activity within the FLuc gene might contribute to the previous determination of the strength of the cryptic promoter found in the cDNA corresponding to the hepatitis C virus internal ribosome entry site. Our findings should appeal to the researchers to be more careful when designing firefly luciferase-based assays as well as open the possibility of performing some experiments with the hepatitis C virus internal ribosome entry site, which could not be considered until now. PMID:18697919

  4. Magnetic nanoparticles supported ionic liquids improve firefly luciferase properties.

    PubMed

    Noori, Ali Reza; Hosseinkhani, Saman; Ghiasi, Parisa; Akbari, Jafar; Heydari, Akbar

    2014-03-01

    Ionic liquids as neoteric solvents, microwave irradiation, and alternative energy source are becoming as a solvent for many enzymatic reactions. We recently showed that the incubation of firefly luciferase from Photinus pyralis with various ionic liquids increased the activity and stability of luciferase. Magnetic nanoparticles supported ionic liquids have been obtained by covalent bonding of ionic liquids-silane on magnetic silica nanoparticles. In the present study, the effects of [γ-Fe2O3@SiO2][BMImCl] and [γ-Fe2O3@SiO2][BMImI] were investigated on the structural properties and function of luciferase using circular dichroism, fluorescence spectroscopy, and bioluminescence assay. Enzyme activity and structural stability increased in the presence of magnetic nanoparticles supported ionic liquids. Furthermore, the effect of ingredients which were used was not considerable on K(m) value of luciferase for adenosine-5'-triphosphate and also K(m) value for luciferin.

  5. Thermostabilization of firefly luciferase by in vivo directed evolution.

    PubMed

    Koksharov, Mikhail I; Ugarova, Natalia N

    2011-11-01

    Firefly luciferase is widely used in a number of areas of biotechnology and molecular biology. However, rapid inactivation of wild-type (WT) luciferases at elevated temperatures often hampers their application. A simple non-lethal in vivo screening scheme was used to identify thermostable mutants of luciferase in Escherichia coli colonies. This scheme allowed carrying out each cycle of mutagenesis in a rapid and efficient manner. Four rounds of directed evolution were conducted on a part of the gene coding for amino acid residues 130-390 of Luciola mingrelica luciferase. The resultant mutant designated 4TS had a half-life of 10 h at 42°C, which is 65-fold higher compared with the WT luciferase. Moreover, the mutant 4TS showed a 1.9-fold increase in specific activity, 5.7-fold reduction of K(m) for ATP and a higher-temperature optimum compared with the WT enzyme. 4TS contains eight mutations, four of which are suggested to be mainly responsible for the enhancement of thermostability: R211L, A217V, E356K and S364C. Thus, directed evolution with non-lethal colony screening for in vivo bioluminescence activity proved to be an effective and efficient approach for increasing thermal stability of luciferase while retaining high catalytic activity.

  6. Identification of mutant firefly luciferases that efficiently utilize aminoluciferins.

    PubMed

    Harwood, Katryn R; Mofford, David M; Reddy, Gadarla R; Miller, Stephen C

    2011-12-23

    Firefly luciferase-catalyzed light emission from D-luciferin is widely used as a reporter of gene expression and enzymatic activity both in vitro and in vivo. Despite the power of bioluminescence for imaging and drug discovery, light emission from firefly luciferase is fundamentally limited by the physical properties of the D-luciferin substrate. We and others have synthesized aminoluciferin analogs that exhibit light emission at longer wavelengths than D-luciferin and have increased affinity for luciferase. However, although these substrates can emit an intense initial burst of light that approaches that of D-luciferin, this is followed by much lower levels of sustained light output. Here we describe the creation of mutant luciferases that yield improved sustained light emission with aminoluciferins in both lysed and live mammalian cells, allowing the use of aminoluciferins for cell-based bioluminescence experiments.

  7. Firefly bioluminescence: a mechanistic approach of luciferase catalyzed reactions.

    PubMed

    Marques, Simone M; Esteves da Silva, Joaquim C G

    2009-01-01

    Luciferase is a general term for enzymes catalyzing visible light emission by living organisms (bioluminescence). The studies carried out with Photinus pyralis (firefly) luciferase allowed the discovery of the reaction leading to light production. It can be regarded as a two-step process: the first corresponds to the reaction of luciferase's substrate, luciferin (LH(2)), with ATP-Mg(2+) generating inorganic pyrophosphate and an intermediate luciferyl-adenylate (LH(2)-AMP); the second is the oxidation and decarboxylation of LH(2)-AMP to oxyluciferin, the light emitter, producing CO(2), AMP, and photons of yellow-green light (550- 570 nm). In a dark reaction LH(2)-AMP is oxidized to dehydroluciferyl-adenylate (L-AMP). Luciferase also shows acyl-coenzyme A synthetase activity, which leads to the formation of dehydroluciferyl-coenzyme A (L-CoA), luciferyl-coenzyme A (LH(2)-CoA), and fatty acyl-CoAs. Moreover luciferase catalyzes the synthesis of dinucleoside polyphosphates from nucleosides with at least a 3'-phosphate chain plus an intact terminal pyrophosphate moiety. The LH(2) stereospecificity is a particular feature of the bioluminescent reaction where each isomer, D-LH(2) or L-LH(2), has a specific function. Practical applications of the luciferase system, either in its native form or with engineered proteins, encloses the analytical assay of metabolites like ATP and molecular biology studies with luc as a reporter gene, including the most recent and increasing field of bioimaging.

  8. Effect of solvents on the fluorescence spectra of bacterial luciferase

    NASA Astrophysics Data System (ADS)

    Sukovataya, Irina E.; Tyulkova, Natalya A.; Kaykova, Elisaveta V.

    2006-08-01

    Bacteria luciferases catalyze the oxidation reaction of the long-chain aliphatic aldehyde and reduced flavinmononucleotide involving molecular oxygen to a respective fatty acid emitting light quanta in the visible spectrum. Fluorescence emission of luciferases from Photobacterium leiognathi dissolved in organic solvent-water mixtures was investigated. Methanol, acetone, dimethyl sulfoxide and formamide were used as organic solvents. As the methanol and acetone concentration is increased the emission maximum peak is decrease. In contrast, with dimethyl sulfoxide and formamide addition induced a increasing of the emission maximum intensity. The values of wavelength maximum (λ max) at the addition of this solvent can shows the spectra shifted to the red by about 12 nm. These increasing in the fluorescence intensity and in the λ max may be due to luciferase denaturation, resulting from the more intensive contact of chromospheres of luciferase with the solvent. At all used concentrations of methanol, acetone and formamide the shape of the fluorescence spectra was not changed. These studies demonstrate that the luciferase tryptophan fluorescence is sensitive to changes of physical-chemical property of enzyme environment. A comparison of activation/inactivation and fluorescence spectra of luciferase in methanol or acetone solutions shows that the extent of inactivation is larger than the extent of fluorescence changes at the same methanol or acetone concentration.

  9. Structure, Mechanism, and Mutation of Bacterial Luciferase.

    PubMed

    Tinikul, Ruchanok; Chaiyen, Pimchai

    2016-01-01

    : Bacterial luciferase is a flavin-dependent monooxygenase found in bioluminescent bacteria. The enzyme catalyzes a light-emitting reaction by using reduced flavin, long chain aldehyde, and oxygen as substrates and yields oxidized flavin, carboxylic acid, and water as products with concomitant emission of blue-green light around 485-490 nm. The enzyme is a heterodimer consisting of two homologous subunits, designated as the α- and β-subunits. The reactive reaction center is located in the α-subunit, whereas the β-subunit is required for maintaining the active conformation of the α-subunit. The enzyme reaction occurs through the generation of a reactive C4a-oxygenflavin adduct, presumably C4a-peroxyflavin, before the light-emitting species is generated from the decomposition of an adduct between the C4a-peroxyflavin and the aldehyde. Because the luciferase reaction generates light, the enzyme has the potential to be used as a bioreporter for a wide variety of applications. With the recent invention of the fusion enzyme that can be expressed in mammalian cells, future possibilities for the development of additional bioreporter applications are promising.

  10. Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift.

    PubMed

    Kheirabadi, Mitra; Sharafian, Zohreh; Naderi-Manesh, Hossein; Heineman, Udo; Gohlke, Ulrich; Hosseinkhani, Saman

    2013-12-01

    Firefly bioluminescence reaction in the presence of Mg(2+), ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350-359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7Å and 2.2Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351-359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission.

  11. Identification of a functional luciferase gene in the non-luminous diurnal firefly, Lucidina biplagiata.

    PubMed

    Oba, Y; Furuhashi, M; Inouye, S

    2010-12-01

    We isolated a luciferase gene (LbLuc) from the non-luminous diurnal firefly, Lucidina biplagiata, with high similarity to that from the nocturnal firefly, Photinus pyralis. The recombinant LbLuc showed luminescence activity comparable to that of the luciferases from P. pyralis and Luciola cruciata. To understand the non-luminosity of L. biplagiata, we determined the amount of luciferase in the adult specimen using the luciferin-luciferase reaction and found that the content of luciferase in L. biplagiata was estimated to be only 0.1% of that in L. cruciata. As previously reported, the content of luciferin in L. biplagiata was less than 0.1% of that in L. cruciata. Thus, the non-luminosity of L. biplagiata might be explained by low levels of both luciferase and luciferin.

  12. Complementation of subunits from different bacterial luciferases. Evidence for the role of the. beta. subunit in the bioluminescent mechanism

    SciTech Connect

    Meighen, E.A.; Bartlet, I.

    1980-12-10

    Complementation of the nonidentical subunits (..cap alpha.. and ..beta..) of luciferases isolated from two different bioluminescent strains, Beneckea harveyi and Photobacterium phosphoreum, has resulted in the formation of a functional hybrid luciferase (..cap alpha../sub h/..beta../sub p/) containing the ..cap alpha.. subunit from B. harveyi luciferase (..cap alpha../sub h/) and the ..beta.. subunit from P. phosphoreum luciferase (..beta../sub p/). The complementation was unidirectional; activity could not be restored by complementing the ..cap alpha.. subunit of P. phosphoreum luciferase with the ..beta.. subunit of B. harveyi luciferase, showing that the subunits from these luciferases were not identical. Kinetic parameters of the hybrid luciferase reflecting the intermediate and later steps of the bioluminescent reaction as well as the overall activity and specificity were essentially identical to the same kinetic parameters for B. harveyi luciferase, the source of the ..cap alpha.. subunit, and quite distinct from those of P. phosphoreum luciferase. However, kinetic parameters that reflected the initial step in the reaction involving interaction of FMNH/sub 2/ and luciferase were altered in the hybrid luciferase compared to both the parental luciferases, the K/sub d/ for FMNH/sub 2/ actually being closer to that observed for the P. phosphoreum luciferase (the source of the ..beta.. subunit). These results provide direct evidence that modification or alteration of the ..beta.. subunit in a dimeric luciferase molecule can affect the kinetic properties and indicates that the ..beta.. subunit plays a functional role in the bioluminescent mechanism. It is proposed that both the ..cap alpha.. and ..beta.. subunits are involved with the initial interaction with FMNH/sub 2/, whereas subsequent steps in the mechanism are dictated exclusively by the ..cap alpha.. subunit and are unaffected by alterations in the ..beta.. subunit.

  13. Luciferase protection against proteolytic degradation: a key for improving signal in nano-system biology.

    PubMed

    Ataei, Farangis; Hosseinkhani, Saman; Khajeh, Khosro

    2009-10-26

    Luciferase is most widely used bioluminescence protein in biotechnological processes, but the enzyme is susceptible to proteolytic degradation, thereby its intracellular half-life decreased. Osmolytes are known to enhance the stability of proteins and protect them in a native folded and functional state. The effects of osmolytes, including sucrose, glycine and DMSO on the stability of luciferase were investigated. To different extents, all osmolytes protected the luciferase towards proteolytic degradation in a concentration-dependent manner. The results showed that 1.5M sucrose, 1.5M glycine and 15% DMSO are the best. The ability of these osmolytes to protect luciferase against proteolysis decreased from sucrose, glycine, and finally DMSO. Enzymatic kinetic data showed that the luciferase activity is significantly kept in the presence of sucrose and glycine compared to DMSO, particularly at high temperatures. Bioluminescence intensity, circular dichroism (CD), intrinsic and ANS fluorescence experiments showed change in secondary and tertiary luciferase structure. These results suggest that osmolytes exert an important effect on stabilization of luciferase conformation; decreasing the unfolding rate, preventing adaptation and binding of luciferase at the active site of proteases, thereby the proteolytic digestion reduced and its active conformation was kept.

  14. Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells.

    PubMed

    Stepanyuk, Galina A; Xu, Hao; Wu, Chia-Kuei; Markova, Svetlana V; Lee, John; Vysotski, Eugene S; Wang, Bi-Cheng

    2008-10-01

    Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence (lambda(max)=480 nm). This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, Escherichia coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2-3 mg/L. This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence, indicates that luciferase does not undergo post-translational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis.

  15. Mass culture of photobacteria to obtain luciferase

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Rich, E., Jr.

    1969-01-01

    Inoculating preheated trays containing nutrient agar with photobacteria provides a means for mass culture of aerobic microorganisms in order to obtain large quantities of luciferase. To determine optimum harvest time, growth can be monitored by automated light-detection instrumentation.

  16. Creation of High Efficient Firefly Luciferase

    NASA Astrophysics Data System (ADS)

    Nakatsu, Toru

    Firefly emits visible yellow-green light. The bioluminescence reaction is carried out by the enzyme luciferase. The bioluminescence of luciferase is widely used as an excellent tool for monitoring gene expression, the measurement of the amount of ATP and in vivo imaging. Recently a study of the cancer metastasis is carried out by in vivo luminescence imaging system, because luminescence imaging is less toxic and more useful for long-term assay than fluorescence imaging by GFP. However the luminescence is much dimmer than fluorescence. Then bioluminescence imaging in living organisms demands the high efficient luciferase which emits near infrared lights or enhances the emission intensity. Here I introduce an idea for creating the high efficient luciferase based on the crystal structure.

  17. Expression and stabilization of bacterial luciferase in mammalian cells

    NASA Astrophysics Data System (ADS)

    Patterson, Stacey S.; Dionisi, Hebe M.; Gupta, Rakesh K.; Sayler, Gary S.

    2004-06-01

    Current mammalian bioreporters using either firefly luciferase (luc) or GFP constructs require lysis and/or exogenous excitation to evoke a measurable response. Consequently, these cells cannot serve as continuous, on-line monitoring devices for in vivo imaging. Bacterial luciferase, lux, produces a photonic reaction that is cyclic, resulting in autonomous signal generation without the requirement for exogenous substrates or external activation. Therefore, lux-based bioluminescent bioreporters are the only truly autonomous light-generating sensors in existence. Unfortunately, the bacterial lux system has not yet been efficiently expressed in mammalian cells. In this research, three approaches for optimal expression of the a and b subunits of the bacterial luciferase protein were compared and reporter signal stability was evaluated from stably transfected human embryonic kidney cells. Maximum light levels were obtained from cells expressing the luciferase subunits linked with an internal ribosomal entry site (IRES). Cells harboring this construct produced bioluminescence equaling 2.6 X 106 photons/sec compared to 7.2 X 104 photons/sec obtained from cells expressing the luciferase from a dual promoter vector and 3.5 X 104 photons/sec from a Lux fusion protein. Furthermore, the bioluminescence levels remained stable for more than forty cell passages (5 months) in the absence of antibiotic selection. After this time, bioluminescence signals dropped at a rate of approximately 5% per cell passage. These data indicate that mammalian cell lines can be engineered to efficiently express the bacterial lux system, thus lending themselves to possible long-term continuous monitoring or imaging applications in vivo.

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

    PubMed

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

    2013-11-01

    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.

  19. Split luciferase-based biosensors for characterizing EED binders.

    PubMed

    Li, Ling; Feng, Lijian; Shi, Minlong; Zeng, Jue; Chen, Zijun; Zhong, Li; Huang, Li; Guo, Weihui; Huang, Ying; Qi, Wei; Lu, Chris; Li, En; Zhao, Kehao; Gu, Justin

    2017-04-01

    The EED (embryonic ectoderm development) subunit of the Polycomb repressive complex 2 (PRC2) plays an important role in the feed forward regulation of the PRC2 enzymatic activity. We recently identified a new class of allosteric PRC2 inhibitors that bind to the H3K27me3 pocket of EED. Multiple assays were developed and used to identify and characterize this type of PRC2 inhibitors. One of them is a genetically encoded EED biosensor based on the EED[G255D] mutant and the split firefly luciferase. This EED biosensor can detect the compound binding in the transfected cells and in the in vitro biochemical assays. Compared to other commonly used cellular assays, the EED biosensor assay has the advantage of shorter compound incubation with cells. The in vitro EED biosensor is much more sensitive than other label-free biophysical assays (e.g. DSF, ITC). Based on the crystal structure, the DSF data as well as the biosensor assay data, it's most likely that compound-induced increase in the luciferase activity of the EED[G255D] biosensor results from the decreased non-productive interactions between the EED subdomain and other subdomains within the biosensor construct. This new insight of the mechanism might help to broaden the use of the split luciferase based biosensors.

  20. Firefly luciferase genes from the subfamilies Psilocladinae and Ototretinae (Lampyridae, Coleoptera).

    PubMed

    Oba, Yuichi; Yoshida, Mayumi; Shintani, Takeru; Furuhashi, Mana; Inouye, Satoshi

    2012-02-01

    Firefly luciferase genes have been isolated from approximately 20 species of Lampyrinae, Luciolinae, and Photurinae. These are mostly nocturnal luminescent species that use light signals for sexual communication. In this study, we isolated three cDNAs for firefly luciferase from Psilocladinae (Cyphonocerus ruficollis) and Ototretinae (Drilaster axillaris and Stenocladius azumai), which are diurnal non-luminescent or weakly luminescent species that may use pheromones for communication. The amino acid sequences deduced from the three cDNAs showed 81-89% identities to each other and 60-81% identities with known firefly luciferases. The three purified recombinant proteins showed luminescence and fatty acyl-CoA synthetic activities, as observed in other firefly luciferases. The emission maxima by the three firefly luciferases (λmax, 545-546nm) were shorter than those by known luciferases from the nocturnal fireflies (λmax, 550-568nm). These results suggest that the primary structures and enzymatic properties of luciferases are conserved in Lampyridae, but the luminescence colors were red-shifted in nocturnal species compared to diurnal species.

  1. Firefly Luciferase Enzyme Fragment Complementation for Imaging in Cells and Living Animals

    PubMed Central

    Paulmurugan, Ramasamy; Gambhir, Sanjiv S.

    2014-01-01

    We identified different fragments of the firefly luciferase gene based on the crystal structure of firefly luciferase. These split reporter genes which encode for protein fragments, unlike the fragments currently used for studying protein–protein interactions, can self-complement and provide luciferase enzyme activity in different cell lines in culture and in living mice. The comparison of the fragment complementation associated recovery of firefly luciferase enzyme activity with intact firefly luciferase was estimated for different fragment combinations and ranged from 0.01 to 4% of the full firefly luciferase activity. Using a cooled optical charge-coupled device camera, the analysis of firefly luciferase fragment complementation in transiently transfected subcutaneous 293T cell implants in living mice showed significant detectable enzyme activity upon injecting D-luciferin, especially from the combinations of fragments identified (Nfluc and Cfluc are the N and C fragments of the firefly luciferase gene, respectively): Nfluc (1–475)/Cfluc (245–550), Nfluc (1–475)/Cfluc (265–550), and Nfluc (1–475)/Cfluc (300–550). The Cfluc (265–550) fragment, upon expression with the nuclear localization signal (NLS) peptide of SV40, shows reduced enzyme activity when the cells are cotransfected with the Nfluc (1–475) fragment expressed without NLS. We also proved in this study that the complementing fragments could be efficiently used for screening macromolecule delivery vehicles by delivering TAT–Cfluc (265–550) to cells stably expressing Nfluc (1–475) and recovering signal. These complementing fragments should be useful for many reporter-based assays including intracellular localization of proteins, studying cellular macromolecule delivery vehicles, studying cell–cell fusions, and also developing intracellular phosphorylation sensors based on fragment complementation. PMID:15732910

  2. Monitoring Protein–Protein Interactions Using Split Synthetic Renilla Luciferase Protein-Fragment-Assisted Complementation

    PubMed Central

    Paulmurugan, R.; Gambhir, S. S.

    2014-01-01

    In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein–protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor α through NFκB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein–protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network. PMID:12705589

  3. Suitability of Macrolampis firefly and Pyrearinus click beetle luciferases for bacterial light off toxicity biosensor.

    PubMed

    Gabriel, Gabriele V M; Lopes, P S; Viviani, V R

    2014-01-15

    Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.

  4. Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein.

    PubMed

    Liu, J; Wang, Y; Szalay, A A; Escher, A

    2000-01-01

    We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells.

  5. Making temporal maps using bacterial luciferase: Bacteriophage

    NASA Astrophysics Data System (ADS)

    Kuhn, Jonathan; Broza, Rachel; Verkin, Ekaterina

    2004-06-01

    A method for making temporal maps in bacteria, plasmids and bacteriophages is described. A cassette containing both the genes for bacterial luciferase and kanamycin resistance can be introduced at precise sites. The technique involves clonging followed by genetic recombination. The result is formation of structures that have the luciferase genes in place of the normal DNA and this allows the very precise measurement of transcription/translation of the substituted regions. Very low levels of transcription as well as the kinetics of induction can be easily ascertained. As a specific demonstration of this general method, the technique was used with bacteriophage λ, one of the best known organisms. By measuring light emission, the expression of luciferase was followed after induction for both early and late genes. The exact timing of initial expression of genes was also determined by sampling at very short intervals. The results show that the early genes express almost without delay implying that the function of the N antitermination system is not temporal regulation.

  6. Increase of segmental mobility through insertion of a flexible linker in split point of firefly luciferase.

    PubMed

    Bahmani, Parisa; Hosseinkhani, Saman

    2017-01-01

    The crystal structure of Photinus pyralis luciferase shows a unique molecular architecture consisting of a large N-terminal domain and a small C-terminal domain which is separated by a wide cleft. Protein engineering methods attempts to design the peptide linkers that make a connection between different protein domains or subunits to allow for separating domains and improve kinetics and structural features of proteins. In regard to this; introduction of a flexible linker at split point of luciferase which has a strong self-association activity, may leads to conformational change and improve general flexibility of protein. In this study, two flexible linkers in the split point of luciferase are introduced in order to test the effect of linker on flexibility of luciferase activity. Glycine-rich linkers are introduced into P. pyralis firefly luciferase to make two separate mutant enzymes. Enzymatic properties of mutant and native forms were measured using luminescence assay. Results show that lengthening of luciferase domains through insertion of a flexible linker did not affect bioluminescence emission spectra. Also adding linkers do not have remarkable effect on thermostability. The Km values of mutants were increased compared to native form, indicating lower affinity of mutants toward substrates.

  7. The fusion Vibrio campbellii luciferase as a eukaryotic gene reporter.

    PubMed

    Tinikul, Ruchanok; Thotsaporn, Kittisak; Thaveekarn, Wichit; Jitrapakdee, Sarawut; Chaiyen, Pimchai

    2012-12-31

    Bacterial luciferase from Vibrio campbellii is a thermostable enzyme with an in vitro thermal inactivation half-life of ~1020 min at 37°C. The enzyme also binds tightly to reduced FMN. In this study, a V. campbellii fusion luciferase construct in which the α and β subunits are linked with a decapeptide was made and characterized. In general, the overall enzymatic properties of the two enzymes are similar. Expression of the enzymes in Escherichia coli demonstrated that the V. campbellii fusion luciferase emits less light than the native luciferase, but still emits a much greater amount of light than native luciferase from Vibrio harveyi and Photobacterium leiognathi TH1. The intensity of light emitted by the V. campbellii fusion luciferase was more than 80-fold greater than that from the V. harveyi native luciferase when expressed at 37°C. Biochemical characterization has shown that the V. campbellii fusion luciferase also retains a high binding affinity for reduced flavin mononucleotide and high thermostability. The levels of bioluminescence emitted by the V. campbellii fusion luciferase expressed in HEK293T cells reached ~1×10(6) Relative Light Units/mg total protein. These findings suggest that the V. campbellii fusion luciferase is a promising candidate for further development as a luciferase-based reporter for eukaryotic systems.

  8. Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

    PubMed

    Verhoef, Lisette G G C; Mattioli, Michela; Ricci, Fernanda; Li, Yao-Cheng; Wade, Mark

    2016-02-01

    Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways.

  9. Fully Codon-Optimized luciferase Uncovers Novel Temperature Characteristics of the Neurospora Clock▿ †

    PubMed Central

    Gooch, Van D.; Mehra, Arun; Larrondo, Luis F.; Fox, Julie; Touroutoutoudis, Melissa; Loros, Jennifer J.; Dunlap, Jay C.

    2008-01-01

    We report the complete reconstruction of the firefly luciferase gene, fully codon optimized for expression in Neurospora crassa. This reporter enhances light output by approximately 4 log orders over that with previously available versions, now producing light that is visible to the naked eye and sufficient for monitoring the activities of many poorly expressed genes. Time lapse photography of strains growing in race tubes, in which the frq or eas/ccg-2 promoter is used to drive luciferase, shows the highest levels of luciferase activity near the growth front and newly formed conidial bands. Further, we have established a sorbose medium colony assay that will facilitate luciferase-based screens. The signals from sorbose-grown colonies of strains in which the frq promoter drives luciferase exhibit the properties of circadian rhythms and can be tracked for many days to weeks. This reporter now makes it possible to follow the clock in real time, even in strains or under conditions in which the circadian rhythm in conidial banding is not expressed. This property has been used to discover short, ca. 15-h period rhythms at high temperatures, at which banding becomes difficult to observe in race tubes, and to generate a high-resolution temperature phase-response curve. PMID:17766461

  10. Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain.

    PubMed

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

    2016-04-11

    Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small-molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno-associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.

  11. Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology

    PubMed Central

    Thorne, Natasha; Inglese, James; Auld, Douglas S.

    2010-01-01

    Summary Understanding luciferase enzymology and the structure of compounds that modulate luciferase activity can be used to improve the design of luminescence-based assays. This review provides an overview of these popular reporters with an emphasis on the commonly used firefly luciferase from Photinus pyralis (FLuc). Large-scale chemical profile studies have identified a variety of scaffolds that inhibit FLuc. In some cell-based assays these inhibitors can act in a counter-intuitive way –leading to a gain in luminescent signal. Although formerly attributed to transcriptional activation, intracellular stabilization of FLuc is the primary mechanism underlying this observation. FLuc inhibition/stabilization can be complex, as illustrated by the compound PTC124, which is converted by FLuc in the presence of ATP to a high affinity multi-substrate-adduct inhibitor, PTC124-AMP. The potential influence these findings can have on drug discovery efforts is provided here. PMID:20609414

  12. A Photinus pyralis and Luciola italica chimeric firefly luciferase produces enhanced bioluminescence.

    PubMed

    Branchini, Bruce R; Southworth, Tara L; Fontaine, Danielle M; Davis, Audrey L; Behney, Curran E; Murtiashaw, Martha H

    2014-10-14

    We report the enhanced bioluminescence properties of a chimeric enzyme (PpyLit) that contains the N-domain of recombinant Photinus pyralis luciferase joined to the C-domain of recombinant Luciola italica luciferase. Compared to the P. pyralis enzyme, the novel PpyLit chimera exhibited 1.8-fold enhanced flash-height specific activity, 2.0-fold enhanced integration-based specific activity, 2.9-fold enhanced catalytic efficiency (kcat/Km), and a 1.4-fold greater bioluminescence quantum yield. The results of this study provide an underlying basis of this unusual example of a chimeric enzyme with enhanced catalytic properties that are not simply the sum of the contributions of the two luciferases.

  13. Bioluminescence is produced from a trapped firefly luciferase conformation predicted by the domain alternation mechanism.

    PubMed

    Branchini, Bruce R; Rosenberg, Justin C; Fontaine, Danielle M; Southworth, Tara L; Behney, Curran E; Uzasci, Lerna

    2011-07-27

    According to the domain alternation mechanism and crystal structure evidence, the acyl-CoA synthetases, one of three subgroups of a superfamily of adenylating enzymes, catalyze adenylate- and thioester-forming half-reactions in two different conformations. The enzymes accomplish this by presenting two active sites through an ~140° rotation of the C-domain. The second half-reaction catalyzed by another subgroup, the beetle luciferases, is a mechanistically dissimilar oxidative process that produces bioluminescence. We have demonstrated that a firefly luciferase variant containing cysteine residues at positions 108 and 447 can be intramolecularly cross-linked by 1,2-bis(maleimido)ethane, trapping the enzyme in a C-domain-rotated conformation previously undocumented in the available luciferase crystal structures. The cross-linked luciferase cannot adenylate luciferin but is nearly fully capable of bioluminescence with synthetic luciferyl adenylate because it retains the ability to carry out the oxidative half-reaction. The cross-linked luciferase is apparently trapped in a conformation similar to those adopted by acyl-CoA synthetases as they convert acyl adenylates into the corresponding CoA thioesters.

  14. Fabrication of Adenosine Triphosphate-Molecule Recognition Chip by Means of Bioluminous Enzyme Luciferase

    NASA Astrophysics Data System (ADS)

    Tanii, Takashi; Goto, Tomomi; Iida, Tomoyuki; Koh-Masahara, Meishoku; Ohdomari, Iwao

    2001-10-01

    We have succeeded in detecting the adenosine triphosphate (ATP) concentration in a solution quantitatively using an ATP-molecule recognition chip. The ATP-molecule recognition chip is composed of a silicon photodiode on which bioluminous enzyme luciferase is immobilized. When the chip was immersed in an ATP-containing solution, the luciferase emitted light and the photoinduced current detected by the photodiode was in proportion to the ATP concentration. We found that the photoinduced current fits the Michaelis-Menten plot. These results indicate that the luciferase is successfully immobilized on the silicon chip without losing the bioluminous activity and that the proposed device enables us to detect the ATP concentration in a solution by measuring the photoinduced current.

  15. Luciferase fragment complementation imaging in preclinical cancer studies

    PubMed Central

    Lake, Madryn C.; Aboagye, Eric O.

    2014-01-01

    The luciferase fragment complementation assay (LFCA) enables molecular events to be non-invasively imaged in live cells in vitro and in vivo in a comparatively cheap and safe manner. It is a development of previous enzyme complementation assays in which reporter genes are split into two, individually enzymatically inactive, fragments that are able to complement one another upon interaction. This complementation can be used to externally visualize cellular activities. In recent years, the number of studies which have used LFCAs to probe questions relevant to cancer have increased, and this review summarizes the most significant and interesting of these. In particular, it focuses on work conducted on the epidermal growth factor, nuclear and chemokine receptor families, and intracellular signaling pathways, including IP3, cAMP, Akt, cMyc, NRF2 and Rho GTPases. LFCAs which have been developed to image DNA methylation and detect RNA transcripts are also discussed. PMID:25594026

  16. Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

    NASA Astrophysics Data System (ADS)

    Branchini, Bruce R.; Southworth, Tara L.; Khattak, Neelum F.; Murtiashaw, Martha H.; Fleet, Sarah E.

    2004-06-01

    Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

  17. Genome-Wide RNAi High-Throughput Screen Identifies Proteins Necessary for the AHR-Dependent Induction of CYP1A1 by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

    PubMed Central

    Hankinson, Oliver

    2013-01-01

    The aryl hydrocarbon receptor (AHR) has a plethora of physiological roles, and upon dysregulation, carcinogenesis can occur. One target gene of AHR encodes the xenobiotic and drug-metabolizing enzyme CYP1A1, which is inducible by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the AHR. An siRNA library targeted against over 5600 gene candidates in the druggable genome was used to transfect mouse Hepa-1 cells, which were then treated with TCDD, and subsequently assayed for CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) activity. Following redundant siRNA activity (RSA) statistical analysis, we identified 93 hits that reduced EROD activity with a p value ≤ .005 and substantiated 39 of these as positive hits in a secondary screening using endoribonuclease-prepared siRNAs (esiRNAs). Twelve of the corresponding gene products were subsequently confirmed to be necessary for the induction of CYP1A1 messenger RNA by TCDD. None of the candidates were deficient in aryl hydrocarbon nuclear translocator expression. However 6 gene products including UBE2i, RAB40C, CRYGD, DCTN4, RBM5, and RAD50 are required for the expression of AHR as well as for induction of CYP1A1. We also found 2 gene products, ARMC8 and TCF20, to be required for the induction of CYP1A1, but our data are ambiguous as to whether they are required for the expression of AHR. In contrast, SIN3A, PDC, TMEM5, and CD9 are not required for AHR expression but are required for the induction of CYP1A1, implicating a direct role in Cyp1a1 transcription. Our methods, although applied to Cyp1a1, could be modified for identifying proteins that regulate other inducible genes. PMID:23997114

  18. A transgenic rat with ubiquitous expression of firefly luciferase gene

    NASA Astrophysics Data System (ADS)

    Hakamata, Yoji; Murakami, Takashi; Kobayashi, Eiji

    2006-02-01

    In vivo imaging strategies provide cellular and molecular events in real time that helps us to understand biological processes in living animals. The development of molecular tags such as green fluorescent proteins and luciferase from the firefly Photinus pyralis has lead to a revolution in the visualization of complex biochemical processes. We developed a novel inbred transgenic rat strain containing firefly luciferase based on the transgenic (Tg) technique in rats. This Tg rat expressed the luciferase gene ubiquitously under control of the ROSA26 promoter. Cellular immune responsiveness against the luciferase protein was evaluated using conventional skin grafting and resulted in the long-term acceptance of Tg rat skin on wild-type rats. Strikingly, organ transplant with heart and small bowel demonstrated organ viability and graft survival, suggesting that cells from luciferase-Tg are transplantable to track their fate. Taking advantage of the less immunogenic luciferase, we also tested the role of hepatocyte-infusion in a liver injury model, and bone marrow-derived cells in a skin defect model. Employed in conjunction with modern advances in optical imaging, this luciferase-Tg rat system provides an innovative animal tool and a new means of facilitating biomedical research such as in the case of regeneration medicine.

  19. Luciferase-dependent oxygen consumption by bioluminescent vibrios.

    PubMed Central

    Makemson, J C

    1986-01-01

    Oxygen uptake due to luciferase in two luminous Vibrio species was estimated in vivo by utilizing inhibitors having specificities for luciferase (decanol) and cytochromes (cyanide). Cyanide titration of respiration revealed a component of oxygen uptake less sensitive to cyanide which was completely inhibitable by low concentrations of decanol. From this it was estimated that in vivo luciferase is responsible for less than 12% (Vibrio harveyi) or 20% (Vibrio fischeri) of the total respiration. From these data in vivo bioluminescent quantum yields are estimated to be not lower than 1.7 and 2.6%, respectively. PMID:3944057

  20. Luciferase-dependent oxygen consumption by bioluminescent vibrios

    SciTech Connect

    Makemson, J.C.

    1986-02-01

    Oxygen uptake due to luciferase in two luminous Vibrio species was estimated in vivo by utilizing inhibitors having specificities for luciferase (decanol) and cytochromes (cyanide). Cyanide titration of respiration revealed a component of oxygen uptake less sensitive to cyanide which was completely inhibitable by low concentrations of decanol. From this it was estimated that in vivo luciferase is responsible for less than 12% (Vibrio harveyi) or 20% (Vibrio fischeri) of the total respiration. From these data in vivo bioluminescent quantum yields are estimated to be not lower than 1.7 and 2.6%, respectively.

  1. Visualization of Malaria Parasites in the Skin Using the Luciferase Transgenic Parasite, Plasmodium berghei.

    PubMed

    Matsuoka, Hiroyuki; Tomita, Hiroyuki; Hattori, Ryuta; Arai, Meiji; Hirai, Makoto

    2015-03-01

    We produced a transgenic rodent malaria parasite (Plasmodium berghei) that contained the luciferase gene under a promoter region of elongation factor-1α. These transgenic (TG) parasites expressed luciferase in all stages of their life cycle, as previously reported. However, we were the first to succeed in observing sporozoites as a mass in mouse skin following their deposition by the probing of infective mosquitoes. Our transgenic parasites may have emitted stronger bioluminescence than previous TG parasites. The estimated number of injected sporozoites by mosquitoes was between 34 and 775 (median 80). Since luciferase activity diminished immediately after the death of the parasites, luciferase activity could be an indicator of the existence of live parasites. Our results indicated that sporozoites survived at the probed site for more than 42 hours. We also detected sporozoites in the liver within 15 min of the intravenous injection. Bioluminescence was not observed in the lung, kidney or spleen. We confirmed the observation that the liver was the first organ in which malaria parasites entered and increased in number.

  2. Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis.

    PubMed Central

    Carrière, C; Riska, P F; Zimhony, O; Kriakov, J; Bardarov, S; Burns, J; Chan, J; Jacobs, W R

    1997-01-01

    TM4 is a lytic mycobacteriophage which infects mycobacteria of clinical importance. A luciferase reporter phage, phAE40, has been constructed from TM4 and was previously shown to be useful for the rapid detection and drug susceptibility testing of Mycobacterium tuberculosis. However, the lytic nature of the phage results in a loss of detectable light output and limits the sensitivity of detection. We describe several strategies aimed at improving the luciferase activity generated by TM4 luciferase phages, including (i) varying the position of the luciferase gene in the phage genome, (ii) isolating host-range mutants of the phage, and (iii) introducing temperature-sensitive mutations in the phage such that it will not replicate at the infecting temperature. Several new phages generated by these methods show increased intensity of luciferase production compared to the first-generation reporter phage phAE40, and one phage, phAE88, also demonstrates an enhanced duration of luciferase activity. This has allowed the detection of as few as 120 BCG cells and the determination of drug susceptibilities of M. tuberculosis in as little as 1 day. PMID:9399525

  3. Engineering an Enhanced, Thermostable, Monomeric Bacterial Luciferase Gene As a Reporter in Plant Protoplasts

    PubMed Central

    Song, Yunhong; Wei, Jinsong; Li, Changfu; Wang, Tietao; Wang, Yao; Zhao, Tianyong; Shen, Xihui

    2014-01-01

    The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future. PMID:25271765

  4. [Nucleotide sequence of genes for alpha- and beta-subunits of luciferase from Photobacterium leiognathi].

    PubMed

    Illarionov, B A; Protopopova, M V; Karginov, V A; Mertvetsov, N P; Gitel'zon, I I

    1988-03-01

    Nucleotide sequence of the Photobacterium leiognathi DNA containing genes of alpha and beta subunits of luciferase has been determined. We also deduced amino acid sequence and molecular mass of luciferase and localized luciferase genes in the sequenced DNA fragment.

  5. The aryl hydrocarbon receptor suppresses osteoblast proliferation and differentiation through the activation of the ERK signaling pathway

    SciTech Connect

    Yu, Haitao; Du, Yuxuan; Zhang, Xulong; Sun, Ying; Li, Shentao; Dou, Yunpeng; Li, Zhanguo; Yuan, Huihui; Zhao, Wenming

    2014-11-01

    Ahr activation is known to be associated with synovitis and exacerbated rheumatoid arthritis (RA), but its contributions to bone loss have not been completely elucidated. Osteoblast proliferation and differentiation are abnormal at the erosion site in RA. Here, we reported that the expression of Ahr was increased in the hind paws' bone upon collagen-induced arthritis (CIA) in mice, and the levels of Ahr were negatively correlated with bone mineral density (BMD). In addition, immunofluorescent staining showed that the high expression of Ahr was mainly localized in osteoblasts from the CIA mice compared to normal controls. Moreover, the luciferase intensity of Ahr in the nucleus increased by 12.5% in CIA osteoblasts compared to that in normal controls. In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activation of the Ahr inhibited pre-osteoblast MC3T3-E1 cellular proliferation and differentiation in a dose-dependent manner. Interestingly, the levels of alkaline phosphatase (ALP) mRNA expression in the osteoblasts of CIA mice were reduced compared to normal controls. In contrast, decreased ALP expression by activated Ahr was completely reversed after pretreatment with an Ahr inhibitor (CH-223191) in MC3T3-E1 cell lines and primary osteoblasts on day 5. Our data further showed that activation of Ahr promoted the phosphorylation of ERK after 5 days. Moreover, Ahr-dependent activation of the ERK signaling pathway decreased the levels of proliferation cells and inhibited ALP activity in MC3T3-E1 cells. These results demonstrated that the high expression of Ahr may suppress osteoblast proliferation and differentiation through activation of the ERK signaling pathway, further enabling bone erosion in CIA mice. - Highlights: • The upregulation of Ahr was localized in osteoblasts of CIA mice. • The overexpression of Ahr suppressed osteoblast development. • The Ahr activated ERK signaling pathway to exacerbate bone erosion.

  6. Delicate balance of electrostatic interactions and disulfide bridges in thermostability of firefly luciferase.

    PubMed

    Karimzadeh, Somayeh; Moradi, Maryam; Hosseinkhani, Saman

    2012-12-01

    The wild type Photinus pyralis luciferase does not have any disulfide bridge. Disulfide bridges are determinant in inherent stability of protein at moderate temperatures. Meanwhile, arginin is responsible for thermostability at higher temperatures. In this study, by concomitant introduction of disulfide bridge and a surface arginin in a mutant (A296C-A326C/I232R), the contribution of disulfide bridge introduction and surface hydrophilic residue on activity and global stability of P. pyralis luciferase is investigated. In addition to the mentioned mutant; I232R, A296C-A326C and wild type luciferases are characterized. Though addition of Arg caused stability against proteolysis but in combination with disulfide bridge resulted in decreased thermal stability compared to A296C-A326C mutant. In spite of long distance of two different mutations (A296C-A326C and I232R) from each other in the three-dimensional structure, combination of their effects on the stability of luciferase was not cumulative.

  7. [Effect of glucose on luciferase expression in Photobacterium leiognathi].

    PubMed

    Watanabe, Toshihiko; Suzuki, Takuya; Yoshikawa, Naoki; Ueno, Yukihiro; Mikami, Takeshi; Matsumoto, Tatsuji

    2004-10-01

    Photobacterium leiognathi cultured in marine broth emits a luminescence that is temporarily enhanced and then extinguished by glucose. Glucose reduces the luciferase level and the expression of lux ABG mRNA in P. leiognathi. The amount of ATP in P. leiognathi is temporarily increased and then declines to the normal level. These results indicate that the extinguishing by glucose in P. leiognathi is induced by the interruption of the translation of luciferase.

  8. The influence of the region between residues 220 and 344 and beyond in Phrixotrix railroad worm luciferases green and red bioluminescence.

    PubMed

    Viviani, Vadim R; Joaquim da Silva Neto, Antonio; Ohmiya, Yoshihiro

    2004-02-01

    To find the regions having a major influence on the bioluminescence spectra of railroad worm luciferases, we constructed new chimeric luciferases switching the fragments from residues 1-219 and from 220-545 between Phrixotrix viviani (PxvGR; lambda(max) = 548 nm) green light-emitting luciferase and Phrixothrix hirtus (PxhRE; lambda(max) = 623 nm) red light-emitting luciferases. The emission spectrum (lambda(max) = 571 nm) and K(M) for luciferin in the chimera PxRE220GR (1-219, PxhRE; 220-545, PxvGR) suggested that the region above residue 220 of PxvGR had a major effect on the active site. However, switching the sequence between the residues 220-344 from PxvGR luciferase into PxhRE (PxREGRRE) luciferase resulted in red light emission (lambda(max) = 603 nm), indicating that the region 220-344 by itself does not determine the emission spectrum. Furthermore, the sequence before residue 220 of the green-emitting luciferase is incompatible for light emission with the sequence above residue 220 of PxhRE. These results suggest that the fragments before and after residue 220, which correspond to distinct subdomains, may fold differently in the green- and red-emitting luciferases, affecting the active site conformation.

  9. Alternative luciferase for monitoring bacterial cells under adverse conditions.

    PubMed

    Wiles, Siouxsie; Ferguson, Kathryn; Stefanidou, Martha; Young, Douglas B; Robertson, Brian D

    2005-07-01

    The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.

  10. A novel luciferase knock-in reporter system for studying transcriptional regulation of the human Sox2 gene.

    PubMed

    Xiao, Dan; Zhang, Weifeng; Li, Yan; Liu, Kuan; Zhao, Junli; Sun, Xiaohong; Shan, Linlin; Mao, Qinwen; Xia, Haibin

    2016-02-10

    Sox2 is an important transcriptional factor that has multiple functions in stem cell maintenance and tumorigenesis. To investigate the transcriptional regulation of the Sox2 gene, a luciferase knock-in reporter system was established in HEK293 cells by placing the luciferase gene in the genome under the control of the Sox2 gene promoter using a transcription activator-like effector nuclease (TALEN)-mediated genome editing technique. PCR and Southern blot results confirmed the site-specific integration of a single copy of the exogenous luciferase gene into the genome. To prove the reliability and sensitivity of this novel luciferase knock-in system, a CRISPR/Cas transcription activation system for the Sox2 gene was constructed and applied to the knock-in system. The results indicated that luciferase activity was directly correlated with the activity of the Sox2 endogenous promoter. This novel system will be a useful tool to study the transcriptional regulation of Sox2, and has great potential in medical and industrial applications.

  11. Structural basis for the spectral difference in luciferase bioluminescence.

    PubMed

    Nakatsu, Toru; Ichiyama, Susumu; Hiratake, Jun; Saldanha, Adrian; Kobashi, Nobuyuki; Sakata, Kanzo; Kato, Hiroaki

    2006-03-16

    Fireflies communicate with each other by emitting yellow-green to yellow-orange brilliant light. The bioluminescence reaction, which uses luciferin, Mg-ATP and molecular oxygen to yield an electronically excited oxyluciferin species, is carried out by the enzyme luciferase. Visible light is emitted during relaxation of excited oxyluciferin to its ground state. The high quantum yield of the luciferin/luciferase reaction and the change in bioluminescence colour caused by subtle structural differences in luciferase have attracted much research interest. In fact, a single amino acid substitution in luciferase changes the emission colour from yellow-green to red. Although the crystal structure of luciferase from the North American firefly (Photinus pyralis) has been described, the detailed mechanism for the bioluminescence colour change is still unclear. Here we report the crystal structures of wild-type and red mutant (S286N) luciferases from the Japanese Genji-botaru (Luciola cruciata) in complex with a high-energy intermediate analogue, 5'-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine (DLSA). Comparing these structures to those of the wild-type luciferase complexed with AMP plus oxyluciferin (products) reveals a significant conformational change in the wild-type enzyme but not in the red mutant. This conformational change involves movement of the hydrophobic side chain of Ile 288 towards the benzothiazole ring of DLSA. Our results indicate that the degree of molecular rigidity of the excited state of oxyluciferin, which is controlled by a transient movement of Ile 288, determines the colour of bioluminescence during the emission reaction.

  12. Enhanced red-emitting railroad worm luciferase for bioassays and bioimaging.

    PubMed

    Li, Xueyan; Nakajima, Yoshihiro; Niwa, Kazuki; Viviani, Vadim R; Ohmiya, Yoshihiro

    2010-01-01

    A luciferase from the railroad worm (Phrixothrix hirtus) is the only red-emitting bioluminescent enzyme in nature that is advantageous in multicolor luciferase assays and in bioluminescence imaging (BLI). However, it is not used widely in scientific or industrial applications because of its low activity and stability. By using site-directed mutagenesis, we produced red-emitting mutants with higher activity and better stability. Compared with the wild-type (WT), the luminescent activities from extracts of cultured mammalian cells expressing mutant luciferase were 9.8-fold in I212L/N351K, 8.4-fold in I212L, and 7.8-fold in I212L/S463R; and the cell-based activities were 3.6-fold in I212L/N351K and 3.4-fold in N351K. The remaining activities after incubation at 37 degrees C for 10 min were 50.0% for I212L/S463R, 31.8% for I212L, and 23.0% for I212L/N351K, but only 5.2% for WT. To demonstrate an application of I212L/N351K, cell-based BLI was performed, and the luminescence signal was 3.6-fold higher than in WT. These results indicate that the mutants might improve the practicability of this signaling in bioassays and BLI.

  13. [Expression of thermostable recombiant Luciola lateralis luciferase and development of heat-stable pyrosequencing system].

    PubMed

    Xu, Shu; Zou, Bingjie; Wang, Jianping; Wu, Haiping; Zhou, Guohua

    2012-06-01

    Pyrosequencing is a tool based on bioluminescence reaction for real-time analyzing DNA sequences. The sensitivity of pyrosequencing mainly depends on luciferase in reaction mixture. However, the instability of pyrosequencing reagents caused by fragile wild Photinus pyralis luciferase (PpL) in conventional pyrosequencing usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, the coding sequences of mutant thermostable Luciola lateralis luciferase (rt-LlL) was synthesized, and inserted into the plasmid of pET28a(+) to express the thermostable rt-LlL with a 6 x His-tag in the N terminal. The purified rt-LlL with the molecular mass of 60 kDa was obtained by Ni-affinity chromatography. The specific activity of rt-LlL was determined as 4.29 x 10(10) RLU/mg. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50 degrees C, and remained 90% of activity after incubated at 40 degrees C for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent. Comparing with conventional pyrosequencing reagent, the thermostable pyrosequencing reagent is more stable, and it's activity would not lose when incubated at 37 degrees C for 1 h. This study laid foundation of establishing reliable and stable pyrosequencing system which would be applied in Point-of-Care Testing.

  14. An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications.

    PubMed

    Branchini, Bruce R; Southworth, Tara L; Fontaine, Danielle M; Kohrt, Dawn; Talukder, Munya; Michelini, Elisa; Cevenini, Luca; Roda, Aldo; Grossel, Martha J

    2015-09-01

    Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega's luc2 for reporter and imaging applications.

  15. Enhanced Beetle Luciferase for High-Resolution Bioluminescence Imaging

    PubMed Central

    Nakajima, Yoshihiro; Yamazaki, Tomomi; Nishii, Shigeaki; Noguchi, Takako; Hoshino, Hideto; Niwa, Kazuki; Viviani, Vadim R.; Ohmiya, Yoshihiro

    2010-01-01

    We developed an enhanced green-emitting luciferase (ELuc) to be used as a bioluminescence imaging (BLI) probe. ELuc exhibits a light signal in mammalian cells that is over 10-fold stronger than that of the firefly luciferase (FLuc), which is the most widely used luciferase reporter gene. We showed that ELuc produces a strong light signal in primary cells and tissues and that it enables the visualization of gene expression with high temporal resolution at the single-cell level. Moreover, we successfully imaged the nucleocytoplasmic shuttling of importin α by fusing ELuc at the intracellular level. These results demonstrate that the use of ELuc allows a BLI spatiotemporal resolution far greater than that provided by FLuc. PMID:20368807

  16. Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-vargeted luciferase from neuronal cells.

    PubMed

    Pathe-Neuschäfer-Rube, Andrea; Neuschäfer-Rube, Frank; Genz, Lara; Püchel, Gerhard P

    2015-01-01

    Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations.Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by of botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use.

  17. Creation of artificial luciferases to expand their analytical potential.

    PubMed

    Frank, Ludmila A

    2015-01-01

    Bioluminescent proteins have been intensively used as high sensitive reporters in all kinds of binding assays (immuno-, nucleic acid hybridization assays, etc.) and in bioimaging. But natural luciferases do not always meet the requirements set for them as the assay reporters: thermostabitity, definite bioluminescence spectral and kinetics characteristics, stability to chemical modifications, etc. Luciferases with different appropriate characteristics as well as various luciferin derivatives were obtained using mutagenesis and chemical synthesis. Thanks to rigorous efforts of many researchers bioluminescencebased analytical techniques offer a great potential for solving analytical tasks in the field of biotechnology, biomedicine, pharmacology, etc.

  18. Implication of an unfavorable residue (Thr346) in intrinsic flexibility of firefly luciferase.

    PubMed

    Moradi, Maryam; Hosseinkhani, Saman; Emamzadeh, Rahman

    2012-09-10

    In order to better understand the functional role of an unusual residue (Thr346) of firefly luciferase mutagenesis at this residue was performed. Firefly luciferase, catalyzes the bioluminescence reaction and is an excellent tool as a reporter in nano-system biology studies. Nonetheless, the enzyme rapidly loses its activity at temperatures above 30 °C and this leads to reduced sensitivity and precision in analytical applications. Residue Thr346 in a connecting loop (341-348) of firefly luciferase is located in a disallowed region of Ramachandran plot. In this study, we have substituted this residue (T346) with anomalous dihedral angles with Val, Gly and Pro to clarify the role of this residue in structure and function of the enzyme using site-directed mutagenesis. Substitution of this unfavorable residue (T346) with atypical dihedral angles (ψ, φ) with other residues brought about an increase of thermostability and decrease of specific activity. Structural and functional properties of the mutants were analyzed using different spectroscopic methods. It seems that this residue is a critically conserved residue to support the functional flexibility for a fast kinetic bioluminescence reaction at the expense of lower stability.

  19. Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN-oxidoreductase.

    PubMed

    Vetrova, E V; Kudryasheva, N S; Visser, A J W G; van Hoek, A

    2005-01-01

    The bioluminescent bacterial enzyme system NAD(P)H:FMN-oxidoreductase-luciferase has been used as a test system for ecological monitoring. One of the modes to quench bioluminescence is the interaction of xenobiotics with the enzymes, which inhibit their activity. The use of endogenous flavin fluorescence for investigation of the interactions of non-fluorescent compounds with the bacterial luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri has been proposed. Fluorescence spectroscopy methods have been used to study characteristics of endogenous flavin fluorescence (fluorophore lifetime, the rotational correlation time). The fluorescence anisotropy behaviour of FMN has been analysed and compared to that of the enzyme-bound flavin. The fluorescence characteristics of endogenous flavin of luciferase and NAD(P)H:FMN-oxidoreductase have been shown to be applicable in studying enzymes' interactions with non-fluorescent compounds.

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

    EPA Science Inventory

    Determination of dioxin-like activity requires knowledge of both the concentration and toxicity to evaluate the risk of adverse human health and environmental effects. The dioxin-like response of several polybrominated dibenzo-p-dioxins/furans (PBDDs/Fs) and polybrominated/chlori...

  1. Development of red-shifted mutants derived from luciferase of Brazilian click beetle Pyrearinus termitilluminans

    NASA Astrophysics Data System (ADS)

    Nishiguchi, Tomoki; Yamada, Toshimichi; Nasu, Yusuke; Ito, Mashiho; Yoshimura, Hideaki; Ozawa, Takeaki

    2015-10-01

    Luciferase, a bioluminescent protein, has been used as an analytical tool to visualize intracellular phenomena. Luciferase with red light emission is particularly useful for bioluminescence imaging because of its high transmittance in mammalian tissues. However, the luminescence intensity of existing luciferases with their emission over 600 nm is insufficient for imaging studies because of their weak intensities. We developed mutants of Emerald luciferase (Eluc) from Brazilian click beetle (Pyrearinus termitilluminans), which emits the strongest bioluminescence among beetle luciferases. We successively introduced four amino acid mutations into the luciferase based on a predicted structure of Eluc using homology modeling. Results showed that quadruple mutations R214K/H241K/S246H/H347A into the beetle luciferase emit luminescence with emission maximum at 626 nm, 88-nm red-shift from the wild-type luciferase. This mutant luciferase is anticipated for application in in vivo multicolor imaging in living samples.

  2. Inhibition of luciferase expression by synthetic hammerhead ribozymes and their cellular uptake.

    PubMed Central

    Bramlage, B; Alefelder, S; Marschall, P; Eckstein, F

    1999-01-01

    Two synthetic hammerhead ribozymes, one unmodified and the other with 2"-modifications and four phosphorothioate groups, targeting a single GUA site in the luciferase mRNA, were compared for their inhibition of gene expression in cell cultureand their cellular uptake was also analysed. A HeLa X1/5 cell line stably expressing luciferase, under an inducible promoter, was treated with these ribozymes by liposome-mediated transfection to determine their activity.Luciferase expression in cells was inhibited to approximately 50% with little difference between the unmodified and the 2"-modified ribozyme. A similar degree of inhibition was observed with two catalytically inactive ribozymes, indicating that inhibition was mainly due to an antisense effect. A ribozyme carrying a cholesterol moiety, applied to the cells without carrier, showed no inhibition. Northern blotting indicated a similar amount of cellular uptake of all ribozymes. The unmodified ribozyme was essentially evenly distributed between cytoplasm and nucleus, whereas a higher proportion of the phosphorothioate-containing ribozyme was observed in the nucleus. Fluorescence microscopy, including confocal microscopy using 5"-fluorescein-labelled ribozymes, showed that the unmodified and 2"-modified ribozymes were present in the cytoplasm and in the nucleus to a similar extent, whereas the fluorescence of the phosphorothioate-containing ribozyme was much stronger in the nucleus. Both ribozymes inhibited luciferase expression to a comparable degree, suggesting that the ribozyme in the nucleus did not contribute significantly to the inhibition. Ribozymes with a cholesterol moiety were predominantly trapped in the cell membrane, explaining their inability to interfere with gene expression. PMID:10454613

  3. The role of proline substitutions within flexible regions on thermostability of luciferase.

    PubMed

    Yu, Haoran; Zhao, Yang; Guo, Chao; Gan, Yiru; Huang, He

    2015-01-01

    Improving the stability of firefly luciferase has been a critical issue for its wider industrial applications. Studies about hyperthermophile proteins show that flexibility could be an effective indicator to find out weak spots to engineering thermostability of proteins. However, the relationship among flexibility, activity and stability in most of proteins is unclear. Proline is the most rigid residue and can be introduced to rigidify flexible regions to enhance thermostability of proteins. We firstly apply three different methods, molecular dynamics (MD) simulation, B-FITTER and framework rigidity optimized dynamics algorithm (FRODA) to determine the flexible regions of Photinus pyralis luciferase: Fragment 197-207; Fragment 471-481 and Fragment 487-495. Then, introduction of proline is used to rigidify these flexible regions. Two mutants D476P and H489P within most flexible regions are finally designed. In the results, H489P mutant shows improved thermostability while maintaining its catalytic efficiency compared to that of wild type luciferase. Flexibility analysis confirms that the overall rigidity and local rigidity of H489P mutant are greatly strengthened. D476P mutant shows decreased thermosatbility and the reason for this is elucidated at the molecular level. S307P mutation is randomly chosen outside the flexible regions as a control. Thermostability analysis shows that S307P mutation has decreased kinetic stability and enhanced thermodynamic stability.

  4. Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors.

    PubMed

    Walker, Joel R; Hall, Mary P; Zimprich, Chad A; Robers, Matthew B; Duellman, Sarah J; Machleidt, Thomas; Rodriguez, Jacquelynn; Zhou, Wenhui

    2017-02-22

    Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42 000 PubChem compound library with a low micromolar IC50 against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC50 shifts calculated from both live and lysed cells expressing Nluc cytosolically. Luminescence imaging further confirmed that cell permeable compounds inhibit both intracellular and extracellular Nluc, whereas less permeable compounds differentially inhibit extracellular Nluc and Nluc on the cell surface. The compounds displayed little to no toxicity to cells and high luciferase specificity, showing no activity against firefly luciferase or even the closely related NanoBit system. Looking forward, the structural motifs used to gain access to the Nluc surface can also be appended with other functional groups, and therefore interesting opportunities for developing assays based on relief-of-inhibition can be envisioned.

  5. Visualization of Oxidative Stress Induced by Experimental Periodontitis in Keap1-Dependent Oxidative Stress Detector-Luciferase Mice

    PubMed Central

    Kataoka, Kota; Ekuni, Daisuke; Tomofuji, Takaaki; Irie, Koichiro; Kunitomo, Muneyoshi; Uchida, Yoko; Fukuhara, Daiki; Morita, Manabu

    2016-01-01

    The aim of this study was to investigate whether a Keap1-dependent oxidative stress detector-luciferase (OKD-LUC) mouse model would be useful for the visualization of oxidative stress induced by experimental periodontitis. A ligature was placed around the mandibular first molars for seven days to induce periodontitis. Luciferase activity was measured with an intraperitoneal injection of d-luciferin on days 0, 1, and 7. The luciferase activity in the periodontitis group was significantly greater than that in the control group at seven days. The expressions of heme oxygenase-1 (HO-1) and malondialdehyde in periodontal tissue were significantly higher in the periodontitis group than in the control group. Immunofluorescent analysis confirmed that the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) occurred more frequently in the periodontitis group than in the control group. This study found that under oxidative stress induced by experimental periodontitis, the Nrf2/antioxidant defense pathway was activated and could be visualized from the luciferase activity in the OKD-LUC model. Thus, the OKD-LUC mouse model may be useful for exploring the mechanism underlying the relationship between the Nrf2/antioxidant defense pathway and periodontitis by enabling the visualization of oxidative stress over time. PMID:27854327

  6. Dual-color bioluminescence imaging assay using green- and red-emitting beetle luciferases at subcellular resolution.

    PubMed

    Yasunaga, Mayu; Nakajima, Yoshihiro; Ohmiya, Yoshihiro

    2014-09-01

    Bioluminescence imaging is widely used to monitor cellular events, including gene expression in vivo and in vitro. Moreover, recent advances in luciferase technology have made possible imaging at the single-cell level. To improve the bioluminescence imaging system, we have developed a dual-color imaging system in which the green-emitting luciferase from a Brazilian click beetle (Emerald Luc, ELuc) and the red-emitting luciferase from a railroad worm (Stable Luciferase Red, SLR) were used as reporters, which were localized to the peroxisome and the nucleus, respectively. We clearly captured simultaneously the subcellular localization of ELuc in the peroxisome and SLR in the nucleus of a single cell using a high-magnification objective lens with 3-min exposure time without binning using a combination of optical filters. Furthermore, to apply this system to quantitative time-lapse imaging, the activation of nuclear factor triggered by tumor necrosis factor α was measured using nuclear-targeted SLR and peroxisome-targeted ELuc as the test and internal control reporters, respectively. We successfully quantified the kinetics of activation of nuclear factor κB using nuclear-targeted SLR and the transcriptional change of the internal control promoter using peroxisome-targeted ELuc simultaneously in a single cell, and showed that the activation kinetics, including activation rate and amplitude, differed among cells. The results demonstrated that this imaging system can visualize the subcellular localization of reporters and track the expressions of two genes simultaneously at subcellular resolution.

  7. A puzzling homology: a brittle star using a putative cnidarian-type luciferase for bioluminescence.

    PubMed

    Delroisse, Jérôme; Ullrich-Lüter, Esther; Blaue, Stefanie; Ortega-Martinez, Olga; Eeckhaut, Igor; Flammang, Patrick; Mallefet, Jérôme

    2017-04-01

    Bioluminescence relies on the oxidation of a luciferin substrate catalysed by a luciferase enzyme. Luciferins and luciferases are generic terms used to describe a large variety of substrates and enzymes. Whereas luciferins can be shared by phylogenetically distant organisms which feed on organisms producing them, luciferases have been thought to be lineage-specific enzymes. Numerous light emission systems would then have co-emerged independently along the tree of life resulting in a plethora of non-homologous luciferases. Here, we identify for the first time a candidate luciferase of a luminous echinoderm, the ophiuroid Amphiura filiformis Phylogenomic analyses identified the brittle star predicted luciferase as homologous to the luciferase of the sea pansy Renilla (Cnidaria), contradicting with the traditional viewpoint according to which luciferases would generally be of convergent origins. The similarity between the Renilla and Amphiura luciferases allowed us to detect the latter using anti-Renilla luciferase antibodies. Luciferase expression was specifically localized in the spines which were demonstrated to be the bioluminescent organs in vivo However, enzymes homologous to the Renilla luciferase but unable to trigger light emission were also identified in non-luminous echinoderms and metazoans. Our findings strongly indicate that those enzymes, belonging to the haloalkane dehalogenase family, might then have been convergently co-opted into luciferases in cnidarians and echinoderms. In these two benthic suspension-feeding species, similar ecological pressures would constitute strong selective forces for the functional shift of these enzymes and the emergence of bioluminescence.

  8. Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance.

    PubMed

    Law, G H Erica; Gandelman, Olga A; Tisi, Laurence C; Lowe, Christopher R; Murray, James A H

    2006-07-15

    Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow-green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 degrees C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

  9. Laboratory procedures manual for the firefly luciferase assay for adenosine triphosphate (ATP)

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Curtis, C. A.; Knust, E. A.; Nibley, D. A.; Vance, R. B.

    1975-01-01

    A manual on the procedures and instruments developed for the adenosine triphosphate (ATP) luciferase assay is presented. Data cover, laboratory maintenance, maintenance of bacterial cultures, bacteria measurement, reagents, luciferase procedures, and determination of microbal susceptibility to antibiotics.

  10. Optimization of the firefly luciferase reaction for analytical purposes.

    PubMed

    Lundin, Arne

    2014-01-01

    The optimization of assays has two purposes: (1) to increase the sensitivity of the assay so that low levels of the analyte can be determined; and (2) to prevent small changes of the reaction conditions from having a large impact on the outcome of the assay. The two purposes are usually equally important, as has been recognized in well-established branches of analytical chemistry, such as clinical chemistry. The firefly luciferase reaction can be used for many types of assays. The way to optimize these assays is not trivial, as there are many parameters to consider. Furthermore, as there are now several types of recombinant luciferases available, one has to decide which is the most suitable for each individual assay. The optimization is influenced by the conditions and requirements under which the assay is performed. Special attention is given to ways to calibrate assays. Examples on optimization are mainly taken from the author's own work during 40 years using assays based on the firefly luciferase reaction.

  11. Engineering luciferase enzymes and substrates for novel assay capabilities

    NASA Astrophysics Data System (ADS)

    Wood, Keith V.

    2004-06-01

    In the development of HTS as a central paradigm of drug discovery, fluorescent reporter molecules have generally been adopted as the favored signal transducer. Nevertheless, luminescence has maintained a prominent position among certain methodologies, most notably genetic reporters. Recently, there has been growing partiality for luminescent assays across a broader range of applications due to their sensitivity, extensive linearity, and robustness to library compounds and complex biological samples. This trend has been fostered by development several new assay designs for diverse targets such as kinases, cytochrome p450's, proteases, apoptosis, and cytotoxicity. This review addresses recent progress made in the use of bioluminescent assays for drug discovery, highlighting new detection capabilities brought about by engineering luciferase enzymes and substrates. In reporter gene applications, modified luciferases have provided greatly improved expression efficiency in mammalian cells, improved responsiveness to changes of transcriptional rate, and increased the magnitude of the reporter response. Highly stabilized luciferase mutants have enabled new assays strategies for high-throughput screening based on detection of ATP and luciferin. Assays based on ATP support rapid analysis of cell metabolism and enzymatic processes coupled to ATP hydrolysis. Although luciferin is found natively only in luminous beetles, coupled assays have been designed using modified forms of luciferin requiring the action of second enzyme to yield luminescence. Due to the very low inherent background and protection of the photon-emitter afforded by the enzyme, bioluminescent assays often outperform the analogous fluorescent assays for analyses performed in multiwell plates.

  12. Visualization and Quantification of Browning Using a Ucp1-2A-Luciferase Knock-in Mouse Model.

    PubMed

    Mao, Liufeng; Nie, Baoming; Nie, Tao; Hui, Xiaoyan; Gao, Xuefei; Lin, Xiaoliang; Liu, Xin; Xu, Yong; Tang, Xiaofeng; Yuan, Ran; Li, Kuai; Li, Peng; Ding, Ke; Wang, Yu; Xu, Aimin; Fei, Jian; Han, Weiping; Liu, Pentao; Madsen, Lise; Kristiansen, Karsten; Zhou, Zhiguang; Ding, Sheng; Wu, Donghai

    2017-02-01

    Both mammals and adult humans possess classic brown adipocytes and beige adipocytes, and the amount and activity of these adipocytes are considered key factors in combating obesity and its associated metabolic diseases. Uncoupling protein 1 (Ucp1) is the functional marker of both brown and beige adipocytes. To facilitate a reliable, easy, and sensitive measurement of Ucp1 expression both in vivo and in vitro, we generated a Ucp1-2A-luciferase knock-in mouse by deleting the stop codon for the mouse Ucp1 gene and replacing it with a 2A peptide. This peptide was followed by the luciferase coding sequence to recapitulate the expression of the Ucp1 gene at the transcriptional and translational levels. With this mouse, we discovered a cold-sensitive brown/beige adipose depot underneath the skin of the ears, which we named uBAT. Because of the sensitivity and high dynamic range of luciferase activity, the Ucp1-2A-luciferase mouse is useful for both in vitro quantitative determination and in vivo visualization of nonshivering thermogenesis. With the use of this model, we identified and characterized axitinib, an oral small-molecule tyrosine kinase inhibitor, as an effective browning agent.

  13. Firefly Luciferase and Rluc8 Exhibit Differential Sensitivity to Oxidative Stress in Apoptotic Cells

    PubMed Central

    Czupryna, Julie; Tsourkas, Andrew

    2011-01-01

    Over the past decade, firefly Luciferase (fLuc) has been used in a wide range of biological assays, providing insight into gene regulation, protein-protein interactions, cell proliferation, and cell migration. However, it has also been well established that fLuc activity can be highly sensitive to its surrounding environment. In this study, we found that when various cancer cell lines (HeLa, MCF-7, and 293T) stably expressing fLuc were treated with staurosporine (STS), there was a rapid loss in bioluminescence. In contrast, a stable variant of Renilla luciferase (RLuc), RLuc8, exhibited significantly prolonged functionality under the same conditions. To identify the specific underlying mechanism(s) responsible for the disparate sensitivity of RLuc8 and fLuc to cellular stress, we conducted a series of inhibition studies that targeted known intracellular protein degradation/modification pathways associated with cell death. Interestingly, these studies suggested that reactive oxygen species, particularly hydrogen peroxide (H2O2), was responsible for the diminution of fLuc activity. Consistent with these findings, the direct application of H2O2 to HeLa cells also led to a reduction in fLuc bioluminescence, while H2O2 scavengers stabilized fLuc activity. Comparatively, RLuc8 was far less sensitive to ROS. These observations suggest that fLuc activity can be substantially altered in studies where ROS levels become elevated and can potentially lead to ambiguous or misleading findings. PMID:21603648

  14. Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate

    PubMed Central

    2012-01-01

    Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes. PMID:22894855

  15. Detection of allergenic compounds using an IL-4/luciferase/CNS-1 transgenic mice model.

    PubMed

    Bae, Chang Joon; Lee, Jae Won; Bae, Hee Sook; Shim, Sun Bo; Jee, Seung Wan; Lee, Su Hae; Lee, Chang Kyu; Hong, Jin Tae; Hwang, Dae Youn

    2011-04-01

    The interleukin-4 (IL-4) signaling cascade has been identified as a potentially important pathway in the development of allergies. The principal objective of this study was to produce novel transgenic (Tg) mice harboring the luciferase gene under the control of the human IL-4 promoter and the enhancer of IL-4 (CNS-1), in an effort to evaluate three types of allergens including a respiratory sensitizer, vaccine additives, and crude extracts of natural allergens in vivo. A new lineage of Tg mice was generated by the microinjection of pIL-4/Luc/CNS-1 constructs into a fertilized mice egg. The luciferase activity was successfully regulated by the IL-4 promoter in splenocytes cultured from IL-4/Luc/CNS-1 Tg mice. From the first five founder lines, one (#57) evidencing a profound response to ovalbumin was selected for use in evaluating the allergens. Additionally, the lungs, thymus, and lymph nodes of IL-4/Luc/CNS-1 Tg mice evidenced high luciferase activity in response to allergens such as phthalic anhydride (PA), trimellitic anhydride, ovalbumin, gelatin, Dermatophagoides pteronyssinus extracts, and Japanese cedar pollen, whereas key allergy-related indicators including ear thickness, Immunoglobulin E concentration, and the infiltration of inflammatory leukocytes in response to PA were unaltered in the Tg mice relative to the non-Tg mice. Furthermore, the expression levels of endogenous type 2 helper T cells cytokines and proinflammatory cytokines were similarly increased in these organs of IL-4/Luc/CNS-1 Tg mice in response to allergens. These results indicate that IL-4/Luc/CNS-1 Tg mice may be used as an animal model for the evaluation and prediction of the human body response to a variety of allergens originating from the environment and from certain industrial products.

  16. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.

    PubMed

    Hall, Mary P; Unch, James; Binkowski, Brock F; Valley, Michael P; Butler, Braeden L; Wood, Monika G; Otto, Paul; Zimmerman, Kristopher; Vidugiris, Gediminas; Machleidt, Thomas; Robers, Matthew B; Benink, Hélène A; Eggers, Christopher T; Slater, Michael R; Meisenheimer, Poncho L; Klaubert, Dieter H; Fan, Frank; Encell, Lance P; Wood, Keith V

    2012-11-16

    Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

  17. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

    PubMed Central

    Omokoko, Tana A.; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard 51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the 51Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  18. Trigger factor assists the refolding of heterodimeric but not monomeric luciferases.

    PubMed

    Melkina, O E; Goryanin, I I; Manukhov, I V; Baranova, A V; Kolb, V A; Svetlov, M S; Zavilgelsky, G B

    2014-01-01

    The refolding of thermally inactivated protein by ATP-independent trigger factor (TF) and ATP-dependent DnaKJE chaperones was comparatively analyzed. Heterodimeric (αβ) bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi, and Vibrio harveyi as well as monomeric luciferases of Vibrio harveyi and Luciola mingrelica (firefly) were used as substrates. In the presence of TF, thermally inactivated heterodimeric bacterial luciferases refold, while monomeric luciferases do not refold. These observations were made both in vivo (Escherichia coli ΔdnaKJ containing plasmids with tig gene) and in vitro (purified TF). Unlike TF, the DnaKJE chaperone system refolds both monomeric and heterodimeric luciferases with equal efficiency.

  19. Genetic modification in organ transplantation and in vivo luciferase imaging

    NASA Astrophysics Data System (ADS)

    Murakami, Takashi; Inoue, Sei-ichiro; Sato, Yuki; Ajiki, Takashi; Ohsawa, Ichiro; Kobayashi, Eiji

    2005-04-01

    The genetic modification for organ transplantation is one of the most promising strategies to regulate allogeneic immune response. Organ-selective gene transfer has especially benefit to control local immune responses. Based on the catheter technique, we tested to deliver naked plasmid DNA to target graft organs of rats (liver and limbs) by a rapid injection (hydrodynamics-based transfection). Recent advances in transplantation have been achieved by visualization of cellular process and delivered gene expression during the inflammatory process by using non-invasive in vivo imaging. Herein, we examined the fate of genetically modified grafts using a firefly luciferase expression plasmid. For liver modification before transplantation, 6.25% of body weight PBS containing plasmid DNA was injected into the liver through the inferior vena cava using a catheter, and the liver was subsequently transplanted to the recipient rat. For limb modification, the femoral caudal epigastric vein was used. In the rat liver transplantation model, substantial luciferase expression was visualized and sustained for only a few days in the grafted liver. We also addressed stress responses by this hydrodynamics procedure using reporter plasmids containing cis-acting enhancer binding site such as NF-kappa B, cAMP, or heat shock response element. In contrast to hepatic transduction, this genetic limb targeting achieved long lasting luciferase expression in the muscle for 2 months or more. Thus, our results suggest that this catheter-based in vivo transfection technique provides an effective strategy for organ-selective gene modification in transplantation, and the bioluminescent imaging is broadening its potential for evaluation to various preclinical studies.

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

    PubMed Central

    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

  1. Rapid and scalable assembly of firefly luciferase substrates†

    PubMed Central

    McCutcheon, David C.; Porterfield, William B.; Prescher, Jennifer A.

    2015-01-01

    Bioluminescence imaging with luciferase-luciferin pairs is a popular method for visualizing biological processes in vivo. Unfortunately, most luciferins are difficult to access and remain prohibitively expensive for some imaging applications. Here we report cost-effective and efficient syntheses of D-luciferin and 6′-aminoluciferin, two widely used bioluminescent substrates. Our approach employs inexpensive anilines and Appel's salt to generate the luciferin cores in a single pot. Additionally, the syntheses are scalable and can provide multi-gram quantities of both substrates. The streamlined production and improved accessibility of luciferin reagents will bolster in vivo imaging efforts. PMID:25525906

  2. Application of luciferase assay for ATP to antimicrobial drug susceptibility

    NASA Technical Reports Server (NTRS)

    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.

  3. In Vivo Imaging of Human MDR1 Transcription in the Brain and Spine of MDR1-Luciferase Reporter Mice.

    PubMed

    Yasuda, Kazuto; Cline, Cynthia; Lin, Yvonne S; Scheib, Rachel; Ganguly, Samit; Thirumaran, Ranjit K; Chaudhry, Amarjit; Kim, Richard B; Schuetz, Erin G

    2015-11-01

    P-glycoprotein (Pgp) [the product of the MDR1 (ABCB1) gene] at the blood-brain barrier (BBB) limits central nervous system (CNS) entry of many prescribed drugs, contributing to the poor success rate of CNS drug candidates. Modulating Pgp expression could improve drug delivery into the brain; however, assays to predict regulation of human BBB Pgp are lacking. We developed a transgenic mouse model to monitor human MDR1 transcription in the brain and spinal cord in vivo. A reporter construct consisting of ∼10 kb of the human MDR1 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line (MDR1-luc). Fluorescence in situ hybridization localized the MDR1-luciferase transgene on chromosome 3. Reporter gene expression was monitored with an in vivo imaging system following D-luciferin injection. Basal expression was detectable in the brain, and treatment with activators of the constitutive androstane, pregnane X, and glucocorticoid receptors induced brain and spinal MDR1-luc transcription. Since D-luciferin is a substrate of ABCG2, the feasibility of improving D-luciferin brain accumulation (and luciferase signal) was tested by coadministering the dual ABCB1/ABCG2 inhibitor elacridar. The brain and spine MDR1-luc signal intensity was increased by elacridar treatment, suggesting enhanced D-luciferin brain bioavailability. There was regional heterogeneity in MDR1 transcription (cortex > cerebellum) that coincided with higher mouse Pgp protein expression. We confirmed luciferase expression in brain vessel endothelial cells by ex vivo analysis of tissue luciferase protein expression. We conclude that the MDR1-luc mouse provides a unique in vivo system to visualize MDR1 CNS expression and regulation.

  4. In Vivo Imaging of Human MDR1 Transcription in the Brain and Spine of MDR1-Luciferase Reporter Mice

    PubMed Central

    Yasuda, Kazuto; Cline, Cynthia; Lin, Yvonne S.; Scheib, Rachel; Ganguly, Samit; Thirumaran, Ranjit K.; Chaudhry, Amarjit; Kim, Richard B.

    2015-01-01

    P-glycoprotein (Pgp) [the product of the MDR1 (ABCB1) gene] at the blood-brain barrier (BBB) limits central nervous system (CNS) entry of many prescribed drugs, contributing to the poor success rate of CNS drug candidates. Modulating Pgp expression could improve drug delivery into the brain; however, assays to predict regulation of human BBB Pgp are lacking. We developed a transgenic mouse model to monitor human MDR1 transcription in the brain and spinal cord in vivo. A reporter construct consisting of ∼10 kb of the human MDR1 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line (MDR1-luc). Fluorescence in situ hybridization localized the MDR1-luciferase transgene on chromosome 3. Reporter gene expression was monitored with an in vivo imaging system following D-luciferin injection. Basal expression was detectable in the brain, and treatment with activators of the constitutive androstane, pregnane X, and glucocorticoid receptors induced brain and spinal MDR1-luc transcription. Since D-luciferin is a substrate of ABCG2, the feasibility of improving D-luciferin brain accumulation (and luciferase signal) was tested by coadministering the dual ABCB1/ABCG2 inhibitor elacridar. The brain and spine MDR1-luc signal intensity was increased by elacridar treatment, suggesting enhanced D-luciferin brain bioavailability. There was regional heterogeneity in MDR1 transcription (cortex > cerebellum) that coincided with higher mouse Pgp protein expression. We confirmed luciferase expression in brain vessel endothelial cells by ex vivo analysis of tissue luciferase protein expression. We conclude that the MDR1-luc mouse provides a unique in vivo system to visualize MDR1 CNS expression and regulation. PMID:26281846

  5. Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo.

    PubMed

    Guse, K; Dias, J D; Bauerschmitz, G J; Hakkarainen, T; Aavik, E; Ranki, T; Pisto, T; Särkioja, M; Desmond, R A; Kanerva, A; Hemminki, A

    2007-06-01

    Oncolytic viruses kill cancer cells by tumor-selective replication. Clinical data have established the safety of the approach but also the need of improvements in potency. Efficacy of oncolysis is linked to effective infection of target cells and subsequent productive replication. Other variables include intratumoral barriers, access to target cells, uptake by non-target organs and immune response. Each of these aspects relates to the location and degree of virus replication. Unfortunately, detection of in vivo replication has been difficult, labor intensive and costly and therefore not much studied. We hypothesized that by coinfection of a luciferase expressing E1-deleted virus with an oncolytic virus, both viruses would replicate when present in the same cell. Photon emission due to conversion of D-Luciferin is sensitive and penetrates tissues well. Importantly, killing of animals is not required and each animal can be imaged repeatedly. Two different murine xenograft models were used and intratumoral coinjections of luciferase encoding virus were performed with eight different oncolytic adenoviruses. In both models, we found significant correlation between photon emission and infectious virus production. This suggests that the system can be used for non-invasive quantitation of the amplitude, persistence and dynamics of oncolytic virus replication in vivo, which could be helpful for the development of more effective and safe agents.

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

    SciTech Connect

    Shi, Junwei; Zhang, Huan; Fang, Liurong; Xi, Yongqiang; Zhou, Yanrong; Luo, Rui; Wang, Dang 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.

  7. Monitoring the dynamics of clonal tumour evolution in vivo using secreted luciferases

    PubMed Central

    Charles, Joël P.; Fuchs, Jeannette; Hefter, Mirjam; Vischedyk, Jonas B.; Kleint, Maximilian; Vogiatzi, Fotini; Schäfer, Jonas A.; Nist, Andrea; Timofeev, Oleg; Wanzel, Michael; Stiewe, Thorsten

    2014-01-01

    Tumours are heterogeneous cell populations that undergo clonal evolution during tumour progression, metastasis and response to therapy. Short hairpin RNAs (shRNAs) generate stable loss-of-function phenotypes and are versatile experimental tools to explore the contribution of individual genetic alterations to clonal evolution. In these experiments tumour cells carrying shRNAs are commonly tracked with fluorescent reporters. While this works well for cell culture studies and leukaemia mouse models, fluorescent reporters are poorly suited for animals with solid tumours—the most common tumour types in cancer patients. Here we develop a toolkit that uses secreted luciferases to track the fate of two different shRNA-expressing tumour cell clones competitively, both in vitro and in vivo. We demonstrate that secreted luciferase activities can be measured robustly in the blood stream of tumour-bearing mice to accurately quantify, in a minimally invasive manner, the dynamic evolution of two genetically distinct tumour subclones in preclinical mouse models of tumour development, metastasis and therapy. PMID:24889111

  8. Monitoring the dynamics of clonal tumour evolution in vivo using secreted luciferases.

    PubMed

    Charles, Joël P; Fuchs, Jeannette; Hefter, Mirjam; Vischedyk, Jonas B; Kleint, Maximilian; Vogiatzi, Fotini; Schäfer, Jonas A; Nist, Andrea; Timofeev, Oleg; Wanzel, Michael; Stiewe, Thorsten

    2014-06-03

    Tumours are heterogeneous cell populations that undergo clonal evolution during tumour progression, metastasis and response to therapy. Short hairpin RNAs (shRNAs) generate stable loss-of-function phenotypes and are versatile experimental tools to explore the contribution of individual genetic alterations to clonal evolution. In these experiments tumour cells carrying shRNAs are commonly tracked with fluorescent reporters. While this works well for cell culture studies and leukaemia mouse models, fluorescent reporters are poorly suited for animals with solid tumours--the most common tumour types in cancer patients. Here we develop a toolkit that uses secreted luciferases to track the fate of two different shRNA-expressing tumour cell clones competitively, both in vitro and in vivo. We demonstrate that secreted luciferase activities can be measured robustly in the blood stream of tumour-bearing mice to accurately quantify, in a minimally invasive manner, the dynamic evolution of two genetically distinct tumour subclones in preclinical mouse models of tumour development, metastasis and therapy.

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

    PubMed Central

    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

  10. Transgenic mouse model harboring the transcriptional fusion ccl20-luciferase as a novel reporter of pro-inflammatory response.

    PubMed

    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.

  11. In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells

    PubMed Central

    Feeney, Kevin A.; Putker, Marrit; Brancaccio, Marco; O’Neill, John S.

    2016-01-01

    Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type. PMID:28112045

  12. A Panel of Trypanosoma brucei Strains Tagged with Blue and Red-Shifted Luciferases for Bioluminescent Imaging in Murine Infection Models

    PubMed Central

    Van Reet, Nick; Van de Vyver, Hélène; Pyana, Patient Pati; Van der Linden, Anne Marie; Büscher, Philippe

    2014-01-01

    Background Genetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients. Methodology/Principal findings We transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT. Conclusions/Significance We established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better

  13. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    SciTech Connect

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James

    2010-04-07

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 {angstrom} cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the 'off-target' effect of a small molecule is mediated by an MAI mechanism.

  14. High-throughput, luciferase-based reverse genetics systems for identifying inhibitors of Marburg and Ebola viruses.

    PubMed

    Uebelhoer, Luke S; Albariño, César G; McMullan, Laura K; Chakrabarti, Ayan K; Vincent, Joel P; Nichol, Stuart T; Towner, Jonathan S

    2014-06-01

    Marburg virus (MARV) and Ebola virus (EBOV), members of the family Filoviridae, represent a significant challenge to global public health. Currently, no licensed therapies exist to treat filovirus infections, which cause up to 90% mortality in human cases. To facilitate development of antivirals against these viruses, we established two distinct screening platforms based on MARV and EBOV reverse genetics systems that express secreted Gaussia luciferase (gLuc). The first platform is a mini-genome replicon to screen viral replication inhibitors using gLuc quantification in a BSL-2 setting. The second platform is complementary to the first and expresses gLuc as a reporter gene product encoded in recombinant infectious MARV and EBOV, thereby allowing for rapid quantification of viral growth during treatment with antiviral compounds. We characterized these viruses by comparing luciferase activity to virus production, and validated luciferase activity as an authentic real-time measure of viral growth. As proof of concept, we adapt both mini-genome and infectious virus platforms to high-throughput formats, and demonstrate efficacy of several antiviral compounds. We anticipate that both approaches will prove highly useful in the development of anti-filovirus therapies, as well as in basic research on the filovirus life cycle.

  15. SpyTag/SpyCatcher Cyclization Enhances the Thermostability of Firefly Luciferase

    PubMed Central

    Si, Meng; Xu, Qing

    2016-01-01

    SpyTag can spontaneously form a covalent isopeptide bond with its protein partner SpyCatcher. Firefly luciferase from Photinus pyralis was cyclized in vivo by fusing SpyCatcher at the N terminus and SpyTag at the C terminus. Circular LUC was more thermostable and alkali-tolerant than the wild type, without compromising the specific activity. Structural analysis indicated that the cyclized LUC increased the thermodynamic stability of the structure and remained more properly folded at high temperatures when compared with the wild type. We also prepared an N-terminally and C-terminally shortened form of the SpyCatcher protein and cyclization using this truncated form led to even more thermostability than the original form. Our findings suggest that cyclization with SpyTag and SpyCatcher is a promising and effective strategy to enhance thermostability of enzymes. PMID:27658030

  16. Enhanced Gaussia luciferase blood assay for monitoring of in vivo biological processes.

    PubMed

    Bovenberg, M Sarah S; Degeling, M Hannah; Tannous, Bakhos A

    2012-01-17

    Secreted Gaussia luciferase (Gluc) has been shown to be a useful tool for ex vivo monitoring of in vivo biological processes. The Gluc level in the blood was used to detect tumor growth, metastasis and response to therapy, gene transfer, and circulating cells viability, as well as transcription factors activation, complementing in vivo bioluminescence imaging. The sensitivity of the Gluc blood assay is limited due to the absorption of blue light by pigmented molecules such as hemoglobin, resulting in quenching of the signal and therefore lower sensitivity. To overcome this problem, we designed an alternative microtiter well-based binding assay in which Gluc is captured first from blood using a specific antibody followed by the addition of coelenterazine and signal acquisition using a luminometer. This assay showed to be over 1 order of magnitude more sensitive in detecting Gluc in the blood as compared to the direct Gluc blood assay enhancing ex vivo monitoring of biological processes.

  17. Full color modulation of firefly luciferase through engineering with unified Stark effect.

    PubMed

    Cai, Duanjun; Marques, Miguel A L; Nogueira, Fernando

    2013-11-07

    The firefly luciferase has been a unique marking tool used in various bioimaging techniques. Extensive color modulation is strongly required to meet special marking demands; however, intentional and accurate wavelength tuning has yet to be achieved. Here, we demonstrate that the color shift of the firefly chromophore (OxyLH2-1) by internal and external fields can be described as a unified Stark shift. Electrostatic microenvironmental effects on fluorescent spectroscopy are modeled in vacuo through effective electric fields by using time-dependent density functional theory. A complete visible fluorescence spectrum of firefly chromophore is depicted, which enables one to control the emission in a specific color. As an application, the widely observed pH-correlated color shift is proved to be associated with the local Stark field generated by the trace water-ions (vicinal hydronium and hydroxide ions) at active sites close to the OxyLH2-1.

  18. Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

    PubMed

    Poutiainen, Pekka K; Rönkkö, Teemu; Hinkkanen, Ari E; Palvimo, Jorma J; Närvänen, Ale; Turhanen, Petri; Laatikainen, Reino; Weisell, Janne; Pulkkinen, Juha T

    2014-01-15

    In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 μM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

  19. Interaction of Photobacterium leiognathi and Vibrio fischeri Y1 luciferases with fluorescent (antenna) proteins: bioluminescence effects of the aliphatic additive.

    PubMed

    Petushkov, V N; Ketelaars, M; Gibson, B G; Lee, J

    1996-09-17

    The kinetics of the bacterial bioluminescence reaction is altered in the presence of the fluorescent (antenna) proteins, lumazine protein (LumP) from Photobacterium or the yellow fluorescence proteins (YFP) having FMN or Rf bound, from Vibrio fischeri strain Y1. Depending on reaction conditions, the bioluminescence intensity and its decay rate may be either enhanced or strongly quenched in the presence of the fluorescent proteins. These effects can be simply explained on the basis of the same protein-protein complex model that accounts for the bioluminescence spectral shifts induced by these fluorescent proteins. In such a complex, where the fluorophore evidently is in proximity to the luciferase active site, it is expected that the on-off rate of certain aliphatic components of the reaction should be altered with a consequent shift in the equilibria among the luciferase intermediates, as recently elaborated in a kinetic scheme. These aliphatic components are the bioluminescence reaction substrate, tetradecanal or other long-chain aldehyde, its carboxylic acid product, or dodecanol used as a stabilizer of the luciferase peroxyflavin. No evidence can be found for the protein-protein interaction in the absence of the aliphatic component.

  20. Identification, characterization and use of two tick promoters for construction of a dual luciferase reporter vector

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dual luciferase reporter systems are valuable tools for functional genomic studies, but have not previously been developed for use in tick cell culture. We evaluated expression of available luciferase constructs in tick cell cultures derived from Rhipicephalus (Boophilus) microplus, an important vec...

  1. Point mutations in firefly luciferase C-domain demonstrate its significance in green color of bioluminescence.

    PubMed

    Modestova, Yulia; Koksharov, Mikhail I; Ugarova, Natalia N

    2014-09-01

    Firefly luciferase is a two-domain enzyme that catalyzes the bioluminescent reaction of firefly luciferin oxidation. Color of the emitted light depends on the structure of the enzyme, yet the exact color-tuning mechanism remains unknown by now, and the role of the C-domain in it is rarely discussed, because a very few color-shifting mutations in the C-domain were described. Recently we reported a strong red-shifting mutation E457K in the C-domain; the bioluminescence spectra of this enzyme were independent of temperature or pH. In the present study we investigated the role of the residue E457 in the enzyme using the Luciola mingrelica luciferase with a thermostabilized N-domain as a parent enzyme for site-directed mutagenesis. We obtained a set of mutants and studied their catalytic properties, thermal stability and bioluminescence spectra. Experimental spectra were represented as a sum of two components (bioluminescence spectra of putative "red" and "green" emitters); λmax of these components were constant for all the mutants, but the ratio of these emitters was defined by temperature and mutations in the C-domain. We suggest that each emitter is stabilized by a specific conformation of the active site; thus, enzymes with two forms of the active site coexist in the reactive media. The rigid structure of the C-domain is crucial for maintaining the conformation corresponding to the "green" emitter. We presume that the emitters are the keto- and enol forms of oxyluciferin.

  2. Diversity of zebrafish peripheral oscillators revealed by luciferase reporting.

    PubMed

    Kaneko, Maki; Hernandez-Borsetti, Nancy; Cahill, Gregory M

    2006-09-26

    In various multicellular organisms, circadian clocks are present not only in the central nervous system, but also in peripheral organs and tissues. In mammals peripheral oscillators are not directly responsive to light, but are entrained by the central oscillator in the suprachiasmatic nucleus. These individual oscillators are diverse in their free-running periods and phases. In contrast, cultured peripheral tissues and cell lines from zebrafish are not only rhythmic, but can also be directly entrained by light. Because of the convenience of studying rhythms in cultured cells, however, little has been known about properties of individual oscillators in intact zebrafish. Here, we show the remarkable diversity and consistency of oscillator properties in various peripheral organs and tissues from the period3-luciferase (per3-luc) transgenic zebrafish. Tissue-dependent differences were found in free-running period, phase, response to light, and temperature compensation. Furthermore, cycling amplitudes were reduced at lower temperatures in some, but not all, of the organs tested. Finally, we found that per3-luc rhythms can free run in both constant dark and constant light with remarkably similar amplitudes, phases, and periods, despite the fact that the mRNA of per2 and per1 has been shown not to oscillate in constant light.

  3. Influenza A virus encoding secreted Gaussia luciferase as useful tool to analyze viral replication and its inhibition by antiviral compounds and cellular proteins.

    PubMed

    Eckert, Nadine; Wrensch, Florian; Gärtner, Sabine; Palanisamy, Navaneethan; Goedecke, Ulrike; Jäger, Nils; Pöhlmann, Stefan; Winkler, Michael

    2014-01-01

    Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1-3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.

  4. [Photoreactivation of UV-irradiated Escherichia coli K12 AB1886 uvrA6 with assistance of luminescence of Photobacterium leiognathi Luciferase].

    PubMed

    Melkina, O E; Kotova, V Yu; Konopleva, M N; Manukhov, I V; Pustovoit, K S; Zavilgelsky, G B

    2015-01-01

    The bioluminescence induced by luciferases of marine bacteria promotes repair of UV damaged DNA of Escherichia coli AB1886 uvrA6. It is shown that bacterial photolyase that implements photoreactivation activity is the major contributor to DNA repair. However, the intensity of bioluminescence increasing induced by UV-irradiation (SOS-induction) in bacterial cells is not enough for efficient photoreactivation.

  5. Proposed ionic bond between Arg300 and Glu270 and Glu271 are not involved in inactivation of a mutant firefly luciferase (LRR).

    PubMed

    Sobhani-Damavandifar, Zahra; Hosseinkhani, Saman; Sajedi, Reza H

    2016-05-01

    The weakness of firefly luciferase is its rapid inactivation. Many studies have been done to develop thermostable luciferases. One of these modifications was LRR mutant in which the Leu300 was substituted with Arg in the E(354)RR(356)Lampyris turkestanicus luciferase as template. LRR was more thermostable than the wild type but with only 0.02% activity. In this study, site-directed mutagenesis was used to change the proposed ionic bond between the Arg and two neighboring residues (Glu270 and Glu271), to understand if the induced interactions were responsible for inactivation in LRR. Our results showed that substitution of Glu270 and 271 with Ala removed the interactions but the activity of enzyme did not return. The E270A mutant was more active than LRR but the E271A and E270A/E271A mutants were inactive. Fluorescence and CD measurements showed that these mutations were accompanied by conformational changes. Extrinsic fluorescence measurement and obtained quenching data by KI and acrylamide also confirmed that the mutants were less compact than the LRR enzyme. In conclusion, in LRR, the interactions between Arg300 and Glu270 and Glu271 were not responsible for the enzyme inactivation and it is proposed that the enzyme inactivation is due to conformational changes of LRR mutant of firefly luciferase.

  6. A destabilized bacterial luciferase for dynamic gene expression studies

    PubMed Central

    Allen, Michael S.; Wilgus, John R.; Chewning, Christopher S.; Sayler, Gary S.

    2006-01-01

    Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression. PMID:19003433

  7. Correlation between luminescence intensity and cytotoxicity in cell-based cytotoxicity assay using luciferase.

    PubMed

    Wakuri, S; Yamakage, K; Kazuki, Y; Kazuki, K; Oshimura, M; Aburatani, S; Yasunaga, M; Nakajima, Y

    2017-04-01

    The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay.

  8. Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene

    PubMed Central

    Shen, Liang; Yang, Yang; Ye, Fei; Liu, Gaoshan; Desforges, Marc

    2016-01-01

    Human coronaviruses (HCoVs) cause 15 to 30% of mild upper respiratory tract infections. However, no specific antiviral drugs are available to prevent or treat HCoV infections to date. Here, we developed four infectious recombinant HCoVs-OC43 (rHCoVs-OC43) which express the Renilla luciferase (Rluc) reporter gene. Among these four rHCoVs-OC43, rOC43-ns2DelRluc (generated by replacing ns2 with the Rluc gene) showed robust luciferase activity with only a slight impact on its growth characteristics. Additionally, this recombinant virus remained stable for at least 10 passages in BHK-21 cells. rOC43-ns2DelRluc was comparable to its parental wild-type virus (HCoV-OC43-WT) with respect to the quantity of the antiviral activity of chloroquine and ribavirin. We showed that chloroquine strongly inhibited HCoV-OC43 replication in vitro, with a 50% inhibitory concentration (IC50) of 0.33 μM. However, ribavirin showed inhibition of HCoV-OC43 replication only at high concentrations which may not be applicable to humans in clinical treatment, with an IC50 of 10 μM. Furthermore, using a luciferase-based small interfering RNA (siRNA) screening assay, we identified double-stranded-RNA-activated protein kinase (PKR) and DEAD box RNA helicases (DDX3X) that exhibited antiviral activities, which were further verified by the use of HCoV-OC43-WT. Therefore, rOC43-ns2DelRluc represents a promising safe and sensitive platform for high-throughput antiviral screening and quantitative analysis of viral replication. PMID:27381385

  9. Firefly luciferase as the reporter for transcriptional response to the environment in Escherichia coli.

    PubMed

    Ryo, Masashi; Oshikoshi, Yuta; Doi, Shosei; Motoki, Shogo; Niimi, Atsuko; Aoki, Setsuyuki

    2013-12-15

    We demonstrate that firefly luciferase is a good reporter in Escherichia coli for transcription dynamics in response to the environment. E. coli strains, carrying a fusion of the promoter of the ycgZ gene and the coding region of the luciferase gene, showed transient bioluminescence on receiving blue light. This response was compromised in mutants lacking known regulators in manners consistent with each regulator's function. We also show that relA, a gene encoding a (p)ppGpp synthetase, affects ycgZ dynamics when nullified. Moreover, two unstable luciferase variants showed improved response dynamics and should be useful to study quick changes of gene expression.

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

    PubMed

    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 Km 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.

  11. High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria

    PubMed Central

    2016-01-01

    In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format. This assay uses the exoerythrocytic stage of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several biased and unbiased compound libraries, including two small sets of molecules (400 and 89 compounds, respectively) with known activity against malaria erythrocytic-stage parasites and a set of 9886 diversity-oriented synthesis (DOS)-derived compounds. Of the compounds screened, we obtain hit rates of 12–13 and 0.6% in preselected and naïve libraries, respectively, and identify 52 compounds with exoerythrocytic-stage activity less than 1 μM and having minimal host cell toxicity. Our data demonstrate the ability of this method to identify compounds known to have causal prophylactic activity in both human and animal models of malaria, as well as novel compounds, including some exclusively active against parasite exoerythrocytic stages. PMID:27275010

  12. Control of luciferase synthesis in a newly isolated strain of Photobacterium leiognathi.

    PubMed

    Katznelson, R; Ulitzur, S

    1977-12-15

    In previous studies with luminous bacteria of all different species it has been reported that the synthesis of luciferase is autoinducible: during growth at low cell densities synthesis is effectively repressed while after induction, at higher cell densities, the rate of synthesis of enzyme is up to five times the growth rate. In this paper we report on newly isolated strains of Photobacterium leiognathi which show continued luciferase synthesis irrespective of the cell density. The specific synthesis rate may nevertheless differ from the rate of growth and depends on the luciferase content of the inoculated cells. A ratio of 1 was established for cells having a maximum luciferase content varying to a ratio of about 2 for cells that contained only 1% of the maximum.

  13. Excitation transfer in the in-vitro reaction of photobacterium luciferase bioluminescence

    NASA Astrophysics Data System (ADS)

    Lee, John W.

    1992-04-01

    Fluorescence dynamics methods are used to probe the mechanism by which the chemi- energized intermediates of the bacterial luciferase catalyzed oxidation of FMNH2 and tetradecanal are able to excite the ligand of lumazine protein to its first excited singlet state. A fluorescence dynamics study of the effect of lumazine protein on the reaction of several types of luciferase has recently been published (Biochemistry 30 6825, 1991). This present report examines the case of the Photobacterium leiognathi luciferase reaction in more detail. The fluorescence anisotropy of a mixture of this luciferase fluorescent transient mixed with lumazine protein decays rapidly with a correlation time of 5 ns, interpreted as due to energy transfer. There is no sign of a longer time corresponding to the rotation of the proteins themselves. No rise time of the lumazine (acceptor) fluorescence on exciting into the fluorescent transient (donor) absorption is measureable, so that no straightforward estimate of the energy transfer rate can be made.

  14. Does pressure antagonize anesthesia? High-pressure stopped-flow study of firefly luciferase and anatomy of initial flash.

    PubMed Central

    Ueda, I; Minami, H; Matsuki, H; Inoue, T

    1999-01-01

    The antagonizing effect of high pressure against anesthesia is well known. With purified firefly luciferase, however,. Biophys. J. 60:1309-1314) reported that high pressure did not affect the initial flash intensity. Firefly luciferase emits a burst of light when the substrates luciferin and ATP are added in the presence of O2. The light intensity decays rapidly and the weak light lasts for hours. The initial flash is a transient event and is not in a steady state. The steady state is represented by the slope of the linear part of the integral of the light output. The present study used a high-pressure stopped-flow system to compare the pressure effects on the initial flash intensity and the steady-state light intensity. The flash intensity did not change by the application of hydrostatic pressure in the presence or absence of chloroform or 1-octanol. In contrast, high pressure increased the steady-state light intensity. The application of 12 MPa pressure increased the steady-state light intensity of firefly luciferase inhibited by 5 mM chloroform or 0.7 mM 1-octanol by 19.7% and 18.8%, respectively. When analyzed by the rapid reaction kinetics of the transition state theory, the initial peak intensity represents the total amount of active enzyme and is unrelated to the reaction rate. Anesthetics inhibited the initial flash by unfolding the protein, thereby decreasing the concentration of the active enzyme. Pressure affected the steady-state light intensity by changing the reaction rates. PMID:9876160

  15. Effect of charge distribution in a flexible loop on the bioluminescence color of firefly luciferases.

    PubMed

    Moradi, Ali; Hosseinkhani, Saman; Naderi-Manesh, Hossein; Sadeghizadeh, Majid; Alipour, Bagher Said

    2009-01-27

    Firefly luciferase is a monooxygenase that catalyzes the ATP-dependent conversion of firefly luciferin into a luciferyl-adenylate, which is oxidized to an electronically excited oxyluciferin in a multistep reaction and produces visible light with a remarkable quantum yield. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetles, only luciferase from Phrixotrix railroad worm (Ph(RE)) emits red bioluminescence, naturally. The presence of Arg353 in Ph(RE) luciferase, which corresponds to the deleted residue in the other luciferases, is an important distinctive structural feature of it. Insertion of Arg356 into a green-emitter luciferase (Lampyris turkestanicus), corresponding to Arg353 in Phrixotrix hirtus, changed the emitted light from green to red. To further clarify the effect of this position on the light shift mechanism, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus pyralis luciferase, using site-specific insertion mutagenesis. Insertion of a residue with a positive side chain (Arg356 and Lys356) changed the light color to red, while insertion of a residue with a negative side chain (Glu356) had little effect on color. Insertion of a neutral residue (Gln356) at this position was performed without any change in bioluminescence spectra. Insertion of positively charged residues in this loop took place with a series of structural changes which were confirmed by fluorescence spectroscopy and homology modeling. Homology modeling reveals the appearance of a bulge in a flexible loop (T352-P359) upon mutation which shifts to the left side with a color change from green to red.

  16. Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line.

    PubMed

    Legler, J; van den Brink, C E; Brouwer, A; Murk, A J; van der Saag, P T; Vethaak, A D; van der Burg, B

    1999-03-01

    Development of an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression (ER-CALUX) assay was attempted by stable transfection of luciferase reporter genes in a number of cell lines. Stable transfection of the chimeric Gal4 estrogen receptor and luciferase gene constructs in MCF-7 breast cancer and Hepa.1c1c7 mouse hepatoma cell lines, as well as transfection of a newly constructed luciferase reporter gene pEREtata-Luc in the ECC-1 human endometrial cell line, resulted in constitutive, non-estradiol-inducible clones. Stable transfection of pEREtata-Luc in the T47D breast cancer cell line, however, resulted in an extremely sensitive, highly responsive cell line. Following a 24-h exposure to estradiol (E2), stably transfected T47D.Luc cells demonstrated a detection limit of 0.5 pM, an EC50 of 6 pM, and a maximum induction of 100-fold relative to solvent controls. No clear reduction in responsiveness has been found over extended culture periods (50 passages). Anti-estrogens ICI 182,780, TCDD, and tamoxifen inhibited the estradiol-mediated luciferase induction. Genistein, nonylphenol, and o,p'DDT were the most potent (pseudo-)estrogens tested in this system (EC50 100, 260, and 660 nM, respectively). Determination of interactive effects of the (pseudo-)estrogens nonylphenol, o,p'DDT, chlordane, endosulfan, dieldrin, and methoxychlor revealed that, in combination with 3 pM E2, (pseudo-)estrogens were additive. Slightly more than additive effects (less than 2-fold) were found for combinations of dieldrin and endosulfan tested in the range of 3 to 6 microM. At these concentrations, the combination of endosulfan and chlordane demonstrated additive interaction. The ER-CALUX assay with T47D cells can provide a sensitive, responsive, and rapid in vitro system to detect and measure substances with potential (anti-)estrogenic activity.

  17. Bifunctional role of leucine 300 of firefly luciferase in structural rigidity.

    PubMed

    Yousefi, Farzad; Ataei, Farangis; Mortazavi, Mojtaba; Hosseinkhani, Saman

    2017-03-18

    Firefly luciferase is susceptible to thermal inactivation, thereby its intracellular half-life decreased. Previous reports indicated that L(300)R mutation (LRR mutant) in E(354)R/Arg(356) double mutant (ERR mutant) from Lampyris turkestanicus luciferase has increased its thermal stability and rigidity through induction of some ionic bonds with Asp 270 and 271. Disruption of the deduced ionic bonds in an ultra-rigid mutant of firefly luciferase did not reverse the flexibility of the protein. In this study, we investigated the effects of this residue to find the truth behind an extraordinary increase in thermal stability and rigidity of luciferase after replacement of leucine 300 by arginine based on previous reports. For this purpose, L(300)R, L(300)K and L(300)E mutations were performed to compare the effects of these mutations on the native firefly luciferase. In spite of increase of intrinsic fluorescence of the mutants a slight increase in thermostability and retention of kinetic properties was observed. Based on our results, we can conclude that L(300)R mutation in LRR mutant accompanying with alteration in a flexible loop (352-359) increased thermostability and rigidity of luciferase.

  18. Sensitive Dual Color in vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase

    DTIC Science & Technology

    2011-04-01

    better separation in deeper tissues [27]. Recently, Hida and colleagues applied multicolor luciferases to study protein-protein interaction and proposed...fluorescence imaging with a multivariate curve resolution spectral unmixing technique. J Biomed Opt 14: 064011. 28. Hida N, Awais M, Takeuchi M, Ueno N

  19. Identifying a Kinase Network Regulating FGF14:Nav1.6 Complex Assembly Using Split-Luciferase Complementation

    PubMed Central

    Hsu, Wei-Chun; Nenov, Miroslav N.; Shavkunov, Alexander; Panova, Neli; Zhan, Ming; Laezza, Fernanda

    2015-01-01

    Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA) is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14). Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC) as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3) and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the normal and

  20. Replication of Subgenomic Hepatitis A Virus RNAs Expressing Firefly Luciferase Is Enhanced by Mutations Associated with Adaptation of Virus to Growth in Cultured Cells

    PubMed Central

    Yi, MinKyung; Lemon, Stanley M.

    2002-01-01

    Replication of hepatitis A virus (HAV) in cultured cells is inefficient and difficult to study due to its protracted and generally noncytopathic cycle. To gain a better understanding of the mechanisms involved, we constructed a subgenomic HAV replicon by replacing most of the P1 capsid-coding sequence from an infectious cDNA copy of the cell culture-adapted HM175/18f virus genome with sequence encoding firefly luciferase. Replication of this RNA in transfected Huh-7 cells (derived from a human hepatocellular carcinoma) led to increased expression of luciferase relative to that in cells transfected with similar RNA transcripts containing a lethal premature termination mutation in 3Dpol (RNA polymerase). However, replication could not be confirmed in either FrhK4 cells or BSC-1 cells, cells that are typically used for propagation of HAV. Replication was substantially slower than that observed with replicons derived from other picornaviruses, as the basal luciferase activity produced by translation of input RNA did not begin to increase until 24 to 48 h after transfection. Replication of the RNA was reversibly inhibited by guanidine. The inclusion of VP4 sequence downstream of the viral internal ribosomal entry site had no effect on the basal level of luciferase or subsequent increases in luciferase related to its amplification. Thus, in this system this sequence does not contribute to viral translation or replication, as suggested previously. Amplification of the replicon RNA was profoundly enhanced by the inclusion of P2 (but not 5′ noncoding sequence or P3) segment mutations associated with adaptation of wild-type virus to growth in cell culture. These results provide a simple reporter system for monitoring the translation and replication of HAV RNA and show that critical mutations that enhance the growth of virus in cultured cells do so by promoting replication of viral RNA in the absence of encapsidation, packaging, and cellular export of the viral genome. PMID

  1. Bioorthogonal Catalysis: A General Method To Evaluate Metal-Catalyzed Reactions in Real Time in Living Systems Using a Cellular Luciferase Reporter System

    PubMed Central

    2015-01-01

    The development of abiological catalysts that can function in biological systems is an emerging subject of importance with significant ramifications in synthetic chemistry and the life sciences. Herein we report a biocompatible ruthenium complex [Cp(MQA)Ru(C3H5)]+PF6–2 (Cp = cyclopentadienyl, MQA = 4-methoxyquinoline-2-carboxylate) and a general analytical method for evaluating its performance in real time based on a luciferase reporter system amenable to high throughput screening in cells and by extension to evaluation in luciferase transgenic animals. Precatalyst 2 activates alloc-protected aminoluciferin 4b, a bioluminescence pro-probe, and releases the active luminophore, aminoluciferin (4a), in the presence of luciferase-transfected cells. The formation and enzymatic turnover of 4a, an overall process selected because it emulates pro-drug activation and drug turnover by an intracellular target, is evaluated in real time by photon counting as 4a is converted by intracellular luciferase to oxyaminoluciferin and light. Interestingly, while the catalytic conversion (activation) of 4b to 4a in water produces multiple products, the presence of biological nucleophiles such as thiols prevents byproduct formation and provides almost exclusively luminophore 4a. Our studies show that precatalyst 2 activates 4b extracellularly, exhibits low toxicity at concentrations relevant to catalysis, and is comparably effective in two different cell lines. This proof of concept study shows that precatalyst 2 is a promising lead for bioorthogonal catalytic activation of pro-probes and, by analogy, similarly activatable pro-drugs. More generally, this study provides an analytical method to measure abiological catalytic activation of pro-probes and, by analogy with our earlier studies on pro-Taxol, similarly activatable pro-drugs in real time using a coupled biological catalyst that mediates a bioluminescent readout, providing tools for the study of imaging signal amplification and

  2. Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters.

    PubMed

    Ramanathan, Chidambaram; Khan, Sanjoy K; Kathale, Nimish D; Xu, Haiyan; Liu, Andrew C

    2012-09-27

    In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere. Individual cells are the functional units for generation and maintenance of circadian rhythms, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms. Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection or stable transduction. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host

  3. Fireflies in the coalmine: luciferase technologies in next-generation toxicity testing.

    PubMed

    Simmons, Steven O

    2011-09-01

    Whole-animal studies have been the mainstay of toxicity testing for decades. These approaches are too expensive and laborious to effectively characterize all of the chemicals currently in commercial use. In addition, there are social and ethical pressures to reduce, refine and replace animal testing in toxicology. The National Research Council (NRC) has outlined a new strategy to transition from animal-based tests to high throughput, cell-based assays and computational modeling approaches to characterize chemical toxicants. Critical to this vision, assays that measure toxicity pathways associated with adverse health effects must be developed. Bioluminescent assays are particularly well suited to the demands of next-generation toxicity testing because they measure a wide range of biological activities in a quantitative and high throughput manner. This review describes the limitations of traditional, animal-based toxicity testing and discusses the current and developing uses of bioluminescent technologies in next-generation testing based on three general assay formats: luciferase-limited assays, ATP-limited assays and luciferin-limited assays.

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

    PubMed

    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.

  5. Interspecific luciferase beta subunit hybrids between Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi.

    PubMed

    Almashanu, S; Gendler, I; Hadar, R; Kuhn, J

    1996-09-01

    Bacterial luciferase (EC 1.14.14.3) is a heterodimer composed of alpha- and beta-chains encoded by luxA and luxB, respectively. Although some interspecific combinations of these subunits lead to active enzyme, others do not. The beta subunits of Vibrio fischeri and Photobacterium leiognathi form active enzyme with the alpha subunits of V.fischeri, P.leiognathi and Vibrio harveyi, while the beta subunit from V.harveyi only complements the alpha subunit of V.harveyi. Inactivity is caused by a lack of dimerization of the beta subunit of V.harveyi with the alpha subunits of V.fischeri and P.leiognathi. These observations served as the basis for a search to discover which segment of the beta polypeptide confers the ability to dimerize with the alpha subunits of V.fischeri and P.leiognathi. Intragenic beta subunit hybrids were made between V.harveyi, V.fischeri and P.leiognathi. Unique restriction sites were introduced into the respective luxB genes to divide them into four roughly equal segments. In all, 78 hybrids were constructed by in vitro techniques. The N-terminal segment of the peptide contains the signals that differentiate between the beta subunits of V.fischeri and P. leiognathi and the beta subunit of V. harveyi, and allow the former to dimerize with their alpha subunits. The second segment has no major effect on enzyme activity but does exhibit some context effects. Important interactions were found between the third and fourth segments of the polypeptide with respect to enzymatic activity.

  6. The Effect of Surface Charge Saturation on Heat-induced Aggregation of Firefly Luciferase.

    PubMed

    Gharanlar, Jamileh; Hosseinkhani, Saman; Sajedi, Reza H; Yaghmaei, Parichehr

    2015-01-01

    We present here the effect of firefly luciferase surface charge saturation and the presence of some additives on its thermal-induced aggregation. Three mutants of firefly luciferase prepared by introduction of surface Arg residues named as 2R, 3R and 5R have two, three and five additional arginine residues substituted at their surface compared to native luciferase; respectively. Turbidimetric study of heat-induced aggregation indicates that all three mutants were reproducibly aggregated at higher rates relative to wild type in spite of their higher thermostability. Among them, 2R had most evaluated propensity to heat-induced aggregation. Therefore, the hydrophilization followed by appearing of more substituted arginine residues with positive charge on the firefly luciferase surface was not reduced its thermal aggregation. Nevertheless, at the same condition in the presence of charged amino acids, e.g. Arg, Lys and Glu, as well as a hydrophobic amino acid, e.g. Val, the heat-induced aggregation of wild type and mutants of firefly luciferases was markedly decelerated than those in the absence of additives. On the basis of obtained results it seems, relinquishment of variety in charge of amino acid side chains, they via local interactions with proteins cause to decrease rate and extent of their thermal aggregation.

  7. Titration-based screening for evaluation of natural product extracts: identification of an aspulvinone family of luciferase inhibitors

    SciTech Connect

    Cruz, P.G.; Auld, D.S.; Schultz, P.J.; Lovell, S.; Battaile, K.P.; MacArthur, R.; Shen, M.; Tamayo-Castillo, G.; Inglese, J.; Sherman, D.H.

    2011-11-28

    The chemical diversity of nature has tremendous potential for the discovery of molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, and macro- and microorganisms has curtailed their use in lead discovery. Here, we describe a process for leveraging the concentration-response curves obtained from quantitative HTS to improve the initial selection of actives from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm improves the probability that labor-intensive subsequent steps of reculturing, extraction, and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by X-ray crystallography.

  8. Firefly Luciferase-Based Sequential Bioluminescence Resonance Energy Transfer (BRET)-Fluorescence Resonance Energy Transfer (FRET) Protease Assays.

    PubMed

    Branchini, Bruce

    2016-01-01

    We describe here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyzes yellow-green (560 nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-Infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. This method successfully employs an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence to assay physiologically important protease activities and should be generally applicable to the measurement of any endoprotease lacking accessible cysteine residues.

  9. Replication Competent Molecular Clones of HIV-1 Expressing Renilla Luciferase Facilitate the Analysis of Antibody Inhibition in PBMC

    PubMed Central

    Edmonds, Tara G.; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S.; Conway, Joan A.; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T.; Montefiori, David C.; Kappes, John C.; Ochsenbauer, Christina

    2010-01-01

    Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. PMID:20863545

  10. Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models

    NASA Astrophysics Data System (ADS)

    Close, Dan M.; Hahn, Ruth E.; Patterson, Stacey S.; Baek, Seung J.; Ripp, Steven A.; Sayler, Gary S.

    2011-04-01

    Bioluminescent and fluorescent reporter systems have enabled the rapid and continued growth of the optical imaging field over the last two decades. Of particular interest has been noninvasive signal detection from mammalian tissues under both cell culture and whole animal settings. Here we report on the advantages and limitations of imaging using a recently introduced bacterial luciferase (lux) reporter system engineered for increased bioluminescent expression in the mammalian cellular environment. Comparison with the bioluminescent firefly luciferase (Luc) system and green fluorescent protein system under cell culture conditions demonstrated a reduced average radiance, but maintained a more constant level of bioluminescent output without the need for substrate addition or exogenous excitation to elicit the production of signal. Comparison with the Luc system following subcutaneous and intraperitoneal injection into nude mice hosts demonstrated the ability to obtain similar detection patterns with in vitro experiments at cell population sizes above 2.5 × 104 cells but at the cost of increasing overall image integration time.

  11. Interaction of aromatic compounds with Photobacterium leiognathi luciferase: fluorescence anisotropy study.

    PubMed

    Kudryasheva, N S; Nemtseva, E V; Visser, A J W G; Van Hoek, A

    2003-01-01

    The time-resolved and steady-state fluorescence techniques were employed to elucidate possible interactions of four aromatic compounds (anthracene, POPOP, MSB and 1,4-naphthalendiol) with bacterial luciferase. Fluorescence spectra and fluorescence anisotropy decays of these compounds were studied in ethanol, water-ethanol solutions and in the presence of bacterial luciferase. Shifts of fluorescent spectra and differences in rotational correlation times are interpreted in terms of weak (hydrophobic) interactions of the molecules with the enzyme. These interactions suggest the feasibility of intermolecular energy transfer by an exchange resonance mechanism with a collision-interaction radius as a way of excitation of these compounds in the reaction catalysed by bacterial luciferase.

  12. Posttranslationally caused bioluminescence burst of the Escherichia coli luciferase reporter strain.

    PubMed

    Ideguchi, Yamato; Oshikoshi, Yuta; Ryo, Masashi; Motoki, Shogo; Kuwano, Takashi; Tezuka, Takafumi; Aoki, Setsuyuki

    2016-01-01

    We continuously monitored bioluminescence from a wild-type reporter strain of Escherichia coli (lacp::luc+/WT), which carries the promoter of the lac operon (lacp) fused with the firefly luciferase gene (luc+). This strain showed a bioluminescence burst when shifted into the stationary growth phase. Bioluminescence profiles of other wild-type reporter strains (rpsPp::luc+ and argAp::luc+) and gene-deletion reporter strains (lacp::luc+/crp- and lacp::luc+/lacI-) indicate that transcriptional regulation is not responsible for generation of the burst. Consistently, changes in the luciferase protein levels did not recapitulate the profile of the burst. On the other hand, dissolved oxygen levels increased over the period across the burst, suggesting that the burst is, at least partially, caused by an increase in intracellular oxygen levels. We discuss limits of the firefly luciferase when used as a reporter for gene expression and its potential utility for monitoring metabolic changes in cells.

  13. Problem areas in the use of the firefly luciferase assay for bacterial detection

    NASA Technical Reports Server (NTRS)

    Picciolo, G. L.; Chappelle, E. W.; Knust, E. A.; Tuttle, S. A.; Curtis, C. A.

    1975-01-01

    By purifying the firefly luciferase extract and adding all necessary chemicals but ATP in excess, an assay for ATP was performed by measuring the amount of light produced when a sample containing soluble ATP is added to the luciferase reaction mixture. Instrumentation, applications, and basic characteristics of the luciferase assay are presented. Effect of the growth medium and length of time grown in this medium on ATP per viable E. coli values is shown in graphic form, along with an ATP concentration curve showing relative light units versus ATP injected. Reagent functions and concentration methods are explored. Efforts to develop a fast automatable system to detect the presence of bacteria in biological fluids, especially urine, resulted in the optimization of procedures for use with different types of samples.

  14. Low-cost system for real-time monitoring of luciferase gene expression.

    PubMed

    Gailey, P C; Miller, E J; Griffin, G D

    1997-03-01

    In some mammalian cells transfected with luciferase reporter genes, the luciferase/luciferin reaction in a cell monolayer produces a very small light flux. While the low light levels are often measurable with single-photon counting cameras, these devices are expensive and may require long averaging times to acquire an image. We describe an approach for real-time monitoring of light produced by luciferase gene expression in intact, cultured cells using readily available and relatively inexpensive components. The system uses a single-photon counting photomultiplier tube with built-in high voltage supply and photon counting circuitry to rapidly measure average light output from growing cells in a 35 mm culture dish. The fast, accurate and highly sensitive response of the system makes it useful for studying the dynamics of gene expression over time periods ranging from minutes to days.

  15. Light without substrate amendment: the bacterial luciferase gene cassette as a mammalian bioreporter

    NASA Astrophysics Data System (ADS)

    Close, Dan M.; Xu, Tingting; Smartt, Abby E.; Jegier, Pat; Ripp, Steven A.; Sayler, Gary S.

    2011-06-01

    Bioluminescent production represents a facile method for bioreporter detection in mammalian tissues. The lack of endogenous bioluminescent reactions in these tissues allows for high signal to noise ratios even at low signal strength compared to fluorescent signal detection. While the luciferase enzymes commonly employed for bioluminescent detection are those from class Insecta (firefly and click beetle luciferases), these are handicapped in that they require concurrent administration of a luciferin compound to elicit a bioluminescent signal. The bacterial luciferase (lux) gene cassette offers the advantages common to other bioluminescent proteins, but is simultaneously capable of synthesizing its own luciferin substrates using endogenously available cellular compounds. The longstanding shortcoming of the lux cassette has been its recalcitrance to function in the mammalian cellular environment. This paper will present an overview of the work completed to date to overcome this limitation and provide examples of mammalian lux-based bioreporter technologies that could provide the framework for advanced, biomedically relevant real-time sensor development.

  16. Quantitative High-throughput Luciferase Screening in Identifying CAR Modulators

    PubMed Central

    Lynch, Caitlin; Zhao, Jinghua; Wang, Hongbing; Xia, Menghang

    2017-01-01

    Summary The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR. PMID:27518621

  17. A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the β-actin promoter and enhancer.

    PubMed

    Lupold, Shawn E; Johnson, Tamara; Chowdhury, Wasim H; Rodriguez, Ronald

    2012-01-01

    Secreted reporter molecules offer a means to evaluate biological processes in real time without the need to sacrifice samples at pre-determined endpoints. Here we have adapted the secreted bioluminescent reporter gene, Metridia luciferase, for use in a real-time viability assay for mammalian cells. The coding region of the marine copepod gene has been codon optimized for expression in human cells (hMLuc) and placed under the control of the human β-actin promoter and enhancer. Metridia luciferase activity of stably transfected cell models corresponded linearly with cell number over a 4-log dynamic range, detecting as few as 40 cells. When compared to standard endpoint viability assays, which measure the mitochondrial dehydrogenase reduction of tetrazolium salts, the hMLuc viability assay had a broader linear range of detection, was applicable to large tissue culture vessels, and allowed the same sample to be repeatedly measured over several days. Additional studies confirmed that MLuc activity was inhibited by serum, but demonstrated that assay activity remained linear and was measurable in the serum of mice bearing subcutaneous hMLuc-expressing tumors. In summary, these comparative studies demonstrate the value of humanized Metridia luciferase as an inexpensive and non-invasive method for analyzing viable cell number, growth, tumor volume, and therapeutic response in real time.

  18. Genomic structure of the luciferase gene and phylogenetic analysis in the Hotaria-group fireflies.

    PubMed

    Choi, Yong Soo; Bae, Jin Sik; Lee, Kwang Sik; Kim, Seong Ryul; Kim, Iksoo; Kim, Jong Gill; Kim, Keun Young; Kim, Sam Eun; Suzuki, Hirobumi; Lee, Sang Mong; Sohn, Hung Dae; Jin, Byung Rae

    2003-02-01

    The luminescent fireflies have species specific flash patterns, being recognized as sexual communication. The luciferase gene is the sole enzyme responsible for bioluminescence. We describe here the complete nucleotide sequence and the exon-intron structure of the luciferase gene of the Hotaria-group fireflies, H. unmunsana, H. papariensis and H. tsushimana. The luciferase gene of the Hotaria-group firefly including the known H. parvula spans 1950 bp and consisted of six introns and seven exons coding for 548 amino acid residues, suggesting highly conserved structure among the Hotaria-group fireflies. Although only one luciferase gene was cloned from H. papariensis, each of the two sequences of the gene was found in H. unmunsana (U1 and Uc) and H. tsushimana (T1 and T2). The amino acid sequence divergence among H. unmunsana, H. papariensis, and H. tsushimana only ranged from zero to three amino acid residues, but H. parvula differed by 10-11 amino acid residues from the other Hotaria-group fireflies, suggesting a divergent relationship of this species. Phylogenetic analysis using the deduced amino acid sequences of the luciferase gene resulted in a monophyletic group in the Hotaria excluding H. parvula, suggesting a close relationship among H. unmunsana, H. papariensis and H. tsushimana. Additionally, we also analyzed the mitochondrial cytochrome oxidase I (COI) gene of the Hotaria-group fireflies. The deduced amino acid sequence of the COI gene of H. unmunsana was identical to that of H. papariensis and H. tsushimana, but different by three positions from H. parvula. In terms of nucleotide sequences of the COI gene, intraspecific sequence divergence was sometimes larger than interspecies level, and phylogenetic analysis placed the three species into monophyletic groups unresolved among them, but excluded H. parvula. In conclusion, our results suggest that H. unmunsana, H. papariensis and H. tsushimana are very closely related or might be an identical species, at

  19. Quantum/molecular mechanics study of firefly bioluminescence on luciferase oxidative conformation

    NASA Astrophysics Data System (ADS)

    Pinto da Silva, Luís; Esteves da Silva, Joaquim C. G.

    2014-07-01

    This is the first report of a computational study of the color tuning mechanism of firefly bioluminescence, using the oxidative conformation of luciferase. The results of these calculations demonstrated that the electrostatic field generated by luciferase is fundamental both for the emission shift and efficiency. Further calculations indicated that a shift in emission is achieved by modulating the energy, at different degrees, of the emissive and ground states. These differences in energy modulation will then lead to changes in the energy gap between the states.

  20. Bioluminescence resonance energy transfer using luciferase-immobilized quantum dots for self-illuminated photodynamic therapy.

    PubMed

    Hsu, Chia-Yen; Chen, Ching-Wen; Yu, Hsiu-Ping; Lin, Yan-Fu; Lai, Ping-Shan

    2013-01-01

    Photodynamic therapy (PDT) is an innovative method for cancer treatment that involves the administration of a photosensitizing agent followed by exposure to visible light. An appreciable amount of a particular light source is a key to activate photosensitizers in PDT. However, the external excitation light source is a problem for clinical application because of the limitation of tissue-penetrating properties. Additionally, the wavelength of laser emission should match the absorption wavelength of each photosensitizer for efficient generation of reactive oxygen species and cell killing. In this study, Renilla luciferase-immobilized quantum dots-655 (QD-RLuc8) was used for bioluminescence resonance energy transfer (BRET)-mediated PDT to resolve these problems. The bioluminescent QD-RLuc8 conjugate exhibits self-illumination at 655 nm after coelenterazine addition, which can activate the photosensitizer, Foscan(®)-loaded micelles for PDT. Our results show that BRET-mediated PDT by QD-RLuc8 plus coelenterazine (20 μg/mL) successfully generated reactive oxygen species (40.8%), killed ~ 50% A549 cells at 2 μg/mL equivalent Foscan(®)in vitro and significantly delayed tumor growth in vivo due to cell apoptosis under TUNEL analysis without obvious weight loss. Based on immunohistochemical observations, the proliferating cell nuclear antigen (PCNA)-negative area of tumor sections after BRET-mediated PDT was obviously increased compared to the PDT-untreated groups without an external light source. We conclude that this nanotechnology-based PDT possesses several clinical benefits, such as overcoming light penetration issues and treating deeper lesions that are intractable by PDT alone.

  1. Titration-based screening for evaluation of natural product extracts: identification of an aspulvinone family of luciferase inhibitors

    PubMed Central

    Cruz, Patricia G.; Auld, Douglas S.; Schultz, Pamela J.; Lovell, Scott; Battaile, Kevin P.; MacArthur, Ryan; Shen, Min; Tamayo-Castillo, Giselle; Inglese, James; Sherman, David H.

    2011-01-01

    The chemical diversity of nature has tremendous potential for discovery of new molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, macro- and microorganisms has curtailed their use in lead discovery efforts. Here we describe a process for leveraging the concentration-response curves (CRCs) obtained from quantitative HTS to improve the initial selection of “actives” from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm aims to improve the probability that labor-intensive subsequent steps of re-culturing, extraction and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by x-ray crystallography. PMID:22118678

  2. 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.

    PubMed

    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

  3. A new humanized in vivo model of KIT D816V+ advanced systemic mastocytosis monitored using a secreted luciferase

    PubMed Central

    Bibi, Siham; Zhang, Yanyan; Hugonin, Caroline; Mangean, Mallorie Depond; He, Liang; Wedeh, Ghaith; Launay, Jean-Marie; Van Rijn, Sjoerd; Würdinger, Thomas; Louache, Fawzia; Arock, Michel

    2016-01-01

    Systemic mastocytosis are rare neoplasms characterized by accumulation of mast cells in at least one internal organ. The majority of systemic mastocytosis patients carry KIT D816V mutation, which activates constitutively the KIT receptor. Patient with advanced forms of systemic mastocytosis, such as aggressive systemic mastocytosis or mast cell leukemia, are poorly treated to date. Unfortunately, the lack of in vivo models reflecting KIT D816V+ advanced disease hampers pathophysiological studies and preclinical development of new therapies for such patients. Here, we describe a new in vivo model of KIT D816V+ advanced systemic mastocytosis developed by transplantation of the human ROSAKIT D816V-Gluc mast cell line in NOD-SCID IL-2R g−/− mice, using Gaussia princeps luciferase as a reporter. Intravenous injection of ROSAKIT D816V-Gluc cells led, in 4 weeks, to engraftment in all injected primary recipient mice. Engrafted cells were found at high levels in bone marrow, and at lower levels in spleen, liver and peripheral blood. Disease progression was easily monitored by repeated quantification of Gaussia princeps luciferase activity in peripheral blood. This quantification evidenced a linear relationship between the number of cells injected and the neoplastic mast cell burden in mice. Interestingly, the secondary transplantation of ROSAKIT D816V-Gluc cells increased their engraftment capability. To conclude, this new in vivo model mimics at the best the features of human KIT D816V+ advanced systemic mastocytosis. In addition, it is a unique and convenient tool to study the kinetics of the disease and the potential in vivo activity of new drugs targeting neoplastic mast cells. PMID:27783996

  4. Protein sterilization method of firefly luciferase using reduced pressure and molecular sieves

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Rich, E., Jr. (Inventor)

    1973-01-01

    The sterilization of the protein fruitfly luciferase under conditions that prevent denaturation is examined. Denaturation is prevented by heating the protein in contact with molecular seives and under a reduced pressure of the order of 0.00005 millimeters of mercury.

  5. Adaptation of a Luciferase Gene Reporter and lac Expression System to Borrelia burgdorferi▿ †

    PubMed Central

    Blevins, Jon S.; Revel, Andrew T.; Smith, Alexandra H.; Bachlani, Gulnaz N.; Norgard, Michael V.

    2007-01-01

    The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi. PMID:17220265

  6. The influence of hypoxia on bioluminescence in luciferase-transfected gliosarcoma tumor cells in vitro.

    PubMed

    Moriyama, Eduardo H; Niedre, Mark J; Jarvi, Mark T; Mocanu, Joseph D; Moriyama, Yumi; Subarsky, Patrick; Li, Buhong; Lilge, Lothar D; Wilson, Brian C

    2008-06-01

    Firefly luciferase catalyzes the emission of light from luciferin in the presence of oxygen and adenosine triphosphate. This bioluminescence is commonly employed in imaging mode to monitor tumor growth and treatment responses in vivo. A potential concern is that, since solid tumors are often hypoxic, either constitutively and/or as a result of treatment, the oxygen available for the bioluminescence reaction could be reduced to limiting levels, leading to underestimation of the actual number of luciferase-labeled cells during in vivo experiments. We present studies of the oxygen dependence of bioluminescence in vitro in rat 9 L gliosarcoma cells tagged with the firefly luciferase gene (9L(luc)). We demonstrate that the bioluminescence signal decreases at pO(2) luciferase expression or oxygen itself.

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

    SciTech Connect

    Wu, Anna M

    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.

  8. Properties of recombinant fluorescent proteins from Photobacterium leiognathi and their interaction with luciferase intermediates.

    PubMed

    Petushkov, V N; Gibson, B G; Lee, J

    1995-03-14

    Ligand binding and luciferase interaction properties of the recombinant protein corresponding to the lumazine protein gene (EMBL X56534) of Photobacterium leiognathi have been determined by fluorescence dynamics, circular dichroism, gel filtration, and SDS-PAGE. Scatchard analysis of a fluorescence titration shows that the apoprotein possess one binding site, and at 30 degrees C the KdS (microM) are as follows: 6,7-dimethyl-8-ribityllumazine, 0.26; riboflavin, 0.53; and much more weakly bound FMN, 30. All holoproteins are highly fluorescent and have absorption spectra distinct from each other and from the free ligands. The longest wavelength absorption maxima are, respectively (nm, 2 degrees C), 420, 463, and 458. Ligand binding produces no change in the far-UV circular dichroism; all have mean residual ellipticity at 210 nm of -6500 deg cm2 dmol-1, the same as the native protein. However, in the bioluminescence reaction only the lumazine holoprotein shows a bioluminescence effect. Fluorescence emission anisotropy decay was used to establish that none of these holoproteins complexed with native luciferase and that the lumazine protein alone formed a 1:1 complex with the luciferase hydroxyflavin fluorescent transient and the luciferase peroxyflavin intermediates, revealed by a dominant channel of anisotropy loss, with rotational correlation time of 2.5 ns, and attributed to excitation transfer from the luciferase flavin donor to the acceptor, the lumazine ligand. The complex stability was sufficient to allow its isolation by FPLC gel filtration and verification by SDS-PAGE. These methods also confirmed the absence of interaction of the holoflavoproteins.

  9. Firefly luciferase in chemical biology: A compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter

    PubMed Central

    Thorne, Natasha; Shen, Min; Lea, Wendy A.; Simeonov, Anton; Lovell, Scott; Auld, Douglas S.; Inglese, James

    2012-01-01

    SUMMARY Firefly luciferase (FLuc) is frequently used as a reporter in high-throughput screening assays owing to the exceptional sensitivity, dynamic range, and rapid measurement that bioluminescence affords. However, interaction of small molecules with FLuc has, to some extent, confounded its use in chemical biology and drug discovery. To identify and characterize chemotypes interacting with FLuc, we determined potency values for 360,864 compounds, found in the NIH Molecular Libraries Small Molecule Repository, available in PubChem. FLuc inhibitory activity was observed for 12% of this library with discernible SAR. Characterization of 151 inhibitors demonstrated a variety of inhibition modes including FLuc-catalyzed formation of multisubstrate-adduct enzyme inhibitor complexes. As in some cell-based FLuc reporter assays compounds acting as FLuc inhibitors yield paradoxical luminescence increases, data on compounds acquired from FLuc-dependent assays requires careful analysis as described in this report. PMID:22921073

  10. Novel application of luciferase assay for the in vitro functional assessment of KAL1 variants in three females with septo-optic dysplasia (SOD)

    PubMed Central

    McCabe, Mark J.; Hu, Youli; Gregory, Louise C.; Gaston-Massuet, Carles; Alatzoglou, Kyriaki S.; Saldanha, José W.; Gualtieri, Angelica; Thankamony, Ajay; Hughes, Ieuan; Townshend, Sharron; Martinez-Barbera, Juan-Pedro; Bouloux, Pierre-Marc; Dattani, Mehul T.

    2015-01-01

    KAL1 is implicated in 5% of Kallmann syndrome cases, a disorder which genotypically overlaps with septo-optic dysplasia (SOD). To date, a reporter-based assay to assess the functional consequences of KAL1 mutations is lacking. We aimed to develop a luciferase assay for novel application to functional assessment of rare KAL1 mutations detected in a screen of 422 patients with SOD. Quantitative analysis was performed using L6-myoblasts stably expressing FGFR1, transfected with a luciferase-reporter vector containing elements of the FGF-responsive osteocalcin promoter. The two variants assayed [p.K185N, p.P291T], were detected in three females with SOD (presenting with optic nerve hypoplasia, midline and pituitary defects). Our novel assay revealed significant decreases in transcriptional activity [p.K185N: 21% (p < 0.01); p.P291T: 40% (p < 0.001)]. Our luciferase-reporter assay, developed for assessment of KAL1 mutations, determined that two variants in females with hypopituitarism/SOD are loss-of-function; demonstrating that this assay is suitable for quantitative assessment of mutations in this gene. PMID:26375424

  11. Photodynamic therapy using luciferase nanoconjugate as a treatment for colon cancer

    NASA Astrophysics Data System (ADS)

    Koritarov, Tamara

    Photodynamic Therapy (PDT) has proven itself in previous studies to be a successful therapeutic treatment for surface tumors, but its effectiveness is limited to only shallow depths that allow for the penetration of light. This study demonstrates that we have improved upon the conventional method of PDT and have overcome the previous depth limitation by creating the light at the location of the tumor in situ. We conjugated a bioluminescent protein, Luciferase, to a semiconductor nanoparticle, TiO2, and with a cell specific antibody, anti-EGFR monoclonal antibody C225. The nanoconjugate, TiDoL-C225, was then activated by ATP and Luciferin in a reaction that creates reactive oxygen species (ROS) and induces apoptosis in the tumor cells. We created the optimal nanoconjugate synthesis protocol to make TiDoL and TiDoL-C225 for use in the PDT treatment. The TiDoL-C225 nanoconjugate is able to bind specifically to colon caner cells as the C225 antibody recognizes EGFR expressed at the surface of the cells, and further, when activated it will react only with the tumor cells. The optimal cell staining protocols were developed to visualize the treatment process and later analyze with the laser confocal microscope. The TiDoL nanoconjugate was found to only be operational and effective at killing tumor cells after being activated by Luciferin and ATP, which then enhances the control we have over the therapy. The TiDoL-C225 nanoconjugate increases the efficacy of binding to tumor cells and the speed of the reaction in the cells to begin apoptosis, even in lower concentrations when compared to the free TiDoL nanoconjugate. Finally, our PDT technique allowed us to monitor the tumor cells as they begin to undergo apoptosis in less than five minutes after the Luciferin was added to activate the reaction. The advantage of our method of PDT with the TiDoL-C225 nanoconjugate is that it can be used for early detection as well as developed into an effective treatment for cancers in all

  12. Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models

    PubMed Central

    Close, Dan M.; Hahn, Ruth E.; Patterson, Stacey S.; Baek, Seung J.; Ripp, Steven A.; Sayler, Gary S.

    2011-01-01

    Bioluminescent and fluorescent reporter systems have enabled the rapid and continued growth of the optical imaging field over the last two decades. Of particular interest has been noninvasive signal detection from mammalian tissues under both cell culture and whole animal settings. Here we report on the advantages and limitations of imaging using a recently introduced bacterial luciferase (lux) reporter system engineered for increased bioluminescent expression in the mammalian cellular environment. Comparison with the bioluminescent firefly luciferase (Luc) system and green fluorescent protein system under cell culture conditions demonstrated a reduced average radiance, but maintained a more constant level of bioluminescent output without the need for substrate addition or exogenous excitation to elicit the production of signal. Comparison with the Luc system following subcutaneous and intraperitoneal injection into nude mice hosts demonstrated the ability to obtain similar detection patterns with in vitro experiments at cell population sizes above 2.5 × 104 cells but at the cost of increasing overall image integration time. PMID:21529093

  13. Luciferase-Based, High-Throughput Assay for Screening and Profiling Transmission-Blocking Compounds against Plasmodium falciparum Gametocytes

    PubMed Central

    Lucantoni, Leonardo; Fidock, David A.

    2016-01-01

    The discovery of new antimalarial drugs able to target both the asexual and gametocyte stages of Plasmodium falciparum is critical to the success of the malaria eradication campaign. We have developed and validated a robust, rapid, and cost-effective high-throughput reporter gene assay to identify compounds active against late-stage (stage IV and V) gametocytes. The assay, which is suitable for testing compound activity at incubation times up to 72 h, demonstrates excellent quality and reproducibility, with average Z′ values of 0.85 ± 0.01. We used the assay to screen more than 10,000 compounds from three chemically diverse libraries. The screening outcomes highlighted the opportunity to use collections of compounds with known activity against the asexual stages of the parasites as a starting point for gametocytocidal activity detection in order to maximize the chances of identifying gametocytocidal compounds. This assay extends the capabilities of our previously reported luciferase assay, which tested compounds against early-stage gametocytes, and opens possibilities to profile the activities of gametocytocidal compounds over the entire course of gametocytogenesis. PMID:26787698

  14. Crystal structure of a flavoprotein related to the subunits of bacterial luciferase.

    PubMed Central

    Moore, S A; James, M N; O'Kane, D J; Lee, J

    1993-01-01

    The molecular structure of the luxF protein from the bioluminescent bacterium Photobacterium leiognathi has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 17.8% at 2.3 A resolution. The 228 amino acid polypeptide exists as a symmetrical homodimer and 33% of the monomer's solvent-accessible surface area is buried upon dimerization. The monomer displays a novel fold that contains a central seven-stranded beta-barrel. The solvent-exposed surface of the monomer is covered by seven alpha-helices, whereas the dimer interface is primarily a flat surface composed of beta-strands. The protein monomer binds two molecules of flavin mononucleotide, each of which has C6 of the flavin isoalloxazine moiety covalently attached to the C3' carbon atom of myristic acid. Both myristyl groups of these adducts are buried within the hydrophobic core of the protein. One of the cofactors contributes to interactions at the dimer interface. The luxF protein displays considerable amino acid sequence homology with both alpha- and beta-subunits of bacterial luciferase, especially the beta-subunit. Conserved amino acid residues shared between luxF and the luciferase subunits cluster predominantly in two distinct regions of the luxF protein molecule. These homologous regions in the luciferase subunits probably share a three-dimensional fold similar to that of the luxF protein. Images PMID:8491169

  15. Detection of luciferase gene sequence in nonluminescent Vibrio cholerae by colony hygridization and polymerase chain reaction

    SciTech Connect

    Palmer, L.M.; Colwell, R.R. )

    1991-05-01

    Bioluminescence is a trait observed among approximately 10% of Vibrio cholerae isolates. We have demonstrated that not only do some strains of V. cholerae produce low levels of light, undetectable by the human eye, but the luciferase gene sequence is present in strains of V. cholerae which emit no detectable light, evidenced by hybridization with a luciferase DNA probe. Comparisons of the amino acid sequences of luciferase regions of amino acid identity. The polymerase chain reaction method of DNA amplification with oligonucleotide primers based on these regions was used to isolate a region of the luxA gene from both luminescent and nonluminescent V. cholerae strains. The nucleotide sequence of this region was determined and reveals that nonluminescent V. cholerae have 99.7% nucleotide sequence similarity in this region with the luminescent biovar V. cholerae by albensis as well as significant similarity to other species of bioluminescent bacteria, a finding that is in accord with the hypothesis that these species have a common luminescent ancestor, most probably from the marine environment.

  16. Expression of firefly luciferase in Candida albicans and its use in the selection of stable transformants.

    PubMed

    Doyle, Timothy C; Nawotka, Kevin A; Purchio, Anthony F; Akin, Ali R; Francis, Kevin P; Contag, Pamela R

    2006-02-01

    The infectious yeast Candida albicans is a model organism for understanding the mechanisms of fungal pathogenicity. We describe the functional expression of the firefly luciferase gene, a reporter commonly used to tag genes in many other cellular systems. Due to a non-standard codon usage by this yeast, the CUG codons were first mutated to UUG to allow functional expression. When integrated into the chromosome of C. albicans with a strong constitutive promoter, cells bioluminesce when provided with luciferin substrate in their media. When fused to the inducible promoter from the HWP1 gene, expression and bioluminescence was only detected in cultures conditioning hyphal growth. We further used the luciferase gene as a selection to isolate transformed cell lines from clinical isolates of C. albicans, using a high-density screening strategy that purifies transformed colonies by virtue of light emission. This strategy requires no drug or auxotrophic selectable marker, and we were thus able to generate stable transformants of clinical isolates that are identical to the parental strain in all aspects tested, other than their bioluminescence. The firefly luciferase gene can, therefore, be used as a sensitive reporter to analyze gene function both in laboratory and clinical isolates of this medically important yeast.

  17. Buffer enhanced bioluminescence resonance energy transfer sensor based on Gaussia luciferase for in vitro detection of protease.

    PubMed

    Li, Fengyun; Yu, Junping; Zhang, Zhiping; Cui, Zongqiang; Wang, Dianbing; Wei, Hongping; Zhang, Xian-En

    2012-04-29

    Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.

  18. Development of a Suite of Luciferase Gene Probes for the Screening and Detection of Marine Bioluminescent Systems and Organisms

    DTIC Science & Technology

    2006-01-01

    microorganisms in order to supplement bathyphotometric luminescence measurements with coincident real-time molecular data. (3) to conduct real - time PCR quantification of luciferase mRNA in dinoflagellates under different growth stages.

  19. Impact of site-directed mutant luciferase on quantitative green and orange/red emission intensities in firefly bioluminescence.

    PubMed

    Wang, Yu; Akiyama, Hidefumi; Terakado, Kanako; Nakatsu, Toru

    2013-01-01

    Firefly bioluminescence has attracted great interest because of its high quantum yield and intriguing modifiable colours. Modifications to the structure of the enzyme luciferase can change the emission colour of firefly bioluminescence, and the mechanism of the colour change has been intensively studied by biochemists, structural biologists, optical physicists, and quantum-chemistry theorists. Here, we report on the quantitative spectra of firefly bioluminescence catalysed by wild-type and four site-directed mutant luciferases. While the mutation caused different emission spectra, the spectra differed only in the intensity of the green component (λmax ~ 560 nm). In contrast, the orange (λmax ~ 610 nm) and red (λmax ~ 650 nm) components present in all the spectra were almost unaffected by the modifications to the luciferases and changes in pH. Our results reveal that the intensity of the green component is the unique factor that is influenced by the luciferase structure and other reaction conditions.

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

    SciTech Connect

    Allegra, Danilo; Mertens, Daniel

    2011-03-25

    Research highlights: {yields} Posttranscriptional regulation of miRNA processing is difficult to quantify. {yields} Our in-vivo processing assay can quantify Drosha cleavage in live cells. {yields} It is based on luciferase reporters fused with pri-miRNAs. {yields} The assay validates the processing defect caused by a mutation in pri-16-1. {yields} 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.

  1. Metabolomics Reveals that Aryl Hydrocarbon Receptor Activation by Environmental Chemicals Induces Systemic Metabolic Dysfunction in Mice

    PubMed Central

    Zhang, Limin; Hatzakis, Emmanuel; Nichols, Robert G.; Hao, Ruixin; Correll, Jared; Smith, Philip B.; Chiaro, Christopher R.; Perdew, Gary H.; Patterson, Andrew D.

    2016-01-01

    Environmental exposure to dioxins and dioxin-like compounds poses a significant health risk for human health. Developing a better understanding of the mechanisms of toxicity through activation of the aryl hydrocarbon receptor (AHR) is likely to improve the reliability of risk assessment. In this study, the AHR-dependent metabolic response of mice exposed to 2,3,7,8-tetrachlorodibenzofuran (TCDF) were assessed using global 1H nuclear magnetic resonance (NMR)-based metabolomics and targeted metabolic profiling of extracts obtained from serum and liver. 1H NMR analyses revealed that TCDF exposure suppressed gluconeogenesis and glycogenolysis, stimulated lipogenesis, and triggered inflammatory gene expression in an Ahr-dependent manner. Targeted analyses using gas chromatography mass spectrometry showed TCDF treatment altered the ratio of unsaturated/saturated fatty acids. Consistent with this observation, an increase in hepatic expression of stearoyl coenzyme A desaturase 1 was also observed. In addition, TCDF exposure resulted in inhibition of de novo fatty acid biosynthesis manifested by down-regulation of acetyl-CoA, malonyl-CoA and palmitoyl-CoA metabolites and related mRNA levels. In contrast, no significant changes in the levels of glucose and lipid were observed in serum and liver obtained from Ahr-null mice following TCDF treatment, thus strongly supporting the important role of the AHR in mediating the metabolic effects seen following TCDF exposure. PMID:26023891

  2. The evolution of the adenylate-forming protein family in beetles: multiple luciferase gene paralogues in fireflies and glow-worms.

    PubMed

    Day, John C; Goodall, Tim I; Bailey, Mark J

    2009-01-01

    Bioluminescence in beetles is dependent upon the enzyme luciferase. It has been hypothesised luciferase evolved from a fatty acyl-CoA synthetase gene deriving a novel bioluminescent function (neofunctionalization) after a gene duplication event. We evaluated this hypothesis within a phylogenetic framework using independent evidence obtained from the genome of Tribolium castaneum, published luciferase genes and novel luciferase and luciferase-like sequences. This phylogenetic study provides evidence for a large gene family of luciferase and luciferase-like paralogues in bioluminescent and non-bioluminescent beetles. All luciferase sequences formed a clade supporting a protoluciferase existing prior to the divergence of the Lampyridae, Elateridae and Phengodidae (Elateroidea). Multiple luciferase genes were identified from members of the Photurinae and the Luciolinae indicating complex gene duplication events within lampyrid genomes. The majority of luciferase residues were identified to be under purifying selection as opposed to positive selection. We conclude that beetle luciferase may have arisen from a process of subfunctionalization as opposed to neofunctionalization early on in the evolution of the Elateroidea.

  3. A high-throughput cell-based Gaussia luciferase reporter assay for identifying modulators of fibulin-3 secretion.

    PubMed

    Hulleman, John D; Brown, Steven J; Rosen, Hugh; Kelly, Jeffery W

    2013-07-01

    An R345W mutation in fibulin-3 causes its inefficient secretion, increased intracellular steady-state levels, and the macular dystrophy, Malattia Leventinese (ML), a disease similar to age-related macular degeneration. It is unknown whether R345W causes ML through increased intracellular levels, by the secretion of a potentially aggregation-prone protein, or both. To identify small molecules that alter the secretion of fibulin-3, we developed ARPE19 retinal cell lines that inducibly express wild-type (WT) or R345W fibulin-3 fused to an enhanced Gaussia luciferase (eGLuc2). Screening of the Library of Pharmacologically Active Compounds demonstrated that these cell lines and the GLuc assay are suitable for high-throughput chemical screening. Two estrogen-related compounds enhanced fibulin-3 secretion, whereas a diverse series of small molecules reduced fibulin-3 secretion. A counterscreen identified compounds that did not substantially alter the secretion of unfused eGLuc2, demonstrating at least partial selectivity for fibulin-3. A secondary assay using untagged fibulin-3 confirmed that the top three inhibitory compounds reduced R345W fibulin-3 secretion. Interestingly, in untagged fibulin-3 studies, one compound, phorbol 12-myristate 13-acetate, reduced R345W fibulin-3 secretion while minimally enhancing WT fibulin-3 secretion, the desired activity and selectivity we sought for ML. The identified compounds could serve as tools for probing the etiology of fibulin-3-related diseases.

  4. A High-Throughput Cell-Based Gaussia Luciferase Reporter Assay for Identifying Modulators of Fibulin-3 Secretion

    PubMed Central

    Hulleman, John D.; Brown, Steven J.; Rosen, Hugh; Kelly, Jeffery W.

    2015-01-01

    An R345W mutation in fibulin-3 causes its inefficient secretion, increased intracellular steady-state levels, and the macular dystrophy, Malattia Leventinese (ML), a disease similar to age-related macular degeneration. It is unknown whether R345W causes ML through increased intracellular levels, by the secretion of a potentially aggregation-prone protein, or both. To identify small molecules that alter the secretion of fibulin-3, we developed ARPE19 retinal cell lines that inducibly express wild-type (WT) or R345W fibulin-3 fused to an enhanced Gaussia luciferase (eGLuc2). Screening of the Library of Pharmacologically Active Compounds demonstrated that these cell lines and the GLuc assay are suitable for high-throughput chemical screening. Two estrogen-related compounds enhanced fibulin-3 secretion, whereas a diverse series of small molecules reduced fibulin-3 secretion. A counterscreen identified compounds that did not substantially alter the secretion of unfused eGLuc2, demonstrating at least partial selectivity for fibulin-3. A secondary assay using untagged fibulin-3 confirmed that the top three inhibitory compounds reduced R345W fibulin-3 secretion. Interestingly, in untagged fibulin-3 studies, one compound, phorbol 12-myristate 13-acetate, reduced R345W fibulin-3 secretion while minimally enhancing WT fibulin-3 secretion, the desired activity and selectivity we sought for ML. The identified compounds could serve as tools for probing the etiology of fibulin-3–related diseases. PMID:23230284

  5. Ultrastructure of granules and immunocytochemical localization of luciferase in photocytes of fireflies.

    PubMed

    Neuwirth, M

    1981-01-01

    Photocyte granules are round to oval, 1-2 micron, contain a peripheral dense area and are of three structural types. Type one granules consist of an amorphous matrix, a bundle of 2-12 microtubules and a flask-shaped vacuole. The type two granule is characterized by a large crystal or several smaller crystals embedded in an amorphous matrix with microtubules lined up along the face of the crystal. The type three granule is filled with a large number of thick-walled tubules (40-50 nm o.d.), usually found in bundles of two to four and a few microtubules. Luciferase has been shown to be localized in these photocyte granules by the immunoferritin technique. Ferritin is not localized over microtubules or flask-shaped vacuoles in type one granules but is randomly distributed over the matrix. In type two granules, ferritin is more densely distributed over the crystals and in type three granules over filamentous structures. There is no ferritin over the microtubules. Other parts of photocytes and of light organs are negative for luciferase. Buffer and anticalliphorin incubated sections showed no ferritin in granules.

  6. Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate.

    PubMed

    Ribeiro, César; Esteves da Silva, Joaquim C G

    2008-09-01

    The inhibition mechanisms of the firefly luciferase (Luc) by the two major products of the reactions catalysed by Luc, oxyluciferin and dehydroluciferyl-adenylate (L-AMP), were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 microM oxyluciferin; 0.0025 to 1.25 microM L-AMP) has been measured in 50 mM Hepes buffer (pH=7.5), 10 nM Luc, 250 microM ATP and D-Luciferin (from 3.75 up to 120 microM). Nonlinear regression analysis with the appropriate kinetic models (Henri-Michaelis-Menten and William-Morrison equations) reveals that oxyluciferin is a competitive inhibitor of luciferase (Ki=0.50+/-0.03 microM) while L-AMP act as a tight-binding competitive inhibitor (Ki=3.8+/-0.7 nM). The Km values obtained both for oxyluciferin and L-AMP were 14.7+/-0.7 and 14.9+/-0.2 microM, respectively. L-AMP is a stronger inhibitor of Luc than oxyluciferin and the major responsible for the characteristic flash profile of in vitro Luc bioluminescence.

  7. Optical imaging of Renilla luciferase reporter gene expression in living mice

    PubMed Central

    Bhaumik, S.; Gambhir, S. S.

    2002-01-01

    Imaging reporter gene expression in living subjects is a rapidly evolving area of molecular imaging research. Studies have validated the use of reporter genes with positron emission tomography (PET), single photon emission computed tomography (SPECT), MRI, fluorescence with wild-type and mutants of green fluorescent protein, as well as bioluminescence using Firefly luciferase enzyme/protein (FL). In the current study, we validate for the first time the ability to image bioluminescence from Renilla luciferase enzyme/protein (RL) by injecting the substrate coelenterazine in living mice. A highly sensitive cooled charge-coupled device camera provides images within a few minutes of photon counting. Cells, transiently expressing the Rluc were imaged while located in the peritoneum, s.c. layer, as well as in the liver and lungs of living mice tail-vein injected with coelenterazine. Furthermore, d-luciferin (a substrate for FL) does not serve as a substrate for RL, and coelenterazine does not serve as a substrate for FL either in cell culture or in living mice. We also show that both Rluc and Fluc expression can be imaged in the same living mouse and that the kinetics of light production are distinct. The approaches validated will have direct applications to various studies where two molecular events need to be tracked, including cell trafficking of two cell populations, two gene therapy vectors, and indirect monitoring of two endogenous genes through the use of two reporter genes. PMID:11752410

  8. Stable Tagging of Rhizobium meliloti with the Firefly Luciferase Gene for Environmental Monitoring

    PubMed Central

    Cebolla, Angel; Ruiz-Berraquero, Francisco; Palomares, Antonio Jose

    1993-01-01

    A system for stable tagging of gram-negative bacteria with the firefly luciferase gene, luc, is described. A previously constructed fusion constitutively expressing luc from the λpR promoter was used. Stable integration into the bacterial genome was achieved by use of mini-Tn5 delivery vectors. The procedure developed was applied for tagging of representative gram-negative bacteria, such as Escherichia coli, Rhizobium meliloti, Pseudomonas putida, and Agrobacterium tumefaciens. The system permitted the detection of tagged R. meliloti in the presence of more than 105 CFU per plate without the use of any selective markers (such as antibiotic resistance genes). No significant differences in growth rates or soil survival were found between the marked strain and the wild-type strain. Studies of bioluminescent R. meliloti also revealed a good correlation between cell biomass and bioluminescence. The firefly luciferase tagging system is an easy, safe, and sensitive method for the detection and enumeration of bacteria in the environment. Images PMID:16349015

  9. High level constitutive expression of luciferase reporter by lsd90 promoter in fission yeast.

    PubMed

    Verma, Hemant Kumar; Shukla, Poonam; Alfatah, Md; Khare, Asheesh Kumar; Upadhyay, Udita; Ganesan, Kaliannan; Singh, Jagmohan

    2014-01-01

    Because of a large number of molecular similarities with higher eukaryotes, the fission yeast Schizosaccharomyces pombe has been considered a potentially ideal host for expressing human proteins having therapeutic and pharmaceutical applications. However, efforts in this direction are hampered by lack of a strong promoter. Here, we report the isolation and characterization of a strong, constitutive promoter from S. pombe. A new expression vector was constructed by cloning the putative promoter region of the lsd90 gene (earlier reported to be strongly induced by heat stress) into a previously reported high copy number vector pJH5, which contained an ARS element corresponding to the mat2P flanking region and a truncated URA3m selectable marker. The resulting vector was used to study and compare the level of expression of the luciferase reporter with that achieved with the known vectors containing regulatable promoter nmt1 and the strong constitutive promoter adh1 in S. pombe and the methanol-inducible AOX1 promoter in Pichia pastoris. Following growth in standard media the new vector containing the putative lsd90 promoter provided constitutive expression of luciferase, at a level, which was 19-, 39- and 10-fold higher than that achieved with nmt1, adh1 and AOX1 promoters, respectively. These results indicate a great potential of the new lsd90 promoter-based vector for commercial scale expression of therapeutic proteins in S. pombe.

  10. A luciferase-based screening method for inhibitors of alphavirus replication applied to nucleoside analogues.

    PubMed

    Pohjala, Leena; Barai, Vladimir; Azhayev, Alex; Lapinjoki, Seppo; Ahola, Tero

    2008-06-01

    Several members of the widespread alphavirus group are pathogenic, but no therapy is available to treat these RNA virus infections. We report here a quantitative assay to screen for inhibitors of Semliki Forest virus (SFV) replication, and demonstrate the effects of 29 nucleosides on SFV and Sindbis virus replication. The anti-SFV assay developed is based on a SFV strain containing Renilla luciferase inserted after the nsP3 coding region, yielding a marker virus in which the luciferase is cleaved out during polyprotein processing. The reporter-gene assay was miniaturized, automated and validated, resulting in a Z' value of 0.52. [3H]uridine labeling for 1 h at the maximal viral RNA synthesis time point was used as a comparative method. Anti-SFV screening and counter-screening for cell viability led to the discovery of several new SFV inhibitors. 3'-amino-3'-deoxyadenosine was the most potent inhibitor in this set, with an IC50 value of 18 microM in the reporter-gene assay and 2 microM in RNA synthesis rate detection. Besides the 3'-substituted analogues, certain N6-substituted nucleosides had similar IC50 values for both SFV and Sindbis replication, suggesting the applicability of this methodology to alphaviruses in general.

  11. Tracking of dendritic cell migration into lymph nodes using molecular imaging with sodium iodide symporter and enhanced firefly luciferase genes.

    PubMed

    Lee, Ho Won; Yoon, Seung Yun; Singh, Thoudam Debraj; Choi, Yoon Ju; Lee, Hong Je; Park, Ji Young; Jeong, Shin Young; Lee, Sang-Woo; Ha, Jeoung-Hee; Ahn, Byeong-Cheol; Jeon, Yong Hyun; Lee, Jaetae

    2015-05-14

    We sought to evaluate the feasibility of molecular imaging using the human sodium iodide symporter (hNIS) gene as a reporter, in addition to the enhanced firefly luciferase (effluc) gene, for tracking dendritic cell (DCs) migration in living mice. A murine dendritic cell line (DC2.4) co-expressing hNIS and effluc genes (DC/NF) was established. For the DC-tracking study, mice received either parental DCs or DC/NF cells in the left or right footpad, respectively, and combined I-124 PET/CT and bioluminescence imaging (BLI) were performed. In vivo PET/CT imaging with I-124 revealed higher activity of the radiotracer in the draining popliteal lymph nodes (DPLN) of the DC/NF injection site at day 1 than DC injection site (p < 0.05). The uptake value further increased at day 4 (p < 0.005). BLI also demonstrated migration of DC/NF cells to the DPLNs at day 1 post-injection, and signals at the DPLNs were much higher at day 4. These data support the feasibility of hNIS reporter gene imaging in the tracking of DC migration to lymphoid organs in living mice. DCs expressing the NIS reporter gene could be a useful tool to optimize various strategies of cell-based immunotherapy.

  12. Development of luciferase expressing Leishmania donovani axenic amastigotes as primary model for in vitro screening of antileishmanial compounds.

    PubMed

    Ravinder; Bhaskar; Gangwar, Sonali; Goyal, Neena

    2012-12-01

    The development of new therapeutic leads against leishmaniasis relies primarily on screening of a large number of compounds on multiplication of clinically irrelevant transgenic promastigotes. The advent of the successful in vitro culture of axenic amastigotes allows the development of transgenic axenic amastigotes as a primary screen which can test compounds in a high throughput mode like promastigotes, still representative of the clinically relevant mammalian amastigotes stage. The present study reports the development of luciferase-tagged axenic amastigotes of Leishmania donovani, the causative agent of Indian Kala-azar, for in vitro drug screening. Luciferase expressing promastigotes were transformed to axenic amastigotes at a low pH and high temperature without the loss of luciferase expression. As compared to transgenic promastigotes, the luciferase expressing axenic amastigotes exhibited more sensitivity to antileishmanial drugs, particularly to pentavalent antimony (~2.8-fold) and also to the test compounds. Hence, the developed luciferase expressing axenic amastigotes make an ideal choice for high throughput drug screening for antileishmanial compounds.

  13. A sensor for adenosine triphosphate fabricated by laser-induced forward transfer of luciferase onto a poly(dimethylsiloxane) microchip

    NASA Astrophysics Data System (ADS)

    Tsuboi, Yasuyuki; Furuhata, Yosuke; Kitamura, Noboru

    2007-08-01

    Laser-induced forward transfer (LIFT) of the enzyme luciferase was explored as a potential technique to be used in the fabrication of a microchip adenosine triphosphate (ATP) sensor. Poly(dimethylsiloxane) (PDMS) was selected as the substrate for deposition of the luciferase. In comparison with other solid substrates, such as glass and polystyrene, it was found that the flexibility of PDMS made it a superior substrate for the immobilization of micro-spots of luciferase. LIFT of luciferase onto a PDMS substrate using a 355 nm laser was successfully carried out, while the bioactivity of the enzyme was maintained. Yellow luminescence ascribed to luciferase was observed from a transferred spot on the PDMS chip from the enzymatic reaction between luciferin and ATP. A microchip ATP sensor was also fabricated by attaching a small photodiode to the PDMS chip. On the basis of the fabricated microchip, the Michaelis-Menten relation between the luminescence intensity from the spot, and the ATP concentration was confirmed. The potential for fabricating biosensors using a combination of the LIFT technique with a PDMS substrate was shown to be very good.

  14. Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein.

    PubMed

    Branchini, Bruce R; Rosenberg, Justin C; Ablamsky, Danielle M; Taylor, Kelsey P; Southworth, Tara L; Linder, Samantha J

    2011-07-15

    We report here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyze yellow-green (560nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41nM for caspase 3, 1.0nM for thrombin, and 58nM for factor Xa were realized with a scanning fluorometer. Our results demonstrate for the first time that an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence can be employed to assay physiologically important protease activities.

  15. Enhanced microbial biomass assay using mutant luciferase resistant to benzalkonium chloride.

    PubMed

    Hattori, Noriaki; Sakakibara, Tatsuya; Kajiyama, Naoki; Igarashi, Toshinori; Maeda, Masako; Murakami, Seiji

    2003-08-15

    In a biomass assay based on adenosine 5(')-triphosphate (ATP) bioluminescence, extracellular ATP is removed; then intracellular ATP is extracted from the microorganism by an ATP extractant and subsequently reacted with luciferase. To provide a highly sensitive assay, the concentration of benzalkonium chloride (BAC) in the ATP extractant was optimized by using a mutant luciferase resistant to BAC. The use of 0.2% BAC, which was acceptable for the luciferase, simultaneously achieved the maximum extraction of intracellular ATP from microorganisms and the inactivation of the ATP-eliminating enzymes for removal of extracellular ATP. The detection limit (blank+3 SD) for ATP was 1.8x10(-14)M (1.8x10(-18)mol/assay) in the presence of the ATP extractant with coefficients of variation of 0.7 to 6.3%. The reagent system coupled with the ATP-eliminating enzymes allowed for the detection of 93 colony-forming units (CFU)/ml of Escherichia coli ATCC 25922, 170CFU/ml of Pseudomonas aeruginosa ATCC 27853, 170CFU/ml of Proteus mirabilis ATCC 29906, 68CFU/ml of Staphylococcus aureus ATCC 25923, and 7.7CFU/ml of Bacillus subtilis ATCC 6051. The yeast cell of Saccharomyces cerevisiae IFO 10217 could be detected at 1CFU/ml. With 54 kinds of microorganisms, the average ATP extraction efficiency compared to the trichloroacetic acid extraction method was 81.0% in 24 strains among gram-negative bacteria, 99.4% in 13 strains among gram-positive bacteria, and 97.0% in 17 strains among yeast. The ATP contents of the gram-negative bacteria, gram-positive bacteria, and yeasts ranged from 0.40 to 2.70x10(-18)mol/CFU (mean=1.5x10(-18)mol/CFU), from 0.41 to 16.7x10(-18)mol/CFU (mean=5.5x10(-18)mol/CFU), and from 0.714 to 54.6x10(-16)mol/CFU (mean=8.00x10(-16)mol/CFU), respectively.

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

    SciTech Connect

    Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng . E-mail: Dongfeng_pan@yahoo.com

    2005-09-09

    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{sup 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.

  17. Luciferase Genes as Reporter Reactions: How to Use Them in Molecular Biology?

    PubMed

    Cevenini, L; Calabretta, M M; Calabria, D; Roda, A; Michelini, E

    2016-01-01

    : The latest advances in molecular biology have made available several biotechnological tools that take advantage of the high detectability and quantum efficiency of bioluminescence (BL), with an ever-increasing number of novel applications in environmental, pharmaceutical, food, and forensic fields. Indeed, BL proteins are being used to develop ultrasensitive binding assays and cell-based assays, thanks to their high detectability and to the availability of highly sensitive BL instruments. The appealing aspect of molecular biology tools relying on BL reactions is their general applicability in both in vitro assays, such as cell cultures or purified proteins, and in vivo settings, such as in whole-animal BL imaging. The aim of this chapter is to provide the reader with an overview of state-of-the-art bioluminescent tools based on luciferase genes, highlighting molecular biology strategies that have been applied so far, together with some selected examples.

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

    PubMed Central

    Masser, Anna E.; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan

    2016-01-01

    Abstract 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 < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat‐shock promoter (PCYC1–HSE), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C‐terminus of a temperature‐sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

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

    PubMed

    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 < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE ), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd.

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

    PubMed

    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.

  1. Evaluation of a bioluminescent mouse model expressing aromatase PII-promoter-controlled luciferase as a tool for the study of endocrine disrupting chemicals

    SciTech Connect

    Rivest, Patricia Devine, Patrick J. Sanderson, J. Thomas

    2010-11-15

    Dysfunction of the enzyme aromatase (CYP19) is associated with endocrine pathologies such as osteoporosis, impaired fertility and development of hormone-dependent cancers. Certain endocrine disrupting chemicals affect aromatase expression and activity in vitro, but little is known about their ability to do so in vivo. We evaluated a bioluminescent mouse model (LPTA (registered)) CD-1-Tg(Cyp19-luc)-Xen) expressing luciferase under control of the gonadal aromatase pII promoter as an in vivo screening tool for chemicals that may affect aromatase expression. We studied the effects of forskolin, pregnant mare serum gonadotropin and atrazine in this model (atrazine was previously shown to induced pII-promoter-driven aromatase expression in H295R human adrenocortical carcinoma cells). About 2-4 out of every group of 10 male or female Cyp19-luc mice injected i.p. with 10 mg/kg forskolin had increased gonadal bioluminescence after 3-5 days compared to controls; the others appeared non-responsive. Similarly, about 4 per group of 9 individual females injected with pregnant mare serum gonadotropin had increased ovarian bioluminescence after 24 h. There was a statistically significant correlation between ovarian bioluminescence and plasma estradiol concentrations (n = 14; p = 0.022). Males exposed to a single dose of 100 mg/kg or males and females exposed to 5 daily injections of 30 mg/kg atrazine showed no change in gonadal bioluminescence over a 7 day period, but a significant interaction was found between atrazine (100 mg/kg) and time in female mice (p < 0.05; two-way ANOVA). Ex vivo luciferase activity in dissected organs was increased by forskolin in testis, epididymis and ovaries. Atrazine (30 mg/kg/day) increased (30%) luciferase activity significantly in epididymis only. In conclusion, certain individual Cyp19-luc mice are highly responsive to aromatase inducers, suggesting this model, with further optimization, may have potential as an in vivo screening tool for

  2. The rapid quantitation of the filamentous blue-green alga plectonema boryanum by the luciferase assay for ATP

    NASA Technical Reports Server (NTRS)

    Bush, V. N.

    1974-01-01

    Plectonema boryanum is a filamentous blue green alga. Blue green algae have a procaryotic cellular organization similar to bacteria, but are usually obligate photoautotrophs, obtaining their carbon and energy from photosynthetic mechanism similar to higher plants. This research deals with a comparison of three methods of quantitating filamentous populations: microscopic cell counts, the luciferase assay for ATP and optical density measurements.

  3. GENERATION OF TWO NOVEL CELL LINES THAT STABLY EXPRESS HAR AND FIREFLY LUCIFERASE GENES FOR ENDOCRINE SCREENING

    EPA Science Inventory

    Generation of Two Novel Cell Lines that Stably Express hAR and Firefly Luciferase Genes for Endocrine Screening
    K.L. Bobseine*1, W.R. Kelce2, P.C. Hartig*1, and L.E. Gray, Jr.1
    1USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC, 2Searle, Reproductive Toxicology Divi...

  4. Ratiometric bioluminescence indicators for monitoring cyclic adenosine 3',5'-monophosphate in live cells based on luciferase-fragment complementation.

    PubMed

    Takeuchi, Masaki; Nagaoka, Yasutaka; Yamada, Toshimichi; Takakura, Hideo; Ozawa, Takeaki

    2010-11-15

    Bioluminescent indicators for cyclic 3',5'-monophosphate AMP (cAMP) are powerful tools for noninvasive detection with high sensitivity. However, the absolute photon counts are affected substantially by adenosine 5'-triphosphate (ATP) and d-luciferin concentrations, limiting temporal analysis in live cells. This report describes a genetically encoded bioluminescent indicator for detecting intracellular cAMP based on complementation of split fragments of two-color luciferase mutants originated from click beetles. A cAMP binding domain of protein kinase A was connected with an engineered carboxy-terminal fragment of luciferase, of which ends were connected with amino-terminal fragments of green luciferase and red luciferase. We demonstrated that the ratio of green to red bioluminescence intensities was less influenced by the changes of ATP and d-luciferin concentrations. We also showed an applicability of the bioluminescent indicator for time-course and quantitative assessments of intracellular cAMP in living cells and mice. The bioluminescent indicator will enable quantitative analysis and imaging of spatiotemporal dynamics of cAMP in opaque and autofluorescent living subjects.

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

    SciTech Connect

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

    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 their

  6. Food-associated estrogenic compounds induce estrogen receptor-mediated luciferase gene expression in transgenic male mice.

    PubMed

    Ter Veld, Marcel G R; Zawadzka, E; van den Berg, J H J; van der Saag, Paul T; Rietjens, Ivonne M C M; Murk, Albertinka J

    2008-07-30

    The present paper aims at clarifying to what extent seven food-associated compounds, shown before to be estrogenic in vitro, can induce estrogenic effects in male mice with an estrogen receptor (ER)-mediated luciferase (luc) reporter gene system. The luc induction was determined in different tissues 8h after dosing the ER-luc male mice intraperitoneally (IP) or 14h after oral dosing. Estradiol-propionate (EP) was used as a positive control at 0.3 and 1mg/kg bodyweight (bw), DMSO as solvent control. The food-associated estrogenic compounds tested at non-toxic doses were bisphenol A (BPA) and nonylphenol (NP) (both at 10 and 50mg/kgbw), dichlorodiphenyldichloroethylene (p,p'-DDE; at 5 and 25mg/kgbw), quercetin (at 1.66 and 16.6mg/kgbw), di-isoheptyl phthalate (DIHP), di-(2-ethylhexyl) phthalate (DEHP) and di-(2-ethylhexyl) adipate (DEHA) all at 30 and 100mg/kgbw. In general IP dosing resulted in higher luc inductions than oral dosing. EP induced luc activity in the liver in a statistically significant dose-related way with the highest induction of all compounds tested which was 20,000 times higher than the induction by the DMSO-control. NP, DDE, DEHA and DIHP did not induce luc activity in any of the tissues tested. BPA induced luc in the liver up to 420 times via both exposure routes. BPA, DEHP and quercetin induced luc activity in the liver after oral exposure. BPA (50mg/kgbw IP) also induced luc activity in the testis, kidneys and tibia. The current study reveals that biomarker-responses in ER-luc male mice occur after a single oral exposure to food-associated estrogenic model compounds at exposure levels 10 to 10(4) times higher than the established TDI's for some of these compounds. Given the facts that (i) the present study did not include chronic exposure and that (ii) simultaneous exposure to multiple estrogenic compounds may be a realistic exposure scenario, it remains to be seen whether this margin is sufficiently high.

  7. Searching for Biomarkers: Humoral Response Profiling with Luciferase Immunoprecipitation Systems (LIPS)

    PubMed Central

    Burbelo, Peter D.; Ching, Kathryn H.; Bren, Kathleen E.; Iadarola, Michael J.

    2013-01-01

    B-cell mediated humoral responses are triggered in many human diseases including autoimmune, cancer, neurologic, and infectious diseases. However, the full exploitation of the information contained within a patient's antibody repertoire, for diagnosis, monitoring and even disease prediction has been limited due to the poor diagnostic performance of many immunoassay formats. We have developed Luciferase immunoprecipitation systems (LIPS) that harnesses light emitting proteins to generate high definition antibody profiles optimal for both diagnostics and biomarker discovery. Here we describe the results and implications from a range of LIPS antibody profiling studies performed in our laboratory. These include highly sensitive diagnostics for domestic and global pathogens, insights into infection-related diseases, discovery of new biomarkers for human diseases, subcategorization of symptoms and identification of pathogenic autoantibodies against self-proteins. These investigations highlight the types of humoral response profiles associated with different diseases, provide new information related to disease pathogenesis, and provide a framework for incorporating LIPS antibody profiling into global health initiatives and disease monitoring. PMID:21679112

  8. Urine and Serum Analysis of Consumed Curcuminoids Using an IκB-Luciferase Surrogate Marker Assay

    PubMed Central

    Ponnurangam, Sivapriya; Mondalek, Fadee G.; Govind, Janita; Subramaniam, Dharmalingam; Houchen, Courtney W.; Anant, Shrikant; Pantazis, Panayotis; Ramanujam, Rama P.

    2011-01-01

    Background Curcumin metabolites are detectable in body fluids such as serum and urine. We have developed a novel assay that can detect metabolites in such body fluids by measuring their effect on the nuclear factor kappa B/inhibitor of kappa B (NF-κB/IκB) pathway. Patients and Methods Fifteen healthy individuals were enrolled in the study and randomly assigned to two groups: control group (five) and curcumin group (ten). The test group ingested 8 g of the curcuminoids (C3-Complex™) with 16 oz of bottled water. Blood and urine were collected at 0, 4, 8, and 24 h after ingestion. Degradation of the NF-κB/IκB complex was detected by the Genetic Expression and Measurement (GEM™) assay using HCT116 cells stably transfected with PGL3-IκB firefly luciferase. Results Using our novel GEM assay, the five controls who had not taken curcumin were identified. Conclusion The GEM assay is a very sensitive and accurate non-invasive assay that could be utilized to detect metabolites in body fluids. It could also serve as a tool to determine participants’ compliance during clinical research studies. PMID:21164045

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

    NASA Technical Reports Server (NTRS)

    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.

  10. Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

    PubMed Central

    Close, Dan M.; Patterson, Stacey S.; Ripp, Steven; Baek, Seung J.; Sanseverino, John; Sayler, Gary S.

    2010-01-01

    Background 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. Methodology/Principal Findings 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 (FMNH2) 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. FMNH2 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. Conclusions/Significance 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. PMID:20805991

  11. Leishmania tropica experimental infection in the rat using luciferase-transfected parasites.

    PubMed

    Talmi-Frank, Dalit; Jaffe, Charles L; Nasereddin, Abedelmajeed; Baneth, Gad

    2012-06-08

    Leishmania tropica is the causative agent of zoonotic cutaneous leishmaniasis in different parts of the Old World. Although it is a common cause of disease in some areas of the world, there is insufficient knowledge on the pathogenicity of this parasite in mammalian hosts and animal models. L. tropica luciferase-transfected metacyclic-stage promastigotes were inoculated into the footpad or ear of Sprague Dawley (SD) rats. Parasite DNA was detected by kDNA real time PCR in the blood at varying levels from 2 days to 5 weeks post infection (PI) in the absence of clinical signs. Parasite DNA was found in the spleen of all rats at the end of the study, and the parasitic load was up to 40 times higher in the spleen when compared with inoculation sites. Parasites were cultured from the spleen, and skin inoculation sites 5 weeks PI. Bioluminescent parasites were observed by in vivo imaging at one day PI, but the technique was not sufficiently sensitive to follow parasite spread after this time. This study provides new evidence for the viscerotropic spread of L. tropica in the rat and demonstrates that the rat can serve as a model for persistent visceralizing infection with this parasite.

  12. A new blue-shifted luciferase from the Brazilian Amydetes fanestratus (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties.

    PubMed

    Viviani, Vadim R; Amaral, Danilo; Prado, Rogilene; Arnoldi, Frederico G C

    2011-12-01

    Firefly luciferases usually produce bioluminescence in the yellow-green region, with colors in the green and yellow-orange extremes of the spectrum being less common. Several firefly luciferases have already been cloned and sequenced, and site-directed mutagenesis studies have already identified important regions and residues for bioluminescence colors. However the structural determinants and mechanisms of bioluminescence colors turned out to be elusive, mainly when comparing luciferases with a high degree of divergence. Thus comparison of more similar luciferases producing colors in the two extremes of the spectrum could be revealing. The South-American fauna of fireflies remains largely unstudied, with some unique taxa that are not found anywhere else in the world and that produce a wide range of bioluminescence colors. Among them, fireflies of the genus Amydetes are especially interesting because its taxonomical status as an independent subfamily or as a tribe is not yet solved, and because they usually produce a continuous bright blue-shifted bioluminescence. In this work we cloned the cDNA for the luciferase of the Atlantic rain forest Amydetes fanestratus firefly, which is found near Sorocaba municipality (São Paulo, Brazil). Despite showing a higher degree of identity with the South-American Cratomorphus, the European Lampyris and the Asiatic Pyrocoelia, phylogenetical analysis of the luciferase sequence support the inclusion of Amydetes as an independent subfamily. Amydetes luciferase displays one of the most blue-shifted emission spectra (λ(max) = 538 nm) among beetle luciferases, with lower pH-sensitivity and higher affinity for ATP when compared to other luciferases, making this luciferase attractive for sensitive ATP and reporter assays.

  13. A novel luciferase fusion protein for highly sensitive optical imaging: from single-cell analysis to in vivo whole-body bioluminescence imaging.

    PubMed

    Mezzanotte, Laura; Blankevoort, Vicky; Löwik, Clemens W G M; Kaijzel, Eric L

    2014-09-01

    Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 10(3) and 5 × 10(4) cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging.

  14. Kinetics of inhibition of firefly luciferase by dehydroluciferyl-coenzyme A, dehydroluciferin and L-luciferin.

    PubMed

    da Silva, Luís Pinto; da Silva, Joaquim C G Esteves

    2011-06-01

    The inhibition mechanisms of the firefly luciferase (Luc) by three of the most important inhibitors of the reactions catalysed by Luc, dehydroluciferyl-coenzyme A (L-CoA), dehydroluciferin (L) and L-luciferin (L-LH(2)) were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 μM) has been measured in 50 mM Hepes buffer (pH = 7.5), 10 nM Luc, 250 μM ATP and D-luciferin (D-LH(2), from 3.75 up to 120 μM). Nonlinear regression analysis with the appropriate kinetic models (Henri-Michaelis-Menten and William-Morrison equations) reveals that L-CoA is a non-competitive inhibitor of Luc (K(i) = 0.88 ± 0.03 μM), L is a tight-binding uncompetitive inhibitor (K(i) = 0.00490 ± 0.00009 μM) and L-LH(2) acts as a mixed-type non-competitive-uncompetitive inhibitor (K(i) = 0.68 ± 0.14 μM and αK(i) = 0.34 ± 0.16 μM). The K(m) values obtained for L-CoA, L and L-LH(2) were 16.1 ± 1.0, 16.6 ± 2.3 and 14.4 ± 0.96 μM, respectively. L and L-LH(2) are strong inhibitors of Luc, which may indicate an important role for these compounds in Luc characteristic flash profile. L-CoA K(i) supports the conclusion that CoA can stimulate the light emission reaction by provoking the formation of a weaker inhibitor.

  15. Robust circadian rhythms in organoid cultures from PERIOD2::LUCIFERASE mouse small intestine.

    PubMed

    Moore, Sean R; Pruszka, Jill; Vallance, Jefferson; Aihara, Eitaro; Matsuura, Toru; Montrose, Marshall H; Shroyer, Noah F; Hong, Christian I

    2014-09-01

    Disruption of circadian rhythms is a risk factor for several human gastrointestinal (GI) diseases, ranging from diarrhea to ulcers to cancer. Four-dimensional tissue culture models that faithfully mimic the circadian clock of the GI epithelium would provide an invaluable tool to understand circadian regulation of GI health and disease. We hypothesized that rhythmicity of a key circadian component, PERIOD2 (PER2), would diminish along a continuum from ex vivo intestinal organoids (epithelial 'miniguts'), nontransformed mouse small intestinal epithelial (MSIE) cells and transformed human colorectal adenocarcinoma (Caco-2) cells. Here, we show that bioluminescent jejunal explants from PERIOD2::LUCIFERASE (PER2::LUC) mice displayed robust circadian rhythms for >72 hours post-excision. Circadian rhythms in primary or passaged PER2::LUC jejunal organoids were similarly robust; they also synchronized upon serum shock and persisted beyond 2 weeks in culture. Remarkably, unshocked organoids autonomously synchronized rhythms within 12 hours of recording. The onset of this autonomous synchronization was slowed by >2 hours in the presence of the glucocorticoid receptor antagonist RU486 (20 μM). Doubling standard concentrations of the organoid growth factors EGF, Noggin and R-spondin enhanced PER2 oscillations, whereas subtraction of these factors individually at 24 hours following serum shock produced no detectable effects on PER2 oscillations. Growth factor pulses induced modest phase delays in unshocked, but not serum-shocked, organoids. Circadian oscillations of PER2::LUC bioluminescence aligned with Per2 mRNA expression upon analysis using quantitative PCR. Concordant findings of robust circadian rhythms in bioluminescent jejunal explants and organoids provide further evidence for a peripheral clock that is intrinsic to the intestinal epithelium. The rhythmic and organotypic features of organoids should offer unprecedented advantages as a resource for elucidating the role

  16. A mouse model of luciferase-transfected stromal cells of giant cell tumor of bone.

    PubMed

    Lau, Carol P Y; Wong, Kwok Chuen; Huang, Lin; Li, Gang; Tsui, Stephen K W; Kumta, Shekhar Madhukar

    2015-11-01

    A major barrier towards the study of the effects of drugs on Giant Cell Tumor of Bone (GCT) has been the lack of an animal model. In this study, we created an animal model in which GCT stromal cells survived and functioned as proliferating neoplastic cells. A proliferative cell line of GCT stromal cells was used to create a stable and luciferase-transduced cell line, Luc-G33. The cell line was characterized and was found that there were no significant differences on cell proliferation rate and recruitment of monocytes when compared with the wild type GCT stromal cells. We delivered the Luc-G33 cells either subcutaneously on the back or to the tibiae of the nude mice. The presence of viable Luc-G33 cells was assessed using real-time live imaging by the IVIS 200 bioluminescent imaging (BLI) system. The tumor cells initially propagated and remained viable on site for 7 weeks in the subcutaneous tumor model. We also tested in vivo antitumor effects of Zoledronate (ZOL) and Geranylgeranyl transferase-I inhibitor (GGTI-298) alone or their combinations in Luc-G33-transplanted nude mice. ZOL alone at 400 µg/kg and the co-treatment of ZOL at 400 µg/kg and GGTI-298 at 1.16 mg/kg reduced tumor cell viability in the model. Furthermore, the anti-tumor effects by ZOL, GGTI-298 and the co-treatment in subcutaneous tumor model were also confirmed by immunohistochemical (IHC) staining. In conclusion, we established a nude mice model of GCT stromal cells which allows non-invasive, real-time assessments of tumor development and testing the in vivo effects of different adjuvants for treating GCT.

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

    PubMed Central

    Close, Dan; Xu, Tingting; Smartt, Abby; Rogers, Alexandra; Crossley, Robert; Price, Sarah; Ripp, Steven; Sayler, Gary

    2012-01-01

    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. PMID:22368493

  18. Two techniques for eliminating luminol interference material and flow system configurations for luminol and firefly luciferase systems

    NASA Technical Reports Server (NTRS)

    Thomas, R. R.

    1976-01-01

    Two methods for eliminating luminol interference materials are described. One method eliminates interference from organic material by pre-reacting a sample with dilute hydrogen peroxide. The reaction rate resolution method for eliminating inorganic forms of interference is also described. The combination of the two methods makes the luminol system more specific for bacteria. Flow system designs for both the firefly luciferase and luminol bacteria detection systems are described. The firefly luciferase flow system incorporating nitric acid extraction and optimal dilutions has a functional sensitivity of 3 x 100,000 E. coli/ml. The luminol flow system incorporates the hydrogen peroxide pretreatment and the reaction rate resolution techniques for eliminating interference. The functional sensitivity of the luminol flow system is 1 x 10,000 E. coli/ml.

  19. A method to rapidly and accurately compare relative efficacies of non-invasive imaging reporter genes in a mouse model, and its application to luciferase reporters

    PubMed Central

    Gil, Jose S.; Machado, Hidevaldo B.; Herschman, Harvey R.

    2013-01-01

    Purpose Our goal is to develop a simple, quantitative, robust method to compare the efficacy of imaging reporter genes in culture and in vivo. We describe an adenoviral vector-liver transduction procedure, and compare the luciferase reporter efficacies. Procedures Alternative reporter genes are expressed in a common adenoviral vector. Vector amounts used in vivo are based on cell culture titrations, ensuring the same transduction efficacy is used for each vector. After imaging, in vivo and in vitro values are normalized to hepatic vector transduction using quantitative real-time PCR. Results We assayed standard firefly luciferase (FLuc), enhanced firefly luciferase (EFLuc), luciferase 2 (Luc2), humanized Renilla luciferase (hRLuc), Renilla luciferase 8.6-535 (RLuc8.6), and a membrane-bound Gaussia luciferase variant (extGLuc) in cell culture and in vivo. We observed a greater that 100-fold increase in bioluminescent signal for both EFLuc and Luc2 when compared to FLuc, and a greater than 106-fold increase for RLuc8.6 when compared to hRLuc. ExtGLuc was not detectable in liver. Conclusions Our findings contrast, in some cases, with conclusions drawn in prior comparisons of these reporter genes, and demonstrate the need for a standardized method to evaluate alternative reporter genes in vivo. Our procedure can be adapted for reporter genes that utilize alternative imaging modalities (fluorescence, bioluminescence, MRI, SPECT, PET). PMID:21850545

  20. A mouse model for studying the clearance of hepatitis B virus in vivo using a luciferase reporter.

    PubMed

    Liang, Sheng-qiang; Du, Juan; Yan, Hu; Zhou, Qian-qian; Zhou, Yong; Yuan, Zhen-nan; Yan, Shao-duo; Fu, Qiu-xia; Wang, Xiao-hui; Jia, Shuai-zheng; Peng, Jian-chun; Zhang, Yang-gen; Zhan, Lin-sheng

    2013-01-01

    Hepatitis B virus(HBV) infection remains a global problem, despite the effectiveness of the Hepatitis B vaccine in preventing infection. The resolution of Hepatitis B virus infection has been believed to be attributable to virus-specific immunity. In vivo direct evaluation of anti-HBV immunity in the liver is currently not possible. We have developed a new assay system that detects HBV clearance in the liver after the hydrodynamic transfer of a reporter gene and over-length, linear HBV DNA into hepatocytes, followed by bioluminescence imaging of the reporter gene (Fluc). We employed bioluminescence detection of luciferase expression in HBV-infected hepatocytes to measure the Hepatitis B core antigen (HBcAg)-specific immune responses directed against these infected hepatocytes. Only HBcAg-immunized, but not mock-treated, animals decreased the amounts of luciferase expression, HBsAg and viral DNA from the liver at day 28 after hydrodynamic infection with over-length HBV DNA, indicating that control of luciferase expression correlates with viral clearance from infected hepatocytes.

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

    NASA Astrophysics Data System (ADS)

    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.

  2. A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Guéren

    PubMed Central

    Igarashi, Masayuki; Doe, Matsumi; Tamaru, Aki; Kinoshita, Naoko; Ogura, Yoshitoshi; Iwamoto, Tomotada; Sawa, Ryuichi; Umekita, Maya; Enany, Shymaa; Nishiuchi, Yukiko; Osada-Oka, Mayuko; Hayashi, Tetsuya; Niki, Mamiko; Tateishi, Yoshitaka; Hatano, Masaki

    2015-01-01

    Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multi-drug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904–1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 μg/ml, 0.5 μg/ml, and 2.0–7.5 μg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904–1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904–1. Our method provides a

  3. Demonstration of protein-fragment complementation assay using purified firefly luciferase fragments

    PubMed Central

    2013-01-01

    Background Human interactome is predicted to contain 150,000 to 300,000 protein-protein interactions, (PPIs). Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI, as well as Förster resonance energy transfer (FRET). To date, successful applications of firefly luciferase (Fluc)-based PCA have been reported in vivo, in cultured cells and in cell-free lysate, owing to its high sensitivity, high signal-to-background (S/B) ratio, and reversible response. Here we show the assay also works with purified proteins with unexpectedly rapid kinetics. Results Split Fluc fragments both fused with a rapamycin-dependently interacting protein pair were made and expressed in E. coli system, and purified to homogeneity. When the proteins were used for PCA to detect rapamycin-dependent PPI, they enabled a rapid detection (~1 s) of PPI with high S/B ratio. When Fn7-8 domains (7 nm in length) that was shown to abrogate GFP mutant-based FRET was inserted between split Fluc and FKBP12 as a rigid linker, it still showed some response, suggesting less limitation in interacting partner’s size. Finally, the stability of the probe was investigated. Preincubation of the probes at 37 degreeC up to 1 h showed marked decrease of the luminescent signal to 1.5%, showing the limited stability of this system. Conclusion Fluc PCA using purified components will enable a rapid and handy detection of PPIs with high S/B ratio, avoiding the effects of concomitant components. Although the system might not be suitable for large-scale screening due to its limited stability, it can detect an interaction over larger distance than by FRET. This would be the first demonstration of Fluc PCA in vitro, which has a distinct advantage over other PPI assays. Our system enables detection of direct PPIs without risk of perturbation by PPI mediators in the complex cellular milieu. PMID:23536995

  4. Interconversion of ketoprofen recognition in firefly luciferase-catalyzed enantioselective thioesterification reaction using from Pylocoeria miyako (PmL) and Hotaria parvura (HpL) just by mutating two amino acid residues.

    PubMed

    Kato, Dai-ichiro; Hiraishi, Yoshihiro; Maenaka, Mika; Yokoyama, Keisuke; Niwa, Kazuki; Ohmiya, Yoshihiro; Takeo, Masahiro; Negoro, Seiji

    2013-11-01

    We identified the critical amino acid residues for substrate recognition using two firefly luciferases from Pylocoeria miyako (PmL) and Hotaria parvura (HpL), as these two luciferase enzymes exhibit different activities toward ketoprofen. Specifically, PmL can catalyze the apparent enantioselective thioesterification reaction, while HpL cannot. By comparing the amino acid sequences around the active site, we identified two residues (I350 and M397 in PmL and F351 and S398 in HpL) that were different between the two enzymes, and the replacement of these amino acids resulted in changing the ketoprofen recognition pattern. The inactive HpL was converted to the active enzyme toward ketoprofen and vice versa for PmL. These residues also affected the enantioselectivity toward ketoprofen; however, the bioluminescent color was not affected. In addition, using molecular dynamics calculations, the replacement of these two amino acids induced changes in the state of hydrogen bonding between ketoprofen and the S349 side chain through the active site water. As S349 is not considered to influence color tuning, these changes specifically caused the differences in ketoprofen recognition in the enzyme.

  5. Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ-ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation.

    PubMed

    Jobe, Timothy O; Sung, Dong-Yul; Akmakjian, Garo; Pham, Allis; Komives, Elizabeth A; Mendoza-Cózatl, David G; Schroeder, Julian I

    2012-06-01

    Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M(2) seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione depletion

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

    PubMed

    Noguchi, Takako; Ikeda, Masaaki; Ohmiya, Yoshihiro; Nakajima, Yoshihiro

    2012-01-01

    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 this co

  7. Optimized chromatographic and bioluminescent methods for inorganic pyrophosphate based on its conversion to ATP by firefly luciferase.

    PubMed

    Marques, Simone M; Peralta, Filipe; Esteves da Silva, Joaquim C G

    2009-02-15

    Two new methods for inorganic pyrophosphate (PPi) quantification are described. They are based on the enzymatic conversion of PPi into ATP by firefly luciferase (Luc, E.C. 1.13.12.7) in the presence of dehydroluciferyl-adenylate (L-AMP) followed by the determination of ATP by one of two different procedures, either UV-monitored (260 nm) ion-pair-HPLC (IP-HPLC) (method A) or luciferase-dependent bioluminescence in the presence of its substrate, firefly luciferin (D-LH(2)) (method B). These methods were subjected to optimization using experimental design methodologies to obtain optimum values for the selected factors: method A-incubation time (t(inc)=15 min), inactivation time of the enzyme (t(inac)=2 min), pH of the reaction mixture (pH 7.50) and the concentrations of L-AMP ([L-AMP]=40 microM) and luciferase ([Luc]=0.1 microM); method B-concentrations of L-AMP ([L-AMP]=2 microM), luciferase ([Luc]=50 nM) and luciferin ([LH(2)]=30 microM). Method A has a linear response over the range of 0.1-20 microM of PPi, with a limit of detection (LOD) of 0.5 microM and a limit of quantitation (LOQ) of 1.8 microM. Precision, expressed as relative standard deviation (R.S.D.), is 7.4% at 1 microM PPi and 5.9% at 8 microM PPi. Method B has a linear response over the range of 0.75-6.0 microM of PPi, with LOD and LOQ of 0.624 and 2.23 microM, respectively, and a R.S.D. of 5.1% at 2.5 microM PPi and 4.9% at 5 microM PPi. Under optimized conditions sensitive and robust methods can be obtained for the analysis of PPi impurities in commercial nucleotides and tripolyphosphate (P(3)).

  8. Identification of circadian-clock-regulated enhancers and genes of Drosophila melanogaster by transposon mobilization and luciferase reporting of cyclical gene expression.

    PubMed Central

    Stempfl, Thomas; Vogel, Marion; Szabo, Gisela; Wülbeck, Corinna; Liu, Jian; Hall, Jeffrey C; Stanewsky, Ralf

    2002-01-01

    A new way was developed to isolate rhythmically expressed genes in Drosophila by modifying the classic enhancer-trap method. We constructed a P element containing sequences that encode firefly luciferase as a reporter for oscillating gene expression in live flies. After generation of 1176 autosomal insertion lines, bioluminescence screening revealed rhythmic reporter-gene activity in 6% of these strains. Rhythmically fluctuating reporter levels were shown to be altered by clock mutations in genes that specify various circadian transcription factors or repressors. Intriguingly, rhythmic luminescence in certain lines was affected by only a subset of the pacemaker mutations. By isolating genes near 13 of the transposon insertions and determining their temporal mRNA expression pattern, we found that four of the loci adjacent to the trapped enhancers are rhythmically expressed. Therefore, this approach is suitable for identifying genetic loci regulated by the circadian clock. One transposon insert caused a mutation in the rhythmically expressed gene numb. This novel numb allele, as well as previously described ones, was shown to affect the fly's rhythm of locomotor activity. In addition to its known role in cell fate determination, this gene and the phosphotyrosine-binding protein it encodes are likely to function in the circadian system. PMID:11861563

  9. A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase tag insertion at the 3′end using CRISPR-Cas9 genome editing technique

    PubMed Central

    Basu, Sambuddha; Adams, Levi; Guhathakurta, Subhrangshu; Kim, Yoon-Seong

    2017-01-01

    α-synuclein (α-SYN) is a major pathologic contributor to Parkinson’s disease (PD). Multiplication of α-SYN encoding gene (SNCA) is correlated with early onset of the disease underlining the significance of its transcriptional regulation. Thus, monitoring endogenous transcription of SNCA is of utmost importance to understand PD pathology. We developed a stable cell line expressing α-SYN endogenously tagged with NanoLuc luciferase reporter using CRISPR/Cas9-mediated genome editing. This allows efficient measurement of transcriptional activity of α-SYN in its native epigenetic landscape which is not achievable using exogenous transfection-based luciferase reporter assays. The NanoLuc activity faithfully monitored the transcriptional regulation of SNCA following treatment with different drugs known to regulate α-SYN expression; while exogenous promoter-reporter assays failed to reproduce the similar outcomes. To our knowledge, this is the first report showing endogenous monitoring of α-SYN transcription, thus making it an efficient drug screening tool that can be used for therapeutic intervention in PD. PMID:28374838

  10. Generation of a recombinant classical swine fever virus stably expressing the firefly luciferase gene for quantitative antiviral assay.

    PubMed

    Shen, Liang; Li, Yongfeng; Chen, Jianing; Li, Chao; Huang, Junhua; Luo, Yuzi; Sun, Yuan; Li, Su; Qiu, Hua-Ji

    2014-09-01

    Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious swine disease leading to significant economic losses worldwide. Vaccines are widely used to control the disease, and no CSFV-specific antivirals are currently available. To facilitate anti-CSFV molecule discovery, we developed a reporter virus CSFV-N(pro)Fluc stably expressing the firefly luciferase (Fluc) gene in the N(pro) gene. The reporter virus enabled more sensitive and convenient detection of the N(pro) protein expression and the viral replication by luciferase reporter assay than by traditional methods. The CSFV N(pro) protein was detectable as early as 4.5h post-infection. As a proof-of-concept for its utility in rapid antiviral screening, this reporter virus was used to quantify anti-CSFV neutralizing antibodies of 50 swine sera and to assess 12 small interfering RNAs targeting different regions of the CSFV genome. The results were comparable to those obtained by traditional methods. Taken together, the reporter virus CSFV-N(pro)Fluc represents a useful tool for rapid and quantitative screening and evaluation of antivirals against CSFV.

  11. Nucleocytoplasmic transport of luciferase gene mRNA requires CRM1/Exportin1 and RanGTPase.

    PubMed

    Kimura, Tominori; Hashimoto, Iwao; Nishikawa, Masao; Yamada, Hisao

    2009-06-01

    Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) protein was shown to reduce the expression level of the co-transfected luciferase reporter gene (luc+) introduced to monitor transfection efficiency. We studied the mechanism of the inhibitory Rev effect. The effect, caused by nuclear retention of luc+ mRNA, was reversed if rev had a point mutation that makes its nuclear export signal (NES) unable to associate with cellular transport factors. The Rev NES receptor CRM1 (chromosome region maintenance 1)-specific inhibitor, leptomycin B, blocked luc+ mRNA export. This finding was also supported by the overexpression of delta CAN, another specific CRM1 inhibitor that caused inhibition of luciferase gene expression. Experiments involving tsBN2 cells, which have a temperature-sensitive RCC1 (regulator of chromosome condensation 1) allele, demonstrated that luc+ expression required generation of the GTP-bound form of RanGTPase (RanGTP) by RCC1. The constitutive transport element (CTE)-mediated nuclear export of luc+ mRNA was found to also depend upon RanGTP. Nuclear export of luc+ mRNA is thus suggested to involve CRM1 and RanGTP, which Rev employs to transport viral mRNA. The Rev effect is therefore considered to involve competition between two molecules for common transport factors.

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

    PubMed

    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.

  13. Quantitative Analysis of Human T-Lymphotropic Virus Type 1 (HTLV-1) Infection Using Co-Culture with Jurkat LTR-Luciferase or Jurkat LTR-GFP Reporter Cells.

    PubMed

    Alais, Sandrine; Dutartre, Hélène; Mahieux, Renaud

    2017-01-01

    Unlike HIV-1, HTLV-1 viral transmission requires cell-to-cell contacts, while cell-free virions are poorly infectious and almost absent from body fluids. Though the virus uses three nonexclusive mechanisms to infect new target cells: (1) MTOC polarization followed by formation of a virological synapse and viral transfer into a synaptic cleft, (2) genesis of a viral biofilm and its transfer of embedded viruses, or (3) HTLV-1 transmission using conduits. The Tax transactivator and the p8 viral proteins are involved in virological synapse and nanotube formation respectively.HTLV-1 transcription from the viral promoter (i.e., LTR) requires the Tax protein that is absent from the viral particle and is expressed after productive infection. The present chapter focuses on a series of protocols used to quantify HTLV-1 de novo infection of target cells. These techniques do not discriminate between the different modes of transmission, but allow an accurate measure of productive infection. We used cell lines that are stably transfected with LTR-GFP or LTR-luciferase plasmids and quantified Green Fluorescent Protein expression or luciferase activity, since both of them reflect Tax expression.

  14. Minicircle HBV cccDNA with a Gaussia luciferase reporter for investigating HBV cccDNA biology and developing cccDNA-targeting drugs

    PubMed Central

    Li, Feng; Cheng, Liang; Murphy, Christopher M.; Reszka-Blanco, Natalia J.; Wu, Yaxu; Chi, Liqun; Hu, Jianming; Su, Lishan

    2016-01-01

    Chronic Hepatitis B Virus (HBV) infection is generally not curable with current anti-viral drugs. Virus rebounds after stopping treatment from the stable HBV covalently-closed-circular DNA (cccDNA). The development of drugs that directly target cccDNA is hampered by the lack of robust HBV cccDNA models. We report here a novel HBV cccDNA technology that will meet the need. We engineered a minicircle HBV cccDNA with a Gaussia Luciferase reporter (mcHBV-GLuc cccDNA), which serves as a surrogate to measure cccDNA activity. The mcHBV-GLuc cccDNA was easily produced in bacteria, and it formed minichromosomes as HBV cccDNA episome DNA does when it was transfected into human hepatocytes. Compared to non-HBV minicircle plasmids, mcHBV-GLuc cccDNA showed persistent HBV-GLuc activity and HBx-dependent gene expression. Importantly, the mcHBV-GLuc cccDNA showed resistance to interferons (IFN) treatment, indicating its unique similarity to HBV cccDNA that is usually resistant to long-term IFN treatment in chronic HBV patients. Most importantly, GLuc illuminates cccDNA as a surrogate of cccDNA activity, providing a very sensitive and quick method to detect trace amount of cccDNA. The mcHBV-GLuc cccDNA model is independent of HBV infection, and will be valuable for investigating HBV cccDNA biology and for developing cccDNA-targeting drugs. PMID:27819342

  15. Construction of a cytosolic firefly luciferase reporter cassette for use in PCR-mediated gene deletion and fusion in Saccharomyces cerevisiae.

    PubMed

    Ainsworth, W B; Rome, C M; Hjortsø, M A; Benton, M G

    2012-12-01

    Monitoring promoter response to environmental changes using reporter systems has provided invaluable information regarding cellular state. With the development of in vivo luciferase reporter systems, inexpensive, sensitive and accurate promoter assays have been developed without the variability reported between in vitro samplings. Current luciferase reporter systems, however, are largely inflexible to modifications to the promoter of interest. To overcome problems in flexibility and stability of these expression vectors, we report the creation of a novel vector system which introduces a cytosol-localized Photinus pyralis luciferase [LUC*(-SKL)] capable of one-step, in vivo measurements into a promoter-reporter system via PCR-based gene deletion and fusion. After introduction of the reporter under HUG1 promoter control, cytosolic localization was confirmed by fluorescence microscopy. The dose-response of this novel construct was then compared with that of a similar HUG1Δ::yEGFP1 promoter-reporter system and shown to give a similar response pattern.

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

    NASA Astrophysics Data System (ADS)

    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

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

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed Central

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

    2016-01-01

    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 that of drug delivery carriers upon administration in the blood circulation, which will complicate the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction of near infrared (NIR) fluorophores to investigate the respective PK profiles of 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 is interesting to uncover 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 a 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. PMID:26818100

  19. Photoactivation of a psoralen-blocked luciferase gene by blue light.

    PubMed

    Stafforst, Thorsten; Stadler, Jens M

    2013-11-18

    A single psoralen cross-link completely blocks expression of a gene. An aminopyrene derivative has been found that allows the efficient light-triggered activation of cross-linked genes by UV or blue light. This paves the way to apply such psoralen cross-links for the photocleavable protection of nucleic acids.

  20. Bacterial Luciferase: Determination of the Structure by X-Ray Diffraction

    DTIC Science & Technology

    1994-05-20

    protein , since it permits distinction between (first- in the refolding mixture and varying the time of refolding order) folding processes of the...We propose that the active confomation of this protein is at only one of several possible energy minima, and that during the process of refolding In... evaluation of the effects of protein - protein interactions on the structures of the individual subunits involved in the oligomer. 2. We performed

  1. Oxyluciferin, a luminescence product of firefly luciferase, is enzymatically regenerated into luciferin.

    PubMed

    Gomi, K; Kajiyama, N

    2001-09-28

    The activity regenerating luciferin from the luminescent product oxyluciferin was found in the protein fraction of a lantern extract from Photinus pyralis. The protein, luciferin-regenerating enzyme (LRE), was purified to homogeneity by ammonium sulfate precipitation followed by successive column chromatography on Ultrogel AcA34, S-Sepharose FF, Q-Sepharose FF, TSKgel super Q 5pw and TSKgel G3000 SW(XL). This enzyme was a single polypeptide with a molecular mass of 38 kDa. LRE converted oxyluciferin to 2-cyano-6-hydroxybenzothiazole and thioglycolic acid. In the presence of d-cysteine, 2-cyano-6-hydroxybenzothiazole was turned over into luciferin. The same activities were detected in the extracts from two Japanese fireflies, Luciola cruciata and Luciola lateralis. We have cloned a cDNA encoding LRE from poly(A)+ RNA of the lantern of P. pyralis using reverse transcription-polymerase chain reaction, 5'-RACE (rapid amplification of cDNA ends) and 3'-RACE. The primary structure of LRE from P. pyralis deduced from the nucleotide sequence was shown to consist of 308 amino acids with a molecular weight of 33,619. The cDNA was successfully expressed under the control of the tac promoter in Escherichia coli.

  2. GENERATION OF TWO STABLE CELL LINES THAT EXPRESS HER-ALPHA OR HER-ALPHA AND -BETA AND FIREFLY LUCIFERASE GENES FOR ENDOCRINE SCREENING

    EPA Science Inventory

    Generation of Two Stable Cell Lines that Express hERa or
    hERa and b and Firefly Luciferase Genes for Endocrine Screening

    K.L. Bobseine*1, W.R. Kelce2, P.C. Hartig*1, and L.E. Gray, Jr.1

    1USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC, 2Searle, Reprod...

  3. A new orange emitting luciferase from the Southern-Amazon Pyrophorus angustus (Coleoptera: Elateridae) click-beetle: structure and bioluminescence color relationship, evolutional and ecological considerations.

    PubMed

    Amaral, Danilo T; Oliveira, Gabriela; Silva, Jaqueline R; Viviani, Vadim R

    2016-09-31

    Bioluminescent click-beetles display a wide variation of bioluminescence colors ranging from green to orange, including an unusual intra-specific color variation in the Jamaican Pyrophorus plagiophthalamus. Recently, we collected individuals of the Pyrophorus angustus species from the Southern Amazon forest, in Brazil, which displays an orange light emitting abdominal lantern. This species was also previously described from Central America, but displaying a bioluminescence spectrum from 536 nm (dorsal) to 578 nm (ventral). The biogeographic variation of the bioluminescence color in this species could be an adaptation to environmental reflectance and inter/intraspecific sexual competition. Here, we cloned, sequenced, characterized and performed site-direct mutagenesis of this new orange emitting luciferase. The in vitro luciferase spectrum displayed a peak at 594 nm, KM values for ATP and d-luciferin of 160 μM and 17 μM, respectively, and an optimum pH of approximately 8.5. Comparative multialignment and site-directed mutagenesis using different color emitting click-beetle luciferases from P. angustus, Fulgeochlizus bruchi and Pyrearinus termitilluminans luciferases cloned by our group showed an integral role of residue 247 in bioluminescence color modulation.

  4. Application of the luciferin-luciferase enzyme system for determination of adenosine triphosphate (ATP) to studies on the mechanisms of herbicide action

    NASA Technical Reports Server (NTRS)

    St.john, J. B.

    1975-01-01

    The luciferin-luciferase enzyme system for determination of ATP is valuable for studies on the mechanisms of herbicide action. Investigations using this system have shown that certain herbicides may act by interfering with ATP production or by blocking ATP use, or by both mechanisms.

  5. The transfer of reduced flavin mononucleotide from LuxG oxidoreductase to luciferase occurs via free diffusion.

    PubMed

    Tinikul, Ruchanok; Pitsawong, Warintra; Sucharitakul, Jeerus; Nijvipakul, Sarayut; Ballou, David P; Chaiyen, Pimchai

    2013-10-01

    Bacterial luciferase (LuxAB) is a two-component flavin mononucleotide (FMN)-dependent monooxygenase that catalyzes the oxidation of reduced FMN (FMNH(-)) and a long-chain aliphatic aldehyde by molecular oxygen to generate oxidized FMN, the corresponding aliphatic carboxylic acid, and concomitant emission of light. The LuxAB reaction requires a flavin reductase to generate FMNH(-) to serve as a luciferin in its reaction. However, FMNH(-) is unstable and can react with oxygen to generate H2O2, so that it is important to transfer it efficiently to LuxAB. Recently, LuxG has been identified as a NADH:FMN oxidoreductase that supplies FMNH(-) to luciferase in vivo. In this report, the mode of transfer of FMNH(-) between LuxG from Photobacterium leiognathi TH1 and LuxABs from both P. leiognathi TH1 and Vibrio campbellii (PlLuxAB and VcLuxAB, respectively) was investigated using single-mixing and double-mixing stopped-flow spectrophotometry. The oxygenase component of p-hydroxyphenylacetate hydroxylase (C2) from Acinetobacter baumannii, which has no structural similarity to LuxAB, was used to measure the kinetics of release of FMNH(-) from LuxG. With all FMNH(-) acceptors used (C2, PlLuxAB, and VcLuxAB), the kinetics of FMN reduction on LuxG were the same, showing that LuxG releases FMNH(-) with a rate constant of 4.5-6 s(-1). Our data showed that the kinetics of binding of FMNH(-)to PlLuxAB and VcLuxAB and the subsequent reactions with oxygen were the same with either free FMNH(-) or FMNH(-) generated in situ by LuxG. These results strongly suggest that no complexes between LuxG and the various species are necessary to transfer FMNH(-) to the acceptors. The kinetics of the overall reactions and the individual rate constants correlate well with a free diffusion model for the transfer of FMNH(-) from LuxG to either LuxAB.

  6. Mass spectrometry analysis and transcriptome sequencing reveal glowing squid crystal proteins are in the same superfamily as firefly luciferase

    PubMed Central

    Gimenez, Gregory; Metcalf, Peter; Paterson, Neil G.; Sharpe, Miriam L.

    2016-01-01

    The Japanese firefly squid Hotaru-ika (Watasenia scintillans) produces intense blue light from photophores at the tips of two arms. These photophores are densely packed with protein microcrystals that catalyse the bioluminescent reaction using ATP and the substrate coelenterazine disulfate. The squid is the only organism known to produce light using protein crystals. We extracted microcrystals from arm tip photophores and identified the constituent proteins using mass spectrometry and transcriptome libraries prepared from arm tip tissue. The crystals contain three proteins, wsluc1–3, all members of the ANL superfamily of adenylating enzymes. They share 19 to 21% sequence identity with firefly luciferases, which produce light using ATP and the unrelated firefly luciferin substrate. We propose that wsluc1–3 form a complex that crystallises inside the squid photophores, and that in the crystal one or more of the proteins catalyses the production of light using coelenterazine disulfate and ATP. These results suggest that ANL superfamily enzymes have independently evolved in distant species to produce light using unrelated substrates. PMID:27279452

  7. Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing.

    PubMed

    Decock, Marie; El Haylani, Laetitia; Stanga, Serena; Dewachter, Ilse; Octave, Jean-Noël; Smith, Steven O; Constantinescu, Stefan N; Kienlen-Campard, Pascal

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β-amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and β C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing.

  8. Construction and testing of a bacterial luciferase reporter gene system for in vivo measurement of nonsense suppression in Streptomyces.

    PubMed

    Weiser, J; Buriánková, K; Kalachová, L; Branny, P; Pernodet, J L

    2006-01-01

    A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.

  9. An investigation of the reaction kinetics of luciferase and the effect of ionizing radiation on the reaction rate.

    PubMed

    Berovic, Nikolas; Parker, David J; Smith, Michael D

    2009-04-01

    The bioluminescence produced by luciferase, a firefly enzyme, requires three substrates: luciferin, ATP and oxygen. We find that ionizing radiation, in the form of a proton beam from a cyclotron, will eliminate dissolved oxygen prior to any damage to other substrates or to the protein. The dose constant for removal of oxygen is 70 +/- 20 Gy, a much smaller dose than required to cause damage to protein. Removal of oxygen, which is initially in excess, leads to a sigmoidal response of bioluminescence to radiation dose, consistent with a Michaelis-Menten relationship to substrate concentration. When excess oxygen is exhausted, the response becomes exponential. Following the irradiation, bioluminescence recovers due to a slow leak of oxygen into the solution. This may also explain previous observations on the response of bioluminescent bacteria to radiation. We have studied the dependence of the reaction rate on enzyme and substrate concentration and propose a model for the reaction pathway consistent with this data. The light output from unirradiated samples decreases significantly with time due to product inhibition. We observe that this inhibition rate changes dramatically immediately after a sample is exposed to the beam. This sudden change of the inhibition rate is unexplained but shows that enzyme regulatory function responds to ionizing radiation at a dose level less than 0.6 Gy.

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

    PubMed

    Berguido, Francisco J; Bodjo, Sanne Charles; Loitsch, Angelika; Diallo, Adama

    2016-01-01

    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 causal agent is peste des petits ruminants virus (PPRV), which belongs to the genus Morbillivirus 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 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.

  11. A Luciferase Gene Driven by an Alphaherpesviral Promoter Also Responds to Immediate Early Antigens of the Betaherpesvirus HCMV, Allowing Comparative Analyses of Different Human Herpesviruses in One Reporter Cell Line

    PubMed Central

    Villinger, Clarissa; Schubert, Axel; Walther, Paul; Sinzger, Christian; Lieber, Diana

    2017-01-01

    Widely used methods for quantification of human cytomegalovirus (HCMV) infection in cell culture such as immunoblotting or plaque reduction assays are generally restricted to low throughput and require time-consuming evaluation. Up to now, only few HCMV reporter cell lines have been generated to overcome these restrictions and they are afflicted with other limitations because permanently expandable cell lines are normally not fully permissive to HCMV. In this work, a previously existing epithelial cell line hosting a luciferase gene under control of a Varicella-zoster virus promoter was adopted to investigate HCMV infection. The cells were susceptible to different HCMV strains at infection efficiencies that corresponded to their respective degree of epithelial cell tropism. Expression of early and late viral antigens, formation of nuclear inclusions, release of infectious virus progeny, and focal growth indicated productive viral replication. However, viral release and spread occurred at lower levels than in primary cell lines which appears to be due to a malfunction of virion morphogenesis during the nuclear stage. Expression of the luciferase reporter gene was specifically induced in HCMV infected cultures as a function of the virus dose and dependent on viral immediate early gene expression. The level of reporter activity accurately reflected infection efficiencies as determined by viral antigen immunostaining, and hence could discriminate the cell tropism of the tested virus strains. As proof-of-principle, we demonstrate that this cell line is applicable to evaluate drug resistance of clinical HCMV isolates and the neutralization capacity of human sera, and that it allows comparative and simultaneous analysis of HCMV and human herpes simplex virus type 1. In summary, the permanent epithelial reporter cell line allows robust, rapid and objective quantitation of HCMV infection and it will be particularly useful in higher throughput analyses as well as in

  12. Remote detection of human toxicants in real time using a human-optimized, bioluminescent bacterial luciferase gene cassette bioreporter

    NASA Astrophysics Data System (ADS)

    Close, Dan; Webb, James; Ripp, Steven; Patterson, Stacey; Sayler, Gary

    2012-06-01

    Traditionally, human toxicant bioavailability screening has been forced to proceed in either a high throughput fashion using prokaryotic or lower eukaryotic targets with minimal applicability to humans, or in a more expensive, lower throughput manner that uses fluorescent or bioluminescent human cells to directly provide human bioavailability data. While these efforts are often sufficient for basic scientific research, they prevent the rapid and remote identification of potentially toxic chemicals required for modern biosecurity applications. To merge the advantages of high throughput, low cost screening regimens with the direct bioavailability assessment of human cell line use, we re-engineered the bioluminescent bacterial luciferase gene cassette to function autonomously (without exogenous stimulation) within human cells. Optimized cassette expression provides for fully endogenous bioluminescent production, allowing continuous, real time monitoring of the bioavailability and toxicology of various compounds in an automated fashion. To access the functionality of this system, two sets of bioluminescent human cells were developed. The first was programed to suspend bioluminescent production upon toxicological challenge to mimic the non-specific detection of a toxicant. The second induced bioluminescence upon detection of a specific compound to demonstrate autonomous remote target identification. These cells were capable of responding to μM concentrations of the toxicant n-decanal, and allowed for continuous monitoring of cellular health throughout the treatment process. Induced bioluminescence was generated through treatment with doxycycline and was detectable upon dosage at a 100 ng/ml concentration. These results demonstrate that leveraging autonomous bioluminescence allows for low-cost, high throughput direct assessment of toxicant bioavailability.

  13. Warm water bath stimulates phase-shifts of the peripheral circadian clocks in PER2::LUCIFERASE mouse.

    PubMed

    Ohnishi, Nobuaki; Tahara, Yu; 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.

  14. Long-term luciferase expression monitored by bioluminescence imaging after adeno-associated virus-mediated fetal gene delivery in rhesus monkeys (Macaca mulatta).

    PubMed

    Tarantal, Alice F; Lee, C Chang I

    2010-02-01

    The safety and efficiency of fetal adeno-associated virus (AAV) gene delivery in rhesus monkeys and long-term monitoring of transgene expression by bioluminescence imaging (BLI) were evaluated. Early second-trimester fetal monkeys were administered AAV2/5, AAV2/9, or AAV2/10 vector supernatant preparations expressing firefly luciferase under the control of the cytomegalovirus promoter, using an intrathoracic (n = 6) or intramyocardial (n = 6) approach and established ultrasound-guided techniques. Postnatal BLI was performed monthly up to 6 months postnatal age (n = 12) and then every 3 months thereafter to monitor transgene expression up to 24 months postnatal age (27 months after gene transfer; n = 6). All AAV serotypes showed greater than 1.0 x 10(9) photons/sec at all time points evaluated with limited biodistribution to nontargeted anatomical sites. The highest levels of bioluminescence (photons per second) observed were noted with AAV2/9 and AAV2/10 when the three vector constructs were compared. To correlate in vivo findings at the tissue level, specimens were collected from selected animals and analyzed. Three-dimensional reconstruction showed that firefly luciferase expression was consistent with imaging and morphometric measures. These findings suggest that (1) high levels of AAV-mediated firefly luciferase expression can be found after fetal AAV gene transfer and without any evidence of adverse effects; (2) the intercostal muscles, myocardium, and muscular component of the diaphragm of developing fetuses are readily transduced with AAV2/5, AAV2/9, or AAV2/10; and (3) postnatal outcomes and long-term luciferase expression can be effectively monitored by BLI in young rhesus monkeys.

  15. Evaluation of a luciferase-based reporter assay as a screen for inhibitors of estrogen-ERα-induced proliferation of breast cancer cells.

    PubMed

    Andruska, Neal; Mao, Chengjian; Cherian, Mathew; Zhang, Chen; Shapiro, David J

    2012-08-01

    Estrogens, acting through estrogen receptor α (ERα), stimulate breast cancer proliferation, making ERα an attractive drug target. Since 384-well format screens for inhibitors of proliferation can be challenging for some cells, inhibition of luciferase-based reporters is often used as a surrogate end point. To identify novel small-molecule inhibitors of 17β-estradiol (E(2))-ERα-stimulated cell proliferation, we established a cell-based screen for inhibitors of E(2)-ERα induction of an estrogen response element (ERE)(3)-luciferase reporter. Seventy-five "hits" were evaluated in tiered follow-up assays to identify where hits failed to progress and evaluate their effectiveness as inhibitors of E(2)-ERα-induced proliferation of breast cancer cells. Only 8 of 75 hits from the luciferase screen inhibited estrogen-induced proliferation of ERα-positive MCF-7 and T47D cells but not control ERα-negative MDA-MB-231 cells. Although 12% of compounds inhibited E(2)-ERα-stimulated proliferation in only one of the ERα-positive cell lines, 40% of compounds were toxic and inhibited growth of all the cell lines, and ~37% exhibited little or no ability to inhibit E(2)-ERα-stimulated cell proliferation. Representative compounds were evaluated in more detail, and a lead ERα inhibitor was identified.

  16. Plasmodium falciparum Transfected with Ultra Bright NanoLuc Luciferase Offers High Sensitivity Detection for the Screening of Growth and Cellular Trafficking Inhibitors

    PubMed Central

    Elsworth, Brendan; Charnaud, Sarah C.; Sanders, Paul R.; Crabb, Brendan S.; Gilson, Paul R.

    2014-01-01

    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. PMID:25392998

  17. 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.

    PubMed

    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.

  18. Non-invasive activation of optogenetic actuators

    NASA Astrophysics Data System (ADS)

    Birkner, Elisabeth; Berglund, Ken; Klein, Marguerita E.; Augustine, George J.; Hochgeschwender, Ute

    2014-03-01

    The manipulation of genetically targeted neurons with light (optogenetics) continues to provide unprecedented avenues into studying the function of the mammalian brain. However, potential translation into the clinical arena faces a number of significant hurdles, foremost among them the need for insertion of optical fibers into the brain to deliver light to opsins expressed on neuronal membranes. In order to overcome these hardware-related problems, we have developed an alternative strategy for delivering light to opsins which does not involve fiber implants. Rather, the light is produced by a protein, luciferase, which oxidizes intravenously applied substrate, thereby emitting bioluminescence. In proof-ofprinciple studies employing a fusion protein of a light-generating luciferase to a light-sensing opsin (luminopsin), we showed that light emitted by Gaussia luciferase is indeed able to activate channelrhodopsin, allowing modulation of neuronal activity when expressed in cultured neurons. Here we assessed applicability of the concept in vivo in mice expressing luminopsins from viral vectors and from genetically engineered transgenes. The experiments demonstrate that intravenously applied substrate reaches neurons in the brain, causing the luciferase to produce bioluminescence which can be imaged in vivo, and that activation of channelrhodopsin by bioluminescence is sufficient to affect behavior. Further developments of such technology based on combining optogenetics with bioluminescence - i.e. combining lightsensing molecules with biologically produced light through luciferases - should bring optogenetics closer to clinical applications.

  19. Replication-Competent Influenza Virus and Respiratory Syncytial Virus Luciferase Reporter Strains Engineered for Co-Infections Identify Antiviral Compounds in Combination Screens

    PubMed Central

    Yan, Dan; Weisshaar, Marco; Lamb, Kristen; Chung, Hokyung K; Lin, Michael Z; Plemper, Richard K

    2016-01-01

    Myxoviruses such as influenza A virus (IAV) and respiratory syncytial virus (RSV) are major human pathogens, mandating the development of novel therapeutics. To establish a high-throughput screening protocol for the simultaneous identification of pathogen- and host-targeted hit candidates against either or both pathogens, we have attempted coinfection of cells with IAV and RSV. However, viral replication kinetics were incompatible, RSV signal window was low, and an IAV-driven minireplicon reporter assay used in initial screens narrowed the host cell range and restricted to single-cycle infections. To overcome these limitations, we developed an RSV strain carrying firefly luciferase fused to an innovative universal small-molecule assisted shut-off domain, which boosted assay signal window, and a hyperactive fusion protein that synchronized IAV and RSV reporter expression kinetics and suppresses the identification of RSV entry inhibitors sensitive to a recently reported RSV pan-resistance mechanism. Combined with a replication-competent recombinant IAV strain harboring nano-luciferase, the assay performed well on a human respiratory cell line and supports multi-cycle infections. Miniaturized to 384-well format, the protocol was validated through screening of a set of the NIH Clinical Collection (NCC) in quadruplicate. These test screens demonstrated favorable assay parameters and reproducibility. Application to a LOPAC library of bioactive compounds in a proof-of-concept campaign detected licensed anti-myxovirus therapeutics, ribavirin and the neuraminidase inhibitor zanamivir, and identified two unexpected RSV-specific hit candidates, Fenretinide and the opioid receptor antagonist BNTX-7. Hits were evaluated in direct and orthogonal dose-response counterscreens using a standard recRSV reporter strain expressing renilla luciferase. PMID:26307636

  20. Strategy of mutual compensation of green and red mutants of firefly luciferase identifies a mutation of the highly conservative residue E457 with a strong red shift of bioluminescence.

    PubMed

    Koksharov, Mikhail I; Ugarova, Natalia N

    2013-11-01

    Bioluminescence spectra of firefly luciferases demonstrate highly pH-sensitive spectra changing the color from green to red light when pH is lowered from alkaline to acidic. This reflects a change of ratio of the green and red emitters in the bimodal spectra of bioluminescence. We show that the mutations strongly stabilizing green (Y35N) or red (H433Y) emission compensate each other leading to the WT color of firefly luciferase. We further used this compensating ability of Y35N to search for strong red-shifting mutations in the C-domain of firefly luciferase by random mutagenesis. The discovered mutation E457K substantially increased the contribution of the red emitter and caused a 12 nm red shift of the green emitter as well. E457 is highly conservative not only in beetle luciferases but also in a whole ANL superfamily of adenylating enzymes and forms a conservative structural hydrogen bond with V471. Our results suggest that the removal of this hydrogen bond only mildly affects luciferase properties and that most of the effect of E457K is caused by the introduction of positive charge. E457 forms a salt bridge with R534 in most ANL enzymes including pH-insensitive luciferases which is absent in pH-sensitive firefly luciferases. The mutant A534R shows that this salt bridge is not important for pH-sensitivity but considerably improves in vivo thermostability. Although E457 is located far from the oxyluciferin-binding site, the properties of the mutant E457K suggest that it affects color by influencing the AMP binding.

  1. [Bioluminescent method of determining antiprotease activity].

    PubMed

    Gitel'zon, I I; Rykov, S A; Kratasiuk, G A; Petushkov, V N; Shvetskiĭ, A G

    1985-11-01

    A method for antiprotease activity measurement based on the use of luminous bacteria luciferase as protein substrate of proteases is suggested. Antiprotease is incubated with protease for 1 to 2 min at 30 degrees C and then it is added to the reaction mixture containing luciferase, NADH: FMN-oxidoreductase and their substrates--myristic aldehyde, FMN and NADH. Biofluorescence is measured in a temperature-controlled cuvette for 1 min. The total time of the measurement is 3 min. The method can be applied both in fine biochemical assays and in medical rapid diagnosis.

  2. Crystal structure of nanoKAZ: The mutated 19 kDa component of Oplophorus luciferase catalyzing the bioluminescent reaction with coelenterazine.

    PubMed

    Tomabechi, Yuri; Hosoya, Takamitsu; Ehara, Haruhiko; Sekine, Shun-ichi; Shirouzu, Mikako; Inouye, Satoshi

    2016-01-29

    The 19 kDa protein (KAZ) of Oplophorus luciferase is a catalytic component, that oxidizes coelenterazine (a luciferin) with molecular oxygen to emit light. The crystal structure of the mutated 19 kDa protein (nanoKAZ) was determined at 1.71 Å resolution. The structure consists of 11 antiparallel β-strands forming a β-barrel that is capped by 4 short α-helices. The structure of nanoKAZ is similar to those of fatty acid-binding proteins (FABPs), even though the amino acid sequence similarity was very low between them. The coelenterazine-binding site and the catalytic site for the luminescence reaction might be in a central cavity of the β-barrel structure.

  3. A comparison of certain extracting agents for extraction of adenosine triphosphate (ATP) from microorganisms for use in the firefly luciferase ATP assay

    NASA Technical Reports Server (NTRS)

    Knust, E. A.; Chappelle, E. W.; Picciolo, G. L.

    1975-01-01

    Firefly luciferase ATP assay is used in clinical and industrial applications, such as determination of urinary infection levels, microbial susceptibility testing, and monitoring of yeast levels in beverages. Three categories of extractants were investigated for their extracting efficiency. They were ionizing organic solvents, nonionizing organic solvents, and inorganic acids. Dimethylsulfoxide and formamide represented the ionizing organic solvents, while n-butanol, chloroform, ethanol, acetone, and methylene chloride were used for the nonionizing organic solvents. Nitric acid and perchloric acid were chosen for the inorganic acids category. Pathogens were tested with each solvent. They included: Saccharomyces carlsbergensis, E. coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter species, Proteus mirabilis, Proteus vulgaris, Staphylococcus epidermidis, Streptococcus faecalis, Pseudomonas aeruginosa, and Candida albicans. These results are shown in graphic representations.

  4. Testing the promiscuity of commercial kinase inhibitors against the AGC kinase group using a split-luciferase screen.

    PubMed

    Jester, Benjamin W; Gaj, Alicia; Shomin, Carolyn D; Cox, Kurt J; Ghosh, Indraneel

    2012-02-23

    Using a newly developed competitive binding assay dependent upon the reassembly of a split reporter protein, we have tested the promiscuity of a panel of reported kinase inhibitors against the AGC group. Many non-AGC targeted kinase inhibitors target multiple members of the AGC group. In general, structurally similar inhibitors consistently exhibited activity toward the same target as well as toward closely related kinases. The inhibition data was analyzed to test the predictive value of either using identity scores derived from residues within 6 Å of the active site or identity scores derived from the entire kinase domain. The results suggest that the active site identity in certain cases may be a stronger predictor of inhibitor promiscuity. The overall results provide general guidelines for establishing inhibitor selectivity as well as for the future design of inhibitors that either target or avoid AGC kinases.

  5. Real-time monitoring of inflammation status in 3T3-L1 adipocytes possessing a secretory Gaussia luciferase gene under the control of nuclear factor-kappa B response element

    SciTech Connect

    Nagasaki, Haruka; Yoshimura, Takeshi; Aoki, Naohito

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Inflammation status in adipocytes can be monitored by the new assay system. Black-Right-Pointing-Pointer Only an aliquot of conditioned medium is required without cell lysis. Black-Right-Pointing-Pointer Inflammation-attenuating compounds can be screened more conveniently. -- Abstract: We have established 3T3-L1 cells possessing a secretory Gaussia luciferase (GLuc) gene under the control of nuclear factor-kappa B (NF-{kappa}B) response element. The 3T3-L1 cells named 3T3-L1-NF-{kappa}B-RE-GLuc could differentiate into adipocyte as comparably as parental 3T3-L1 cells. Inflammatory cytokines such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1{beta} induced GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes in a concentration- and time-dependent manner. GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes was also induced when cultured with RAW264.7 macrophages and was dramatically enhanced by lipopolysaccharide (LPS)-activated macrophages. An NF-{kappa}B activation inhibitor BAY-11-7085 and an antioxidant N-acetyl cysteine significantly suppressed GLuc secretion induced by macrophages. Finally, we found that rosemary-derived carnosic acid strongly suppressed GLuc secretion induced by macrophages and on the contrary up-regulated adiponectin secretion. Collectively, by using 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes, inflammation status can be monitored in real time and inflammation-attenuating compounds can be screened more conveniently.

  6. Dynamic Imaging of Pancreatic Nuclear Factor κB (NF-κB) Activation in Live Mice Using Adeno-associated Virus (AAV) Infusion and Bioluminescence*

    PubMed Central

    Orabi, Abrahim I.; Sah, Swati; Javed, Tanveer A.; Lemon, Kathryn L.; Good, Misty L.; Guo, Ping; Xiao, Xiangwei; Prasadan, Krishna; Gittes, George K.; Jin, Shunqian; Husain, Sohail Z.

    2015-01-01

    Nuclear factor κB (NF-κB) is an important signaling molecule that plays a critical role in the development of acute pancreatitis. Current methods for examining NF-κB activation involve infection of an adenoviral NF-κB-luciferase reporter into cell lines or electrophoretic mobility shift assay of lysate. The use of adeno-associated viruses (AAVs) has proven to be an effective method of transfecting whole organs in live animals. We examined whether intrapancreatic duct infusion of AAV containing an NF-κB-luciferase reporter (AAV-NF-κB-luciferase) can reliably measure pancreatic NF-κB activation. We confirmed the infectivity of the AAV-NF-κB-luciferase reporter in HEK293 cells using a traditional luciferase readout. Mice were infused with AAV-NF-κB-luciferase 5 weeks before induction of pancreatitis (caerulein, 50 μg/kg). Unlike transgenic mice that globally express NF-κB-luciferase, AAV-infused mice showed a 15-fold increase in pancreas-specific NF-κB bioluminescence following 12 h of caerulein compared with baseline luminescence (p < 0.05). The specificity of the NF-κB-luciferase signal to the pancreas was confirmed by isolating the pancreas and adjacent organs and observing a predominant bioluminescent signal in the pancreas compared with liver, spleen, and stomach. A complementary mouse model of post-ERCP-pancreatitis also induced pancreatic NF-κB signals. Taken together these data provide the first demonstration that NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. We believe this technique offers a valuable tool to study real-time activation of NF-κB in vivo. PMID:25802340

  7. Adaptation of a Gaussia princeps Luciferase reporter system in Candida albicans for in vivo detection in the Galleria mellonella infection model.

    PubMed

    Delarze, Eric; Ischer, Françoise; Sanglard, Dominique; Coste, Alix T

    2015-01-01

    For the past 10 years, mini-host models and in particular the greater wax moth Galleria mellonella have tended to become a surrogate for murine models of fungal infection mainly due to cost, ethical constraints and ease of use. Thus, methods to better assess the fungal pathogenesis in G. mellonella need to be developed. In this study, we implemented the detection of Candida albicans cells expressing the Gaussia princeps luciferase in its cell wall in infected larvae of G. mellonella. We demonstrated that detection and quantification of luminescence in the pulp of infected larvae is a reliable method to perform drug efficacy and C. albicans virulence assays as compared to fungal burden assay. Since the linearity of the bioluminescent signal, as compared to the CFU counts, has a correlation of R(2) = 0.62 and that this method is twice faster and less labor intensive than classical fungal burden assays, it could be applied to large scale studies. We next visualized and followed C. albicans infection in living G. mellonella larvae using a non-toxic and water-soluble coelenterazine formulation and a CCD camera that is commonly used for chemoluminescence signal detection. This work allowed us to follow for the first time C. albicans course of infection in G. mellonella during 4 days.

  8. In Vivo Determination of Mitochondrial Function Using Luciferase-Expressing Caenorhabditis elegans: Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant-Induced Dysfunction.

    PubMed

    Luz, Anthony L; Lagido, Cristina; Hirschey, Matthew D; Meyer, Joel N

    2016-08-01

    Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant-induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be developed. Here, using the model organism Caenorhabditis elegans, we describe how to rapidly assess the in vivo role of the electron transport chain, glycolysis, or fatty acid oxidation in energy metabolism following toxicant exposure, using a luciferase-expressing ATP reporter strain. Alterations in mitochondrial function subsequent to toxicant exposure are detected by depleting steady-state ATP levels with inhibitors of the mitochondrial electron transport chain, glycolysis, or fatty acid oxidation. Differential changes in ATP following short-term inhibitor exposure indicate toxicant-induced alterations at the site of inhibition. Because a microplate reader is the only major piece of equipment required, this is a highly accessible method for studying toxicant-induced mitochondrial dysfunction in vivo. © 2016 by John Wiley & Sons, Inc.

  9. Bioluminescence of a firefly pupa: involvement of a luciferase isotype in the dim glow of pupae and eggs in the Japanese firefly, Luciola lateralis.

    PubMed

    Oba, Yuichi; Furuhashi, Mana; Bessho, Manabu; Sagawa, Shingo; Ikeya, Haruyoshi; Inouye, Satoshi

    2013-05-01

    We isolated cDNA for a luciferase isotype, LlLuc2, from the ovary of the Japanese firefly, Luciola lateralis. The gene product LlLuc2 showed 59% amino acid identity with LlLuc1, which had been isolated from the adult L. lateralis lantern. Molecular phylogenetic analysis indicated that LlLuc2 is an orthologue of LcLuc2 from Luciola cruciata. The spectral maxima of the luminescence by recombinant LlLuc1 and LlLuc2 were 550 and 539 nm, respectively. Quantitative PCR analysis revealed that LlLuc1 was expressed predominantly in larvae and adults, and LlLuc2 was expressed in eggs and pupae, which glow dimly, and we found that the in vivo luminescence spectra of the egg and pupa in L. lateralis were in good agreement with the in vitro luminescence spectrum by LlLuc2. These results suggest that, in L. lateralis, LlLuc1 is responsible for the yellowish luminescence of larval and adult lanterns, and LlLuc2 is responsible for the dim, greenish glow of eggs and whole pupae. Similar results were obtained in L. cruciata.

  10. Transgenic mouse model expressing P53R172H, luciferase, EGFP, and KRASG12D in a single open reading frame for live imaging of tumor

    PubMed Central

    Ju, Hye-Lim; Calvisi, Diego F.; Moon, Hyuk; Baek, Sinhwa; Ribback, Silvia; Dombrowski, Frank; Cho, Kyung Joo; Chung, Sook In; Han, Kwang-Hyub; Ro, Simon Weonsang

    2015-01-01

    Genetically engineered mouse cancer models allow tumors to be imaged in vivo via co-expression of a reporter gene with a tumor-initiating gene. However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay. To overcome this limitation, we developed a transgenic mouse in which two oncogenes, encoding P53R172H and KRASG12D, are expressed together with two reporter genes, encoding enhanced green fluorescent protein (EGFP) and firefly luciferase, in a single open reading frame following Cre-mediated DNA excision. Systemic administration of adenovirus encoding Cre to these mice induced specific transgene expression in the liver. Repeated bioluminescence imaging of the mice revealed a continuous increase in the bioluminescent signal over time. A strong correlation was found between the bioluminescent signal and actual tumor size. Interestingly, all liver tumors induced by P53R172H and KRASG12D in the model were hepatocellular adenomas. The mouse model was also used to trace cell proliferation in the epidermis via live fluorescence imaging. We anticipate that the transgenic mouse model will be useful for imaging tumor development in vivo and for investigating the oncogenic collaboration between P53R172H and KRASG12D. PMID:25623590

  11. Potent Effects of Flavonoid Nobiletin on Amplitude, Period, and Phase of the Circadian Clock Rhythm in PER2::LUCIFERASE Mouse Embryonic Fibroblasts

    PubMed Central

    Shinozaki, Ayako; Misawa, Kenichiro; Ikeda, Yuko; Haraguchi, Atsushi; Kamagata, Mayo; Tahara, Yu; Shibata, Shigenobu

    2017-01-01

    Flavonoids are natural polyphenols that are widely found in plants. The effects of flavonoids on obesity and numerous diseases such as cancer, diabetes, and Alzheimer’s have been well studied. However, little is known about the relationships between flavonoids and the circadian clock. In this study, we show that continuous or transient application of flavonoids to the culture medium of embryonic fibroblasts from PER2::LUCIFERASE (PER2::LUC) mice induced various modifications in the circadian clock amplitude, period, and phase. Transient application of some of the tested flavonoids to cultured cells induced a phase delay of the PER2::LUC rhythm at the down slope phase. In addition, continuous application of the polymethoxy flavonoids nobiletin and tangeretin increased the amplitude and lengthened the period of the PER2::LUC rhythm. The nobiletin-induced phase delay was blocked by co-treatment with U0126, an ERK inhibitor. In summary, among the tested flavonoids, polymethoxy flavones increased the amplitude, lengthened the period, and delayed the phase of the PER2::LUC circadian rhythm. Therefore, foods that contain polymethoxy flavones may have beneficial effects on circadian rhythm disorders and jet lag. PMID:28152057

  12. Regulation of cytochrome P4501A1 expression by hyperoxia in human lung cell lines: Implications for hyperoxic lung injury

    SciTech Connect

    Bhakta, Kushal Y. Jiang, Weiwu; Couroucli, Xanthi I.; Fazili, Inayat S.; Muthiah, Kathirvel; Moorthy, Bhagavatula

    2008-12-01

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

  13. Time-restricted feeding of rapidly digested starches causes stronger entrainment of the liver clock in PER2::LUCIFERASE knock-in mice.

    PubMed

    Itokawa, Misa; Hirao, Akiko; Nagahama, Hiroki; Otsuka, Makiko; Ohtsu, Teiji; Furutani, Naoki; Hirao, Kazuko; Hatta, Tamao; Shibata, Shigenobu

    2013-02-01

    Restricting feeding to daytime can entrain circadian clocks in peripheral organs of rodents, and nutrients that rapidly increase the blood glucose level are suitable for inducing entrainment. However, dietetic issues, for example, whether or not the diet comprises heated food, have not been fully explored. We therefore hypothesized that rapidly digested starch causes stronger entrainment than slowly digested starch. The entrainment ability of the liver clock in PER2::LUCIFERASE knock-in mice, blood glucose levels, insulin levels, and acute changes in liver clock gene expression were compared between a β-starch (native)-substituted AIN-93M standard diet and an α-starch (gelatinized)-substituted diet. β-Corn and β-rice starch induced larger phase delays of the liver clock, larger blood glucose increases, and higher Per2 gene expression in the liver compared with β-potato starch. Starch granule size, as examined by electron microscopy, was larger for β-potato starch than for β-corn or β-rice starch. After heating, we obtained gelatinized α-potato, α-corn, and α-rice starch, which showed destruction of the crystal structure and a high level of gelatinization. No difference in the increase of blood glucose or insulin levels was observed between β-corn and α-corn starch, or between β-rice and α-rice starch. In contrast, α-potato starch caused higher levels of glucose and insulin compared with β-potato starch. An α-potato starch-substituted diet induced larger phase delays of the liver clock than did β-potato starch. Therefore, rapidly digested starch is appropriate for peripheral clock entrainment. Dietetic issues (heated vs unheated) are important when applying basic mouse data to humans.

  14. A high throughput system for the evaluation of protein kinase C inhibitors based on Elk1 transcriptional activation in human astrocytoma cells.

    PubMed

    Sharif, T R; Sharif, M

    1999-02-01

    Protein kinase C (PKC) designates a family of kinases that regulate many essential functions including cell growth and differentiation. The tight regulation of PKC activity is crucial for maintaining normal cellular proliferation and excessive activity leads to abnormal or uncontrolled cell growth. Recent reports indicate that malignant glioma cell lines express 100 to 1000-fold higher PKC activity when compared to non-neoplastic astrocytes. This high activity correlates well with the proliferation of tumor cells in vitro. We recently reported on the anti-proliferative properties of selective PKC inhibitors on the growth of U-373MG human astrocytoma cell line, and their ability to block mitogen-activated protein (MAP) kinase pathway activated by substance P (SP) neuropeptide receptor signaling via a PKC-dependent mechanism. Therefore, inhibiting PKC activity by selective PKC inhibitors may present a promising approach for improving astroglial brain tumor therapy. For this purpose, we constructed a high throughput model cell system to evaluate the efficacy of PKC inhibitors. This system is based on the measurement of light production in U-373MG cells stably transfected with the luciferase reporter gene whose expression depends on the transcriptional activation of GAL4-Elk1 fusion protein by enzyme components of the MAP kinase pathway and the upstream activation of PKC (PKC activation-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity). In brief, we have demonstrated that the PKC activator 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced luciferase activity in this cell system is mediated via the MAP kinase pathway and can be blocked in the presence of MEK1 selective inhibitors (PD 098059 or U0126). We also demonstrated that TPA-induced luciferase activity in U-373MG stable clones can be blocked by PKC inhibitors (CGP 41251, Go 6976, and GF 109203X) in a concentration dependent manner. In contrast, epidermal growth factor (EGF

  15. Activator protein 1 promotes the transcriptional activation of IRAK-M.

    PubMed

    Jin, Peipei; Bo, Lulong; Liu, Yongjian; Lu, Wenbin; Lin, Shengwei; Bian, Jinjun; Deng, Xiaoming

    2016-10-01

    Interleukin-1 receptor-associated kinase M (IRAK-M) is a well-known negative regulator for Toll-like receptor signaling, which can regulate immune homeostasis and tolerance in a number of pathological settings. However, the mechanism for IRAK-M regulation at transcriptional level remains largely unknown. In this study, a 1.4kb upstream sequence starting from the major IRAK-M transcriptional start site was cloned into luciferase reporter vector pGL3-basic to construct the full-length IRAK-M promoter. Luciferase reporter plasmids harboring the full-length and the deletion mutants of IRAK-M were transfected into 293T and A549 cells, and their relative luciferase activity was measured. The results demonstrated that activator protein 1(AP-1) cis-element plays a crucial role in IRAK-M constitutive gene transcription. Silencing of c-Fos and/or c-Jun expression suppressed the IRAK-M promoter activity as well as its mRNA and protein expressions. As a specific inhibitor for AP-1 activation, SP600125 also significantly suppressed the basal transcriptional activity of IRAK-M, the binding activity of c-Fos/c-Jun with IRAK-M promoter, and IRAK-M protein expression. Taken together, the result of this study highlights the importance of AP-1 in IRAK-M transcription, which offers more information on the role of IRAK-M in infectious and non-infectious diseases.

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

    PubMed Central

    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

  17. In Silico Identification of an Aryl Hydrocarbon Receptor Antagonist with Biological Activity In Vitro and In Vivo

    PubMed Central

    Parks, Ashley J.; Pollastri, Michael P.; Hahn, Mark E.; Stanford, Elizabeth A.; Novikov, Olga; Franks, Diana G.; Haigh, Sarah E.; Narasimhan, Supraja; Ashton, Trent D.; Hopper, Timothy G.; Kozakov, Dmytro; Beglov, Dimitri; Vajda, Sandor; Schlezinger, Jennifer J.

    2014-01-01

    The aryl hydrocarbon receptor (AHR) is critically involved in several physiologic processes, including cancer progression and multiple immune system activities. We, and others, have hypothesized that AHR modulators represent an important new class of targeted therapeutics. Here, ligand shape–based virtual modeling techniques were used to identify novel AHR ligands on the basis of previously identified chemotypes. Four structurally unique compounds were identified. One lead compound, 2-((2-(5-bromofuran-2-yl)-4-oxo-4H-chromen-3-yl)oxy)acetamide (CB7993113), was further tested for its ability to block three AHR-dependent biologic activities: triple-negative breast cancer cell invasion or migration in vitro and AHR ligand–induced bone marrow toxicity in vivo. CB7993113 directly bound both murine and human AHR and inhibited polycyclic aromatic hydrocarbon (PAH)– and TCDD-induced reporter activity by 75% and 90% respectively. A novel homology model, comprehensive agonist and inhibitor titration experiments, and AHR localization studies were consistent with competitive antagonism and blockade of nuclear translocation as the primary mechanism of action. CB7993113 (IC50 3.3 × 10−7 M) effectively reduced invasion of human breast cancer cells in three-dimensional cultures and blocked tumor cell migration in two-dimensional cultures without significantly affecting cell viability or proliferation. Finally, CB7993113 effectively inhibited the bone marrow ablative effects of 7,12-dimethylbenz[a]anthracene in vivo, demonstrating drug absorption and tissue distribution leading to pharmacological efficacy. These experiments suggest that AHR antagonists such as CB7993113 may represent a new class of targeted therapeutics for immunomodulation and/or cancer therapy. PMID:25159092

  18. 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

    NASA Technical Reports Server (NTRS)

    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.

  19. Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice

    PubMed Central

    Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

    2016-01-01

    Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

  20. The Effects of Chronic Lifelong Activation of the AHR Pathway by Industrial Chemical Pollutants on Female Human Reproduction

    PubMed Central

    Vacca, Margherita; Nardelli, Claudia; Castegna, Alessandra; Arnesano, Fabio; Carella, Nicola; Depalo, Raffaella

    2016-01-01

    Environmental chemicals, such as heavy metals, affect female reproductive function. A biological sensor of the signals of many toxic chemical compounds seems to be the aryl hydrocarbon receptor (AHR). Previous studies demonstrated the environmental of heavy metals in Taranto city (Italy), an area that has been influenced by anthropogenic factors such as industrial activities and waste treatments since 1986. However, the impact of these elements on female fertility in this geographic area has never been analyzed. Thus, in the present study, we evaluated the AHR pathway, sex steroid receptor pattern and apoptotic process in granulosa cells (GCs) retrieved from 30 women, born and living in Taranto, and 30 women who are living in non-contaminated areas (control group), who were undergoing in vitro fertilization (IVF) protocol. In follicular fluids (FFs) of both groups the toxic and essential heavy metals, such as chromiun (Cr), Manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), were also analyzed. Higher levels of Cr, Fe, Zn and Pb were found in the FFs of the women from Taranto as compared to the control group, as were the levels of AHR and AHR-dependent cytochrome P450 1A1 and 1B1; while CYP19A1 expression was decreased. The anti-apoptotic process found in the GCs of women fromTaranto was associated with the highest levels of progesterone receptor membrane component 1 (PGRMC1), a novel progesterone receptor, the expression of which is subjected to AHR activated by its highest affinity ligands (e.g., dioxins) or indirectly by other environmental pollutants, such as heavy metals. In conclusion, decreased production of estradiol and decreased number of retrieved mature oocytes found in women from Taranto could be due to chronic exposure to heavy metals, in particular to Cr and Pb. PMID:27008165

  1. DETECTION OF ANDROGENIC ACTIVITY IN EMISSIONS FROM DIESEL FUEL AND BIOMASS COMBUSTION

    EPA Science Inventory

    The present study evaluated both diesel fuel exhaust and biomass (wood) burn extracts for androgen receptor¿mediated activity using MDA-kb2 cells, which contain an androgen-responsive promoter-luciferase reporter gene construct. This assay and analytical fractionization of the sa...

  2. Persistent synchronized oscillations in prolactin gene promoter activity in living pituitary cells.

    PubMed

    McFerran, D W; Stirland, J A; Norris, A J; Khan, R A; Takasuka, N; Seymour, Z C; Gill, M S; Robertson, W R; Loudon, A S; Davis, J R; White, M R

    2001-07-01

    PRL gene expression in the anterior pituitary gland responds rapidly to different hormonal signals. We have investigated the long-term timing of transcriptional activation from the PRL, GH, and cytomegalovirus promoters in response to different stimulus duration, using real-time imaging of luciferase expression in living stably transfected GH3 cells. Long-term stimulation of serum-starved cells with 50% serum induced a homogeneous rise in PRL promoter activity, with subsequent heterogeneous fluctuations in luciferase activity in individual cells. When cells were subjected to a 2-h pulse of 50% serum, followed by serum-free medium, there were long-term (approximately 50 h) synchronized, homogeneous oscillations in PRL promoter activity. This response was PRL-specific, because in GH3 cells expressing luciferase from the GH or cytomegalovirus promoters, a serum pulse elicited no oscillations in luciferase expression after an initial transient response to serum. The PRL promoter may therefore be a template for an unstable transcription complex subject to stochastic regulation, allowing an oscillatory transcriptional response to physiological signals. This suggests that precise timing and coordination of cell responses to different signal-duration may represent a novel mechanism for coordinating long-term dynamic changes in transcription in cell populations.

  3. Activity.

    ERIC Educational Resources Information Center

    Clearing: Nature and Learning in the Pacific Northwest, 1984

    1984-01-01

    Presents three activities: (1) investigating succession in a schoolground; (2) investigating oak galls; and (3) making sun prints (photographs made without camera or darkroom). Each activity includes a list of materials needed and procedures used. (JN)

  4. The activity of the HIV-1 IRES is stimulated by oxidative stress and controlled by a negative regulatory element

    PubMed Central

    Gendron, Karine; Ferbeyre, Gerardo; Heveker, Nikolaus; Brakier-Gingras, Léa

    2011-01-01

    Initiation of translation of the full-length messenger RNA of HIV-1, which generates the viral structural proteins and enzymes, is cap-dependent but can also use an internal ribosome entry site (IRES) located in the 5′ untranslated region. Our aim was to define, through a mutational analysis, regions of HIV-1 IRES that are important for its activity. A dual-luciferase reporter construct where the Renilla luciferase (Rluc) translation is cap-dependent while the firefly luciferase (Fluc) translation depends on HIV-1 IRES was used. The Fluc/Rluc ratio was measured in lysates of Jurkat T cells transfected with the dual-luciferase plasmid bearing either the wild-type or a mutated IRES. Deletions or mutations in three regions decreased the IRES activity but deletion or mutations of a stem-loop preceding the primer binding site increased the IRES activity. The wild-type IRES activity, but not that of an IRES with a mutated stem-loop, was increased when cells were treated with agents that induce oxidative stress. Such stress is known to be caused by HIV-1 infection and we propose that this stem-loop is involved in a switch that stimulates the IRES activity in cells infected with HIV-1, supporting the suggestion that the IRES activity is up-regulated in the course of HIV-1 replication cycle. PMID:20935056

  5. Ah Receptor Activation by Dioxin Disrupts Activin, BMP, and WNT Signals During the Early Differentiation of Mouse Embryonic Stem Cells and Inhibits Cardiomyocyte Functions.

    PubMed

    Wang, Qin; Kurita, Hisaka; Carreira, Vinicius; Ko, Chia-I; Fan, Yunxia; Zhang, Xiang; Biesiada, Jacek; Medvedovic, Mario; Puga, Alvaro

    2016-02-01

    The AHR is a ligand-activated transcription factor that mediates gene-environment interactions. Genome-wide expression profiling during differentiation of mouse ES cells into cardiomyocytes showed that AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin; Dioxin (TCDD), its prototypical ligand, disrupted the expression of multiple homeobox transcription factors and inhibited cardiomyocyte contractility. Here we treated ES cells with TCDD at daily differentiation intervals to investigate whether TCDD-induced loss of contractility had a developmental window of sensitivity. Surprisingly, contractility was an AHR-dependent TCDD target solely between differentiation days 0 and 3 during the period of panmesoderm development, when TCDD also disrupted expression of genes in the TGFβ/BMP2/4 and wingless-type MMTV integration site (WNT)signaling pathways, suppressed the secretion of bone morphogenetic protein (BMP4), WNT3a, and WNT5a and elevated the secretion of Activin A, as determined by ELISA of the secreted proteins in the culture medium. Supplementing the culture medium with BMP4, WNT3a, or WNT5a during the first 3 days of differentiation successfully countered TCDD-induced impairment of contractility, while anti-WNT3a, or anti-WNT5a antibodies or continuous Noggin (a BMP4 antagonist) or Activin A treatment inhibited the contractile phenotype. In Ahr(+/+), but not in Ahr(-) (/) (-) ES cells, TCDD treatment significantly increased mitochondrial copy number, suggestive of mitochondrial stress and remodeling. Sustained AHR activation during ES cell differentiation appears to disrupt the expression of signals critical to the ontogeny of cardiac mesoderm and cause the loss of contractility in the resulting cardiomyocyte lineage.

  6. Ah Receptor Activation by Dioxin Disrupts Activin, BMP, and WNT Signals During the Early Differentiation of Mouse Embryonic Stem Cells and Inhibits Cardiomyocyte Functions

    PubMed Central

    Wang, Qin; Kurita, Hisaka; Carreira, Vinicius; Ko, Chia-I; Fan, Yunxia; Zhang, Xiang; Biesiada, Jacek; Medvedovic, Mario; Puga, Alvaro

    2016-01-01

    The AHR is a ligand-activated transcription factor that mediates gene-environment interactions. Genome-wide expression profiling during differentiation of mouse ES cells into cardiomyocytes showed that AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin; Dioxin (TCDD), its prototypical ligand, disrupted the expression of multiple homeobox transcription factors and inhibited cardiomyocyte contractility. Here we treated ES cells with TCDD at daily differentiation intervals to investigate whether TCDD-induced loss of contractility had a developmental window of sensitivity. Surprisingly, contractility was an AHR-dependent TCDD target solely between differentiation days 0 and 3 during the period of panmesoderm development, when TCDD also disrupted expression of genes in the TGFβ/BMP2/4 and wingless-type MMTV integration site (WNT)signaling pathways, suppressed the secretion of bone morphogenetic protein (BMP4), WNT3a, and WNT5a and elevated the secretion of Activin A, as determined by ELISA of the secreted proteins in the culture medium. Supplementing the culture medium with BMP4, WNT3a, or WNT5a during the first 3 days of differentiation successfully countered TCDD-induced impairment of contractility, while anti-WNT3a, or anti-WNT5a antibodies or continuous Noggin (a BMP4 antagonist) or Activin A treatment inhibited the contractile phenotype. In Ahr+/+, but not in Ahr−/− ES cells, TCDD treatment significantly increased mitochondrial copy number, suggestive of mitochondrial stress and remodeling. Sustained AHR activation during ES cell differentiation appears to disrupt the expression of signals critical to the ontogeny of cardiac mesoderm and cause the loss of contractility in the resulting cardiomyocyte lineage. PMID:26572662

  7. Phytochemicals Mediate the Expression and Activity of OCTN2 as Activators of the PPARγ/RXRα Pathway

    PubMed Central

    Luo, Jian; Qu, Jian; Yang, Rui; Ge, Meng-Xue; Mei, Yin; Zhou, Bo-Ting; Qu, Qiang

    2016-01-01

    Many phytochemicals exert activities as agonists of peroxisome proliferator-activated receptor gamma (PPARγ). This study aims to investigate whether phytochemicals are agonists of the PPARγ/RXRα pathway and modulate the target gene OCTN2. In this study, a luciferase reporter gene system was used to screen novel OCTN2 activators from 39 phytochemicals. Kaempferol, curcumin, and puerarin were found to show the significant PPRE-mediated luciferase activities (>150%) at 20 μM and showed a dose-dependent manner. Phytochemicals also elevated the mRNA and protein expression of OCTN2 in a dose-dependent fashion in colorectal cancer SW480 cells. These induction effects were gradually inhibited by PPARγ antagonist GW9662 in the luciferase reporter gene system and in SW480 cells. Moreover, the results of cell viability assay imply that three phytochemicals probably induce OCTN2 expression leading to the enhanced uptake of its substrate, oxaliplatin, thereby making cells more sensitive to oxaliplatin. The molecular docking study showed the possible binding sites of phytochemicals in PPARγ protein, and all of the docked phytochemicals fitted the same active pocket in PPARγ as troglitazone. All three phytochemicals exhibited hydrogen bonds between their polar moieties and the amino acid residues. Thus, we identified three phytochemicals as PPARγ ligands, which potentiated the expression and activity of OCTN2. PMID:27445823

  8. Activation of human peroxisome-activated receptor-gamma ...

    EPA Pesticide Factsheets

    Obesity in children has become an epidemic and recent research suggests a possible contribution from exposure to environmental chemicals. Several chemicals, such as phthalates, brominated flame retardants, and perfluorinated chemicals, are common in house dust on floors where children play and are suspected obesogens. Obesogens can act via a mechanism that involves activation of peroxisome proliferator-activated receptor-gamma (PPARy). A previous study found that dust collected from children’s homes binds to PPARy. Here, we investigated the ability of house dust to activate PPARy in a transiently transfected cell assay. Dust samples were collected in 2012 from carpeted and hardwood floors in children’s homes using thimbles fitted into a vacuum cleaner hose (“TEO” samples), or from homes in an adult cohort NIEHS study. Dust was extracted with 50:50 hexane:acetone, sonicated, centrifuged, and the organic layer collected. This was repeated 2X. The extracts were filtered to remove particulates, dried with purified nitrogen, and reconstituted in DMS0 at 200 ug/ul. COS-1 cells were transfected for 24 hrs with a human PPARy vector containing a luciferase reporter, and exposed for 24 hrs to negative controls water or DMSO (0.1%), positive controls Troglitazone (3 uM in water) or Rosiglitazone (100 nM in DMSO), or dust extracts serially diluted in DMEM at 50, 100, and 200 ug/ml in 0.1% DMSO. Cells were lysed and luciferase activity was measured. Data were log-tra

  9. Prenylation inhibitors stimulate both estrogen receptor α transcriptional activity through AF-1 and AF-2 and estrogen receptor β transcriptional activity

    PubMed Central

    Cestac, Philippe; Sarrabayrouse, Guillaume; Médale-Giamarchi, Claire; Rochaix, Philippe; Balaguer, Patrick; Favre, Gilles; Faye, Jean-Charles; Doisneau-Sixou, Sophie

    2005-01-01

    Introduction We showed in a previous study that prenylated proteins play a role in estradiol stimulation of proliferation. However, these proteins antagonize the ability of estrogen receptor (ER) α to stimulate estrogen response element (ERE)-dependent transcriptional activity, potentially through the formation of a co-regulator complex. The present study investigates, in further detail, how prenylated proteins modulate the transcriptional activities mediated by ERα and by ERβ. Methods The ERE-β-globin-Luc-SV-Neo plasmid was either stably transfected into MCF-7 cells or HeLa cells (MELN cells and HELN cells, respectively) or transiently transfected into MCF-7 cells using polyethylenimine. Cells deprived of estradiol were analyzed for ERE-dependent luciferase activity 16 hours after estradiol stimulation and treatment with FTI-277 (a farnesyltransferase inhibitor) or with GGTI-298 (a geranylgeranyltransferase I inhibitor). In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERα, the full-length ERβ, the AF-1-deleted ERα or the AF-2-deleted ERα. The presence of ERα was then detected by immunocytochemistry in either the nuclei or the cytoplasms of MCF-7 cells. Finally, Clostridium botulinum C3 exoenzyme treatment was used to determine the involvement of Rho proteins in ERE-dependent luciferase activity. Results FTI-277 and GGTI-298 only stimulate ERE-dependent luciferase activity in stably transfected MCF-7 cells. They stimulate both ERα-mediated and ERβ-mediated ERE-dependent luciferase activity in HELN cells, in the presence of and in the absence of estradiol. The roles of both AF-1 and AF-2 are significant in this effect. Nuclear ERα is decreased in the presence of prenyltransferase inhibitors in MCF-7 cells, again in the presence of and in the absence of estradiol. By contrast, cytoplasmic ERα is mainly decreased after treatment with FTI

  10. Effect of 60 Hz magnetic fields on the activation of hsp70 promoter in cultured INER-37 and RMA E7 cells.

    PubMed

    Heredia-Rojas, J Antonio; Rodríguez de la Fuente, Abraham Octavio; Alcocer González, Juan Manuel; Rodríguez-Flores, Laura E; Rodríguez-Padilla, Cristina; Santoyo-Stephano, Martha A; Castañeda-Garza, Esperanza; Taméz-Guerra, Reyes S

    2010-10-01

    It has been reported that 50-60 Hz magnetic fields (MF) with flux densities ranging from microtesla to millitesla are able to induce heat shock factor or heat shock proteins in various cells. In this study, we investigated the effect of 60 Hz sinusoidal MF at 8 and 80 μT on the expression of the luciferase gene contained in a plasmid labeled as electromagnetic field-plasmid (pEMF). This gene construct contains the specific sequences previously described for the induction of hsp70 expression by MF, as well as the reporter for the luciferase gene. The pEMF vector was transfected into INER-37 and RMA E7 cell lines that were later exposed to either MF or thermal shock (TS). Cells that received the MF or TS treatments and their controls were processed according to the luciferase assay system for evaluate luciferase activity. An increased luciferase gene expression was observed in INER-37 cells exposed to MF and TS compared with controls (p < 0.05), but MF exposure had no effect on the RMA E7 cell line.

  11. 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.

    PubMed

    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.

  12. Constitutive Activation of the Aromatic Hydrocarbon Receptor

    PubMed Central

    Chang, Ching-Yi; Puga, Alvaro

    1998-01-01

    The ligand-activated aromatic hydrocarbon receptor (AHR) dimerizes with the AHR nuclear translocator (ARNT) to form a functional complex that transactivates expression of the cytochrome P-450 CYP1A1 gene and other genes in the dioxin-inducible [Ah] gene battery. Previous work from this laboratory has shown that the activity of the CYP1A1 enzyme negatively regulates this process. To study the relationship between CYP1A1 activity and Ah receptor activation we used CYP1A1-deficient mouse hepatoma c37 cells and CYP1A1- and AHR-deficient African green monkey kidney CV-1 cells. Using gel mobility shift and luciferase reporter gene expression assays, we found that c37 cells that had not been exposed to exogenous Ah receptor ligands already contained transcriptionally active AHR-ARNT complexes, a finding that we also observed in wild-type Hepa-1 cells treated with Ellipticine, a CYP1A1 inhibitor. In CV-1 cells, transient expression of AHR and ARNT leads to high levels of AHR–ARNT-dependent luciferase gene expression even in the absence of an agonist. Using a green fluorescent protein-tagged AHR, we showed that elevated reporter gene expression correlates with constitutive nuclear localization of the AHR. Transcriptional activation of the luciferase reporter gene observed in CV-1 cells is significantly decreased by (i) expression of a functional CYP1A1 enzyme, (ii) competition with chimeric or truncated AHR proteins containing the AHR ligand-binding domain, and (iii) treatment with the AHR antagonist α-naphthoflavone. These results suggest that a CYP1A1 substrate, which accumulates in cells lacking CYP1A1 enzymatic activity, is an AHR ligand responsible for endogenous activation of the Ah receptor. PMID:9418899

  13. Activities.

    ERIC Educational Resources Information Center

    Bippert, Judy

    1993-01-01

    Presents activities designed to give students an opportunity to solve concrete problems involving spatial relationships and logical thinking utilizing hands-on manipulatives. Provides teacher instructions and four reproducible worksheets. (MDH)

  14. Allele-specific transcriptional activity of the variable number of tandem repeats in 5' region of the DRD4 gene is stimulus specific in human neuronal cells.

    PubMed

    Paredes, U M; Quinn, J P; D'Souza, U M

    2013-03-01

    The dopamine receptor D4 (DRD4) gene includes several variable number of tandem repeat loci that have been suggested to modulate DRD4 gene expression patterns. Previous studies showed differential basal activity of the two most common variants of a tandem repeat (120 bp per repeat unit) located in the 5' region adjacent to the DRD4 promoter in human cell lines. In this communication, we further characterized the ability of this polymorphic repeat to elicit tissue-, allele- and stimuli-specific transcriptional activity in vitro. The short and long variants of the DRD4 5' tandem repeat were cloned into a luciferase reporter gene construct containing the SV40 promoter. The luciferase constructs were cotransfected with expression vectors of two ubiquitously expressed human transcription factors (TFs), CCCTC-binding factor (CTCF) and upstream stimulatory factor 2 (USF2), into human cell lines and primary cultures of neonate rat cortex and luciferase activity measured. Overexpression with these TFs resulted in differential cell- and allele-specific transcriptional activities of the luciferase constructs. The results of our experiments show that variants of this tandem repeat in the 5' promoter of the DRD4 gene will direct differential reporter gene transcriptional activity in a cell-type-specific manner dependent on the signal pathways activated.

  15. A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONISTS AND ANTAGONISTS

    EPA Science Inventory

    The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

  16. A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONIST AND ANTAGONISTS

    EPA Science Inventory

    The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

  17. Activities.

    ERIC Educational Resources Information Center

    Kincaid, Charlene; And Others

    1993-01-01

    Presents an activity in which students collect and organize data from a real-world simulation of the scientific concept of half life. Students collect data using a marble sifter, analyze the data using a graphing calculator, and determine an appropriate mathematical model. Includes reproducible worksheets. (MDH)

  18. Anti-HIV activity of some synthetic lignanolides and intermediates.

    PubMed

    Sancho, Rocío; Medarde, Manuel; Sánchez-Palomino, Sonsoles; Madrigal, Blanca M; Alcamí, José; Muñoz, Eduardo; San Feliciano, Arturo

    2004-09-06

    The evaluation of the anti-HIV-1 activity of synthetic lignanolides and their intermediates is reported. The antiviral activity was studied through luciferase-based assays targeting the HIV-1 promoter activation induced by either, the HIV-1 Tat protein or the cellular transcription factor NF-kappaB, both known as crucial factors in HIV-1 replication. Among the compounds tested, three of them 2, 4 and 13 were further analysed for their anti-HIV-1 activity by recombinant virus assays, showing a suitable profile for development of novel anti-HIV-1 drugs.

  19. Phytol directly activates peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and regulates gene expression involved in lipid metabolism in PPAR{alpha}-expressing HepG2 hepatocytes

    SciTech Connect

    Goto, Tsuyoshi; Takahashi, Nobuyuki; Kato, Sota; Egawa, Kahori; Ebisu, Shogo; Moriyama, Tatsuya; Fushiki, Tohru; Kawada, Teruo . E-mail: fat@kais.kyoto-u.ac.jp

    2005-11-18

    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 PPAR{alpha}-specific activator. Phytol induced the increase in PPAR{alpha}-dependent luciferase activity and the degree of in vitro binding of a coactivator, SRC-1, to GST-PPAR{alpha}. Moreover, the addition of phytol upregulated the expression of PPAR{alpha}-target genes at both mRNA and protein levels in PPAR{alpha}-expressing HepG2 hepatocytes. These findings indicate that phytol is functional as a PPAR{alpha} ligand and that it stimulates the expression of PPAR{alpha}-target genes in intact cells. Because PPAR{alpha} activation enhances circulating lipid clearance, phytol may be important in managing abnormalities in lipid metabolism.

  20. Screening for novel human genes associated with CRE pathway activation with cell microarray.

    PubMed

    Tian, Linjie; Wang, Pingzhang; Guo, Jinhai; Wang, Xinyu; Deng, Weiwei; Zhang, Chenying; Fu, Dongxu; Gao, Xia; Shi, Taiping; Ma, Dalong

    2007-07-01

    In this study, cell microarray technology is used to identify novel human genes associated with CRE pathway activation. By reverse transfection, expression plasmids containing full-length cDNAs were cotransfected with the reporter plasmid pCRE-d2EGFP to monitor the activation of the CRE pathway via enhanced green fluorescence protein (EGFP) expression. Of the 575 predominantly novel genes screened, 22 exhibited relatively higher EGFP fluorescence compared with a negative control. After a functional validation with a dual luciferase reporter system that included both cis- and trans-luciferase assays, 4 of the 22 genes (RNF41, C8orf32, C6orf208, and MEIS3P1) were confirmed as CRE-pathway activators. Western blot analysis revealed that RNF41 can promote CREB phosphorylation. These results demonstrate the successful combination of cell microarray technology with this reporting system and the potential of this tool to characterize functions of novel genes in a highly parallel format.

  1. LIGAND STRUCTURE-DEPENDENT ACTIVATION OF ESTROGEN RECEPTOR α/Sp BY ESTROGENS AND XENOESTROGENS

    PubMed Central

    Wu, Fei; Khan, Shaheen; Wu, Qian; Barhoumi, Rola; Burghardt, Robert; Safe, Stephen

    2008-01-01

    This study investigated the effects of E2, diethylstilbestrol (DES), antiestrogens, the phytoestrogen resveratrol, and the xenoestrogens octylphenol (OP), nonylphenol (NP), endosulfan, kepone, 2,3,4,5-tetrachlorobiphenyl-4-ol (HO-PCB-Cl4), bisphenol-A(BPA), and 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on induction of luciferase activity in breast cancer cells transfected with a construct (pSp13) containing three tandem GC-rich Sp binding sites linked to luciferase and wild-type or variant ERα. The results showed that induction of luciferase activity was highly structure-dependent in both MCF-7 and MDA-MB-231 cells. Moreover, RNA interference assays using small inhibitory RNAs for Sp1, Sp3 and Sp4 also demonstrated structure-dependent differences in activation of ERα/Sp1, ERα/Sp3 and ERα/Sp4. These results demonstrate for the first time that various structural classes of ER ligands differentially activate wild-type and variant ERα/Sp-dependent transactivation, selectively use different Sp proteins, and exhibit selective ER modulator (SERM)-like activity. PMID:18400491

  2. Plasma-activated air mediates plasmid DNA delivery in vivo

    PubMed Central

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets. PMID:27110584

  3. Polyphenols in alcoholic beverages activating constitutive androstane receptor CAR.

    PubMed

    Yao, Ruiqing; Yasuoka, Akihito; Kamei, Asuka; Kitagawa, Yoshinori; Rogi, Tomohiro; Taieishi, Norifumi; Tsuruoka, Nobuo; Kiso, Yoshionobu; Misaka, Takumi; Abe, Keiko

    2011-01-01

    The constitutive androstane receptor CAR is a xenosensing nuclear receptor that can be activated by natural polyphenols such as flavonoids and catechins. We examined alcoholic beverage phytochemicals for their ability to activate CAR. HepG2 cells were transfected with CAR expression vector and its reporter gene, and then treated with trans-resveratrol, ellagic acid, β-caryophyllene, myrcene, and xanthohumol. A luciferase assay revealed that ellagic acid and trans-resveratrol activated both human and mouse CAR. Since CAR regulates many genes involved in energy metabolism, the possibility exists that these polyphenols would reduce the risk of certain alcohol-induced metabolic disorders with the help of CAR.

  4. A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus

    DOE PAGES

    Lund, C. H.; Bromley, J. R.; Stenbaek, A.; ...

    2014-10-18

    A growing body of evidence suggests that protein–protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. Wemore » tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. In conclusion, our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.« less

  5. A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus

    SciTech Connect

    Lund, C. H.; Bromley, J. R.; Stenbaek, A.; Rasmussen, R. E.; Scheller, H. V.; Sakuragi, Y.

    2014-10-18

    A growing body of evidence suggests that protein–protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. In conclusion, our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.

  6. A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus.

    PubMed

    Lund, Christian H; Bromley, Jennifer R; Stenbæk, Anne; Rasmussen, Randi E; Scheller, Henrik V; Sakuragi, Yumiko

    2015-01-01

    A growing body of evidence suggests that protein-protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. Our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.

  7. Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-kB, Calcium Signaling, and Calcineurin

    PubMed Central

    Jin, Shunqian; Orabi, Abrahim I.; Le, Tianming; Javed, Tanveer A.; Sah, Swati; Eisses, John F.; Bottino, Rita; Molkentin, Jeffery D.; Husain, Sohail Z.

    2015-01-01

    Background & Aims Radiocontrast agents are required for radiographic procedures, but these agents can injure tissues by unknown mechanisms. We investigated whether exposure of pancreatic tissues to radiocontrast agents during endoscopic retrograde cholangiopancreatography (ERCP) causes pancreatic inflammation, and studied the effects of these agents on human cell lines and in mice. Methods We exposed mouse and human acinar cells to the radiocontrast agent iohexol (Omnipaque) and measured intracellular release of Ca2+, calcineurin activation (using a luciferase reporter), activation of nuclear factor-κB (NF-κB, using a luciferase reporter), and cell necrosis (via propidium iodide uptake). We infused the radiocontrast agent into the pancreatic ducts of wild type mice (C57BL/6) to create a mouse model of post-ERCP pancreatitis; some mice were given intraperitoneal injections of the calcineurin inhibitor FK506 before and after infusion of the radiocontrast agent. CnAβ−/− mice were also used. This experiment was also performed in mice given infusions of AAV6-NF-κB-luciferase, to assess activation of this transcription factor in vivo. Results Incubation of mouse and human acinar cells, but not HEK293 or COS7 cells, with iohexol led to a peak and then plateau in Ca2+ signaling, along with activation of the transcription factors NF-κB and NFAT. Suppressing Ca2+ signaling or calcineurin with BAPTA, cyclosporine A, or FK506 prevented activation of NF-κB and acinar cell injury. Calcineurin Aβ-deficient mice were protected against induction of pancreatic inflammation by iohexol. The calcineurin inhibitor FK506 prevented contrast-induced activation of NF-κB in pancreata of mice; this was observed by live imaging of mice given infusions of AAV6- NF-kB-luciferase. Conclusions Radiocontrast agents cause pancreatic inflammation in mice, via activation of NF-κB, Ca2+ signaling, and calcineurin. Calcineurin inhibitors might be developed to prevent post-ERCP pancreatitis

  8. Pterostilbene-mediated Nrf2 activation: Mechanistic insights on Keap1:Nrf2 interface.

    PubMed

    Bhakkiyalakshmi, Elango; Dineshkumar, Kesavan; Karthik, Suresh; Sireesh, Dornadula; Hopper, Waheeta; Paulmurugan, Ramasamy; Ramkumar, Kunka Mohanram

    2016-08-15

    The discovery of Keap1-Nrf2 protein-protein interaction (PPI) inhibitors has become a promising strategy to develop novel lead molecules against variety of stress. Hence, Keap1-Nrf2 system plays an important role in oxidative/electrophilic stress associated disorders. Our earlier studies identified pterostilbene (PTS), a natural analogue of resveratrol, as a potent Nrf2 activator and Keap1-Nrf2 PPI inhibitor as assessed by luciferase complementation assay. In this study, we further identified the potential of PTS in Nrf2 activation and ARE-driven downstream target genes expression by nuclear translocation experiments and ARE-luciferase reporter assay, respectively. Further, the luciferase complementation assay identified that PTS inhibits Keap1-Nrf2 PPI in both dose and time-dependent manner. Computational studies using molecular docking and dynamic simulation revealed that PTS directly interacts with the basic amino acids of kelch domain of Keap1 and perturb Keap1-Nrf2 interaction pattern. This manuscript not only shows the binding determinants of Keap1-Nrf2 proteins but also provides mechanistic insights on Nrf2 activation potential of PTS.

  9. Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity.

    PubMed

    Legler, Juliette; Zeinstra, Laura M; Schuitemaker, Femke; Lanser, Peter H; Bogerd, Jan; Brouwer, Abraham; Vethaak, A Dick; De Voogt, Pim; Murk, Albertinka J; Van der Burg, Bart

    2002-10-15

    Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p'-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent Although the xenoestrogens o,p'-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p'-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes alpha, beta, and gamma were cloned, and transactivation by (xeno)estrogens was compared to human ERalpha and ERbeta. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERalpha and ERbeta compared to human ERalpha and ERbeta. Zebrafish ERbeta and ERgamma showed higher transactivation by (xeno)estrogens relative to E2 than human ERbeta.

  10. Comparison of Cell-Labeling Methods with 124I-FIAU and 64Cu-PTSM for Cell Tracking Using Chronic Myelogenous Leukemia Cells Expressing HSV1-tk and Firefly Luciferase

    PubMed Central

    Park, Jae-Jun; Son, Jin-Ju; Chun, Kwon-Soo; Song, In-Ho; Park, Yong-Serk; Kim, Kwang-Il; Lee, Yong-Jin; Kang, Joo-Hyun

    2012-01-01

    Abstract Cell-tracking methods with molecular-imaging modality can monitor the biodistribution of cells. In this study, the direct-labeling method with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM), indirect cell-labeling methods with herpes simplex virus type 1-thymidine kinase (HSV1-tk)-mediated 124I-2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (124I-FIAU) were comparatively investigated in vitro and in vivo for tracking of human chronic myelogenous leukemia cells. K562-TL was established by retroviral transduction of the HSV1-tk and firefly luciferase gene in the K562 cell. K562-TL cells were labeled with 64Cu-PTSM or 124I-FIAU. Cell labeling efficiency, viability, and radiolabels retention were compared in vitro. The biodistribution of radiolabeled K562-TL cells with each radiolabel and small-animal positron emission tomography imaging were performed. Additionally, in vivo and ex vivo bioluminescence imaging (BLI) and tissue reverse transcriptase–polymerase chain reaction (RT-PCR) analysis were used for confirming those results. K562-TL cells were efficiently labeled with both radiolabels. The radiolabel retention (%) of 124I-FIAU (95.2%±1.1%) was fourfold higher than 64Cu-PTSM (23.6%±0.7%) at 24 hours postlabeling. Viability of radiolabeled cells was statistically nonsignificant between 124I-FIAU and 64Cu-PTSM. The radioactivity of each radiolabeled cells was predominantly accumulated in the lungs and liver at 2 hours. Both the radioactivity of 64Cu-PTSM- and 124I-FIAU-labeled cells was highly accumulated in the liver at 24 hours. However, the radioactivity of 124I-FIAU-labeled cells was markedly decreased from the body at 24 hours. The K562-TL cells were dominantly localized in the lungs and liver, which also verified by BLI and RT-PCR analysis at 2 and 24 hours postinjection. The 64Cu-PTSM-labeled cell-tracking method is more efficient than 124I-FIAU-labeled cell tracking, because of markedly decrease of radioactivity and

  11. L-arginine attenuates Interleukin-1β (IL-1β) induced Nuclear Factor Kappa-Beta (NF-κB) activation in Caco-2 cells

    PubMed Central

    Meng, Qinghe; Cooney, Mitchell; Yepuri, Natesh; Cooney, Robert N.

    2017-01-01

    Background Specific nutrients like L-arginine (L-Arg) ameliorate intestinal inflammation, however the exact mechanisms of this effect are unclear. We hypothesized the anti-inflammatory effects of L-Arg require active transport and metabolism by inducible nitric oxide synthase (iNOS) to generate nitric oxide (NO). To test this hypothesis we examined the effects of L-Arg, L-Arg transport activity, NO production and iNOS inhibitor on IL-1β-mediated NF-κB-activation in Caco-2 cells. Methods Caco-2 cells were cultured, transfected with a NF-κB promoter luciferase vector, incubated ± L-Arg, ± IL-1β and luciferase activity was measured. Using siRNA we inhibited the L-Arg cationic amino acid transporter system y+ (CAT1) expression and examined its effects on L-Arg transport activity and IL-1β-mediated NF-κB-activation. Finally, the effects of sodium nitroprusside (SNP, a NO donor) and Nω-nitro-L-arginine (NNA, an iNOS inhibitor) on IL-1β-mediated NF-κB-activation were examined. Results IL-1β increased NF-κB luciferase activity (8-fold) and NF-κB expression (mRNA and protein), both of these were significantly decreased by L-Arg. System y+ CAT1 siRNA decreased CAT1 expression, L-Arg transport activity and attenuated the inhibitory effects of L-Arg on NF- κB activity. SNP attenuated the IL-1β-induced increase in NF-κB luciferase activity and expression, whereas NNA diminished the inhibitory effects of L-Arg on IL-1β-inducible NF- κB luciferase activity. Conclusion The inhibitory effects of L-Arg on IL-1β-mediated NF-κB-activation in Caco-2 cells involve L-Arg transport activity by CAT1, regulation of IL-1β-mediated increases in NF-κB expression, changes in iNOS expression and NO production. Our data suggest the inhibitory effects of L-Arg on NF-κB activation are mediated in part by iNOS since SNP preserves and NNA attenuates the effects of L-Arg on IL-1β-mediated NF-κB-activation and expression. PMID:28334039

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

    PubMed

    González, Raúl; De la Rosa, Ángel J; Romero-Brufau, Santiago; Barrera-Pulido, Lydia; Gallardo-Chamizo, Francisco; Pereira, Sheila; Marín, Luís M; Álamo, José M; Rodríguez-Hernández, Ángeles; Padillo, Francisco J; Muntané, Jordi

    2015-08-01

    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.

  13. Early STAT1 activation after systemic delivery of HSV amplicon vectors suppresses transcription of the vector-encoded transgene.

    PubMed

    Suzuki, Masataka; Chiocca, E Antonio; Saeki, Yoshinaga

    2007-11-01

    The herpes simplex virus (HSV) amplicon vector is a powerful gene delivery vehicle that can accommodate up to 150 kilobase of exogenous DNA. However, amplicon-mediated transgene expression is often transient outside the nervous system. In order to define the role of host immune responses in silencing amplicon-encoded transgenes, we evaluated the kinetics of cytokine-/chemokine-expression after tail-vein injection of a luciferase-encoding amplicon into mice. Type I interferons (IFNs) were induced earliest, within an hour after injection, and several other cytokines/chemokines were subsequently upregulated in the livers of wild-type (WT) mice. When the same experiment was performed in signal transducers and activators of transcription 1 (STAT1)-knockout (KO) mice, the levels of type I IFN expression were significantly lower and chemokine induction was almost non-existent. Importantly, STAT1-KO mice exhibited significantly higher and more sustained luciferase activity than did the WT mice, which is attributable to increased transcriptional activity rather than increased copy numbers of luciferase-encoding vector DNA. Further studies using primary cultured fibroblasts derived from WT and STAT1-KO mice revealed the significance of STAT1 signaling in transcriptional silencing of the amplicon-encoded transgene in vitro. Our results indicate that type I IFNs induced by systemic delivery of HSV amplicon vectors initiate a cascade of immune responses and suppress transgene expression at the transcriptional level by activation of STAT1.

  14. A new liposomal formulation of Gemcitabine is active in an orthotopic mouse model of pancreatic cancer accessible to bioluminescence imaging.

    PubMed

    Bornmann, C; Graeser, R; Esser, N; Ziroli, V; Jantscheff, P; Keck, T; Unger, C; Hopt, U T; Adam, U; Schaechtele, C; Massing, U; von Dobschuetz, E

    2008-03-01

    Despite its rapid enzymatic inactivation and therefore limited activity in vivo, Gemcitabine is the standard drug for pancreatic cancer treatment. To protect the drug, and achieve passive tumor targeting, we developed a liposomal formulation of Gemcitabine, GemLip (Ø: 36 nm: 47% entrapment). Its anti-tumoral activity was tested on MIA PaCa-2 cells growing orthotopically in nude mice. Bioluminescence measurement mediated by the stable integration of the luciferase gene was employed to randomize the mice, and monitor tumor growth. GemLip (4 and 8 mg/kg), Gemcitabine (240 mg/kg), and empty liposomes (equivalent to 8 mg/kg GemLip) were injected intravenously once weekly for 5 weeks. GemLip (8 mg/kg) stopped tumor growth, as measured via in vivo bioluminescence, reducing the primary tumor size by 68% (SD +/- 8%; p < 0.02), whereas Gemcitabine hardly affected tumor size (-7%; +/- 1.5%). In 80% of animals, luciferase activity in the liver indicated the presence of metastases. All treatments, including the empty liposomes, reduced the metastatic burden. Thus, GemLip shows promising antitumoral activity in this model. Surprisingly, empty liposomes attenuate the spread of metastases similar to Gemcitabine and GemLip. Further, luciferase marked tumor cells are a powerful tool to observe tumor growth in vivo, and to detect and quantify metastases.

  15. The refolding activity of the yeast heat shock proteins Ssa1 and Ssa2 defines their role in protein translocation

    PubMed Central

    1996-01-01

    Ssa1/2p, members of one of the yeast cytosolic hsp70 subfamilies, have been implicated in the translocation of secretory proteins into the lumen of the ER. The involvement of these hsp70s in translocation was tested directly by examining the effect of immunodepleting Ssa1/2p from yeast cytosol and subsequently testing the cytosol for its ability to support co- and post-translational translocation of prepro-alpha- factor. Depletion of Ssa1/2p had no effect on the efficiency of translocation in this in vitro assay. The system was used to examine the effect of the absence of Ssa1/2p on two other putative hsp70 functions: cotranslational folding of nascent luciferase and refolding of denatured luciferase. Depletion of Ssa1/2p had no effect on the ability of the yeast lysate to synthesize enzymatically active luciferase, but had a dramatic effect on the ability of the lysate to refold chemically denatured luciferase. These results demonstrate, for the first time, the refolding activity of Ssa1/2p in the context of the yeast cytosol, and define refolding activity as a chaperone function specific to Ssa1/2p, aprt from other cytosolic hsp70s. They also suggest that Ssa1/2p do not play a significant role in chaperoning the folding of nascent polypeptides. The implications of these findings for Ssa1/2p activity on their proposed role in the process of translocation are discussed. PMID:8947547

  16. Remotely activated protein-producing nanoparticles.

    PubMed

    Schroeder, Avi; Goldberg, Michael S; Kastrup, Christian; Wang, Yingxia; Jiang, Shan; Joseph, Brian J; Levins, Christopher G; Kannan, Sneha T; Langer, Robert; Anderson, Daniel G

    2012-06-13

    The development of responsive nanomaterials, nanoscale systems that actively respond to stimuli, is one general goal of nanotechnology. Here we develop nanoparticles that can be controllably triggered to synthesize proteins. The nanoparticles consist of lipid vesicles filled with the cellular machinery responsible for transcription and translation, including amino acids, ribosomes, and DNA caged with a photolabile protecting group. These particles served as nanofactories capable of producing proteins including green fluorescent protein (GFP) and enzymatically active luciferase. In vitro and in vivo, protein synthesis was spatially and temporally controllable, and could be initiated by irradiating micrometer-scale regions on the time scale of milliseconds. The ability to control protein synthesis inside nanomaterials may enable new strategies to facilitate the study of orthogonal proteins in a confined environment and for remotely activated drug delivery.

  17. TGEV infection up-regulates FcRn expression via activation of NF-κB signaling

    PubMed Central

    Guo, Jinyue; Li, Fei; Qian, Shaoju; Bi, Dingren; He, Qigai; Jin, Hui; Luo, Rui; Li, Shaowen; Meng, Xianrong; Li, Zili

    2016-01-01

    It has been well characterized that the neonatal Fc receptor (FcRn) transports maternal IgG to a fetus or newborn and protects IgG from degradation. We previously reported that FcRn is expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2). Transmissible gastroenteritis is an acute enteric disease of swine that is caused by transmissible gastroenteritis virus (TGEV). How porcine FcRn (pFcRn) expression is regulated by pathogenic infection remains unknown. Our research shows that IPEC-J2 cells infected with TGEV had up-regulated pFcRn expression. In addition, the NF-κB signaling pathway was activated in IPEC-J2 cells by TGEV infection. Furthermore, treatment of TGEV-infected IPEC-J2 cells with the NF-κB-specific inhibitor BAY 11-7082 resulted in down-regulation of pFcRn expression. Transient transfection of pFcRn promoter luciferase report plasmids with overexpression of NF-κB p65 transcription factor enhanced the activation of the luciferase report plasmids. We identified four NF-κB transcription factor binding sites in the promoter region of this gene using luciferase reporter system, chromatin immunoprecipitation, electromobility shift assay, and supershift analysis. Together, the data provide the first evidence that TGEV infection up-regulates pFcRn expression via activation of NF-κB signaling. PMID:27555521

  18. Aryl hydrocarbon receptor-mediated activity of particulate organic matter from the Paso del Norte airshed along the U.S.-Mexico border.

    PubMed

    Arrieta, Daniel E; Ontiveros, Cynthia C; Li, Wen-Whai; Garcia, Jose H; Denison, Michael S; McDonald, Jacob D; Burchiel, Scott W; Washburn, Barbara Shayne

    2003-08-01

    In this study, we determined the biologic activity of dichloromethane-extracted particulate matter < 10 micro m in aerodynamic diameter (PM10) obtained from filters at three sites in the Paso del Norte airshed, which includes El Paso, Texas, USA; Juarez, Chihuahua, Mexico, and Sunland Park, New Mexico, USA. The extracts were rich in polycyclic aromatic hydrocarbons (PAHs) and had significant biologic activity, measured using two in vitro assay systems: ethoxyresorufin-(O-deethylase (EROD) induction and the aryl hydrocarbon-receptor luciferase reporter system. In most cases, both EROD (5.25 pmol/min/mg protein) and luciferase activities (994 relative light units/mg) were highest in extracts from the Advance site located in an industrial neighborhood in Juarez. These values represented 58% and 55%, respectively, of induction associated with 1 micro M ss-naphthoflavone exposures. In contrast, little activity was observed at the Northeast Clinic site in El Paso, the reference site. In most cases, luciferase and EROD activity from extracts collected from the Tillman Health Center site, situated in downtown El Paso, fell between those observed at the other two sites. Overall, a statistically significant correlation existed between PM10 and EROD and luciferase activities. Chemical analysis of extracts collected from the Advance site demonstrated that concentrations of most PAHs were higher than those reported in most other metropolitan areas in the United States. Calculations made with these data suggest a cancer risk of 5-12 cases per 100,000 people. This risk estimate, as well as comparisons with the work of other investigators, raises concern regarding the potential for adverse health effects to the residents of this airshed. Further work is needed to understand the sources, exposure, and effects of PM10 and particulate organic material in the Paso del Norte airshed.

  19. Aryl hydrocarbon receptor-mediated activity of particulate organic matter from the Paso del Norte airshed along the U.S.-Mexico border.

    PubMed Central

    Arrieta, Daniel E; Ontiveros, Cynthia C; Li, Wen-Whai; Garcia, Jose H; Denison, Michael S; McDonald, Jacob D; Burchiel, Scott W; Washburn, Barbara Shayne

    2003-01-01

    In this study, we determined the biologic activity of dichloromethane-extracted particulate matter < 10 micro m in aerodynamic diameter (PM10) obtained from filters at three sites in the Paso del Norte airshed, which includes El Paso, Texas, USA; Juarez, Chihuahua, Mexico, and Sunland Park, New Mexico, USA. The extracts were rich in polycyclic aromatic hydrocarbons (PAHs) and had significant biologic activity, measured using two in vitro assay systems: ethoxyresorufin-(O-deethylase (EROD) induction and the aryl hydrocarbon-receptor luciferase reporter system. In most cases, both EROD (5.25 pmol/min/mg protein) and luciferase activities (994 relative light units/mg) were highest in extracts from the Advance site located in an industrial neighborhood in Juarez. These values represented 58% and 55%, respectively, of induction associated with 1 micro M ss-naphthoflavone exposures. In contrast, little activity was observed at the Northeast Clinic site in El Paso, the reference site. In most cases, luciferase and EROD activity from extracts collected from the Tillman Health Center site, situated in downtown El Paso, fell between those observed at the other two sites. Overall, a statistically significant correlation existed between PM10 and EROD and luciferase activities. Chemical analysis of extracts collected from the Advance site demonstrated that concentrations of most PAHs were higher than those reported in most other metropolitan areas in the United States. Calculations made with these data suggest a cancer risk of 5-12 cases per 100,000 people. This risk estimate, as well as comparisons with the work of other investigators, raises concern regarding the potential for adverse health effects to the residents of this airshed. Further work is needed to understand the sources, exposure, and effects of PM10 and particulate organic material in the Paso del Norte airshed. PMID:12896850

  20. An estrogen-responsive element-targeted histone deacetylase enzyme has an antiestrogen activity that differs from that of hydroxytamoxifen.

    PubMed

    Demirpence, Ediz; Semlali, Abdelhabib; Oliva, Joan; Balaguer, Patrick; Badia, Eric; Duchesne, Marie-Josèphe; Nicolas, Jean-Claude; Pons, Michel

    2002-11-15

    We showed previously that prolonged treatment of a MCF-7-derived cell line with hydroxytamoxifen (OHT) induces the irreversible silencing of some estrogen-responsive genes, whereas OHT-resistant cell growth appears simultaneously (E. Badia et al., Cancer Res., 60: 4130-4138, 2000). Based on the hypothesis that particular gene silencings could be involved in triggering the resistance phenomenon, we focused our study on the mechanism of OHT-induced silencing. More precisely, we wished to determine to what extent the recruited histone deacetylase (HDAC) activity, which is known to be involved in the repressive effect induced by antagonist ligands of nuclear receptors, could participate in various aspects of OHT effects, particularly in gene silencing. A fusion protein (HDAC-EG) of human HDAC1 fused with the estrogen receptor DNA-binding domain and the glucocorticoid receptor ligand-binding domain allowed targeting of chimeric HDAC1 activity on estrogen-responsive elements (EREs) in the presence of glucocorticoid ligands. When HDAC-EG was transiently expressed in HeLa cells together with estrogen receptor, an antiestrogen-like effect was obtained on an ERE-controlled luciferase reporter gene in the presence of agonist or antagonist glucocorticoids. In MCF-7-derived cells stably expressing HDAC-EG and an estrogen-regulated luciferase, liganded HDAC-EG again produced an antiestrogenic effect on expression of natural estrogen-regulated genes such as pS2, progesterone receptor, and cathepsin D and cell growth together with chimeric luciferase gene expression. However, a prolonged HDAC-EG-mediated antiestrogen effect did not lead to irreversible luciferase gene silencing, as OHT does. It nevertheless accelerated the OHT-driven phenomenon. The antiestrogen effect of OHT thus differs from that of an ERE-targeted HDAC1 activity that might participate in irreversible silencing but is not sufficient to trigger it.

  1. Estrogenic/antiestrogenic activity of selected selective serotonin reuptake inhibitors

    PubMed Central

    POP, ANCA; LUPU, DIANA IOANA; CHERFAN, JULIEN; KISS, BELA; LOGHIN, FELICIA

    2015-01-01

    Background and aims Selective serotonin reuptake inhibitors (SSRIs) are one of the most prescribed classes of psychotropics. Even though the SSRI class consists of 6 molecules (citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine and sertraline), only fluoxetine was intensively studied for endocrine disruptive effects, while the other SSRIs received less attention. This study was designed to evaluate the estrogenic/antiestrogenic effect of fluoxetine, sertraline and paroxetine. Methods The in vitro (anti)estrogenic activity was assessed using a firefly luciferase reporter construct in the T47D-KBluc breast cancer cell line. These cells express nuclear estrogen receptors that can activate the transcription of the luciferase reporter gene upon binding of estrogen receptor agonists. Results All three compounds were found to interact with the estrogen receptor. Fluoxetine had dual properties, weak estrogenic at lower concentrations and antiestrogenic effect at higher concentrations. Sertraline shared the same properties with fluoxetine, but also increased the estradiol-mediated transcriptional activity. Paroxetine presented only one type of effect, the ability to increase the estradiol-mediated transcriptional activity. Conclusions Overall, our results indicate a possible interaction of SSRIs with the estrogen receptor. As SSRIs are being used by all categories of population, including pregnant women or children, establishing whether they can affect the endocrine mediated mechanisms should be a priority. PMID:26609273

  2. Induction of cytochromes P450 1A1 and 1A2 by tanshinones in human HepG2 hepatoma cell line

    SciTech Connect

    Zhang Rong; Sun Jianguo; Ma Liping; Wu Xiaolan; Pan Guoyu; Hao Haiping; Zhou Fang; Jiye, A; Liu Changhui; Ai Hua; Shang Lili; Gao Haiyan; Peng Ying; Wan Ping; Wu Hui; Wang Guangji

    2011-04-01

    Diterpenoid tanshinones including tanshinone IIA (TIIA), cryptotanshinone (CTS), tanshinone I (TI) and dihydrotanshinone I (DHTI) are the major bioactive components from Danshen. The major aim of our present study was to investigate the induction potential of these four main components of tanshinones (TIIA, CTS, TI, and DHTI) on the expression of CYP1A1 and CYP1A2 in HepG2 cells. Our results showed that all of these four tanshinones caused a significant time- and concentration-dependent increase in the amount of CYP1A1/2 expression in HepG2 cells. These induction effects were further characterized through transcriptional regulation: the induction of CYP1A1/2 mRNA level by tanshinones was completely blocked by the transcription inhibitor actinomycin D; the expression of CYP1A1/2 heterogeneous nuclear RNA was induced by tanshinone treatment; and CYP1A1 mRNA stability was not influenced by these tanshinones. Interestingly, tanshinones plus B[a]P produced additive/synergistic effect on CYP1A1/2 induction. In addition, the tanshinone-induced CYP1A1/2 expression was abolished by the aryl hydrocarbon receptor (AhR) antagonist resveratrol, suggesting an AhR dependent transcription mechanism. In the reporter gene assay, while TI and DHTI significantly induced AhR-dependent luciferase activity, TIIA and CTS failed to induce this activity. Collectively, the tanshinones could induce CYP1A1 and CYP1A2 expression through transcriptional activation mechanism and exert differential effects on activating AhR in HepG2 cells. Our findings suggest that rational administration of tanshinones should be considered with respect to their effect on AhR and CYP1A1/2 expression.

  3. Immobilized alpha-melanocyte stimulating hormone 10-13 (GKPV) inhibits tumor necrosis factor-alpha stimulated NF-kappaB activity.

    PubMed

    Kelly, J M; Moir, A J G; Carlson, K; Yang, Y; MacNeil, S; Haycock, J W

    2006-02-01

    alpha-MSH is an anti-inflammatory peptide which signals by binding to the melanocortin-1 receptor (MC1R) and elevating cyclic AMP in several different cells and tissues. The carboxyl terminal peptides of alpha-MSH (KPV/GKPV) are the smallest minimal sequences that prevent inflammation, but it is not known if they operate via MC1R or cyclic AMP. The aim of this study was to examine the intracellular signaling potential of the GKPV peptide sequence when immobilized to polystyrene beads via a polyethylene glycol moiety. Beads containing an immobilized GKPV peptide were investigated for their ability to inhibit proinflammatory tumor necrosis factor-alpha (TNF-alpha) stimulated activation of NF-kappaB in HBL cells stably transfected with an NF-kappaB-luciferase reporter construct. Peptide functionalized beads were compared with the ability of soluble peptide alone (alpha-MSH or GKPV) or non-functionalized beads to inhibit TNF-alpha stimulated activation of NF-kappaB. GKPV peptide functionalized beads significantly inhibited NF-kappaB-luciferase activity in comparison to beads containing no peptide moiety in one of two growths conditions investigated. Soluble alpha-MSH and GKPV peptides were also confirmed to inhibit NF-kappaB-luciferase. The present study suggests that the carboxyl terminal MSH peptide acts via a cell receptor-based mechanism and furthermore may support the potential use of such immobilized ligands for anti-inflammatory therapeutic use.

  4. Data describing the effect of DRD4 promoter polymorphisms on promoter activity.

    PubMed

    Tei, Shoin; Mitsuhashi, Hiroaki; Ishiura, Shoichi

    2016-06-01

    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.

  5. Regulation of Activator Protein-1 by 8-iso-Prostaglandin E{sub 2} in a Thromboxane A{sub 2} Receptor-Dependent and -Independent Manner

    SciTech Connect

    Weber, Thomas J.; Markillie, Lye MENG.

    2003-05-01

    The thromboxane A{sub 2} (TXA{sub 2}) receptor (TP) is represented by two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP isoforms may be regulated by novel ligands termed the isoprostanes, which paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes (8-iso-PG{sub 2{sub {alpha}}} and 8-iso-PGE{sub 2}) regulate the activity of TP isoforms expressed in Chinese Hamster Ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist (U46619) in CHO cells transfected with the human TP-P and TP-E receptors and this response was fully inhibited by TP antagonists (ISAP, SQ29,548). AP-1-luciferase activity was potently (nM) increased by 8-iso-PGE2 in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, while 8-iso-PGF{sub 2{sub {alpha}}} was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE{sub 2} mediated AP-1-luciferase activity, indicating that this response is not dependent on de novo TXA2 biosynthesis. Interestingly, 8-iso-PGE{sub 2}-mediated AP-1-luciferase activity was near maximal in naive cells between 1-10 nM concentrations, and this response was not inhibited by TP antagonist or reproduced by agonists for TP or EP1/EP3 receptors. These observations (1) support a role for novel ligands in the regulation of TP-dependent signaling, (2) indicate that TP-P and TP-E couple to AP-1, (3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific fashion, and (4) indicate that additional targets regulated by 8-iso-PGE{sub 2} couple to AP-1.

  6. A novel mutation F826L in the human androgen receptor in partial androgen insensitivity syndrome; increased NH2-/COOH-terminal domain interaction and TIF2 co-activation.

    PubMed

    Wong, Hao Yun; Hoogerbrugge, Jos W; Pang, Kar Lok; van Leeuwen, Marije; van Royen, Martin E; Molier, Michel; Berrevoets, Cor A; Dooijes, Dennis; Dubbink, Hendrikus Jan; van de Wijngaart, Dennis J; Wolffenbuttel, Katja P; Trapman, Jan; Kleijer, Wim J; Drop, Stenvert L S; Grootegoed, J Anton; Brinkmann, Albert O

    2008-09-24

    A novel mutation F826L located within the ligand binding domain (LBD) of the human androgen receptor (AR) was investigated. This mutation was found in a boy with severe penoscrotal hypospadias (classified as 46,XY DSD). The AR mutant F826L appeared to be indistinguishable from the wild-type AR, with respect to ligand binding affinity, transcriptional activation of MMTV-luciferase and ARE2-TATA-luciferase reporter genes, protein level in genital skin fibroblasts (GSFs), and sub-cellular distribution in transfected cells. However, an at least two-fold higher NH2-/COOH-terminal domain interaction was found in luciferase and GST pull-down assays. A two-fold increase was also observed for TIF2 (transcription intermediary factor 2) co-activation of the AR F826L COOH-terminal domain. This increase could not be explained by a higher stability of the mutant protein, which was within wild-type range. Repression of transactivation by the nuclear receptor co-repressor (N-CoR) was not affected by the AR F826L mutation. The observed properties of AR F826L would be in agreement with an increased activity rather than with a partial defective AR transcriptional activation. It is concluded that the penoscrotal hypospadias in the present case is caused by an as yet unknown mechanism, which still may involve the mutant AR.

  7. Measurement of estrogenic activity in sediments from Haihe and Dagu River, China.

    PubMed

    Song, Maoyong; Xu, Yan; Jiang, Qinting; Lam, Paul K S; O'Toole, Desmond K; Giesy, John P; Jiang, Guibin

    2006-07-01

    Sediments from two rivers in China, the Haihe and Dagu Rivers, were examined for estrogenic activity using an estrogen receptor (ER)-mediated in vitro bioassay system. ER-active compounds were isolated from sediments by Soxhlet extraction, and the crude extracts were fractionated using a florisil column into three fractions. The estrogenic activity of each extract was detected by measuring luciferase activity in the human breast cancer cell line MCF-7 transfected with a luciferase receptor gene. Significant estrogenic activity was observed in each total extract. The 17beta-estradiol equivalents (E2-EQs) ranged from 8.24 to 95.28 ng E2 g(-1) dw. As a result, the relative estrogenic potencies of three fractions in this study descended in the order of Fraction 3>Fraction 2>Fraction 1. The results of the bioassay analysis indicated the heavy pollution status of these sites with estrogenic contaminants. In this study, five selected chemicals, the natural estrogens 17beta-estradiol (E2) and estrone (E1), and the xeno-estrogens 4-octylphenol (OP), 4-nonylphenol (NP), and Bisphenol A (BPA) were also analyzed using the in vitro bioassay. The estrogenic activity of these chemicals were E2>E1>NP>OP>BPA.

  8. Anti-Tat and anti-HIV activities of trimers of n-alkylglycines.

    PubMed

    Márquez, Nieves; Sancho, Rocío; Macho, Antonio; Moure, Alejandra; Masip, Isabel; Messeguer, Angel; Muñoz, Eduardo

    2006-02-28

    Transcription of human immunodeficiency virus (HIV-1) is activated by viral Tat protein which regulates HIV-LTR transcription and elongation. In the present report, the evaluation of the anti-Tat activity of a combinatorial library composed of 5120 N-trialkylglycines is reported. The antiviral activity was studied through luciferase-based assays targeting the HIV-1 promoter activation induced by the HIV-1 Tat protein. We identified five peptoids with specific anti-HIV-1 Tat activity; none of these peptoids affected the binding of HIV-1 Tat protein to the viral TAR RNA. Using a recombinant-virus assay in which luciferase activity correlates with the rate of HIV-1 transcription we have detected that one of the five selected peptoids, NC37-37-15C, is a potent inhibitor of HIV-1-LTR transcription in both primary T lymphocytes and transformed cell lines. The inhibitory effect of NC37-37-15C, which is additive with azidothymidine (AZT), correlates with its ability to inhibit CTD phosphorylation and shows a suitable profile for development of novel anti-HIV-1 drugs. Likewise, the structural simplicity of N-alkylglycine oligomers makes these peptidomimetics amenable to structural manipulation, thus facilitating the optimisation of lead molecules for drug-like properties.

  9. AroER tri-screen is a biologically relevant assay for endocrine disrupting chemicals modulating the activity of aromatase and/or the estrogen receptor.

    PubMed

    Chen, Shiuan; Zhou, Dujin; Hsin, Li-Yu; Kanaya, Noriko; Wong, Cynthie; Yip, Richard; Sakamuru, Srilatha; Xia, Menghang; Yuan, Yate-Ching; Witt, Kristine; Teng, Christina

    2014-05-01

    Endocrine disrupting chemicals (EDCs) interfere with the biosynthesis, metabolism, and functions of steroid hormones, including estrogens and androgens. Aromatase enzyme converts androgen to estrogen. Thus, EDCs against aromatase significantly impact estrogen- and/or androgen-dependent functions, including the development of breast cancer. The current study aimed to develop a biologically relevant cell-based high-throughput screening assay to identify EDCs that act as aromatase inhibitors (AIs), estrogen receptor (ER) agonists, and/or ER antagonists. The AroER tri-screen assay was developed by stable transfection of ER-positive, aromatase-expressing MCF-7 breast cancer cells with an estrogen responsive element (ERE) driven luciferase reporter plasmid. The AroER tri-screen can identify: estrogenic EDCs, which increase luciferase signal without 17β-estradiol (E2); anti-estrogenic EDCs, which inhibit the E2-induced luciferase signal; and AI-like EDCs, which suppress a testosterone-induced luciferase signal. The assay was first optimized in a 96-well plate format and then miniaturized into a 1536-well plate format. The AroER tri-screen was demonstrated to be suitable for high-throughput screening in the 1536-well plate format, with a 6.9-fold signal-to-background ratio, a 5.4% coefficient of variation, and a screening window coefficient (Z-factor) of 0.78. The assay suggested that bisphenol A (BPA) functions mainly as an ER agonist. Results from screening the 446 drugs in the National Institutes of Health Clinical Collection revealed 106 compounds that modulated ER and/or aromatase activities. Among these, two AIs (bifonazole and oxiconazole) and one ER agonist (paroxetine) were confirmed through alternative aromatase and ER activity assays. These findings indicate that AroER tri-screen is a useful high-throughput screening system for identifying ER ligands and aromatase-inhibiting chemicals.

  10. Nrf2 activity as a potential biomarker for the pan-epigenetic anticancer agent, RRx-001

    PubMed Central

    Ning, Shoucheng; Sekar, Thillai Veerapazham; Scicinski, Jan; Oronsky, Bryan; Peehl, Donna M.; Knox, Susan J.; Paulmurugan, Ramasamy

    2015-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulatory transcription factor that plays an important role in the antioxidant response pathway against anticancer drug-induced cytotoxic effects. RRx-001 is a new anticancer agent that generates reactive oxygen and nitrogen species, and leads to epigenetic alterations in cancer cells. Here we report the RRx-001 mediated nuclear translocation of Nrf2 and the activation of expression of its downstream enzymes HO-1 and NQO1 in tumor cells. Inhibition of intrinsic Nrf2 expression by Nrf2-specific siRNA increased cell sensitivity to RRx-001. Molecular imaging of tumor cells co-expressing pARE-Firefly luciferase and pCMV-Renilla luciferase-mRFP in vitro and in vivo in mice revealed that RRx-001 significantly increased ARE-FLUC signal in cells in a dose- and time-dependent manner, suggesting that RRx-001 is an effective activator of the Nrf2-ARE signaling pathway. The pre-treatment level of ARE-FLUC signal in cells, reflecting basal activity of Nrf2, negatively correlated with the tumor response to RRx-001. The results support the concept that RRx-001 activates Nrf2-ARE antioxidant signaling pathways in tumor cells. Hence measurement of Nrf2-mediated activation of downstream target genes through ARE signaling may constitute a useful molecular biomarker for the early prediction of response to RRx-001 treatment, and thereby guide therapeutic decision-making. PMID:26280276

  11. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    PubMed

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS.

  12. Anticancer activity of stoppin based on a novel peptide delivery system.

    PubMed

    Gao, Yan-Fang; Wei, Xue-Ni; Ye, Xiao-Lei; Weng, Guo-Bin; Chen, Yi-Chen; Zhao, Ya-Rong; Ji, Hui

    2015-10-01

    Stoppin (L1) is a newly identified anticancer peptide, which is a potent p53‑MDM2/MDMX inhibitor. Due to its limitation in cell delivery efficiency, a new peptide delivery system was developed based on a nucleic acid‑polypeptide‑liposome complex and its stability and effectiveness in vitro was investigated. The nucleic acid‑stoppin‑liposome complex was prepared and characterization of the complex was conducted. The stability of the complex was evaluated by enzyme digestion. Following transfection of the A549 cells with the complex, detection of green fluorescent protein (GFP) and luciferase activity was conducted to evaluate transfection efficiency. In addition, the anticancer activity of the complex was determined by 3‑(4,5‑dimethyl‑thiazolyl‑2)‑2,5 diphenyltetrazolium bromide assay and apoptosis was detected by flow cytometry. The results indicated that the particle size of the complex was 102±10 nm and the encapsulation rate was ~100% when the ratio of liposome, L1 and plasmid was: 4 µl:1 µg:2 µg. The enzyme digestion experiment demonstrated that the complex was resistant to pancreatic and DNA enzyme degradation, indicating that the complex had biological stability. Cell transfection demonstrated that it had a mutual promotion effect on delivery, which could be confirmed by GFP fluorescence and luciferase assay. The cell‑killing efficiency of this novel delivery system was three times higher than with stoppin alone at a low concentration. In conclusion, this novel stoppin peptide delivery system was stable. The nucleic acid‑peptide‑liposome complex can protect the internal component from the degradation of enzymes, promote entry of the peptide into the cells and enhance the anti‑tumor activity of stoppin. Therefore, it is a promising approach for peptide delivery, which can be characterized and visualized using plasmids with GFP or luciferase.

  13. Peroxisome proliferator-activated receptor gamma regulates expression of signal transducer and activator of transcription 5A

    SciTech Connect

    Olsen, Hanne; Haldosen, Lars-Arne . E-mail: Lars-Arne.Haldosen@mednut.ki.se

    2006-05-01

    Signal transducer and activator of transcription 5A (STAT5A) has been shown to be important for terminal differentiation of mammary epithelial cells. In order to understand regulation of expression of STAT5A, the 5' end of the mouse Stat5a gene was isolated. Putative regulatory elements was searched for and several peroxisome proliferator response elements (PPREs) were found, one with high (12/13 nucleotides) and three with less (8-10/13) similarity to the reported consensus sequence. Mouse mammary epithelial HC11 cells were treated with peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligand, the thiazolidinedione (TZD) troglitazone, and an increase in STAT5A protein expression was seen. The 5' flank of Stat5a gene was cloned in a luciferase reporter vector. A concentration dependent activation of the STAT5A-luciferase reporter was detected, when transiently transfected HC11 cells were treated with TZD. The activation could be inhibited by treatment with a PPAR{gamma} antagonist. It has earlier been shown that epidermal growth factor (EGF) induces MAPK phosphorylation of PPAR{gamma} resulting in a less transcriptionally active receptor. In HC11 cells, EGF inhibited TZD induced STAT5A-reporter activity suggesting that our previously reported EGF-mediated suppression of STAT5A expression is mediated in all or partly through inhibition of PPAR{gamma} activity. Furthermore, the MEK inhibitor PD98059 inhibited the EGF effect. All together, data presented suggest that PPAR{gamma} participates in regulation of STAT5A expression.

  14. Molecular mechanisms of p21 and p27 induction by 3-methylcholanthrene, an aryl-hydrocarbon receptor agonist, involved in antiproliferation of human umbilical vascular endothelial cells.

    PubMed

    Pang, Pai-Huei; Lin, Ying-Hsi; Lee, Yi-Hsuan; Hou, Hsing-Han; Hsu, Sung-Po; Juan, Shu-Hui

    2008-04-01

    We previously reported that 3-methylcholanthrene (3MC), an aryl-hydrocarbon receptor (AhR) agonist, inhibits the proliferation of human umbilical vascular endothelial cells (HUVECs; Juan et al., 2006, Eur J Pharmacol 530: 1-8). Herein, pretreatment of HUVECs with p21 or p27 small interfering (si)RNA reduced 3MC-induced elimination of [(3)H]thymidine incorporation, demonstrating their essential roles in the antiproliferation of HUVECs. The molecular mechanisms of p21 and p27 involved in the antiproliferative effects of 3MC were elucidated in this study. 3MC time- and concentration-dependently increased p21 and p27 levels, and decreased the protein level of CDK2 with no apparent alteration of p53. Interestingly, 3MC-mediated p21 and p27 inductions were eliminated by resveratrol, an AhR antagonist, suggesting their AhR dependency, further confirmed by AhR siRNA. Among the relevant pathways, p38MAPK activation sustained the levels of p21 and p27 induced by 3MC, which was eliminated by AhR antagonists and N-acetylcysteine (NAC), an antioxidant. 3MC concentration-dependently enhanced not only the consensus dioxin-responsive element (DRE)-driven luciferase activity, but also the binding activity of the AhR to the putative DRE derived from the p21 and p27 promoters. A deletion of the DRE (-285/-270) in p21 (-2,300/+8) only partially alleviated the 3MC-induced luciferase activity unless NAC was added, suggesting that there may be a DRE-independent mechanism associated with oxidative stress. However, a deletion of the DRE (-660/-645) in p27 (-1,358/-100) almost completely abrogated the activation. Our study demonstrated that both the functional DRE and the phosphorylation of p38MAPK are essential for the induction of p21 and p27, resulting in the antiproliferative action of 3MC in HUVECs.

  15. The novel secretory protein CGREF1 inhibits the activation of AP-1 transcriptional activity and cell proliferation.

    PubMed

    Deng, Weiwei; Wang, Lan; Xiong, Ying; Li, Jing; Wang, Ying; Shi, Taiping; Ma, Dalong

    2015-08-01

    The transcription factor AP-1 plays an important role in inflammation and cell survival. Using a dual-luciferase reporter assay system and a library of 940 candidate human secretory protein cDNA clones, we identified that CGREF1 can inhibit the transcriptional activity of AP-1. We demonstrated that CGREF1 is secreted via the classical secretory pathway through the ER-to-Golgi apparatus. Functional investigations revealed that overexpression of CGREF1 can significantly inhibit the phosphorylation of ERK and p38 MAPK, and suppress the proliferation of HEK293T and HCT116 cells. Conversely, specific siRNAs against CGREF1 can increase the transcriptional activity of AP-1. These results clearly indicated that CGREF1 is a novel secretory protein, and plays an important role in regulation of AP-1 transcriptional activity and cell proliferation.

  16. Polyacetylenes from Notopterygium incisum–New Selective Partial Agonists of Peroxisome Proliferator-Activated Receptor-Gamma

    PubMed Central

    Liu, Xin; Noha, Stefan M.; Malainer, Clemens; Kramer, Matthias P.; Cocic, Amina; Kunert, Olaf; Schinkovitz, Andreas; Heiss, Elke H.; Schuster, Daniela

    2013-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism and therefore an important pharmacological target to combat metabolic diseases. Since the currently used full PPARγ agonists display serious side effects, identification of novel ligands, particularly partial agonists, is highly relevant. Searching for new active compounds, we investigated extracts of the underground parts of Notopterygium incisum, a medicinal plant used in traditional Chinese medicine, and observed significant PPARγ activation using a PPARγ-driven luciferase reporter model. Activity-guided fractionation of the dichloromethane extract led to the isolation of six polyacetylenes, which displayed properties of selective partial PPARγ agonists in the luciferase reporter model. Since PPARγ activation by this class of compounds has so far not been reported, we have chosen the prototypical polyacetylene falcarindiol for further investigation. The effect of falcarindiol (10 µM) in the luciferase reporter model was blocked upon co-treatment with the PPARγ antagonist T0070907 (1 µM). Falcarindiol bound to the purified human PPARγ receptor with a Ki of 3.07 µM. In silico docking studies suggested a binding mode within the ligand binding site, where hydrogen bonds to Cys285 and Glu295 are predicted to be formed in addition to extensive hydrophobic interactions. Furthermore, falcarindiol further induced 3T3-L1 preadipocyte differentiation and enhanced the insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes confirming effectiveness in cell models with endogenous PPARγ expression. In conclusion, we identified falcarindiol-type polyacetylenes as a novel class of natural partial PPARγ agonists, having potential to be further explored as pharmaceutical leads or dietary supplements. PMID:23630612

  17. Thrombin-induced CCAAT/enhancer-binding protein β activation and IL-8/CXCL8 expression via MEKK1, ERK, and p90 ribosomal S6 kinase 1 in lung epithelial cells.

    PubMed

    Lin, Chien-Huang; Nai, Po-Ling; Bien, Mauo-Ying; Yu, Chung-Chi; Chen, Bing-Chang

    2014-01-01

    Thrombin, a serine protease, is a well-known coagulation factor generated during vascular injury and plays an important role in lung inflammation. We previously showed that the c-Src- and Rac/PI3K/Akt-dependent NF-κB pathways are involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells (A549). In this study, we investigated the role of the MEK kinase (MEKK)1/ERK/p90 ribosomal S6 kinase (RSK)1-dependent C/EBPβ signaling pathway in thrombin-induced IL-8/CXCL8 expression. Thrombin-induced IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity were attenuated by small interfering RNA (siRNA) of C/EBPβ and by cells transfected with the C/EBPβ site mutation of the IL-8/CXCL8 construct. Moreover, thrombin-induced κB-luciferase activity was also inhibited by C/EBPβ siRNA. The thrombin-induced increases in IL-8/CXCL8 release and IL-8/CXCL8-luciferase were also inhibited by MEKK1 siRNA, PD98059 (an MEK inhibitor), U0126 (an ERK inhibitor), and RSK1 siRNA. Treatment of cells with thrombin caused an increase in C/EBPβ phosphorylation at Thr(235), C/EBPβ-luciferase activity, recruitment of C/EBPβ to the IL-8/CXCL8 promoter, and C/EBPβ-specific DNA complex formation. Furthermore, thrombin-mediated C/EBPβ phosphorylation and C/EBPβ-luciferase activity were inhibited by MEKK1 siRNA, PD98059, and RSK1 siRNA. Stimulation of cells with thrombin resulted in an increase in RSK1 phosphorylation at Thr(359)/Ser(363), and this effect was inhibited by MEKK1 siRNA and PD98059. The thrombin-induced increase in ERK activation was inhibited by MEKK1 siRNA. These results imply that thrombin activates the MEKK1/ERK/RSK1 signaling pathway, which in turn initiates C/EBPβ activation, recruitment of C/EBPβ to the IL-8/CXCL8 promoter, and C/EBPβ-specific DNA complex formation, and ultimately induces IL-8/CXCL8 expression and release in lung epithelial cells.

  18. Biological Activity of Masked Endotoxin

    PubMed Central

    Schwarz, Harald; Gornicec, Jan; Neuper, Theresa; Parigiani, Maria Alejandra; Wallner, Michael; Duschl, Albert; Horejs-Hoeck, Jutta

    2017-01-01

    Low endotoxin recovery (LER) is a recently discovered phenomenon describing the inability of limulus amebocyte lysate (LAL)-based assays to detect lipopolysaccharide (LPS) because of a “masking effect” caused by chelators or detergents commonly used in buffer formulations for medical products and recombinant proteins. This study investigates the masking capacities of different buffer formulations and whether masked endotoxin is biologically active. We show that both naturally occurring endotoxin as well as control standard endotoxin can be affected by LER. Furthermore, whereas masked endotoxin cannot be detected in Factor C based assays, it is still detectable in a cell-based TLR4-NF-κB-luciferase reporter gene assay. Moreover, in primary human monocytes, masked LPS induces the expression of pro-inflammatory cytokines and surface activation markers even at very low concentrations. We therefore conclude that masked LPS is a potent trigger of immune responses, which emphasizes the potential danger of masked LPS, as it may pose a health threat in pharmaceutical products or compromise experimental results. PMID:28317862

  19. Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state

    PubMed Central

    Hume, David A; Underhill, David M; Sweet, Matthew J; Ozinsky, Adrian O; Liew, Foo Y; Aderem, Alan

    2001-01-01

    Background Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive. Results RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1β promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6. Conclusions These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a

  20. Activity of a Py-Im polyamide targeted to the estrogen response element.

    PubMed

    Nickols, Nicholas G; Szablowski, Jerzy O; Hargrove, Amanda E; Li, Benjamin C; Raskatov, Jevgenij A; Dervan, Peter B

    2013-05-01

    Pyrrole-imidazole (Py-Im) polyamides are a class of programmable DNA minor groove binders capable of modulating the activity of DNA-binding proteins and affecting changes in gene expression. Estrogen receptor alpha (ERα) is a ligand-activated hormone receptor that binds as a homodimer to estrogen response elements (ERE) and is a driving oncogene in a majority of breast cancers. We tested a selection of structurally similar Py-Im polyamides with differing DNA sequence specificity for activity against 17β-estadiol (E2)-induced transcription and cytotoxicity in ERα positive, E2-stimulated T47DKBluc cells, which express luciferase under ERα control. The most active polyamide targeted the sequence 5'-WGGWCW-3' (W = A or T), which is the canonical ERE half site. Whole transcriptome analysis using RNA-Seq revealed that treatment of E2-stimulated breast cancer cells with this polyamide reduced the effects of E2 on the majority of those most strongly affected by E2 but had much less effect on the majority of E2-induced transcripts. In vivo, this polyamide circulated at detectable levels following subcutaneous injection and reduced levels of ER-driven luciferase expression in xenografted tumors in mice after subcutaneous compound administration without significant host toxicity.

  1. 4(1H)-Quinolones with liver stage activity against Plasmodium berghei.

    PubMed

    Lacrue, Alexis N; Sáenz, Fabián E; Cross, R Matthew; Udenze, Kenneth O; Monastyrskyi, Andrii; Stein, Steven; Mutka, Tina S; Manetsch, Roman; Kyle, Dennis E

    2013-01-01

    With the exception of primaquine, tafenoquine, and atovaquone, there are very few antimalarials that target liver stage parasites. In this study, a transgenic Plasmodium berghei parasite (1052Cl1; PbGFP-Luc(con)) that expresses luciferase was used to assess the anti-liver stage parasite activity of ICI 56,780, a 7-(2-phenoxyethoxy)-4(1H)-quinolone (PEQ), as well as two 3-phenyl-4(1H)-quinolones (P4Q), P4Q-146 and P4Q-158, by using bioluminescent imaging (BLI). Results showed that all of the compounds were active against liver stage parasites; however, ICI 56,780 and P4Q-158 were the most active, with low nanomolar activity in vitro and causal prophylactic activity in vivo. This potent activity makes these compounds ideal candidates for advancement as novel antimalarials.

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

    SciTech Connect

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

    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.

  3. Activation of mouse and human peroxisome proliferator-activated receptor alpha by perfluoroalkyl acids of different functional groups and chain lengths.

    PubMed

    Wolf, Cynthia J; Takacs, Margy L; Schmid, Judith E; Lau, Christopher; Abbott, Barbara D

    2008-11-01

    Perfluoroalkyl acids (PFAAs) are surfactants used in consumer products and persist in the environment. Some PFAAs elicit adverse effects on rodent development and survival. PFAAs can activate peroxisome proliferator-activated receptor alpha (PPARalpha) and may act via PPARalpha to produce some of their effects. This study evaluated the ability of numerous PFAAs to induce mouse and human PPARalpha activity in a transiently transfected COS-1 cell assay. COS-1 cells were transfected with either a mouse or human PPARalpha receptor-luciferase reporter plasmid. After 24 h, cells were exposed to either negative controls (water or dimethyl sulfoxide, 0.1%); positive control (WY-14643, PPARalpha agonist); perfluorooctanoic acid or perfluorononanoic acid at 0.5-100 microM; perfluorobutanoic acid, perfluorohexanoic acid, perfluorohexane sulfonate, or perfluorodecanoic acid (PFDA) at 5-100 microM; or perfluorobutane sulfonate or perfluorooctane sulfonate at 1-250 microM. After 24 h of exposure, luciferase activity from the plasmid was measured. Each PFAA activated both mouse and human PPARalpha in a concentration-dependent fashion, except PFDA with human PPARalpha. Activation of PPARalpha by PFAA carboxylates was positively correlated with carbon chain length, up to C9. PPARalpha activity was higher in response to carboxylates compared to sulfonates. Activation of mouse PPARalpha was generally higher compared to that of human PPARalpha. We conclude that, in general, (1) PFAAs of increasing carbon backbone chain lengths induce increasing activity of the mouse and human PPARalpha with a few exceptions, (2) PFAA carboxylates are stronger activators of mouse and human PPARalpha than PFAA sulfonates, and (3) in most cases, the mouse PPARalpha appears to be more sensitive to PFAAs than the human PPARalpha in this model.

  4. Cloning and transcriptional activity analysis of the porcine cofilin 2 gene promoter.

    PubMed

    Wang, Jia-Mei; Lang, Bin; Zhu, Hong-yan; Du, Hai-ting; Tian, Yu-min; Su, Yu-hong

    2014-09-01

    Cofilins (CFL), including CFL1 and CFL2, are members of the family of actin-binding proteins in eukaryote. CFL2 is predominantly expressed in mammalian skeletal muscle and heart and is important to muscle fiber formation and muscular regeneration. To study transcriptional regulation of porcine CFL2, a 2.5 kb upstream sequence starting from the major CFL2 transcriptional start site was cloned by genome walking. Twelve DNA fragments of the 5' flank region of the porcine CFL2 gene were further isolated from porcine genomic DNA via PCR and inserted into the luciferase reporter vector pGL4.10 to make 12 CFL2 reporter constructs. All reporter vectors were transfected into C2C12, NIH3T3, or Hela cells and their relative luciferase activity measured after 48 h, respectively. Bioinformatics analysis suggested that there were two TATA-boxes at the -508 bp and -453 bp, as well as a GC-box and a CAAT-box in this sequence. Additional transcription factor binding sites including SP1, AP1, AP2, and GATA-1 sites were also predicted. The transcriptional activity of pGL4.10-1554 (1502 bp to +51 bp) was the strongest, and the promoter's active region was mapped to a region from -1502 bp to -1317 bp. Our data provide a foundation for future studies into transcriptional regulation of CFL2.

  5. Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner*

    PubMed Central

    Tirodkar, Tejas S.; Lu, Ping; Bai, Aiping; Scheffel, Matthew J.; Gencer, Salih; Garrett-Mayer, Elizabeth; Bielawska, Alicja; Ogretmen, Besim; Voelkel-Johnson, Christina

    2015-01-01

    A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14–26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3–5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity. PMID:25839235

  6. Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner.

    PubMed

    Tirodkar, Tejas S; Lu, Ping; Bai, Aiping; Scheffel, Matthew J; Gencer, Salih; Garrett-Mayer, Elizabeth; Bielawska, Alicja; Ogretmen, Besim; Voelkel-Johnson, Christina

    2015-05-22

    A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14-26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3-5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity.

  7. The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites.

    PubMed

    Fernández-Calero, Tamara; Flouriot, Gilles; Marín, Mónica

    2016-01-01

    Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene.

  8. Hepatitis C virus non-structural protein-2 activates CXCL-8 transcription through NF-kappaB.

    PubMed

    Oem, J-K; Jackel-Cram, C; Li, Y-P; Kang, H-N; Zhou, Y; Babiuk, L A; Liu, Q

    2008-01-01

    Hepatitis C is a devastating disease worldwide. Proteins encoded by the etiologic agent, hepatitis C virus (HCV), are believed to play important roles in HCV-associated pathogenesis. However, the biological functions of the non-structural protein-2 (NS2) encoded by HCV are not well characterized. Here, we show that HCV NS2 protein activates CXCL-8 (interleukin-8, IL-8) transcription in HepG2 cells as measured by reverse transcription-polymerase chain reaction and IL-8 promoter-luciferase reporter assays. Furthermore, when the kappaB site on the IL-8 promoter was eliminated by mutagenesis or when intracellular NF-kappaB activity was suppressed by an inhibitor, NS2 did not activate the IL-8 promoter, suggesting a role of NF-kappaB in this process. These results prompted us to hypothesize that HCV NS2 might be able to activate NF-kappaB. This hypothesis was tested by determination of NF-kappaB-driven reporter gene expression and NF-kappaB p65 subunit subcellular localization after HCV NS2 expression. Indeed, NS2 could up-regulate NF-kappaB-driven luciferase activity and was associated with p65 nuclear localization. These results demonstrate that HCV NS2 up-regulates IL-8 transcription through NF-kappaB. This newly identified function increases our understanding of the role of HCV NS2 protein in virus-host interactions.

  9. CerS6 Is a Novel Transcriptional Target of p53 Protein Activated by Non-genotoxic Stress.

    PubMed

    Fekry, Baharan; Jeffries, Kristen A; Esmaeilniakooshkghazi, Amin; Ogretmen, Besim; Krupenko, Sergey A; Krupenko, Natalia I

    2016-08-05

    Our previous study suggested that ceramide synthase 6 (CerS6), an enzyme in sphingolipid biosynthesis, is regulated by p53: CerS6 was elevated in several cell lines in response to transient expression of p53 or in response to folate stress, which is known to activate p53. It was not clear, however, whether CerS6 gene is a direct transcriptional target of p53 or whether this was an indirect effect through additional regulatory factors. In the present study, we have shown that the CerS6 promoter is activated by p53 in luciferase assays, whereas transcriptionally inactive R175H p53 mutant failed to induce the luciferase expression from this promoter. In vitro immunoprecipitation assays and gel shift analyses have further demonstrated that purified p53 binds within the CerS6 promoter sequence spanning 91 bp upstream and 60 bp downstream of the transcription start site. The Promo 3.0.2 online tool for the prediction of transcription factor binding sites indicated the presence of numerous putative non-canonical p53 binding motifs in the CerS6 promoter. Luciferase assays and gel shift analysis have identified a single motif upstream of the transcription start as a key p53 response element. Treatment of cells with Nutlin-3 or low concentrations of actinomycin D resulted in a strong elevation of CerS6 mRNA and protein, thus demonstrating that CerS6 is a component of the non-genotoxic p53-dependent cellular stress response. This study has shown that by direct transcriptional activation of CerS6, p53 can regulate specific ceramide biosynthesis, which contributes to the pro-apoptotic cellular response.

  10. Acylcarnitines activate proinflammatory signaling pathways.

    PubMed

    Rutkowsky, Jennifer M; Knotts, Trina A; Ono-Moore, Kikumi D; McCoin, Colin S; Huang, Shurong; Schneider, Dina; Singh, Shamsher; Adams, Sean H; Hwang, Daniel H

    2014-06-15

    Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed D,L isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25 μM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.

  11. Acylcarnitines activate proinflammatory signaling pathways

    PubMed Central

    Rutkowsky, Jennifer M.; Knotts, Trina A.; Ono-Moore, Kikumi D.; McCoin, Colin S.; Huang, Shurong; Schneider, Dina; Singh, Shamsher; Hwang, Daniel H.

    2014-01-01

    Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed d,l isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant l-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. l-C14 carnitine (5–25 μM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, l-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, l-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified. PMID:24760988

  12. Detection of estrogen- and dioxin-like activity in pulp and paper mill black liquor and effluent using in vitro bioassays

    SciTech Connect

    Zacharewski, T.; Berhane, K.; Gillesby, B.; Burnison, K. |

    1995-12-31

    Pulp and paper mill effluent contains a complex mixture of compounds which adversely affect fish physiologically and at the population level. These effects include compromised reproductive fitness and the induction of mixed-function oxidase activities; two classic responses mediated by the estrogen and/or Ah receptor. In vitro recombinant receptor/reporter gene assays were used to examine pulp and paper mill black liquor and effluent for estrogenic, dioxin-like and antiestrogenic activities. Using MCF7 cells transiently transfected with a Gal4-estrogen receptor chimeric construct (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc), it was estimated that black liquor contains 4 {+-} 2 ppb ``estrogen equivalents``, while negligible estrogenic activity was observed in a methanol-extracted pulp and paper mill effluent fraction (MF). A dioxin response element (DRE)-regulated luciferase reporter gene (pGudLucl.1) transiently transfected into Hepalclc7 wild type cells exhibited a dose-dependent increase in luciferase activity following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDO), black liquor and MF. Based on the dose response curves, black liquor and MF contain 10 {+-} 4 ppb and 20 {+-} 6 ppt ``TCDD equivalents``, respectively. Moreover, MF exhibited significant AhR-mediated antiestrogenic activity. These results demonstrate the utility of these bioassays and suggest that the effects observed in fish exposed to pulp and paper mill effluent may be due to unidentified ER and AhR ligands not detected by conventional chemical analysis due to the lack of appropriate chemical standards.

  13. Microglial phenotype is regulated by activity of the transcription factor, NFAT

    PubMed Central

    Nagamoto-Combs, Kumi

    2010-01-01

    The transcription factor family, nuclear factor of activated T cells (NFAT), regulates immune cell phenotype. Four different calcium/calmodulin-regulated isoforms have been identified in the periphery, but isoform expression in microglia, the resident immune cells of the central nervous system, has not been fully defined. In this study microglial NFAT isoform expression and involvement in regulating inflammatory responses in murine primary microglia culture was examined. Western blot analysis demonstrated robust detection of NFATc1 and c2 isoforms in microglia. Electrophoretic mobility shift assays demonstrated increased NFAT-DNA binding from nuclear extracts of lipopolysaccharide (LPS) stimulated microglia. Moreover, LPS-stimulated microglia behaved similarly to T cell receptor agonist antibody-stimulated Jurkat cells demonstrating a transient increase in NFAT-driven luciferase reporter gene expression. LPS-induced NFAT-luciferase activity in microglia was attenuated by pretreatment with tat-VIVIT, a cell-permeable NFAT inhibitory peptide. Furthermore, LPS-mediated secretion of microglial cytokines, TNF-α and MCP-1, was decreased by treatment with tat-VIVIT but not with tat-VEET, a negative control peptide. These results demonstrate that NFAT plays a role in regulating proinflammatory responses in cultured murine microglia. PMID:20631193

  14. Molecular mechanisms underlying the regulation of the MFG-E8 gene promoter activity in physiological and inflammatory conditions

    PubMed Central

    Wang, Xiao; Bu, Heng-Fu; Liu, Shirley XL; De Plaen, Isabelle G.; Tan, Xiao-Di

    2015-01-01

    Milk fat globule-EGF factor 8 (MFG-E8) is expressed by macrophages and plays an important role in attenuating inflammation and maintaining tissue homeostasis. Previously, we and others found that LPS inhibits MFG-E8 gene expression in macrophages. Here, we characterized the 5′-flanking region of the mouse MFG-E8 gene. To functionally analyze the upstream regulatory region of the MFG-E8 gene, a series of luciferase reporter gene constructs containing deleted or mutated regulatory elements were prepared. Using the luciferase assay, we revealed that Sp1 binding motifs within the proximal promoter region were necessary for full activity of the MFG-E8 promoter, whereas AP-1 like binding sequence at −372 played a role in governing the promoter activity at a homeostatic level. With chromatin immunoprecipitation assay, we showed that Sp1 and c-Jun physically interact with the MFG-E8 promoter region in vivo. In addition, Sp1 was found to regulate the MFG-E8 promoter activity positively and c-Jun negatively. Furthermore, we demonstrated that LPS inhibited MFG-E8 promoter activity via targeting Sp1 and AP-1-like motifs in the 5′-flanking region. Collectively, our data indicate that Sp1 and AP-1-related factors are involved in the regulation of MFG-E8 gene transcription by targeting their binding sites in the 5′-flanking region under physiological and inflammatory states. PMID:25711369

  15. The early-onset torsion dystonia-associated protein, torsinA, displays molecular chaperone activity in vitro

    PubMed Central

    Burdette, Alexander J.; Churchill, Perry F.; Caldwell, Guy A.

    2010-01-01

    TorsinA is a member of the AAA+ ATPase family of proteins and, notably, is the only known ATPase localized to the ER lumen. It has been suggested to act as a molecular chaperone, while a mutant form associated with early-onset torsion dystonia, a dominantly inherited movement disorder, appears to result in a net loss of function in vivo. Thus far, no studies have examined the chaperone activity of torsinA in vitro. Here we expressed and purified both wild-type (WT) and mutant torsinA fusion proteins in bacteria and examined their ability to function as molecular chaperones by monitoring suppression of luciferase and citrate synthase (CS) aggregation. We also assessed their ability to hold proteins in an intermediate state for refolding. As measured by light scattering and SDS-PAGE, both WT and mutant torsinA effectively, and similarly, suppressed protein aggregation compared to controls. This function was not further enhanced by the presence of ATP. Further, we found that while neither form of torsinA could protect CS from heat-induced inactivation, they were both able to reactivate luciferase when ATP and rabbit reticulocyte lysate were added. This suggests that torsinA holds luciferase in an intermediate state, which can then be refolded in the presence of other chaperones. These data provide conclusive evidence that torsinA acts as a molecular chaperone in vitro and suggests that early-onset torsion dystonia is likely not a consequence of a loss in torsinA chaperone activity but might be an outcome of insufficient torsinA localization at the ER to manage protein folding or trafficking. PMID:20169475

  16. Molecular Imaging of the ATM Kinase Activity

    SciTech Connect

    Williams, Terence M.; Nyati, Shyam; Ross, Brian D.; Rehemtulla, Alnawaz

    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.

  17. Mitochondrial-targeted aryl hydrocarbon receptor and the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cellular respiration and the mitochondrial proteome.

    PubMed

    Hwang, Hye Jin; Dornbos, Peter; Steidemann, Michelle; Dunivin, Taylor K; Rizzo, Mike; LaPres, John J

    2016-08-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor within the Per-Arnt-Sim (PAS) domain superfamily. Exposure to the most potent AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is associated with various pathological effects including metabolic syndrome. While research over the last several years has demonstrated a role for oxidative stress and metabolic dysfunction in AHR-dependent TCDD-induced toxicity, the role of the mitochondria in this process has not been fully explored. Our previous research suggested that a portion of the cellular pool of AHR could be found in the mitochondria (mitoAHR). Using a protease protection assay with digitonin extraction, we have now shown that this mitoAHR is localized to the inter-membrane space (IMS) of the organelle. TCDD exposure induced a degradation of mitoAHR similar to that of cytosolic AHR. Furthermore, siRNA-mediated knockdown revealed that translocase of outer-mitochondrial membrane 20 (TOMM20) was involved in the import of AHR into the mitochondria. In addition, TCDD altered cellular respiration in an AHR-dependent manner to maintain respiratory efficiency as measured by oxygen consumption rate (OCR). Stable isotope labeling by amino acids in cell culture (SILAC) identified a battery of proteins within the mitochondrial proteome influenced by TCDD in an AHR-dependent manner. Among these, 17 proteins with fold changes≥2 are associated with various metabolic pathways, suggesting a role of mitochondrial retrograde signaling in TCDD-mediated pathologies. Collectively, these studies suggest that mitoAHR is localized to the IMS and AHR-dependent TCDD-induced toxicity, including metabolic dysfunction, wasting syndrome, and hepatic steatosis, involves mitochondrial dysfunction.

  18. The New 4-O-Methylhonokiol Analog GS12021 Inhibits Inflammation and Macrophage Chemotaxis: Role of AMP-Activated Protein Kinase α Activation

    PubMed Central

    Kim, Sora; Ka, Sun-O; Lee, Youngyi; Park, Byung-Hyun; Fei, Xiang; Jung, Jae-Kyung; Seo, Seung-Yong; Bae, Eun Ju

    2015-01-01

    Preventing pathologic tissue inflammation is key to treating obesity-induced insulin resistance and type 2 diabetes. Previously, we synthesized a series of methylhonokiol analogs and reported that compounds with a carbamate structure had inhibitory function against cyclooxygenase-2 in a cell-free enzyme assay. However, whether these compounds could inhibit the expression of inflammatory genes in macrophages has not been investigated. Here, we found that a new 4-O-methylhonokiol analog, 3′,5-diallyl-4′-methoxy-[1,1′-biphenyl]-2-yl morpholine-4-carboxylate (GS12021) inhibited LPS- or TNFα-stimulated inflammation in macrophages and adipocytes, respectively. LPS-induced phosphorylation of nuclear factor-kappa B (NF-κB)/p65 was significantly decreased, whereas NF-κB luciferase activities were slightly inhibited, by GS12021 treatment in RAW 264.7 cells. Either mitogen-activated protein kinase phosphorylation or AP-1 luciferase activity was not altered by GS12021. GS12021 increased the phosphorylation of AMP-activated protein kinase (AMPK) α and the expression of sirtuin (SIRT) 1. Inhibition of mRNA expression of inflammatory genes by GS12021 was abolished in AMPKα1-knockdown cells, but not in SIRT1 knockout cells, demonstrating that GS12021 exerts anti-inflammatory effects through AMPKα activation. The transwell migration assay results showed that GS12021 treatment of macrophages prevented the cell migration promoted by incubation with conditioned medium obtained from adipocytes. GS12021 suppression of p65 phosphorylation and macrophage chemotaxis were preserved in AMPKα1-knockdown cells, indicating AMPK is not required for these functions of GS12021. Identification of this novel methylhonokiol analog could enable studies of the structure-activity relationship of this class of compounds and further evaluation of its in vivo potential for the treatment of insulin-resistant states and other chronic inflammatory diseases. PMID:25706552

  19. Dual-color bioluminescent bioreporter for forensic analysis: evidence of androgenic and anti-androgenic activity of illicit drugs.

    PubMed

    Cevenini, Luca; Michelini, Elisa; D'Elia, Marcello; Guardigli, Massimo; Roda, Aldo

    2013-01-01

    Bioassays represent promising complementary techniques to conventional analytical approaches used in doping analysis to detect illicit drugs like anabolic-androgenic steroids (AAS). The fact that all AAS share a common mechanism of action via the human androgen receptor (hAR) enables the use of bioassays, relying on the activation of hAR as antidoping screening tools. Previously, we developed a dual-color bioreporter based on yeast cells engineered to express hAR and androgen response elements driving the expression of the bioluminescent (BL) reporter protein Photinus pyralis luciferase. A second reporter protein, the red-emitting luciferase PpyRE8, was introduced in the bioreporter as internal viability control. Here, we report the first forensic application of a straightforward, accurate, and cost-effective bioassay, relying on spectral resolution of the two BL signals, in 96-microwell format. The bioreporter responds to dihydrotestosterone as reference androgen in a concentration-dependent manner from 0.08 to 1,000 nM with intra- and inter-assay variation coefficients of 11.4 % and 13.1 %, respectively. We also demonstrated the suitability of this dual-color bioreporter to assess (anti)-androgenic activity of pure AAS, mixtures of AAS, and other illicit drugs provided by the Scientific Police. Significant anti-androgenic activity was observed in samples labeled as marijuana and hashish, containing Δ(9)-tetrahydrocannabinol as major constituent.

  20. Genetic Variants in the STMN1 Transcriptional Regulatory Region Affect Promoter Activity and Fear Behavior in English Springer Spaniels

    PubMed Central

    Zhang, Hanying; Xu, Yinxue

    2016-01-01

    Stathmin 1 (STMN1) is a neuronal growth-associated protein that is involved in microtubule dynamics and plays an important role in synaptic outgrowth and plasticity. Given that STMN1 affects fear behavior, we hypothesized that genetic variations in the STMN1 transcriptional regulatory region affect gene transcription activity and control fear behavior. In this study, two single nucleotide polymorphisms (SNPs), g. -327 A>G and g. -125 C>T, were identified in 317 English Springer Spaniels. A bioinformatics analysis revealed that both were loci located in the canine STMN1 putative promoter region and affected transcription factor binding. A statistical analysis revealed that the TT genotype at g.-125 C>T produced a significantly greater fear level than that of the CC genotype (P < 0.05). Furthermore, the H4H4 (GTGT) haplotype combination was significantly associated with canine fear behavior (P < 0.01). Using serially truncated constructs of the STMN1 promoters and the luciferase reporter, we found that a 395 bp (−312 nt to +83 nt) fragment constituted the core promoter region. The luciferase assay also revealed that the H4 (GT) haplotype promoter had higher activity than that of other haplotypes. Overall, our results suggest that the two SNPs in the canine STMN1 promoter region could affect canine fear behavior by altering STMN1 transcriptional activity. PMID:27390866

  1. Neuroinflammation activates efflux transport by NFκB

    PubMed Central

    Yu, Chuanhui; Argyropoulos, George; Zhang, Yan; Kastin, Abba J.; Hsuchou, Hung; Pan, Weihong

    2009-01-01

    Background/aims Although it is known that drug delivery across the blood-brain barrier (BBB) may be hampered by efflux transport activity of the multidrug resistance (mdr) gene product P-glycoprotein, it is not clear how inflammation regulates efflux transporters. In rat brain endothelial (RBE4) cells of BBB origin, the proinflammatory cytokine TNF mainly induces transcriptional upregulation of mdr1b, and to a lesser extent mdr1a, resulting in greater efflux of the substrates (Yu C et al., Cell Physiol Biochem, 2007). This study further determined the mechanisms by which TNF activates mdr1b promoter activity. Methods/Results Luciferase reporter assays and DNA binding studies show that (a) maximal basal promoter activity was conferred by a 476 bp sequence upstream to the mdr1b transcriptional initiation site; (2) TNF induced upregulation of promoter activity by NFkB nuclear translocation; and (3) the NFκB binding site of the mdr1b promoter was solely responsible for basal and TNF-activated gene transcription, whereas the p53 binding site was not involved. Binding of the p65 subunit of NFκB to nuclear DNA from RBE4 cells was shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. Conclusion Thus, NFκB mediated TNF-induced upregulation of mdr1b promoter activity, illustrating how inflammation activates BBB efflux transport. PMID:19088456

  2. Vagal nerve stimulation protects against burn-induced intestinal injury through activation of enteric glia cells

    PubMed Central

    Costantini, Todd W.; Bansal, Vishal; Krzyzaniak, Michael; Putnam, James G.; Peterson, Carrie Y.; Loomis, William H.; Wolf, Paul; Baird, Andrew; Eliceiri, Brian P.

    2010-01-01

    The enteric nervous system may have an important role in modulating gastrointestinal barrier response to disease through activation of enteric glia cells. In vitro studies have shown that enteric glia activation improves intestinal epithelial barrier function by altering the expression of tight junction proteins. We hypothesized that severe injury would increase expression of glial fibrillary acidic protein (GFAP), a marker of enteric glial activation. We also sought to define the effects of vagal nerve stimulation on enteric glia activation and intestinal barrier function using a model of systemic injury and local gut mucosal involvement. Mice with 30% total body surface area steam burn were used as model of severe injury. Vagal nerve stimulation was performed to assess the role of parasympathetic signaling on enteric glia activation. In vivo intestinal permeability was measured to assess barrier function. Intestine was collected to investigate changes in histology; GFAP expression was assessed by quantitative PCR, by confocal microscopy, and in GFAP-luciferase transgenic mice. Stimulation of the vagus nerve prevented injury-induced intestinal barrier injury. Intestinal GFAP expression increased at early time points following burn and returned to baseline by 24 h after injury. Vagal nerve stimulation prior to injury increased GFAP expression to a greater degree than burn alone. Gastrointestinal bioluminescence was imaged in GFAP-luciferase transgenic animals following either severe burn or vagal stimulation and confirmed the increased expression of intestinal GFAP. Injection of S-nitrosoglutathione, a signaling molecule released by activated enteric glia cells, following burn exerts protective effects similar to vagal nerve stimulation. Intestinal expression of GFAP increases following severe burn injury. Stimulation of the vagus nerve increases enteric glia activation, which is associated with improved intestinal barrier function. The vagus nerve may mediate the

  3. Differential effect of pure isoflavones and soymilk on estrogen receptor activity in mice

    SciTech Connect

    Rando, Gianpaolo; Ramachandran, Balaji; Rebecchi, Monica; Ciana, Paolo; Maggi, Adriana

    2009-06-15

    Background: Because of the complexity of estrogen receptor (ER) physiological activity, the interaction of pure isoflavones or soy-based diets on ER needs to be clearly demonstrated. Objectives: To investigate the effects of the administration of isoflavones as a pure compound or as a component of diet on the ER transcriptional activity in adult mice. Methods: Effects of acute (6 h) and chronic (21 days) oral administration of soy milk, pure genistein and a mix of genistein and daidzein was studied in living ERE-Luc mice. In this animal model, the synthesis of luciferase is under the state of ER transcriptional activity. Luciferase activity was measured in living mice by daily bioluminescence imaging sessions and in tissue extracts by enzymatic assay. Results: Acute, oral administration of genistein or soymilk caused a significant increase of ER activity in liver. In a 20 day long treatment, soymilk was more potent than genistein in liver and appeared to extend its influence on ER transcriptional activity in other tissues, such as the digestive tract. A mixture of pure genistein and daidzein at the same concentration as in soymilk failed to induce significant changes during acute and chronic studies suggesting an important, uncharacterized role of the soymilk matrix. Consistent with this observation, synergistic effects of the matrix plus isoflavones were observed in MCF-7 cells stably transfected with the ERE-luc construct. Conclusions: This study underlines the limitations of the analysis of single food components in the evaluation of their effects on estrogen receptor activity and advocates the necessity to use complex organisms for the full comprehension of the effects of compounds altering the endocrine balance.

  4. Regulation of activator protein-1 by 8-iso-prostaglandin E2 in a thromboxane A2 receptor-dependent and -independent manner

    SciTech Connect

    Weber, Thomas J.; Markillie, Lye MENG.

    2003-05-01

    The thromboxane (TX) A{sub 2} receptor (TP) encompasses two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP activity may be modulated by novel ligands, termed the isoprostanes, that paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes 8-iso-prostaglandin (PG)F{sub 2{alpha}} and 8-iso-PGE{sub 2} regulate the activity of TP isoforms expressed in Chinese hamster ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist [9,11-dideoxy-9{alpha},11{alpha}-methanoepoxy PGF{sub 2{alpha}} (U46619)] in CHO cells transfected with the human TP-P and TP-E receptors, and this response was fully inhibited by TP antagonists [1S-[1{alpha},2{beta}(Z),3{alpha},5{alpha}

  5. Human ZCCHC12 activates AP-1 and CREB signaling as a transcriptional co-activator.

    PubMed

    Li, Hong; Liu, Qian; Hu, Xiang; Feng, Du; Xiang, Shuanglin; He, Zhicheng; Hu, Xingwang; Zhou, Jianlin; Ding, Xiaofeng; Zhou, Chang; Zhang, Jian

    2009-07-01

    Mouse zinc finger CCHC domain containing 12 gene (ZCCHC12) has been identified as a transcriptional co-activator of bone morphogenetic protein (BMP) signaling, and human ZCCHC12 was reported to be related to non-syndromic X-linked mental retardation (NS-XLMR). However, the details of how human ZCCHC12 involve in the NS-XLMR still remain unclear. In this study, we identified a novel nuclear localization signal (NLS) in the middle of human ZCCHC12 protein which is responsible for the nuclear localization. Multiple-tissue northern blot analysis indicated that ZCCHC12 is highly expressed in human brain. Furthermore, in situ hybridization showed that ZCCHC12 is specifically expressed in neuroepithelium of forebrain, midbrain, and diencephalon regions of mouse E10.5 embryos. Luciferase reporter assays demonstrated that ZCCHC12 enhanced the transcriptional activities of activator protein 1 (AP-1) and cAMP response element binding protein (CREB) as a coactivator. In conclusion, we identified a new NLS in ZCCHC12 and figured out that ZCCHC12 functions as a transcriptional co-activator of AP-1 and CREB.

  6. Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells.

    PubMed

    Duvoisin, Raphaël; Ayuk, Mary A; Rinaldi, Gabriel; Suttiprapa, Sutas; Mann, Victoria H; Lee, Clarence M; Harris, Nicola; Brindley, Paul J

    2012-06-01

    Blood flukes or schistosomes are the causative agents of human schistosomiasis, one of the major neglected tropical diseases. Draft genome sequences have been reported for schistosomes, but functional genomics tools are needed to investigate the role and essentiality of the newly reported genes. Vector based RNA interference can contribute to functional genomics analysis for schistosomes. Using mRNA encoding reporter firefly luciferase as a model target, we compared the performance of a schistosome and a human promoter from the U6 gene in driving shRNA in human fibrosarcoma cells and in cultured schistosomes. Further, both a retroviral [Murine leukemia virus (MLV)] and plasmid (piggyBac, pXL-Bac II) vector were utilized. The schistosome U6 gene promoter was 270 bp in length, the human U6 gene promoter was 264 bp; they shared 41% identity. Following transduction of both HT1080 fibrosarcoma cells and schistosomules of Schistosoma mansoni with pseudotyped MLV virions, stronger knockdown of luciferase activity was seen with the virions encoding the human U6 promoter driven shRNA than the schistosome U6 promoter. A similar trend was seen after transfection of HT1080 cells and schistosomules with the pXL-Bac-II constructs-stronger knockdown of luciferase activity was seen with constructs encoding the human compared to schistosome U6 promoter. The findings indicate that a human U6 gene promoter drives stronger shRNA activity than its schistosome orthologue, not only in a human cancer cell line but also in larval schistosomes. This RNA polymerase III promoter represents a potentially valuable component for vector based RNA interference studies in schistosomes and related platyhelminth parasites.

  7. Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.

    PubMed

    Fairey, E R; Edmunds, J S; Deamer-Melia, N J; Glasgow, H; Johnson, F M; Moeller, P R; Burkholder, J M; Ramsdell, J S

    1999-09-01

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

  8. One reporter for in-cell activity profiling of majority of protein kinase oncogenes.

    PubMed

    Gudernova, Iva; Foldynova-Trantirkova, Silvie; Ghannamova, Barbora El; Fafilek, Bohumil; Varecha, Miroslav; Balek, Lukas; Hruba, Eva; Jonatova, Lucie; Jelinkova, Iva; Kunova Bosakova, Michaela; Trantirek, Lukas; Mayer, Jiri; Krejci, Pavel

    2017-02-15

    In-cell profiling enables the evaluation of receptor tyrosine activity in a complex environment of regulatory networks that affect signal initiation, propagation and feedback. We used FGF-receptor signaling to identify EGR1 as a locus that strongly responds to the activation of a majority of the recognized protein kinase oncogenes, including 30 receptor tyrosine kinases and 154 of their disease-associated mutants. The EGR1 promoter was engineered to enhance trans-activation capacity and optimized for simple screening assays with luciferase or fluorescent reporters. The efficacy of the developed, fully synthetic reporters was demonstrated by the identification of novel targets for two clinically used tyrosine kinase inhibitors, nilotinib and osimertinib. A universal reporter system for in-cell protein kinase profiling will facilitate repurposing of existing anti-cancer drugs and identification of novel inhibitors in high-throughput screening studies.

  9. Synthesis of PPAR-γ Activators Inspired by the Marine Natural Product, Paecilocin A

    PubMed Central

    Xiao, Bin; Su, Mingzhi; Kim, Eun La; Hong, Jongki; Chung, Hae Young; Kim, Hyung Sik; Yin, Jun; Jung, Jee H.

    2014-01-01

    A series of N-substituted phthalimide derivatives were synthesized based on a pharmacophore study of paecilocin A (a natural PPAR-γ agonist) and synthetic leads. The introduction of hydrophilic and hydrophobic groups to the phthalimide skeleton yielded compounds 3–14. Compound 7 showed significant PPAR-γ activation in a luciferase assay using rat liver Ac2F cells. Docking simulations showed that a free hydroxyl group on the phthalimide head and a suitable hydrophilic tail, including a phenyl linker, were beneficial for PPAR-γ activation. Compound 7 and rosiglitazone concentration-dependently activated PPAR-γ with EC50 values of 0.67 μM and 0.028 μM, respectively. These phthalimide derivatives could be further investigated as a new class of PPAR-γ ligands. PMID:24531188

  10. One reporter for in-cell activity profiling of majority of protein kinase oncogenes

    PubMed Central

    Gudernova, Iva; Foldynova-Trantirkova, Silvie; Ghannamova, Barbora El; Fafilek, Bohumil; Varecha, Miroslav; Balek, Lukas; Hruba, Eva; Jonatova, Lucie; Jelinkova, Iva; Bosakova, Michaela Kunova; Trantirek, Lukas; Mayer, Jiri; Krejci, Pavel

    2017-01-01

    In-cell profiling enables the evaluation of receptor tyrosine activity in a complex environment of regulatory networks that affect signal initiation, propagation and feedback. We used FGF-receptor signaling to identify EGR1 as a locus that strongly responds to the activation of a majority of the recognized protein kinase oncogenes, including 30 receptor tyrosine kinases and 154 of their disease-associated mutants. The EGR1 promoter was engineered to enhance trans-activation capacity and optimized for simple screening assays with luciferase or fluorescent reporters. The efficacy of the developed, fully synthetic reporters was demonstrated by the identification of novel targets for two clinically used tyrosine kinase inhibitors, nilotinib and osimertinib. A universal reporter system for in-cell protein kinase profiling will facilitate repurposing of existing anti-cancer drugs and identification of novel inhibitors in high-throughput screening studies. DOI: http://dx.doi.org/10.7554/eLife.21536.001 PMID:28199182

  11. Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.

    PubMed Central

    Fairey, E R; Edmunds, J S; Deamer-Melia, N J; Glasgow, H; Johnson, F M; Moeller, P R; Burkholder, J M; Ramsdell, J S

    1999-01-01

    Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:10464070

  12. Activated air produced by shielded sliding discharge plasma mediates plasmid DNA delivery to mammalian cells.

    PubMed

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Heller, Richard

    2015-12-01

    Cold plasma is emerging as a potential method for medical applications. The current study assessed the efficacy of a novel cold plasma reactor based on shielded sliding discharge producing cathode-directed streamers generated in ambient air for the delivery of plasmid DNA. Experiments were performed with mouse melanoma cells (B16F10) and human keratinocyte cells (HaCaT) inoculated with plasmid DNA encoding luciferase. Quantitative results measured over a 72-h period displayed luciferase expression levels as high as 5-fold greater in cells exposed to plasma-activated air (PAA) than levels obtained from the inoculation of plasmid DNA alone (P < 0.05, P < 0.01). No effect on cell viability was observed. Delivery of plasmid encoding GFP to HaCaT cells seeded on polycaprolactone (PCL) scaffolds was confirmed by immunostaining. The use of cold plasma for DNA delivery is attractive as it provides a non-viral, non-invasive method where the electrode or the plasma itself never directly contacts the exposed site. The current device design provides localized DNA transfer using a novel technology. Our report suggests PAA warrants further exploration as an alternative or supplemental approach for DNA transfer.

  13. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    PubMed Central

    Israeli, Ma’ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  14. Transcriptional Regulation of the Sodium-activated Potassium Channel SLICK (KCNT2) Promoter by Nuclear Factor-κB*

    PubMed Central

    Tomasello, Danielle L.; Gancarz-Kausch, Amy M.; Dietz, David M.; Bhattacharjee, Arin

    2015-01-01

    Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene. PMID:26100633

  15. Transcriptional Regulation of the Sodium-activated Potassium Channel SLICK (KCNT2) Promoter by Nuclear Factor-κB.

    PubMed

    Tomasello, Danielle L; Gancarz-Kausch, Amy M; Dietz, David M; Bhattacharjee, Arin

    2015-07-24

    Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene.

  16. Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.

    PubMed

    Karpurapu, Manjula; Ranjan, Ravi; Deng, Jing; Chung, Sangwoon; Lee, Yong Gyu; Xiao, Lei; Nirujogi, Teja Srinivas; Jacobson, Jeffrey R; Park, Gye Young; Christman, John W

    2014-01-01

    The role of different lineage specific transcription factors in directing hematopoietic cell fate towards myeloid lineage is well established but the status of epigenetic modifications has not been defined during this important developmental process. We used non proliferating, PU.1 inducible myeloid progenitor cells and differentiating bone marrow derived macrophages to study the PU.1 dependent KLF4 transcriptional regulation and its promoter demethylation during monocyte/macrophage differentiation. Expression of KLF4 was regulated by active demethylation of its promoter and PU.1 specifically bound to KLF4 promoter oligo harboring the PU.1 consensus sequence. Methylation specific quantitative PCR and Bisulfite sequencing indicated demethylation of CpG residues most proximal to the transcription start site of KLF4 promoter. Cloned KLF4 promoter in pGL3 Luciferase and CpG free pcpgf-bas vectors showed accentuated reporter activity when co-transfected with the PU.1 expression vector. In vitro methylation of both KLF4 promoter oligo and cloned KLF4 promoter vectors showed attenuated in vitro DNA binding activity and Luciferase/mouse Alkaline phosphotase reporter activity indicating the negative influence of KLF4 promoter methylation on PU.1 binding. The Cytosine deaminase, Activation Induced Cytidine Deaminase (AICDA) was found to be critical for KLF4 promoter demethylation. More importantly, knock down of AICDA resulted in blockade of KLF4 promoter demethylation, decreased F4/80 expression and other phenotypic characters of macrophage differentiation. Our data proves that AICDA mediated active demethylation of the KLF4 promoter is necessary for transcriptional regulation of KLF4 by PU.1 during monocyte/macrophage differentiation.

  17. Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

    NASA Technical Reports Server (NTRS)

    Mukhopadhyay, C. K.; Mazumder, B.; Fox, P. L.

    2000-01-01

    A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these

  18. 4-Ketopinoresinol, a novel naturally occurring ARE activator, induces the Nrf2/HO-1 axis and protects against oxidative stress-induced cell injury via activation of PI3K/AKT signaling.

    PubMed

    Chen, Huang-Hui; Chen, Yu-Tsen; Huang, Yen-Wen; Tsai, Hui-Ju; Kuo, Ching-Chuan

    2012-03-15

    The Nrf2/ARE pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and has been considered a potential target for cancer chemoprevention because it eliminates harmful reactive oxygen species or reactive intermediates generated from carcinogens. The objectives of this study were to identify novel Nrf2/ARE activators and to investigate the mechanistic signaling pathway involved in the activation of Nrf2-mediated cytoprotective effects against oxidative-induced cell injury. A stable ARE-driven luciferase reporter cell line was established to screen a potentially cytoprotective compound. 4-Ketopinoresinol (4-KPR), the (α-γ) double-cyclized type of lignan obtained from adlay (Coix lachryma-jobi L. var. ma-yuen Stapf), activates ARE-driven luciferase activity more effectively than the classical ARE activator tert-butylhydroquinone. 4-KPR treatment resulted in a transient increase in AKT phosphorylation and subsequent phosphorylation and nuclear translocation of Nrf2, along with increased expression of ARE-dependent cytoprotective genes, such as heme oxygenase-1 (HO-1), aldo-keto reductases, and glutathione synthetic enzyme. 4-KPR suppresses oxidative stress-induced DNA damage and cell death via upregulation of HO-1. Inhibition of PI3K/AKT signaling by chemical inhibitors or RNA interference not only suppressed 4-KPR-induced Nrf2/HO-1 activation, but also eliminated the cytoprotective effect against oxidative damage. These observations in an ARE-regulated gene system suggest that 4-KPR is a novel Nrf2/ARE-mediated transcription activator, activates the Nrf2/HO-1 axis, and protects against oxidative stress-induced cell injury via activation of PI3K/AKT signaling.

  19. Environmental Chemicals Modulate Polar Bear (Ursus maritimus) Peroxisome Proliferator-Activated Receptor Gamma (PPARG) and Adipogenesis in Vitro.

    PubMed

    Routti, Heli; Lille-Langøy, Roger; Berg, Mari K; Fink, Trine; Harju, Mikael; Kristiansen, Kurt; Rostkowski, Pawel; Rusten, Marte; Sylte, Ingebrigt; Øygarden, Lene; Goksøyr, Anders

    2016-10-04

    We studied interactions between polar bear peroxisome proliferator-activated receptor gamma (pbPPARG) and selected compounds using a luciferase reporter assay and predictions through molecular docking. Furthermore, we studied adipogenesis by liver and adipose tissue extracts from a polar bear and three synthetic mixtures of contaminants in murine 3T3-L1 preadipocytes and polar bear adipose tissue-derived stem cells (pbASCs). PCB153 and p,p'-DDE antagonized pbPPARG, although their predicted receptor-ligand affinity was weak. PBDEs, tetrabromobisphenol A, and PCB170 had a weak agonistic effect on pbPPARG, while hexabromocyclododecane, bisphenol A, oxychlordane, and endosulfan were weak antagonists. pbPPARG-mediated luciferase activity was suppressed by synthetic contaminant mixtures reflecting levels measured in polar bear adipose tissue, as were transcript levels of PPARG and the PPARG target gene fatty acid binding protein 4 (FABP4) in pbASCs. Contaminant extracts from polar bear tissues enhanced triglyceride accumulation in murine 3T3-L1 cells and pbASCs, whereas triglyceride accumulation was not affected by the synthetic mixtures. Chemical characterization of extracts using nontarget methods revealed presence of exogenous compounds that have previously been reported to induce adipogenesis. These compounds included phthalates, tonalide, and nonylphenol. In conclusion, major legacy contaminants in polar bear adipose tissue exert antagonistic effects on PPARG, but adipogenesis by a mixture containing emerging compounds may be enhanced through PPARG or other pathways.

  20. Enzymatic activity of albumin shown by coelenterazine chemiluminescence.

    PubMed

    Vassel, N; Cox, C D; Naseem, R; Morse, V; Evans, R T; Power, R L; Brancale, A; Wann, K T; Campbell, A K

    2012-01-01

    Bioluminescence, the emission of light from live organisms, occurs in 18 phyla and is the major communication system in the deep sea. It has appeared independently many times during evolution but its origins remain unknown. Coelenterazine bioluminescence discovered in luminous jellyfish is the most common chemistry causing bioluminescence in the sea, occurring in seven phyla. Sequence similarities between coelenterazine luciferases and photoproteins from different phyla are poor (often < 5%). The aim of this study was to examine albumin that binds organic substances as a coelenterazine luciferase to test the hypothesis that the evolutionary origin of a bioluminescent protein was the result of the formation of a solvent cage containing just a few key amino acids. The results show for the first time that bovine and human albumin catalysed coelenterazine chemiluminescence consistent with a mono-oxygenase, whereas gelatin and haemoglobin, an oxygen carrier, had very weak activity. Insulin also catalysed coelenterazine chemiluminescence and was increased by Zn(2+). Albumin chemiluminescence was heat denaturable, exhibited saturable substrate characteristics and was inhibited by cations that bound these proteins and by drugs that bind to human albumin drug site I. Molecular modelling confirmed the coelenterazine binding site and identified four basic amino acids: lys195, arg222, his242 and arg257, potentially important in binding and catalysis similar to naturally occurring coelenterazine bioluminescent proteins. These results support the 'solvent cage' hypothesis for the evolutionary origin of enzymatic coelenterazine bioluminescent proteins. They also have important consequences in diseases such as diabetes, gut disorders and food intolerance where a mono-oxygenase could affect cell surface proteins.

  1. Leptin induces CYP1B1 expression in MCF-7 cells through ligand-independent activation of the ERα pathway

    SciTech Connect

    Khanal, Tilak; Kim, Hyung Gyun; Do, Minh Truong; Choi, Jae Ho; Won, Seong Su; Kang, Wonku; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-05-15

    Leptin, a hormone with multiple biological actions, is produced predominantly by adipose tissue. Among its functions, leptin can stimulate tumour cell growth. Oestrogen receptor α (ERα), which plays an essential role in breast cancer development, can be transcriptionally activated in a ligand-independent manner. In this study, we investigated the effect of leptin on CYP1B1 expression and its mechanism in breast cancer cells. Leptin induced CYP1B1 protein, messenger RNA expression and promoter activity in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells. Additionally, leptin increased 4-hydroxyoestradiol in MCF-7 cells. Also, ERα knockdown by siRNA significantly blocked the induction of CYP1B1 expression by leptin, indicating that leptin induced CYP1B1 expression via an ERα-dependent mechanism. Transient transfection with CYP1B1 deletion promoter constructs revealed that the oestrogen response element (ERE) plays important role in the up-regulation of CYP1B1 by leptin. Furthermore, leptin stimulated phosphorylation of ERα at serine residues 118 and 167 and increased ERE-luciferase activity, indicating that leptin induced CYP1B1 expression by ERα activation. Finally, we found that leptin activated ERK and Akt signalling pathways, which are upstream kinases related to ERα phosphorylation induced by leptin. Taken together, our results indicate that leptin-induced CYP1B1 expression is mediated by ligand-independent activation of the ERα pathway as a result of the activation of ERK and Akt in MCF-7 cells. - Highlights: • Leptin increased 4-hydroxyoestradiol in MCF-7 breast cancer cells. • Leptin activated ERK and Akt kinases related to ERα phosphorylation. • Leptin induces phosphorylation of ERα at serine residues 118 and 167. • Leptin induces ERE-luciferase activity.

  2. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation

    PubMed Central

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease. PMID:27570485

  3. 10-oxo-12(Z)-octadecenoic acid, a linoleic acid metabolite produced by gut lactic acid bacteria, potently activates PPARγ and stimulates adipogenesis.

    PubMed

    Goto, Tsuyoshi; Kim, Young-Il; Furuzono, Tomoya; Takahashi, Nobuyuki; Yamakuni, Kanae; Yang, Ha-Eun; Li, Yongjia; Ohue, Ryuji; Nomura, Wataru; Sugawara, Tatsuya; Yu, Rina; Kitamura, Nahoko; Park, Si-Bum; Kishino, Shigenobu; Ogawa, Jun; Kawada, Teruo

    2015-04-17

    Our previous study has shown that gut lactic acid bacteria generate various kinds of fatty acids from polyunsaturated fatty acids such as linoleic acid (LA). In this study, we investigated the effects of LA and LA-derived fatty acids on the activation of peroxisome proliferator-activated receptors (PPARs) which regulate whole-body energy metabolism. None of the fatty acids activated PPARδ, whereas almost all activated PPARα in luciferase assays. Two fatty acids potently activated PPARγ, a master regulator of adipocyte differentiation, with 10-oxo-12(Z)-octadecenoic acid (KetoA) having the most potency. In 3T3-L1 cells, KetoA induced adipocyte differentiation via the activation of PPARγ, and increased adiponectin production and insulin-stimulated glucose uptake. These findings suggest that fatty acids, including KetoA, generated in gut by lactic acid bacteria may be involved in the regulation of host energy metabolism.

  4. Calcineurin/nuclear factors of activated T cells (NFAT)-activating and immunoreceptor tyrosine-based activation motif (ITAM)-containing protein (CNAIP), a novel ITAM-containing protein that activates the calcineurin/NFAT-signaling pathway.

    PubMed

    Yang, Jianhua; Hu, Guanghui; Wang, Shen-Wu; Li, Yucheng; Martin, Rachel; Li, Kang; Yao, Zhengbin

    2003-05-09

    We report in this study the identification and characterization of a novel protein that we designated as calcineurin/NFAT-activating and immunoreceptor tyrosine-based activation motif (ITAM)-containing protein (CNAIP). The predicted 270-amino acid sequence contains an N-terminal signal peptide, an immunoglobin domain in the extracellular region, a transmembrane domain and an ITAM in the cytoplasmic tail. Quantitative reverse transcription-PCR showed that CNAIP was preferentially expressed in neutrophils, monocytes, mast cells, and other immune-related cells. Co-transfection of CNAIP expression constructs with luciferase reporter plasmids in HMC-1 cells resulted in activation of interleukin-13 and tumor necrosis factor-alpha promoters, which was mediated through the calcineurin/NFAT-signaling pathway. Mutation of either or both tyrosines in the ITAM abolished transcriptional activation induced by CNAIP, indicating that the ITAM is indispensable for CNAIP function in activating cytokine gene promoters. Thus, it is concluded that CNAIP is a novel ITAM-containing protein that activates the calcineurin/NFAT-signaling pathway and the downstream cytokine gene promoters.

  5. DUX4 promotes transcription of FRG2 by directly activating its promoter in facioscapulohumeral muscular dystrophy

    PubMed Central

    2014-01-01

    Background The most common form of facioscapulohumeral muscular dystrophy (FSHD) is caused by a genetic contraction of the polymorphic D4Z4 macrosatellite repeat array in the subtelomeric region of chromosome 4q. In some studies, genes centromeric to the D4Z4 repeat array have been reported to be over-expressed in FSHD, including FRG1 and FRG2, presumably due to decreased long-distance repression by the shorter array through a mechanism similar to position-effect variegation. Differential regulation of FRG1 in FSHD has never been unequivocally proven, however, FRG2 has been reproducibly shown to be induced in primary FSHD-derived muscle cells when differentiated in vitro. The molecular function of FRG2 and a possible contribution to FSHD pathology remain unclear. Recent evidence has identified the mis-expression of DUX4, located within the D4Z4 repeat unit, in skeletal muscle as the cause of FSHD. DUX4 is a double homeobox transcription factor that has been shown to be toxic when expressed in muscle cells. Methods We used a combination of expression analysis by qRT/PCR and RNA sequencing to determine the transcriptional activation of FRG2 and DUX4. We examined this in both differentiating control and FSHD derived muscle cell cultures or DUX4 transduced control cell lines. Next, we used ChIP-seq analysis and luciferase reporter assays to determine the potential DUX4 transactivation effect on the FRG2 promoter. Results We show that DUX4 directly activates the expression of FRG2. Increased expression of FRG2 was observed following expression of DUX4 in myoblasts and fibroblasts derived from control individuals. Moreover, we identified DUX4 binding sites at the FRG2 promoter by chromatin immunoprecipitation followed by deep sequencing and confirmed the direct regulation of DUX4 on the FRG2 promoter by luciferase reporter assays. Activation of luciferase was dependent on both DUX4 expression and the presence of the DUX4 DNA binding motifs in the FRG2 promoter

  6. Low level laser therapy activates NF-kB via generation of reactive oxygen species in mouse embryonic fibroblasts

    NASA Astrophysics Data System (ADS)

    Chen, Aaron Chih-Hao; Arany, Praveen R.; Huang, Ying-Ying; Tomkinson, Elizabeth M.; Saleem, Taimur; Yull, Fiona E.; Blackwell, Timothy S.; Hamblin, Michael R.

    2009-02-01

    Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation remain unclear. In this study, we isolated murine embryonic fibroblasts (MEF) from transgenic NF-kB luciferase reporter mice and studied their response to 810-nm laser radiation. Significant activation of NFkB was observed for fluences higher than 0.003 J/cm2. NF-kB activation by laser was detectable at 1-hour time point. Moreover, we demonstrated that laser phosphorylated both IKK α/β and NF-kB 15 minutes after irradiation, which implied that laser activates NF-kB via phosphorylation of IKK α/β. Suspecting mitochondria as the source of NF-kB activation signaling pathway, we demonstrated that laser increased both intracellular reactive oxygen species (ROS) by fluorescence microscopy with dichlorodihydrofluorescein and ATP synthesis by luciferase assay. Mitochondrial inhibitors, such as antimycin A, rotenone and paraquat increased ROS and NF-kB activation but had no effect on ATP. The ROS quenchers N-acetyl-L-cysteine and ascorbic acid abrogated laser-induced NF-kB and ROS but not ATP. These results suggested that ROS might play an important role in the signaling pathway of laser induced NF-kB activation. However, the western blot showed that antimycin A, a mitochondrial inhibitor, did not activate NF-kB via serine phosphorylation of IKK α/β as the laser did. On the other hand, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that light also upregulates mitochondrial respiration. ATP upregulation reached a maximum at 0.3 J/cm2 or higher. We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive transcription factor NF-kB by generating ROS as signaling molecules.

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

    PubMed

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-03-01

    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.

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

    PubMed Central

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-01-01

    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. PMID:26950874

  9. Isolation of a galactomannan that enhances macrophage activation from the edible fungus Morchella esculenta.

    PubMed

    Duncan, Christine J G; Pugh, Nirmal; Pasco, David S; Ross, Samir A

    2002-09-25

    The edible mushroom Morchella esculenta is among the most highly prized and morphologically recognizable fungi in the world. We describe the isolation from a polar extract of M. esculenta carpophores of a high-molecular-weight galactomannan, about 1.0 million Da, that exhibits immunostimulatory activity. At 3.0 microg/mL the galactomannan polysaccharide increased NF-kappa B directed luciferase expression in THP-1 human monocytic cells to levels 50% of those achieved by maximal activating concentration (10 microg/mL) of lipopolysaccharide. This galactomannan comprises about 2.0% of the dry fungal material weight, and its glycosyl components include mannose (62.9%) and galactose (20.0%).

  10. An exploration of the estrogen receptor transcription activity of capsaicin analogues via an integrated approach based on in silico prediction and in vitro assays.

    PubMed

    Li, Juan; Ma, Duo; Lin, Yuan; Fu, Jianjie; Zhang, Aiqian

    2014-06-16

    Capsaicin has been considered as an alternative template of dichlorodiphenyl trichloroethane (DDT) in antifouling paint. However, information regarding the estrogenic activity of capsaicin analogues is rather limited in comparison to that of DDT analogues and their metabolites. We here explore the ER transcription activity of selected capsaicin analogues via an integrated approach based on in silico prediction and in vitro assays. Molecular simulation and the agonist/antagonist differential-docking screening identified 6-iodonordihydrocapsaicin (6-I-CPS) as a weak ERα agonist, while anti-estrogenicity was expected for N-arachidonoyldopamine, capsazepine, dihydrocapsaicin, trichostatin A, and capsaicin. On the contrary, the large volume of analogues, such as phorbol 12-phenylacetate 13-acetate 20-homovanillate and phorbol 12,13-dinonanoate 20-homovanillate, cannot fit well with the ER cavity. The result of MVLN assay was in accord with the in silico prediction. 6-I-CPS was demonstrated to induce luciferase gene expression, while the other analogues of relatively small molecular volume reduced luciferase gene expression in MVLN cells, both in the absence and presence of estradiol. This finding suggested that the ER transcription activity of capsaicin analogues is generated at least partly through the ERα-mediated pathway. Moreover, receptor polymorphism analysis indicated that capsaicin analogues may exhibit diverse species selectivity for human beings and marine species.

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

    PubMed Central

    Geng, Deyu; Zhang, Zhixia; Guo, Huarong

    2012-01-01

    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. PMID:25585933

  12. Intermedin/adrenomedullin 2 is a stress-inducible gene controlled by activating transcription factor 4.

    PubMed

    Kovaleva, Irina E; Garaeva, Alisa A; Chumakov, Peter M; Evstafieva, Alexandra G

    2016-09-15

    Intermedin or adrenomedullin 2 is a set of calcitonin-related peptides with a putative tumor angiogenesis promoting activity that are formed by proteolytic processing of the ADM2 gene product. It has been proposed that the ADM2 gene is regulated by the estrogen response element (ERE) and hypoxia response elements (HRE) found within its promoter region. In the present study we reveal a functional mechanism by which ADM2 participates in the unfolded protein response (UPR) and in responses to the mitochondrial respiration chain inhibition. We show that the ADM2 gene is controlled by activating transcription factor 4 (ATF4), the principal regulator of the integrated stress response (ISR). The upregulation of ADM2 mRNA could be prevented by the pharmacological ISR inhibitor ISRIB and by the downregulation of ATF4 with specific shRNA, while ectopic expression of ATF4 cDNA resulted in a notable increase in ADM2 gene transcription. A potential ATF4-binding site was identified in the coding region of the ADM2 gene and the requirement of this site during the ATF4-mediated ADM2 gene promoter activation was validated by the luciferase reporter assay. Mutagenesis of the putative ATF4-response element prevented the induction of luciferase activity in response to ATF4 overproduction, as well as in response to mitochondrial electron transfer chain inhibition by piericidin A and ER stress induction by tunicamycin and brefeldin A. Since ADM2 was shown to inhibit ATF4 expression during myocardial ER stress, a feedback mechanism could be proposed for the ADM2 regulation under ER stress conditions.

  13. CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

    PubMed

    Yu, Zhiyuan; Kong, Qun; Kone, Bruce C

    2010-03-01

    Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

  14. Food-anticipatory activity and liver per1-luc activity in diabetic transgenic rats

    NASA Technical Reports Server (NTRS)

    Davidson, Alec J.; Stokkan, Karl-Arne; Yamazaki, Shin; Menaker, Michael

    2002-01-01

    The mammalian Per1 gene is an important component of the core cellular clock mechanism responsible for circadian rhythms. The rodent liver and other tissues rhythmically express Per1 in vitro but typically damp out within a few cycles. In the liver, the peak of this rhythm occurs in the late subjective night in an ad lib-fed rat, but will show a large phase advance in response to restricted availability of food during the day. The relationship between this shift in the liver clock and food-anticipatory activity (FAA), the circadian behavior entrained by daily feeding, is currently unknown. Insulin is released during feeding in mammals and could serve as an entraining signal to the liver. To test the role of insulin in the shift in liver Per1 expression and the generation of FAA, per-luciferase transgenic rats were made diabetic with a single injection of streptozotocine. Following 1 week of restricted feeding and locomotor activity monitoring, liver was collected for per-luc recording. In two separate experiments, FAA emerged and liver Per1 phase-shifted in response to daytime 8-h food restriction. The results rule out insulin as a necessary component of this system.

  15. The dipeptide H-Trp-Glu-OH (WE) shows agonistic activity to peroxisome proliferator-activated protein-α and reduces hepatic lipid accumulation in lipid-loaded H4IIE cells.

    PubMed

    Jia, Yaoyao; Kim, Jong-Ho; Nam, Bora; Kim, Jiyoung; Lee, Ji Hae; Hwang, Kwang-Yeon; Lee, Sung-Joon

    2014-07-01

    Dipeptides digested from dietary proteins can be directly absorbed by the intestine and delivered to the circulatory system. However, the dipeptides' metabolic roles and biological activities are largely unknown. Lipid-loaded HII4E cells stimulated with H-Trp-Glu-OH (WE) exhibited reduced lipid accumulation, of which the effect was abolished by peroxisome proliferator-activated receptor (PPAR) α gene knock down. A luciferase assay showed that the WE dipeptide induced PPARα transactivation in a dose-dependent manner. Surface plasmon resonance and time-resolved fluorescence resonance energy transfer analyses demonstrated that WE interacts directly with the PPARα ligand binding domain (KD, 120 μM; EC50, 83 μM). Cells stimulated with WE induced PPARα and its responsive genes and increased cellular fatty acid uptake. In conclusion, WE reduces hepatic lipid accumulation in lipid-loaded hepatocytes via the activation of PPARα by a direct interaction.

  16. Caffeic acid phenethyl ester-mediated Nrf2 activation and IkappaB kinase inhibition are involved in NFkappaB inhibitory effect: structural analysis for NFkappaB inhibition.

    PubMed

    Lee, Youna; Shin, Dong-Ha; Kim, Ji-Hye; Hong, Sungchae; Choi, Daekyu; Kim, Yung-Jin; Kwak, Mi-Kyoung; Jung, Yunjin

    2010-09-15

    Caffeic acid phenethyl ester (CAPE) is an active component of propolis from honeybee. We investigated potential molecular mechanisms underlying CAPE-mediated nuclear factor kappa beta (NFkappaB) inhibition and analyzed structure of CAPE for its biological effect. CAPE attenuated expression of NFkappaB dependent luciferase stimulated with TNF-alpha or LPS and suppressed LPS-mediated induction of iNOS, a target gene product of NFkappaB. In HCT116 cells, CAPE interfered with TNF-alpha dependent IkappaBalpha degradation and subsequent nuclear accumulation of p65, which occurred by direct inhibition of inhibitory protein kappaB kinase (IKK). CAPE increased the expression of Nrf2-dependent luciferase and heme oxygenase-1, a target gene of Nrf2, and elevated the nuclear level of Nrf2 protein, indicating that CAPE activated the Nrf2 pathway. In HCT116 cells with stable expression of Nrf2 shRNA, CAPE elicited a reduced inhibitory effect on TNF-alpha-activated NFsmall ka, CyrillicB compared to scramble RNA expressing control cells. On the other hand, the NFkappaB inhibitory effect of CAPE was diminished by removal or modification of the Michael reaction acceptor, catechol or phenethyl moiety in CAPE. These data suggest that CAPE inhibits TNF-alpha-dependent NFkappaB activation via direct inhibition of IKK as well as activation of Nrf2 pathway, in which the functional groups in CAPE may be involved.

  17. Activation of p53 Transcriptional Activity by SMRT: a Histone Deacetylase 3-Independent Function of a Transcriptional Corepressor

    PubMed Central

    Adikesavan, Anbu Karani; Karmakar, Sudipan; Pardo, Patricia; Wang, Liguo; Liu, Shuang; Li, Wei

    2014-01-01

    The silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) is an established histone deacetylase 3 (HDAC3)-dependent transcriptional corepressor. Microarray analyses of MCF-7 cells transfected with control or SMRT small interfering RNA revealed SMRT regulation of genes involved in DNA damage responses, and the levels of the DNA damage marker γH2AX as well as poly(ADP-ribose) polymerase cleavage were elevated in SMRT-depleted cells treated with doxorubicin. A number of these genes are established p53 targets. SMRT knockdown decreased the activity of two p53-dependent reporter genes as well as the expression of p53 target genes, such as CDKN1A (which encodes p21). SMRT bound directly to p53 and was recruited to p53 binding sites within the p21 promoter. Depletion of GPS2 and TBL1, components of the SMRT corepressor complex, but not histone deacetylase 3 (HDAC3) decreased p21-luciferase activity. p53 bound to the SMRT deacetylase activation domain (DAD), which mediates HDAC3 binding and activation, and HDAC3 could attenuate p53 binding to the DAD region of SMRT. Moreover, an HDAC3 binding-deficient SMRT DAD mutant coactivated p53 transcriptional activity. Collectively, these data highlight a biological role for SMRT in mediating DNA damage responses and suggest a model where p53 binding to the DAD limits HDAC3 interaction with this coregulator, thereby facilitating SMRT coactivation of p53-dependent gene expression. PMID:24449765

  18. Synthesis of coumaperine derivatives: Their NF-κB inhibitory effect, inhibition of cell migration and their cytotoxic activity.

    PubMed

    Nandakumar, Natarajan; Muthuraman, Subramani; Gopinath, Pushparathinam; Nithya, Pattusamy; Gopas, Jacob; Kumar, Rajendran Saravana

    2017-01-05

    Coumaperine (an amide alkaloid, present in white piper) and its derivatives were synthesized and investigated for their cytotoxicity against L428 and A549 cells and their NF-κB inhibitory activity. It was found that the coumaperine derivatives CP-9 and CP-38 suppress NF-κB subunits p50 and p65 in nuclear fractions by western blot and by NF-κB luciferase reporter gene assay in a dose dependent manner. Confirmation of these results was obtained by confocal microscopy. CP-9, CP-32 and CP-38 also exhibited dose dependent cell cytotoxicity in a L428 cells expressing constitutively active NF-κB and in A549 cells, with an IC50 value of 43.25 μg/ml, 0.39 μg/ml and 16.85 μg/ml respectively against L428 cells and 57.15 μg/ml, 69.1 μg/ml and 63.2 μg/ml respectively against A549 cells. In addition, the coumaperine derivatives show remarkable inhibitory activity on the cancer cell migration assay against A549 lung adenocarcinoma cells at the concentrations of 5 μg/ml, 10 μg/ml, and 5 μg/ml of CP-9, CP-32 and CP-38 respectively. Aromatic substituents and number of olefinic double bond in coumaperine derivatives found to influence the inhibitory activity. In luciferase reporter gene assay, di-olefin conjugated coumaperine derivatives, CP-38, CP-32 and PIP exhibited higher inhibitory activity than their corresponding tri-olefin conjugated coumaperine derivatives, CP-102, CP-146 and PIP-155 respectively. CP-32 with a stronger electron donating group (-N(CH3)2) showed better inhibitory activity in luciferase reporter gene assay and in cell proliferation of L428 cells. Simple coumaperine derivative (CP-9, with no substituent) effectively inhibited A549 cells proliferation and migration than the other coumaperine derivatives. CP-9 and CP-38 diminish significantly the NF-κB subunits (p50 and p65) of L428 cells in nuclear fractions at the dosage of 10 μg/ml and 30 μg/ml respectively. Which clearly shows that CP-9 and CP-38 inactivate the NF-κB pathway

  19. Angular-type furocoumarins from the roots of Angelica atropurpurea and their inhibitory activity on the NFAT signal transduction pathway.

    PubMed

    Nagasawa, Azumi; Sakasai, Mitsuyoshi; Sakaguchi, Daishi; Moriwaki, Shigeru; Nishizawa, Yoshinori; Kitahara, Takashi

    2014-12-01

    One new (1) and two known angular-type (2,3) furocoumarins, isoarchangelicin (1), archangelicin (2), and 2'-angeloyl-3'-isovaleryl vaginate (3), were isolated from the roots of Angelica atropurpurea. The structure of the new compound was established on the basis of one- and two-dimensional NMR spectra and other spectroscopic studies. The inhibitory activity of these three compounds and a deacylated form of archangelicin (4) on the nuclear factor of activated T cells (NFAT) signal transduction pathway was tested by NFAT-responsive luciferase reporter gene assay in cultured cells. Although 4 did not exhibit inhibitory activity on NFAT signaling, 1-3 exhibited dose-dependent inhibition with IC50 values of 16.5 (1), 9.0 (2), and 9.2 (3) μM.

  20. The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids.

    PubMed

    Briscoe, Celia P; Tadayyon, Mohammad; Andrews, John L; Benson, William G; Chambers, Jon K; Eilert, Michelle M; Ellis, Catherine; Elshourbagy, Nabil A; Goetz, Aaron S; Minnick, Dana T; Murdock, Paul R; Sauls, Howard R; Shabon, Usman; Spinage, Lisa D; Strum, Jay C; Szekeres, Philip G; Tan, Kong B; Way, James M; Ignar, Diane M; Wilson, Shelagh; Muir, Alison I

    2003-03-28

    GPR40 is a member of a subfamily of homologous G protein-coupled receptors that include GPR41 and GPR43 and that have no current function or ligand ascribed. Ligand fishing experiments in HEK293 cells expressing human GPR40 revealed that a range of saturated and unsaturated carboxylic acids with carbon chain lengths greater than six were able to induce an elevation of [Ca(2+)](i), measured using a fluorometric imaging plate reader. 5,8,11-Eicosatriynoic acid was the most potent fatty acid tested, with a pEC(50) of 5.7. G protein coupling of GPR40 was examined in Chinese hamster ovary cells expressing the G alpha(q/i)-responsive Gal4-Elk1 reporter system. Expression of human GPR40 led to a constitutive induction of luciferase activity, which was further increased by exposure of the cells to eicosatriynoic acid. Neither the constitutive nor ligand-mediated luciferase induction was inhibited by pertussis toxin treatment, suggesting that GPR40 was coupled to G alpha(q/11.) Expression analysis by quantitative reverse transcription-PCR showed that GPR40 was specifically expressed in brain and pancreas, with expression in rodent pancreas being localized to insulin-producing beta-cells. These data suggest that some of the physiological effects of fatty acids in pancreatic islets and brain may be mediated through a cell-surface receptor.

  1. RGD-based active targeting of novel polycation liposomes bearing siRNA for cancer treatment.

    PubMed

    Yonenaga, Norihito; Kenjo, Eriya; Asai, Tomohiro; Tsuruta, Atsushi; Shimizu, Kosuke; Dewa, Takehisa; Nango, Mamoru; Oku, Naoto

    2012-06-10

    For the purpose of systemic delivery of siRNA, we previously developed polycation liposomes (PCLs) containing dicetylphosphate-tetraethylenepentamine (DCP-TEPA) as an effective siRNA carrier. In the present study, to endow these PCLs (TEPA-PCL) actively target cancer cells and angiogenic vessels, we decorated the PCLs with cyclic RGD, by using cyclic RGD-grafted distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and investigated the usefulness of this type of carrier (RGD-PEG-PCL) for active targeting. Firstly, the gene-silencing efficacy of siRNA for luciferase (siLuc2) formulated in RGD-PEG-PCL (RGD-PEG-PCL/siLuc2) was examined in vitro by using B16F10-luc2 murine melanoma cells stably expressing the luciferase 2 gene, where the siRNA was grafted with cholesterol at the 3'-end of the sense strand (siRNA-C) for the stable association of the siRNA with the PCL. RGD-PEG-PCL/siLuc2 showed high knockdown efficiency compared with siLuc2 formulated in PEGylated TEPA-PCL without cyclic RGD (PEG-PCL). Next, the gene-silencing efficacy of RGD-PEG-PCL/siLuc2 was examined in vivo by use of B16F10-luc2 lung metastatic model mice. The intravenous injection of RGD-PEG-PCL/siLuc2 showed high knockdown efficiency against metastatic B16F10-luc2 tumors in the lungs of the mice, as assessed with an in vivo imaging system. These data strongly suggest that systemic and active targeting siRNA delivery using RGD-PEG-PCL is useful for cancer RNAi therapy.

  2. Physical association with WWOX suppresses c-Jun transcriptional activity.

    PubMed

    Gaudio, Eugenio; Palamarchuk, Alexey; Palumbo, Tiziana; Trapasso, Francesco; Pekarsky, Yuri; Croce, Carlo M; Aqeilan, Rami I

    2006-12-15

    WWOX is a tumor suppressor that functions as a modular protein partner of transcription factors. WWOX contains two WW domains that mediate protein-protein interactions. In this report, we show that WWOX, via its first WW domain, specifically associates with the proline-rich motif of c-Jun proto-oncogene. Our data show that phosphorylation of c-Jun caused by overexpression of mitogen-activated protein kinase kinase kinase 1 (Mekk1), an upstream activator of c-Jun, enhances the interaction of c-Jun with WWOX. Furthermore, exposure of HaCaT keratinocytes to UVC radiation resulted in the association of endogenous WWOX and c-Jun. The WWOX-c-Jun complexes mainly occur in the cytoplasm. Expression of WWOX attenuates the ability of MEKK1 to increase the activity of a c-Jun-driven activating protein-1 (AP-1)-luciferase reporter plasmid. In contrast, a point mutation in the first WW domain of WWOX has no effect on transactivation of AP-1 when coexpressed with c-Jun protein. Our findings reveal a novel functional cross-talk between c-Jun transcription factor and WWOX tumor suppressor protein.

  3. [Regulation of myostatin promoter activity by myocyte enhancer factor 2].

    PubMed

    Li, Jia; Deng, Jie; Zhang, Junlin; Cheng, De; Wang, Huayan

    2012-08-01

    Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.

  4. The oncoprotein HBXIP up-regulates FGF4 through activating transcriptional factor Sp1 to promote the migration of breast cancer cells.

    PubMed

    Shi, Hui; Li, Yinghui; Feng, Guoxing; Li, Leilei; Fang, Runping; Wang, Zhen; Qu, Jie; Ding, Peijian; Zhang, Xiaodong; Ye, Lihong

    2016-02-26

    We have reported that the oncoprotein hepatitis B X-interacting protein (HBXIP) is able to promote migration of breast cancer cells. Fibroblast growth factor 4 (FGF4) is a multipotent growth factor and is highly expressed in various human cancers. However, the regulatory mechanism of FGF4 in breast cancer remains poorly understood. In the present study, we report that HBXIP is able to up-regulate FGF4 to enhance the migration of breast cancer cells. Immunohistochemistry staining showed that HBXIP and FGF4 were highly expressed in clinical metastatic lymph nodes of breast tumor. The expression levels of HBXIP were positively related to those of FGF4 in clinical breast cancer tissues. Then, we validated that HBXIP up-regulated the expression of FGF4 at the levels of promoter, mRNA and protein by luciferase reporter gene assays, reverse transcription-polymerase chain reaction and Western blot analysis. Moreover, we found that HBXIP was able to activate FGF4 promoter through transcriptional factor Sp1 by luciferase reporter gene assays. Chromatin immunoprecipitation assays confirmed that HBXIP coactivated Sp1 to stimulate FGF4 promoter. In function, we showed that HBXIP promoted breast cancer cell migration through FGF4 by wound healing and transwell cell migration assays. Thus, we conclude that the oncoprotein HBXIP up-regulates FGF4 through activating transcriptional factor Sp1 to promote the migration of breast cancer cells. Therapeutically, HBXIP may serve as a novel target in breast cancer.

  5. MicroRNA-200a mediates nasopharyngeal carcinoma cell proliferation through the activation of nuclear factor-κB.

    PubMed

    Shi, Zhuliang; Hu, Zhiqiang; Chen, Delu; Huang, Jie; Fan, Jie; Zhou, Subo; Wang, Xin; Hu, Jiandao; Huang, Fei

    2016-02-01

    In nasopharyngeal carcinoma (NPC), the nuclear factor-κB (NF-κB) signaling pathway is highly active. The constitutive activation of NF-κB prompts malignant cell proliferation, and microRNAs are considered an important mediator in regulating the NF-κB signaling pathway. The current study investigated the effect of microRNA-200a (miR-200a) on NF-κB activation. Reverse transcription-quantitative polymerase chain reaction was used to quantify the relative level of miR-200a in NPC tissue samples and CNE2 cells. An MTT assay was used to investigate the effect of miR-200a on cell proliferation. To investigate the activation of NF-κB, western blotting was used to measure the protein levels of NF-κB and its downstream targets. To identify the target genes of miR-200a, a luciferase reporter assay was used. The current study demonstrated that miR-200a was upregulated in NPC tissue samples and cell lines. Overexpression of miR-200a resulted in the proliferation of CNE2 cells. Western blot analysis indicated that the protein levels of p65 increased when CNE2 cells were transfected with miR-200a mimics. Additionally, the downstream targets of miR-200a were upregulated, including vascular cell adhesion molecule, intercellular adhesion molecule and monocyte chemoattractant protein-1. The luciferase assay indicated that IκBα was the target gene of miR-200a. In conclusion, miR-200a was demonstrated to enhance NPC cell proliferation by activating the NF-κB signaling pathway.

  6. MicroRNA-19a mediates gastric carcinoma cell proliferation through the activation of nuclear factor-κB.

    PubMed

    Yang, Fan; Wang, Hongjian; Jiang, Zhenyu; Hu, Anxiang; Chu, Lisha; Sun, Yiling; Han, Junqing

    2015-10-01

    In gastric carcinoma, the nuclear factor‑κB (NF‑κB) signaling pathway is highly active, and the constitutive activation of NF‑κB prompts malignant cell proliferation. MicroRNAs are considered to be important mediators in the regulation of the NF‑κB signaling pathway. The present study predominantly focussed on the effects of microRNA (miR)‑19a on NF‑κB activation. Reverse transcription‑quantitative polymerase chain reaction was used to quantify the relative levels of miR‑19a in gastric carcinoma cells. MTT assays were used to determine the effect of miR‑19a on cellular proliferation. To detect the activation of NF‑κB, western blotting was performed to measure the protein levels of NF‑κB and the products of its downstream target genes. To define the target genes, luciferase reporter assays were used. miR‑19a was found to be markedly upregulated in gastric carcinoma cells. The overexpression of miR‑19a resulted in proliferation and enhanced migratory capabilities of the MGC‑803 gastric carcinoma cell line. The results of the western blot analysis demonstrated that the protein levels of p65 increased when the MGC‑803 cells were transfected with miR‑19a mimics. In addition, the downstream target genes of miR‑19a, including intercellular adhesion molecule, vascular cell adhesion molecule and monocyte chemoattractant protein‑1, were upregulated. The results of the luciferase assay indicated that IκB‑α was the target gene of miR‑19a. Therefore, the results of the present study suggested that miR‑19a enhances malignant gastric cell proliferation by constitutively activating the NF‑κB signaling pathway.

  7. Induction of phase 2 enzymes by serum oxidized polyamines through activation of Nrf2: effect of the polyamine metabolite acrolein.

    PubMed

    Kwak, Mi-Kyoung; Kensler, Thomas W; Casero, Robert A

    2003-06-06

    The naturally occurring polycationic polyamines including putrescine, spermidine, and spermine play an important role in cell growth, differentiation, and gene expression. However, circulating polyamines are potential substrates for several oxidizing enzymes including copper-containing serum amine oxidase. These enzymes are capable of oxidizing serum polyamines to several toxic metabolites including aldehydes and H(2)O(2). In this study, we investigated the effects of polyamines as inducers of phase 2 enzymes and other genes that promote cell survival in a cell culture system in the presence of bovine serum. Spermidine and spermine (50 microM) increased NAD(P)H quinone oxidoreductase (NQO1) activity up to 3-fold in murine keratinocyte PE cells. Transcript levels for glutathione S-transferase (GST) A1, GST M1, NQO1, gamma-glutamylcysteine ligase regulatory subunit, and UDP-glucuronyltransferase 1A6 were significantly increased by spermidine and this effect was mediated through the antioxidant response element (ARE). The ARE from the mouse GST A1 promoter was activated about 9-fold by spermine and 5-fold by spermidine treatment, but could be inhibited by the amine oxidase inhibitor, aminoguanidine, suggesting that acrolein or hydrogen peroxide generated from polyamines by serum amine oxidase may be mediators for phase 2 enzyme induction. Elevations of ARE-luciferase expression and NQO1 enzyme activity by spermidine were not affected by catalase, while both were completely repressed by aldehyde dehydrogenase treatment. Direct addition of acrolein to PE cells induced multiple phase 2 genes and elevated nuclear levels of Nrf2, a transcription factor that binds to the ARE. Expression of mutant Nrf2 repressed the activation of the ARE-luciferase reporter by polyamines and acrolein. These results indicate that spermidine and spermine increase the expression of phase 2 genes in cells grown in culture through activation of the Nrf2-ARE pathway by generating the sulfhydryl

  8. PLCz functional haplotypes modulating promoter transcriptional activity are associated with semen quality traits in Chinese Holstein bulls.

    PubMed

    Pan, Qing; Ju, Zhihua; Huang, Jinming; Zhang, Yan; Qi, Chao; Gao, Qin; Zhou, Lei; Li, Qiuling; Wang, Lingling; Zhong, Jifeng; Liu, Mei; Wang, Changfa

    2013-01-01

    The sperm-specific phospholipase C zeta (PLCz) is a candidate sperm-borne oocyte-activating factor that triggers a characteristic series of physiological stimuli via cytoplasmic Ca(2+) oscillations during fertilization. The molecular mechanisms involved in the regulation of PLCz gene expression remain largely unknown. To explore the genetic variations in the 5'-flanking region of the PLCz gene and their common haplotypes in Chinese Holstein bulls, as well as to determine whether these variations affect bovine semen quality traits and transcriptional activity, DNA samples were collected from Chinese Holstein bulls and sequenced for the identification of genetic variants in the 5'-flanking region of PLCz. Two genetic variants were identified, and their haplotypic profiles were constructed. The two novel genetic variations (g. -456 G>A and g. +65 T>C) were genotyped in 424 normal Chinese Holstein bulls. Bioinformatics analysis revealed that both loci are in transcription factor binding sites of the core promoter region. The association studies revealed that the two genetic variations and their haplotype combinations significantly affected semen quality traits. Using serially truncated constructs of the bovine PLCz promoters and the luciferase reporter, we found that a 726 bp (-641 nt to +112 nt) fragment constitutes the core promoter region. Furthermore, four haplotypes, H1H1 (GTGT), H2H2 (GCGC), H3H3 (ATAT), and H4H4 (ACAC), were significantly associated with semen quality traits and successfully transfected into MLTC-1 cell lines. The luciferase reporter assay showed that the different haplotypes exhibited distinct promoter activities. Maximal promoter activity was demonstrated by the H2H2 haplotypes, as compared with the other haplotypes. To the best of our knowledge, this study is the first report on genetic variants and their respective haplotypes in the 5'-flanking region of PLCz gene that can influence the semen quality of Chinese Holstein bulls as well as

  9. IKKβ-Targeted Anti-Inflammatory Activities of a Butanol Fraction of Artificially Cultivated Cordyceps pruinosa Fruit Bodies

    PubMed Central

    Kim, Han Gyung; Yang, Woo Seok; Sung, Gi-Ho; Kim, Ji Hye; Kim, Eunji; Yang, Sungjae; Park, Yung Chul; Sung, Jae Mo; Yoon, Deok Hyo; Kim, Tae Woong; Hong, Sungyoul; Kim, Jong-Hoon

    2014-01-01

    The inhibitory activities of the Cordyceps pruinosa butanol fraction (Cp-BF) were investigated by determining inflammatory responses of lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells and by evaluating HCl/ethanol (EtOH)-triggered gastric ulcers in mice. The molecular mechanisms of the inhibitory effects of Cp-BF were investigated by identifying target enzymes using biochemical and molecular biological approaches. Cp-BF strongly inhibited the production of NO and TNF-α, release of reactive oxygen species (ROS), phagocytic uptake of FITC-dextran, and mRNA expression levels of interleukin (IL)-6, inducible NO synthase (iNOS), and tumour necrosis factor-alpha (TNF)-α in activated RAW264.7 cells. Cp-BF also strongly downregulated the NF-κB pathway by suppressing IKKβ according to luciferase reporter assays and immunoblot analysis. Furthermore, Cp-BF blocked both increased levels of NF-κB-mediated luciferase activities and phosphorylation of p65/p50 observed by IKKβ overexpression. Finally, orally administered Cp-BF was found to attenuate gastric ulcer and block the phosphorylation of IκBα induced by HCl/EtOH. Therefore, these results suggest that the anti-inflammatory activity of Cp-BF may be mediated by suppression of IKKα and its downstream NF-κB activation. Since our group has established the mass cultivation conditions by developing culture conditions for Cordyceps pruinosa, the information presented in this study may be useful for developing new anti-inflammatory agents. PMID:25132860

  10. Sensitive, Rapid, Quantitative and in Vitro Method for the Detection of Biologically Active Staphylococcal Enterotoxin Type E

    PubMed Central

    Rasooly, Reuven; Do, Paula; Hernlem, Bradley

    2016-01-01

    Staphylococcus aureus is a major bacterial cause of clinical infections and foodborne illnesses through its production of a group of enterotoxins (SEs) which cause gastroenteritis and also function as superantigens to massively activate T cells. In the present study, we tested Staphylococcal enterotoxin type E (SEE), which was detected in 17 of the 38 suspected staphylococcal food poisoning incidents in a British study and was the causative agent in outbreaks in France, UK and USA. The current method for detection of enterotoxin activity is an in vivo monkey or kitten bioassay; however, this expensive procedure has low sensitivity and poor reproducibility, requires many animals, is impractical to test on a large number of samples, and raises ethical concerns with regard to the use of experimental animals. The purpose of this study is to develop rapid sensitive and quantitative bioassays for detection of active SEE. We apply a genetically engineered T cell-line expressing the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element (NFAT-RE), combined with a Raji B-cell line that presents the SEE-MHC (major histocompatibility complex) class II to the engineered T cell line. Exposure of the above mixed culture to SEE induces differential expression of the luciferase gene and bioluminescence is read out in a dose dependent manner over a 6-log range. The limit of detection of biologically active SEE is 1 fg/mL which is 109 times more sensitive than the monkey and kitten bioassay. PMID:27187474

  11. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase

    PubMed Central

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana; Gritsenko, Natalia; Rask, Lene; Mainbakh, Yuli; Zilberstein, Yael; Yagil, Ezra; Kolot, Mikhail

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system. PMID:27117628

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

    SciTech Connect

    Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki . E-mail: yk-kim@korea.ac.kr

    2007-05-18

    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.

  13. Modulation of Receptor Phosphorylation Contributes to Activation of Peroxisome Proliferator Activated Receptor α by Dehydroepiandrosterone and Other Peroxisome Proliferators

    PubMed Central

    Tamasi, Viola; Miller, Kristy K. Michael; Ripp, Sharon L.; Vila, Ermin; Geoghagen, Thomas E.; Prough, Russell A.

    2008-01-01

    Dehydroepiandrosterone (DHEA), a C19 human adrenal steroid, activates peroxisome proliferator-activated receptor α (PPARα) in vivo but does not ligand-activate PPARα in transient transfection experiments. We demonstrate that DHEA regulates PPARα action by altering both the levels and phosphorylation status of the receptor. Human hepatoma cells (HepG2) were transiently transfected with the expression plasmid encoding PPARα and a plasmid containing two copies of fatty acyl coenzyme oxidase (FACO) peroxisome-proliferator activated receptor responsive element consensus oligonucleotide in a luciferase reporter gene. Nafenopin treatment increased reporter gene activity in this system, whereas DHEA treatment did not. Okadaic acid significantly decreased nafenopin-induced reporter activity in a concentration-dependent manner. Okadaic acid treatment of primary rat hepatocytes decreased both DHEA- and nafenopin-induced FACO activity in primary rat hepatocytes. DHEA induced both PPARα mRNA and protein levels, as well as PP2A message in primary rat hepatocytes. Western blot analysis showed that the serines at positions 12 and 21 were rapidly dephosphorylated upon treatment with DHEA and nafenopin. Results using specific protein phosphatase inhibitors suggested that protein phosphatase 2A (PP2A) is responsible for DHEA action, and protein phosphatase 1 might be involved in nafenopin induction. Mutation of serines at position 6, 12, and 21 to an uncharged alanine residue significantly increased transcriptional activity, whereas mutation to negative charged aspartate residues (mimicking receptor phosphorylation) decreased transcriptional activity. DHEA action involves induction of PPARα mRNA and protein levels as well as increased PPARα transcriptional activity through decreasing receptor phosphorylation at serines in the AF1 region. PMID:18079279

  14. Detection of androgenic activity in emissions from diesel fuel and biomass combustion.

    PubMed

    Owens, Clyde V; Lambright, Christy; Cardon, Mary; Gray, L Earl; Gullett, Brian K; Wilson, Vickie S

    2006-08-01

    The present study evaluated both diesel fuel exhaust and biomass (wood) burn extracts for androgen receptor-mediated activity using MDA-kb2 cells, which contain an androgen-responsive promoter-luciferase reporter gene construct. This assay and analytical fractionization of the samples were used as tools to separate active from inactive fractions, with the goal of identifying the specific compounds responsible for the activity. A significant androgenic response was detected from the diesel emission. High-performance liquid chromatographic fractionation of the sample indicated that significant androgenic activity was retained in three fractions. 4-Hydroxybiphenyl was identified from the most active fraction using gas chromatography/mass spectroscopy. This purified compound was then tested at doses from 1 nM to 100 microM. 4-Hydroxybiphenol exhibited antagonist activity at low concentrations and agonist activity at high concentrations. A competitive-binding assay confirmed binding to the androgen receptor, with a median inhibitory concentration for radioligand binding of approximately 370 nM. Significant androgenic activity also was detected in the wood burn samples, but we were unable to identify the specific chemicals responsible for this endocrine activity. The present study demonstrates that in vitro bioassays can serve as sensitive bioanalytical tools to aid in characterization of complex environmental mixtures.

  15. Optimization and prevalidation of the in vitro ERalpha CALUX method to test estrogenic and antiestrogenic activity of compounds.

    PubMed

    van der Burg, Bart; Winter, Roos; Weimer, Marc; Berckmans, Pascale; Suzuki, Go; Gijsbers, Linda; Jonas, Arjen; van der Linden, Sander; Witters, Hilda; Aarts, Jac; Legler, Juliette; Kopp-Schneider, Annette; Bremer, Susanne

    2010-08-01

    Estrogenicity of chemicals has received significant attention and is linked to endocrine-disrupting activities. However, there is a paucity of validated methods to assess estrogenicity in vitro. We have established a robust method to test estrogenic and antiestrogenic activity of compounds in vitro, as an alternative to using animal models such as the uterotrophic assay. To this end we optimized protocols to be used in combination with CALUX reporter gene assays and carried out an in house prevalidation, followed by two rounds of tests to establish transferability. Problems in the initial test with transferability were solved by isolation of a novel cell clone of the ERalpha CALUX line with greatly improved stability and luciferase levels. This cell line proved to be a very suitable and reliable predictor of estrogenicity of chemicals and was able to readily rank a range of chemicals on the basis of their EC50 values.

  16. AhR-mediated and antiestrogenic activity of humic substances.

    PubMed

    Janosek, J; Bittner, M; Hilscherová, K; Bláha, L; Giesy, J P; Holoubek, I

    2007-04-01

    Humic substances (HS) were for decades regarded as inert in the ecosystems with respect to their possible toxicity. However, HS have been recently shown to elicit various adverse effects generally attributed to xenobiotics. In our study, we used MVLN and H4IIE-luc cell lines stably transfected with luciferase gene under control of estrogen receptor (ER) and Ah receptor (AhR; receptor connected with so-called dioxin-like toxicity) for assessment of anti/estrogenic and AhR-mediated effects of 12 commercially available humic substances. Out of those, five humic acids were shown to induce AhR-mediated activity with relative potencies related to TCDD 2.6 x 10(-8)-7.4 x 10(-8). Organic extracts of HS solutions also elicited high activities what means that lipophilic molecules are responsible for a great part of effect. However, relatively high activity remaining in extracted solution suggests also presence of polar AhR-agonists. Contribution of persistent organic compounds to the observed effects was ruled out by H(2)SO(4) treatment. Eight out of twelve HS elicited significant antiestrogenic effects with IC(50) ranging from 40 to 164 mg l(-1). The possible explanations of the antiestrogenic effect include sorption of 17-beta-estradiol (E2) on HS, changes in membrane permeability for E2 or another specific mechanism.

  17. Armadillo/Pangolin regulates PCNA and DREF promoter activities.

    PubMed

    Kwon, Eunjeong; Hayashi, Yuko; Otsuki, Kyoko; Hirose, Fumiko; Nishida, Yasuyoshi; Yoo, Mi-Ae; Yamaguchi, Masamitsu

    2004-09-17

    Here we show that Armadillo and Pangolin (dTCF), downstream effectors of the Wingless (Wg) signal transduction pathway, activate transcription of the important DNA replication-related genes encoding Drosophila proliferating cell nuclear antigen (PCNA) and DNA replication-related element-binding factor (DREF). By transient luciferase expression assays and band mobility shift assays, we demonstrated the PCNA gene to be a direct target gene for the Armadillo/Pangolin complex. Using a GAL4-UAS system, stimulation of the PCNA gene by Armadillo/Pangolin was confirmed in adult females. From the published reports of an inhibitory role, we expected that Drosophila CREB-binding protein (dCBP) would interfere with activation. However, effects were only observed with the DREF but not the PCNA gene. In the latter case, as in mammals, dCBP could potentiate Armadillo-mediated activation. These results suggest that first, PCNA and DREF genes are targets of the Armadillo/Pangolin complex and second, dCBP modulates Wg signaling in a gene-specific manner.

  18. Optimization of Lactobacillus acidophilus cultivation using taro waste and evaluation of its biological activity.

    PubMed

    Hsieh, Shu-Chen; Liu, Jui-Ming; Pua, Xiao-Hui; Ting, Yuwen; Hsu, Ren-Jun; Cheng, Kuan-Chen

    2016-03-01

    In this study, taro waste (TW) was utilized for Lactobacillus acidophilus BCRC 14079 cultivation and the anti-tumor and immune-modulatory properties of heat-killed cells (HKCs), cytoplasmic fraction (CF), and exopolysaccharide (EPS) were evaluated. The optimum liquefaction enzyme dosage, temperature, and time determined by Box-Behnken design response surface methodology (BBD-RSM) were 9 mL/L of α-amylase, 79.2 °C, and 5 h of reaction, respectively. The optimum temperature and reaction time for saccharification were determined as 60 °C and 3 h. The optimum medium, CGMY1 medium, constitutes of TW hydrolysate containing 37 g/L of glucose, 25 g/L of corn gluten meal (CGM), and 1 g/L of yeast extract (YE). Results of MTT assay showed that HKCs and EPS from CGM medium exhibited the highest anti-proliferative in HT-29 (IC50 of HKCs, 467.25 μg/mL; EPS, 716.10 μg/mL) and in Caco-2 cells (IC50 of EPS, 741.60 μg/mL). Luciferase-based NF-ΚB and COX-2 systems indicated HKCs from CGM medium stimulated the highest expression of luciferin in both systems. The luciferase activities by using 100 and 500 μg/mL of HKCs from CGM were 24.30- and 45.83-fold in NF-ΚB system and 11.54- and 4.93-fold in COX-2 system higher than the control. In conclusion, this study demonstrated the potential of TW medium for L. acidophilus cultivation and the production of non-viable probiotics with enhanced biological activities.

  19. Gastrin stimulates expression of plasminogen activator inhibitor-1 in gastric epithelial cells.

    PubMed

    Nørsett, Kristin G; Steele, Islay; Duval, Cedric; Sammut, Stephen J; Murugesan, Senthil V M; Kenny, Susan; Rainbow, Lucille; Dimaline, Rod; Dockray, Graham J; Pritchard, D Mark; Varro, Andrea

    2011-09-01

    Plasminogen activator inhibitor (PAI)-1 is associated with cancer progression, fibrosis and thrombosis. It is expressed in the stomach but the mechanisms controlling its expression there, and its biological role, are uncertain. We sought to define the role of gastrin in regulating PAI-1 expression and to determine the relevance for gastrin-stimulated cell migration and invasion. In gastric biopsies from subjects with elevated plasma gastrin, the abundances of PAI-1, urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNAs measured by quantitative PCR were increased compared with subjects with plasma concentrations in the reference range. In patients with hypergastrinemia due to autoimmune chronic atrophic gastritis, there was increased abundance of PAI-1, uPA, and uPAR mRNAs that was reduced by octreotide or antrectomy. Immunohistochemistry revealed localization of PAI-1 to parietal cells and enterochromaffin-like cells in micronodular neuroendocrine tumors in hypergastrinemic subjects. Transcriptional mechanisms were studied by using a PAI-1-luciferase promoter-reporter construct transfected into AGS-G(R) cells. There was time- and concentration-dependent increase of PAI-1-luciferase expression in response to gastrin that was reversed by inhibitors of the PKC and MAPK pathways. In Boyden chamber assays, recombinant PAI-1 inhibited gastrin-stimulated AGS-G(R) cell migration and invasion, and small interfering RNA treatment increased responses to gastrin. We conclude that elevated plasma gastrin concentrations are associated with increased expression of gastric PAI-1, which may act to restrain gastrin-stimulated cell migration and invasion.

  20. Effect of apple extracts on NF-kappaB activation in human umbilical vein endothelial cells.

    PubMed

    Davis, Paul A; Polagruto, John A; Valacchi, Giuseppe; Phung, Anh; Soucek, Karel; Keen, Carl L; Gershwin, M Eric

    2006-05-01

    The mechanisms by which foods, such as fruit, are able to reduce the risk of chronic disease are still unclear. Several fruit products, including apples and apple juice, that are flavonoid-rich are reported to increase antioxidant levels in human subjects. This is supported by the finding from our previous studies that the chronic consumption of apple juice by human subjects reduced ex vivo low-density lipoprotein (LDL) oxidation; we hypothesized that this was due to the flavonoid in the apple juice, which, as we reported earlier, reduced in vitro LDL oxidation. To further explore whether the mixture of flavonoids and other phytochemicals in apples are biologically relevant antioxidants, we tested the effects of this flavonoid-rich apple extract (AE) on oxidant-related pathways in a model of the endothelium: human umbilical vascular endothelial cells (HU-VECs). The effects of AE on oxidant-responsive (i.e., tumor necrosis factor [TNF]-alpha-induced) nuclear factor (NF)- kappaB signaling in cell culture were assessed in transfected HUVECs by using a construct that expressed luciferase under the control of NF-kappaB. Incubation of HUVEC for 24 hrs with up to 10 mM (as gallic acid equivalents) of AE demonstrated no cytotoxicity, as determined by lactate dehydrogenase release, caspase 3 activation, and apoptosis marker-based FACS analysis. AE after a 24-hr incubation period at either 200 or 2000 nM showed a complex pattern of decreased basal and TNF-alpha-stimulated NF-kappaB signaling (63% maximal decrease) as assessed by luciferase activity in the transfected HUVECs, as well as by reduced levels of IkappaBalpha protein phosphorylation detected by Western blot analysis. We suggest that AE downregulates NF-kappaB signaling and that this is indicative of an antioxidant effect of the flavonoids present in AE.

  1. Thymoquinone: An IRAK1 inhibitor with in vivo and in vitro anti-inflammatory activities

    PubMed Central

    Hossen, Muhammad Jahangir; Yang, Woo Seok; Kim, Daewon; Aravinthan, Adithan; Kim, Jong-Hoon; Cho, Jae Youl

    2017-01-01

    Thymoquinone (TQ) is a bioactive component of black seed (Nigella sativa) volatile oil and has been shown to have anti-oxidative, anti-inflammatory, and anti-cancer properties. In the present study, we explored the molecular mechanisms that underlie the anti-inflammatory effect of TQ and its target proteins using lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 and human monocyte-like U937 cells, together with LPS/D-galactosamine (GalN)-induced acute hepatitis and HCl/EtOH-induced gastritis mouse models. TQ strongly inhibited the production of nitric oxide (NO) and repressed NO synthase (iNOS), tumor necrosis factor (TNF)-α, cyclooxygenase (COX)−2, interleukin (IL)−6, and IL-1β expression in LPS-activated RAW264.7 cells. Treatment of LPS/D-GalN–induced hepatitis and EtOH/HCl–induced gastritis mouse models with TQ significantly ameliorated disease symptoms. Using luciferase reporter gene assays, we also showed that the nuclear levels of transcription factors and phosphorylation patterns of signaling proteins, activator protein (AP)−1, and nuclear factor (NF)-κB pathways were all affected by TQ treatment. Finally, we used additional kinase and luciferase validation assays with interleukin-1 receptor-associated kinase 1 (IRAK1) to show that IRAK1 is directly suppressed by TQ treatment. Together, these findings strongly suggest that the anti-inflammatory actions of TQ are caused by suppression of IRAK-linked AP-1/NF-κB pathways. PMID:28216638

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

    SciTech Connect

    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

  3. Active-R filter

    DOEpatents

    Soderstrand, Michael A.

    1976-01-01

    An operational amplifier-type active filter in which the only capacitor in the circuit is the compensating capacitance of the operational amplifiers, the various feedback and coupling elements being essentially solely resistive.

  4. Repression of activated aryl hydrocarbon receptor-induced transcriptional activation by 5alpha-dihydrotestosterone in human prostate cancer LNCaP and human breast cancer T47D cells.

    PubMed

    Sanada, Noriko; Gotoh, Yuka; Shimazawa, Rumiko; Klinge, Carolyn M; Kizu, Ryoichi

    2009-03-01

    Polycyclic aromatic hydrocarbons (PAHs) and dioxins are ubiquitous environmental pollutants and activate the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. It has been reported that testosterone represses 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced transcription of the cytochrome P450 (CYP) 1A1 gene in LNCaP cells. In this study, we investigated the mechanism for the repression of 3-methylcholanthrene (3MC)-induced transcription of AhR-regulated genes, CYP1A1, CYP1A2, CYP1B1, and AhR repressor (AhRR), by 5alpha-dihydroteststerone (DHT) in LNCaP and T47D cells, which are androgen receptor (AR)- and AhR-positive. Real-time PCR analysis showed that DHT repressed 3MC-induced mRNA expression of the CYP1 family and AhRR genes. DHT repressed 3MC-induced luciferase activity in an AhR response element-driven luciferase reporter assay in LNCaP and T47D cells. The inhibitory effect of DHT was abolished by knockdown of AR protein with siRNA. The protein levels of AhR and AhR nuclear translocator (Arnt), the AhR-dimerizing partner, were not affected by DHT. Co-immunoprecipitation assay showed that DHT significantly facilitated the complex formation between AR and AhR in 3MC-treated cells. These results suggest that complex formation between activated AR and AhR plays an important role in the suppression of 3MC-induced transcription of CYP1 family genes by DHT.

  5. Computer-aided discovery, validation, and mechanistic characterization of novel neolignan activators of peroxisome proliferator-activated receptor gamma.

    PubMed

    Fakhrudin, Nanang; Ladurner, Angela; Atanasov, Atanas G; Heiss, Elke H; Baumgartner, Lisa; Markt, Patrick; Schuster, Daniela; Ellmerer, Ernst P; Wolber, Gerhard; Rollinger, Judith M; Stuppner, Hermann; Dirsch, Verena M

    2010-04-01

    Peroxisome proliferator-activated receptor gamma (PPAR gamma) agonists are used for the treatment of type 2 diabetes and metabolic syndrome. However, the currently used PPAR gamma agonists display serious side effects, which has led to a great interest in the discovery of novel ligands with favorable properties. The aim of our study was to identify new PPARgamma agonists by a PPAR gamma pharmacophore-based virtual screening of 3D natural product libraries. This in silico approach led to the identification of several neolignans predicted to bind the receptor ligand binding domain (LBD). To confirm this prediction, the neolignans dieugenol, tetrahydrodieugenol, and magnolol were isolated from the respective natural source or synthesized and subsequently tested for PPAR gamma receptor binding. The neolignans bound to the PPAR gamma LBD with EC(50) values in the nanomolar range, exhibiting a binding pattern highly similar to the clinically used agonist pioglitazone. In intact cells, dieugenol and tetrahydrodieugenol selectively activated human PPAR gamma-mediated, but not human PPAR alpha- or -beta/delta-mediated luciferase reporter expression, with a pattern suggesting partial PPAR gamma agonism. The coactivator recruitment study also demonstrated partial agonism of the tested neolignans. Dieugenol, tetrahydrodieugenol, and magnolol but not the structurally related eugenol induced 3T3-L1 preadipocyte differentiation, confirming effectiveness in a cell model with endogenous PPAR gamma expression. In conclusion, we identified neolignans as novel ligands for PPAR gamma, which exhibited interesting activation profiles, recommending them as potential pharmaceutical leads or dietary supplements.

  6. Molecular screening of Chinese medicinal plants for progestogenic and anti-progestogenic activity.

    PubMed

    Ahmed, H M Manir; Yeh, Jan-Ying; Tang, Yi-Chia; Cheng, Winston Teng-Kuei; Ou, Bor-Rung

    2014-06-01

    Estrogen and progestins have adverse effects, and many of these adverse effects are caused by progestins. Due to this, many women choose to use botanical alternatives for hormone replacement therapy, which does not trigger steroidogenic properties. Therefore, it is necessary to screen these herbs for progestogenic and anti-progestogenic properties. Extract of 13 Chinese medicinal plants were analysed for progestogenic and anti-progestogenic activities by using progesterone response element-driven luciferase reporter gene bioassay. MTT assay was carried out to investigate the cytotoxic effect of herb extract on PAE cells. Among the 13 herbs, Dipsacus asperoides extract exhibited progestogenic activity, and 10 species - Cortex eucommiae, Folium artemisiae argyi, Glycyrrhiza uralensis, Angelica sinensis, Atractylodes macrocephala koidz, Scutellaria baicalensis, Cuscuta chinensis, Euscaphis japonica, Ailanthus altissima, and Dioscorea opposita - were recognized to have anti-progestogenic like activities. Extract of Dipsacus asperoides demonstrated dose-dependent progestogenic activity, and the progestogenic activity of 100 (mu)g/mL extracts was equivalent to 31.45 ng/mL progesterone activity. Herbs extracts that exhibited anti-progestogenic-like activity also inhibited the 314.46 ng/mL progesterone activity in a dose-response manner. None of the herb extracts shown significant toxic effect on PAE cells at 40-100 (mu)g/mL compared to control. This discovery will aid selection of suitable herbs for hormone replacement therapy.

  7. Antiandrogenic activity and metabolism of the organophosphorus pesticide fenthion and related compounds.

    PubMed Central

    Kitamura, Shigeyuki; Suzuki, Tomoharu; Ohta, Shigeru; Fujimoto, Nariaki

    2003-01-01

    We investigated the endocrine-disrupting actions of the organophosphorus pesticide fenthion and related compounds and the influence of metabolic transformation on the activities of these compounds. Fenthion acted as an antagonist of the androgenic activity of dihydrotestosterone (10(-7)M) in the concentration range of 10(-6)-10(-4)M in an androgen-responsive element-luciferase reporter-responsive assay using NIH3T3 cells. The antiandrogenic activity of fenthion was similar in magnitude to that of flutamide. Fenthion also tested positive in the Hershberger assay using castrated male rats. Marked estrogenic and antiestrogenic activities of fenthion and related compounds were not observed in MCF-7 cells. When fenthion was incubated with rat liver microsomes in the presence of NADPH, the antiandrogenic activity markedly decreased, and fenthion sulfoxide was detected as a major metabolite. The oxidase activity toward fenthion was exhibited by cytochrome P450 and flavin-containing monooxygenase. Fenthion sulfoxide was negative in the screening test for antiandrogens, as was fenthion sulfone. However, when fenthion sulfoxide was incubated with liver cytosol in the presence of 2-hydroxypyrimidine, an electron donor of aldehyde oxidase, the extract of the incubation mixture exhibited antiandrogenic activity. In this case, fenthion was detected as a major metabolite of the sulfoxide. Metabolic interconversion between fenthion and fenthion sulfoxide in the body seems to maintain the antiandrogenic activity. PMID:12676606

  8. Evaluation of estrogenic activities of pesticides using an in vitro reporter gene assay.

    PubMed

    Kojima, Mihoko; Fukunaga, Kenji; Sasaki, Mari; Nakamura, Masafumi; Tsuji, Motohiro; Nishiyama, Toshimasa

    2005-08-01

    The estrogenic activities of 32 pesticides in agricultural products were evaluated using the E-CALUX assay system developed by Xenobiotic Detection Systems Inc (North Carolina, USA). This system utilizes human ovarian carcinoma cells (BG1) stably transfected with an estrogen-responsive luciferase reporter gene plasmid. It was found that tolclofos-methyl, prothiofos, diazinon, Thiabenclazole (TBZ) and pyriproxyfen had estrogenic activity. Several pesticides are often present in agricultural products. Therefore the estrogenicity of the mixtures of two kinds of pesticides was evaluated. The activity of diazinon/tolclofos-methyl, pyriproxyfen/prothiofos and TBZ/o-phenylphenol (OPP) was increased up to 1.2-5.3 fold. On the other hand, chlorfluazuron, imazalil and chlorfenapyr had anti-estrogenic activity. Further, to evaluate the change in the estrogenic activity of pesticide metabolites, an experimental system was established using a rat S9 mixture. Metabolites of permethrin and OPP had no estrogenic activity, but they had weak activity after the metabolism. On the other hand, the metabolites of TBZ exhibited less estrogenic activity than the original compounds.

  9. Exposure of airway epithelial cells to Pseudomonas aeruginosa biofilm-derived quorum sensing molecules decrease the activity of the anti-oxidant response element bound by NRF2.

    PubMed

    Roussel, Lucie; Rousseau, Simon

    2017-02-05

    Chronic bacterial infections in cystic fibrosis lung disease are often characterized by Pseudomonas aeruginosa biofilms that are regulated by bacterial intercellular signals termed quorum sensing (QS), such as N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL). This study reports that biofilm-derived exoproducts decrease the transcriptional activity of the anti-oxidant response element in bronchial epithelial cells. In a live co-culture assay of BEAS-2B cells and P. aeruginosa biofilm, the QS molecule 3OC12-HSL was an important but not sole contributor to the inhibition of basal NRF2 luciferase reporter activity. Moreover, biofilm-derived exoproducts and 3OC12-HSL decrease the expression of endogenous antioxidant response element-regulated genes hemeoxygenase-1 (HO-1) and NAD(P)H Quinone Dehydrogenase-1 (NQO-1) while they increase IL-8 expression. As previously reported, IL-8 expression is partially dependent on p38 MAPK activity, but the inhibitory effect of biofilm QS molecules on HO-1 and NQO-1 expression occurs independently of this protein kinase. Finally, the transfection of CFTRdelF508 but not its wild type counterpart decreases basal, planktonic PsaDM and sulforaphane-driven NRF2 luciferase reporter activity in BEAS-2B cells. Therefore, the presence of quorum sensing molecules derived from bacterial biofilms lowers the transcriptional activity of the anti-oxidant response element, which may contribute to the establishment of chronic bacterial infections, especially in the presence of mutated CFTR. Increasing NRF2 activity may thus be a promising strategy to potentiate anti-biofilm activity in cystic fibrosis lung disease.

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

    PubMed

    DeSmet, Marsha L; Fleet, James C

    2017-01-16

    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)2D)-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)2D-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)2D-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