Generation of a recombinant West Nile virus stably expressing the Gaussia luciferase for neutralization assay.

PubMed

Zhang, Pan-Tao; Shan, Chao; Li, Xiao-Dan; Liu, Si-Qing; Deng, Cheng-Lin; Ye, Han-Qing; Shang, Bao-Di; Shi, Pei-Yong; Lv, Ming; Shen, Bei-Fen; Qin, Cheng-Feng; Zhang, Bo

2016-01-04

West Nile virus (WNV) is a neurotropic human pathogen that has caused increasing infected cases over recent years. There is currently no licensed vaccine or effective drug for prevention and treatment of WNV infection in humans. To facilitate antiviral drug discovery and neutralizing antibody detection, a WNV cDNA clone containing a luciferase reporter gene was constructed through incorporating Gaussia luciferase (Gluc) gene within the capsid-coding region of WNV genome. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated luciferase reporter WNV (WNV-Gluc) and the stable WNV-Gluc with high titers (>10(7)PFU/ml) was obtained through plaque purification. Luciferase activity was used to effectively quantify the viral production of WNV-Gluc. Using the reporter virus WNV-Gluc, we developed a luciferase based assay in a 12-well format for evaluating neutralizing antibodies. The reporter virus could be a powerful tool for epidemiological investigation of WNV, vaccine evaluation, antiviral drug screening, and the study of WNV replication and pathogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

High-Throughput Screening of a Luciferase Reporter of Gene Silencing on the Inactive X Chromosome.

PubMed

Keegan, Alissa; Plath, Kathrin; Damoiseaux, Robert

2018-01-01

Assays of luciferase gene activity are a sensitive and quantitative reporter system suited to high-throughput screening. We adapted a luciferase assay to a screening strategy for identifying factors that reactivate epigenetically silenced genes. This epigenetic luciferase reporter is subject to endogenous gene silencing mechanisms on the inactive X chromosome (Xi) in primary mouse cells and thus captures the multilayered nature of chromatin silencing in development. Here, we describe the optimization of an Xi-linked luciferase reactivation assay in 384-well format and adaptation of the assay for high-throughput siRNA and chemical screening. Xi-luciferase reactivation screening has applications in stem cell biology and cancer therapy. We have used the approach described here to identify chromatin-modifying proteins and to identify drug combinations that enhance the gene reactivation activity of the DNA demethylating drug 5-aza-2'-deoxycytidine.

A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

PubMed

Xie, Wensheng; Silvers, Robert; Ouellette, Michael; Wu, Zining; Lu, Quinn; Li, Hu; Gallagher, Kathleen; Johnson, Kathy; Montoute, Monica

2016-01-01

Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.

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

A cooled CCD camera-based protocol provides an effective solution for in vitro monitoring of luciferase.

PubMed

Afshari, Amirali; Uhde-Stone, Claudia; Lu, Biao

2015-03-13

Luciferase assay has become an increasingly important technique to monitor a wide range of biological processes. However, the mainstay protocols require a luminometer to acquire and process the data, therefore limiting its application to specialized research labs. To overcome this limitation, we have developed an alternative protocol that utilizes a commonly available cooled charge-coupled device (CCCD), instead of a luminometer for data acquiring and processing. By measuring activities of different luciferases, we characterized their substrate specificity, assay linearity, signal-to-noise levels, and fold-changes via CCCD. Next, we defined the assay parameters that are critical for appropriate use of CCCD for different luciferases. To demonstrate the usefulness in cultured mammalian cells, we conducted a case study to examine NFκB gene activation in response to inflammatory signals in human embryonic kidney cells (HEK293 cells). We found that data collected by CCCD camera was equivalent to those acquired by luminometer, thus validating the assay protocol. In comparison, The CCCD-based protocol is readily amenable to live-cell and high-throughput applications, offering fast simultaneous data acquisition and visual and quantitative data presentation. In conclusion, the CCCD-based protocol provides a useful alternative for monitoring luciferase reporters. The wide availability of CCCD will enable more researchers to use luciferases to monitor and quantify biological processes. Copyright © 2015 Elsevier Inc. All rights reserved.

The nuclear factor κB inhibitor (E)-2-fluoro-4'-methoxystilbene inhibits firefly luciferase.

PubMed

Braeuning, Albert; Vetter, Silvia

2012-12-01

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4'-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4'-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4'-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4'-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase

PubMed Central

Braeuning, Albert; Vetter, Silvia

2012-01-01

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays. PMID:22789175

Split-luciferase complementary assay: applications, recent developments, and future perspectives.

PubMed

Azad, Taha; Tashakor, Amin; Hosseinkhani, Saman

2014-09-01

Bioluminescent systems are considered as potent reporter systems for bioanalysis since they have specific characteristics, such as relatively high quantum yields and photon emission over a wide range of colors from green to red. Biochemical events are mostly accomplished through large protein machines. These molecular complexes are built from a few to many proteins organized through their interactions. These protein-protein interactions are vital to facilitate the biological activity of cells. The split-luciferase complementation assay makes the study of two or more interacting proteins possible. In this technique, each of the two domains of luciferase is attached to each partner of two interacting proteins. On interaction of those proteins, luciferase fragments are placed close to each other and form a complemented luciferase, which produces a luminescent signal. Split luciferase is an effective tool for assaying biochemical metabolites, where a domain or an intact protein is inserted into an internally fragmented luciferase, resulting in ligand binding, which causes a change in the emitted signals. We review the various applications of this novel luminescent biosensor in studying protein-protein interactions and assaying metabolites involved in analytical biochemistry, cell communication and cell signaling, molecular biology, and the fate of the whole cell, and show that luciferase-based biosensors are powerful tools that can be applied for diagnostic and therapeutic purposes.

Androgen responsiveness of Renilla luciferase reporter vectors is promoter, transgene, and cell line dependent.

PubMed

Mulholland, David J; Cox, Michael; Read, Jason; Rennie, Paul; Nelson, Colleen

2004-05-01

Renilla based reporters are frequently used as transfection controls for luciferase transcriptional reporter assays. However, recent evidence suggests that a commonly used reporter (HSV-thymidine kinase driven Renilla) is responsive to androgen receptor (AR) and glucocorticoid receptors in the presence of the cognate ligands, dihydrotestosterone (DHT) and dexamethasone (DEX), respectively [1]. We further validate this important technical difficulty by illustrating that in LNCaP prostate cancer cells, spurious Renilla luciferase activity is a function of (a) the promoter driving Renilla expression, (b) the presence of co-transfected transgenes, and (c) the androgen responsiveness of the cell line used. Using inhibitors of transcription and translation we showed that transcript interference or translational modulation is not a major means by which androgens affect Renilla luciferase activity. As luciferase reporter assays are a frequent means of studying transcriptional co-regulation in the highly androgen dependent LNCaP cell line, our data serves as a cautionary note that alternative normalization techniques should be employed to avoid misinterpretation of data. Copyright 2004 Wiley-Liss, Inc.

Molecular Targeting of Prostate Cancer During Androgen Ablation: Inhibition of CHES1/FOXN3

DTIC Science & Technology

2011-05-01

activity (Firefly luciferase) was normalized to Renilla luciferase activity. Results are presented as fold-change in PSA reporter activity...reporter (DLR) assays performed. In each sample, the CHES1-RR1/3.5 reporter activity (Firefly luciferase) was normalized to Renilla luciferase...4.0) reporter activity (Firefly luciferase) was normalized to Renilla luciferase activity. Results are presented as fold-change in BNIP3 reporter

Luciferase Protein Complementation Assays for Bioluminescence Imaging of Cells and Mice

PubMed Central

Luker, Gary D.; Luker, Kathryn E.

2015-01-01

Summary Protein fragment complementation assays (PCAs) with luciferase reporters currently are the preferred method for detecting and quantifying protein-protein interactions in living animals. At the most basic level, PCAs involve fusion of two proteins of interest to enzymatically inactive fragments of luciferase. Upon association of the proteins of interest, the luciferase fragments are capable of reconstituting enzymatic activity to generate luminescence in vivo. In addition to bi-molecular luciferase PCAs, unimolecular biosensors for hormones, kinases, and proteases also have been developed using target peptides inserted between inactive luciferase fragments. Luciferase PCAs offer unprecedented opportunities to quantify dynamics of protein-protein interactions in intact cells and living animals, but successful use of luciferase PCAs in cells and mice involves careful consideration of many technical factors. This chapter discusses the design of luciferase PCAs appropriate for animal imaging, including construction of reporters, incorporation of reporters into cells and mice, imaging techniques, and data analysis. PMID:21153371

Different Types of Luciferase Reporters Show Distinct Susceptibility to T3-Evoked Downregulation.

PubMed

Kollár, Anna; Kvárta-Papp, Zsuzsanna; Egri, Péter; Gereben, Balázs

2016-01-01

The firefly luciferase reporter protein is a crucial tool for studies targeting a broad range of biological questions. Importantly, luciferase assays are also widely used to explore mechanisms underlying thyroid hormone dependent regulation of gene expression. However, it was demonstrated that the firefly luciferase reporter is subject to triiodothyronine (T3)-evoked, promoter independent downregulation that is mediated by the thyroid hormone receptor. Since this effect can interfere with readout accuracy, the study aimed to find luciferase reporters that are not susceptible to this phenomenon. Luciferase reporter constructs were generated under the control of a minimal thymidine kinase (TK) promoter and transiently transfected into JEG-3 cells to test their activity upon T3 treatment. Activity of the TK-(dCpG)Luc encoding a synthetic (dCpG)Luciferase and TK-NanoLuc expressing the NanoLuc reporter was not significantly changed by T3 treatment while the firefly luciferase control was suppressed by ∼2.6-fold. T3 also downregulated the activity of Renilla luciferase by ∼30%. Novel types of luciferase reporters, especially the synthetic (dCpG)Luciferase, can be more accurate to study T3-regulated gene expression than the classical firefly luciferase reporter. Renilla luciferase, a popular transfection control of dual luciferase assays, should be used with caution in conditions with T3 treatment.

Secreted dual reporter assay with Gaussia luciferase and the red fluorescent protein mCherry

PubMed Central

Wider, Diana

2017-01-01

The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters. This requirement has been very conveniently met with the popular dual luciferase assay. Its disadvantages are the requirement for cell lysis, the inability to analyze the same cells repeatedly, and the cost, at least in its most commonly used commercial format. Here we describe a novel dual reporter assay with the naturally secreted luciferase from Gaussia princeps as the main reporter protein and a secreted version of the red fluorescent protein mCherry as internal standard. After first measuring mCherry fluorescence in the medium, an enzyme buffer with coelenterazine as substrate is added to the same sample to trigger a glow-type luminescence of the luciferase. The simple and cheap assay can easily be adapted to a variety of experimental situations. As a case in point, we have developed a panel of Gaussia luciferase reporter genes for transcriptional activation assays with estrogen and glucocorticoid response elements, and with response elements for fusion proteins with the Gal4 DNA binding domain for use in mammalian cells. Our secreted dual reporter assay should be an attractive alternative to the currently available commercial kits. PMID:29220385

Secreted dual reporter assay with Gaussia luciferase and the red fluorescent protein mCherry.

PubMed

Wider, Diana; Picard, Didier

2017-01-01

The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters. This requirement has been very conveniently met with the popular dual luciferase assay. Its disadvantages are the requirement for cell lysis, the inability to analyze the same cells repeatedly, and the cost, at least in its most commonly used commercial format. Here we describe a novel dual reporter assay with the naturally secreted luciferase from Gaussia princeps as the main reporter protein and a secreted version of the red fluorescent protein mCherry as internal standard. After first measuring mCherry fluorescence in the medium, an enzyme buffer with coelenterazine as substrate is added to the same sample to trigger a glow-type luminescence of the luciferase. The simple and cheap assay can easily be adapted to a variety of experimental situations. As a case in point, we have developed a panel of Gaussia luciferase reporter genes for transcriptional activation assays with estrogen and glucocorticoid response elements, and with response elements for fusion proteins with the Gal4 DNA binding domain for use in mammalian cells. Our secreted dual reporter assay should be an attractive alternative to the currently available commercial kits.

Characterization of BRCA2 Transcriptional Regulation

DTIC Science & Technology

2000-08-01

Renilla luciferase vector (Promega) with 4 ll of Fugene-6 was used for each transfection. The pRL-TK Renilla luciferase activity was used to control for...experiments, cells received 0.5 jig of BRCA2 promoter construct, 0.1 jig of pRL-TK Renilla luciferase vector, and 0.5 jig of the indicated expression...Myc, and pCMV-Max. Firefly lucifer- ase and renilla luciferase assays were performed using the Dual-Luciferase Reporter Assay Sys- tem (Promega

Renilla luciferase-labeled Annexin V: a new probe for detection of apoptotic cells.

PubMed

Nazari, Mahboobeh; Emamzadeh, Rahman; Hosseinkhani, Saman; Cevenini, Luca; Michelini, Elisa; Roda, Aldo

2012-11-07

The Ca(2+)-dependent binding of Annexin V to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this paper, we report a new class of Annexin V-based probes for apoptosis. Luciferase from Renilla reniformis (RLuc) was linked to Annexin V and expressed successfully in a soluble form in Escherichia coli BL21 (DE3). The new probe, Rluc/Annexin V, was purified and functionally assayed for detection of apoptosis in actinomycin D-induced apoptotic Jurkat cells. Moreover, the spontaneous apoptosis in neutrophils was shown using the new probe. The results indicate that Rluc/Annexin V can bind to the apoptotic cells, and the signal of Renilla luciferase can be detected by luminometric measurements. The availability of Rluc/Annexin V may be of potential commercial interest for improving current apoptosis assays.

Role of Cyclin D1 and cdk Inhibitors in Breast Cancer Pathogenesis

DTIC Science & Technology

2001-10-01

0.1 [tg of a Renilla luciferase expression plasmid (pRL-SV40 Promega) to normalize for transfection efficiency. After an overnight recovery, the...saline, collected, lysed, and analyzed for luciferase and Renilla luciferase activity by using the Dual-Luciferase reporter assay system (Promega...Cyclin A promoter-driven luciferase activity was then normalized to a constant activity of Renilla luciferase. 7 Results In our previous reports we

Modulation of Beta-catenin Activity With PKD1 Prostate Cancer

DTIC Science & Technology

2009-04-01

mutated site as a negative control (FOPFlash) with pRL-TK ( Renilla luciferase) in C4-2- PKD1-GFP cells activated with Bryostatin 1 or DMSO. The...firefly and Renilla luciferase activities were measured with the Dual-Luciferase Reporter (DLR) Assay System. After normalizing the firefly luciferase...activity to that of Renilla luciferase, the FOPFlash reporter plasmid luciferase values were subtracted from the normalized values obtained with the

Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation.

PubMed

Paulmurugan, R; Gambhir, S S

2003-04-01

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 alpha through NFkappaB-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.

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

Development of a Sensitive Luciferase-Based Sandwich ELISA System for the Detection of Human Extracellular Matrix 1 Protein.

PubMed

Li, Ya; Li, Yanqing; Zhao, Junli; Zheng, Xiaojing; Mao, Qinwen; Xia, Haibin

2016-12-01

Enzyme-linked immunosorbent assay (ELISA) has been one of the main methods for detecting an antigen in an aqueous sample for more than four decades. Nowadays, one of the biggest concerns for ELISA is still how to improve the sensitivity of the assay, and the luciferase-luciferin reaction system has been noticed as a new detection method with high sensitivity. In this study, a luciferin-luciferase reaction system was used as the detection method for a sandwich ELISA system. It was shown that this new system led to an increase in the detection sensitivity of at least two times when compared with the traditional horseradish peroxidase (HRP) detection method. Lastly, the serum levels of the human extracellular matrix 1 protein of breast cancer patients were determined by the new system, which were overall similar to the HRP chemiluminescent system. Furthermore, this new luciferase reporter can be implemented into other ELISA systems for the purpose of increasing the assay sensitivity.

Regulation of AR and (Beta)-Catenin Signaling by Pin 1 in Prostate Cancer

DTIC Science & Technology

2005-10-01

Renilla luciferase activities were determined using a dual-luciferase reporter assay kit (Promega, Madison, WI), and Renilla activities were not...consistently affected by any of the cotransfected vectors. The firefly luciferase was divided by the control Renilla luciferase and the results, given as...transfected with pTopflash (50 ng) and CMV- Renilla (2.5 ng) reporter plasmids. Firefly versus Renilla luciferase activities were determined and

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. Copyright © 2016 Elsevier Inc. All rights reserved.

Polymer nanoassemblies with hydrophobic pendant groups in the core induce false positive siRNA transfection in luciferase reporter assays.

PubMed

Rheiner, Steven; Reichel, Derek; Rychahou, Piotr; Izumi, Tadahide; Yang, Hsin-Sheng; Bae, Younsoo

2017-08-07

Poly(ethylene glycol)-conjugated polyethylenimine (PEG-PEI) is a widely studied cationic polymer used to develop non-viral vectors for siRNA therapy of genetic disorders including cancer. Cell lines stably expressing luciferase reporter protein typically evaluate the transfection efficacy of siRNA/PEG-PEI complexes, however recent findings revealed that PEG-PEI can reduce luciferase expression independent of siRNA. This study elucidates a cause of the false positive effect in luciferase assays by using polymer nanoassemblies (PNAs) made from PEG, PEI, poly-(l-lysine) (PLL), palmitate (PAL), and deoxycholate (DOC): PEG-PEI (2P), PEG-PEI-PAL (3P), PEG-PLL (2P'), PEG-PLL-PAL (3P'), and PEG-PEI-DOC (2PD). In vitro transfection and western blot assays of luciferase using a colorectal cancer cell line expressing luciferase (HT29/LUC) concluded that 2P and 2P' caused no luciferase expression reduction while hydrophobically modified PNAs induced a 35-50% reduction (3P'<2PD<3P). Although cell viability remained stagnant, 3P triggered cellular stress responses including increased membrane porosity and decreased ATP and cellular protein concentrations. Raman spectroscopy suggested that hydrophobic groups influence PNA conformation changes, which may have caused over-ubiquitination and degradation of luciferase in the cells. These results indicate that hydrophobically modified PEG-PEI induces cellular distress causing over-ubiquitination of the luciferase protein, producing false positive siRNA transfection in the luciferase assay. Copyright © 2017 Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huysseune, Sandra; Kienlen-Campard, Pascal; Octave, Jean-Noel

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 ofmore » 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.« less

Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3.

PubMed

Yasunaga, Mayu; Murotomi, Kazutoshi; Abe, Hiroko; Yamazaki, Tomomi; Nishii, Shigeaki; Ohbayashi, Tetsuya; Oshimura, Mitsuo; Noguchi, Takako; Niwa, Kazuki; Ohmiya, Yoshihiro; Nakajima, Yoshihiro

2015-01-20

Reporter assays that use luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of luciferase protein in the cells, which results in an increase of sensitivity of the luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST (t1/2=9.8 to 2.8h), the half-life of C9-fused ELuc was significantly shorter (t1/2=1.0h) than that of PEST-fused ELuc when measurements were conducted at 37°C. In addition, firefly luciferase (luc2) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence. Copyright © 2014 Elsevier B.V. All rights reserved.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR).

PubMed

Crosby, Priya; Hoyle, Nathaniel P; O'Neill, John S

2017-12-13

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Development of HSPA1A promoter-driven luciferase reporter gene assays in human cells for assessing the oxidative damage induced by silver nanoparticles.

PubMed

Xin, Lili; Wang, Jianshu; Zhang, Leshuai W; Che, Bizhong; Dong, Guangzhu; Fan, Guoqiang; Cheng, Kaiming

2016-08-01

The exponential increase in the total number of engineered nanoparticles in consumer products requires novel tools for rapid and cost-effective toxicology screening. In order to assess the oxidative damage induced by nanoparticles, toxicity test systems based on a human HSPA1A promoter-driven luciferase reporter in HepG2, LO2, A549, and HBE cells were established. After treated with heat shock and a group of silver nanoparticles (AgNPs) with different primary particle sizes, the cell viability, oxidative damage, and luciferase activity were determined. The time-dependent Ag(+) ions release from AgNPs in cell medium was also evaluated. Our results showed that heat shock produced a strong time-dependent induction of relative luciferase activity in the four luciferase reporter cells. Surprisingly, at 4h of recovery, the relative luciferase activity was >98× the control level in HepG2-luciferase cells. Exposure to different sizes of AgNPs resulted in activation of the HSPA1A promoter in a dose-dependent manner, even at low cytotoxic or non-cytotoxic doses. The smaller (5nm) AgNPs were more potent in luciferase induction than the larger (50 and 75nm) AgNPs. These results were generally in accordance with the oxidative damage indicated by malondialdehyde concentration, reactive oxygen species induction and glutathione depletion, and Ag(+) ions release in cell medium. Compared with the other three luciferase reporter cells, the luciferase signal in HepG2-luciferase cells is obviously more sensitive and stable. We conclude that the luciferase reporter cells, especially the HepG2-luciferase cells, could provide a valuable tool for rapid screening of the oxidative damage induced by AgNPs. Copyright © 2016 Elsevier Inc. All rights reserved.

P53 Mutation Analysis to Predict Tumor Response in Patients Undergoing Neoadjuvant Treatment for Locally Advanced Breast Cancer

DTIC Science & Technology

2006-10-01

then sequenced (for GeneChip- positiv SSCP (for GeneChip-negative). We have received a total of 43 core breast biopsy DNA samples from the UNC... quantitative luciferase reporter. Both reporters exploit a “rheostatable” promoter for p53 expression and utilize the “delitto perfetto” in vivo... quantitative luciferase-based assay is also being used to characterize the altered function sistent an tion T mutants in greater detail. Preliminary

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

Quenching the firefly bioluminescence by various ions.

PubMed

Zhang, Huateng; Bai, Haixiu; Jiang, Tianyu; Ma, Zhao; Cheng, Yanna; Zhou, Yubin; Du, Lupei; Li, Minyong

2016-02-01

The luciferase reporter gene assay system is broadly applied in various biomedical aspects, including signaling pathway dissection, transcriptional activity analysis, and genetic toxicity testing. It significantly improves the experimental accuracy and reduces the experimental error by the addition of an internal control. In the current research, we discovered some specific ions that could selectively inhibit firefly luciferase while having a negligible effect on renilla luciferase in vitro in the dual-reporter gene assay. We showed that these ionic compounds had a high potential of being utilized as quench-and-activate reagents in the dual-reporter assay. Furthermore, results from kinetic studies on ion-mediated quenching effects indicated that different ions have distinct inhibition modes. Our study is anticipated to guide a more affordable design of quench-and-activate reagents in biomedicine and pharmaceutical analysis.

The development of GADD45α luciferase reporter assays in human cells for assessing the genotoxicity of environmental pollutants.

PubMed

Xin, Lili; Wang, Jianshu; Wu, Yanhu; Guo, Sifan

2015-02-01

In order to assess the potential carcinogenic and genotoxic responses induced by environmental pollutants, genotoxicity test systems based on a GADD45α promoter-driven luciferase reporter in human A549 and HepG2 cells were established. Four different types of environmental toxicants including DNA alkylating agents, precarcinogenic agents, DNA cross-linking agents and non-carcinogenic agents, and three environmental samples collected from a coke oven plant were used to evaluate the test systems. After treated with the tested agents and environmental samples for 12 h, the cell viabilities and luciferase activities of the luciferase reporter cells were determined, respectively. Methyl methanesulfonate, benzo[a]pyrene, formaldehyde and the extractable organic matter (EOM) from coke oven emissions in ambient air generally produced significant induction of relative luciferase activity in a similar dose-dependent manner in A549- and HepG2-luciferase cells. No significant increases in relative luciferase activity were observed in pyrene-treated A549- or HepG2-luciferase cells. Significant increase in relative luciferase activity was already evident after 2.5 µM benzo[a]pyrene, 5 µM formaldehyde, 0.006 µg/L bottom-EOM, 0.10 µg/L side-EOM or 0.06 µg/L top-EOM, where no cytotoxic damage was observed. Compared with the A549-luciferase cells, the tested pollutants produced higher induction of relative luciferase activity in HepG2-luciferase cells. Therefore, the new genotoxicity test systems can detect different types of genotoxic agents and low concentrations of environmental samples. The luciferase reporter cells, especially the HepG2-luciferase cells, could provide a valuable tool for rapid screening of the genotoxic damage of environmental pollutants and their complex mixtures.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Auld, Douglas S.; Lovell, Scott; Thorne, Natasha

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 tomore » 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.« less

miR-Sens--a retroviral dual-luciferase reporter to detect microRNA activity in primary cells.

PubMed

Beillard, Emmanuel; Ong, Siau Chi; Giannakakis, Antonis; Guccione, Ernesto; Vardy, Leah A; Voorhoeve, P Mathijs

2012-05-01

MicroRNA-mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3' UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3' UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3' UTR-mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs.

miR-Sens—a retroviral dual-luciferase reporter to detect microRNA activity in primary cells

PubMed Central

Beillard, Emmanuel; Ong, Siau Chi; Giannakakis, Antonis; Guccione, Ernesto; Vardy, Leah A.; Voorhoeve, P. Mathijs

2012-01-01

MicroRNA–mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3′ UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3′ UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3′ UTR–mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs. PMID:22417692

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 by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane. PMID:24708613

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

PubMed

Chou, Ting-Chun; Moyle, Richard L

2014-04-08

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. 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. 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 by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane.

Approaches for Investigating Translational Regulation Controlled by PARP1: Biotin-Based UV Cross-Linking and Luciferase Reporter Assay.

PubMed

Ji, Yingbiao

2017-01-01

The RNA-binding proteins (RBPs) play a pivotal role in controlling gene expression through posttranscriptional processes. As the trans-acting factors, RBPs interact with the cis-regulatory elements located within mRNAs to regulate mRNA translational efficiency. Adding a new-layer regulation, recent studies suggest that poly(ADP-ribosyl)ation of the RNA-binding proteins often inhibit the RNA-binding ability of RBPs, thus regulating RBP-dependent mRNA metabolism including translational control. Here, we describe a biotin-based UV cross-linking method to determine if excessive accumulation of pADPr in the cell disrupts the interaction between RBPs and their target mRNAs. In addition, we illustrate the protocol of using the luciferase reporter assay to determine the effect of poly(ADP-ribosyl)ation on mRNA translation.

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

PubMed

Herbst, Katie J; Allen, Michael D; Zhang, Jin

2009-05-21

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

A Luciferase Functional Quantitative Assay for Measuring NF-ĸB Promoter Transactivation Mediated by HTLV-1 and HTLV-2 Tax Proteins.

PubMed

Bergamo, Elisa; Diani, Erica; Bertazzoni, Umberto; Romanelli, Maria Grazia

2017-01-01

HTLV-1 and HTLV-2 viruses express Tax transactivator proteins required for viral genome transcription and capable of transforming cells in vivo and in vitro. Although Tax oncogenic potential needs to be further elucidated, it is well established that Tax proteins activate, among others, transcription factors of the NF-ĸB family, which are involved in immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis. Here, we describe a reporter gene assay applied for quantitative analysis of Tax-dependent NF-ĸB activation. The procedure is based on co-transfection of two individual vectors containing the cDNA for firefly and Renilla luciferase enzymes and vectors expressing Tax proteins. The luciferase expression is driven by cis-NF-ĸB promoter regulatory elements responsive to Tax transactivating factor. This assay is particularly useful to investigate Tax influence on NF-ĸB activation mediated by viral or host factors.

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. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Novel Cell-Based Assays for Detecting Low Levels of Active Ricin Following Decontamination

DTIC Science & Technology

2011-12-01

fluorescent protein, are powerful tools, which have been used for detection assays for ricin protein ( DeWet , 1987). Zhao et al. (2005) have reported a...toxic Type 2 Ribosome-Inactivating Proteins. FEBS Lett. 2004, 563, pp 219–222. DeWet , J.R. et al. Firefly Luciferase Gene: Structure and

An Optimized Transient Dual Luciferase Assay for Quantifying MicroRNA Directed Repression of Targeted Sequences

PubMed Central

Moyle, Richard L.; Carvalhais, Lilia C.; Pretorius, Lara-Simone; Nowak, Ekaterina; Subramaniam, Gayathery; Dalton-Morgan, Jessica; Schenk, Peer M.

2017-01-01

Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA-target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach. The presented method combines transient expression via agroinfiltration of Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA-target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. benthamiana leaf extracts and dilution factor to apply in order to avoid inhibition of firefly LUC activity. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined. The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miR529b, with as few as six technical replicates. The assay can be extended to assess other small RNA-target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence. PMID:28979287

Utility of an appropriate reporter assay: Heliotrine interferes with GAL4/upstream activation sequence-driven reporter gene systems.

PubMed

Luckert, Claudia; Hessel, Stefanie; Lampen, Alfonso; Braeuning, Albert

2015-10-15

Reporter gene assays are widely used for the assessment of transcription factor activation following xenobiotic exposure of cells. A critical issue with such assays is the possibility of interference of test compounds with the test system, for example, by direct inhibition of the reporter enzyme. Here we show that the pyrrolizidine alkaloid heliotrine interferes with reporter signals derived from GAL4-based nuclear receptor transactivation assays by a mechanism independent of luciferase enzyme inhibition. These data highlight the necessity to conduct proper control experiments in order to avoid perturbation of reporter assays by test chemicals. Copyright © 2015 Elsevier Inc. All rights reserved.

Detecting protein-protein interactions using Renilla luciferase fusion proteins.

PubMed

Burbelo, Peter D; Kisailus, Adam E; Peck, Jeremy W

2002-11-01

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

Development of HSPA1A promoter-driven luciferase reporter gene assays in human cells for assessing the oxidative damage induced by silver nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xin, Lili, E-mail: llxin@suda.edu.cn

The exponential increase in the total number of engineered nanoparticles in consumer products requires novel tools for rapid and cost-effective toxicology screening. In order to assess the oxidative damage induced by nanoparticles, toxicity test systems based on a human HSPA1A promoter-driven luciferase reporter in HepG2, LO2, A549, and HBE cells were established. After treated with heat shock and a group of silver nanoparticles (AgNPs) with different primary particle sizes, the cell viability, oxidative damage, and luciferase activity were determined. The time-dependent Ag{sup +} ions release from AgNPs in cell medium was also evaluated. Our results showed that heat shock producedmore » a strong time-dependent induction of relative luciferase activity in the four luciferase reporter cells. Surprisingly, at 4 h of recovery, the relative luciferase activity was > 98 × the control level in HepG2-luciferase cells. Exposure to different sizes of AgNPs resulted in activation of the HSPA1A promoter in a dose-dependent manner, even at low cytotoxic or non-cytotoxic doses. The smaller (5 nm) AgNPs were more potent in luciferase induction than the larger (50 and 75 nm) AgNPs. These results were generally in accordance with the oxidative damage indicated by malondialdehyde concentration, reactive oxygen species induction and glutathione depletion, and Ag{sup +} ions release in cell medium. Compared with the other three luciferase reporter cells, the luciferase signal in HepG2-luciferase cells is obviously more sensitive and stable. We conclude that the luciferase reporter cells, especially the HepG2-luciferase cells, could provide a valuable tool for rapid screening of the oxidative damage induced by AgNPs. - Highlights: • We established the stable HSPA1A promoter-driven luciferase reporter cells. • Silver nanoparticles induced dose-dependent increases in luciferase activity. • HSPA1A promoter activity is a sensitive and responsive indicator of oxidative stress. • HepG2-luciferase cells can be used to assess the toxicity of silver nanoparticles.« less

Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity

PubMed Central

Fan, Shaoan; Wu, Feng; Martiniuk, Frank; Hale, Martha L; Ellington, Andrew D; Tchou-Wong, Kam-Meng

2008-01-01

AIM: To investigate the therapeutic potential of an RNA ligand (aptamer) specific for the catalytic ricin A-chain (RTA), the protective effects of a 31-nucleotide RNA aptamer (31RA), which formed a high affinity complex with RTA, against ricin-induced toxicity in cell-based luciferase translation and cell cytotoxicity assays were evaluated. METHODS: To test the therapeutic potential of anti-RTA aptamers in Chinese hamster ovary (CHO) AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured using luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. RESULTS: Inhibition of protein synthesis by ricin in CHO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against deglycosylated RTA (dgA) neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CHO AA8 cells. CONCLUSION: We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell-based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication. PMID:19009652

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

PubMed Central

Frau, Aldo; Sgarbanti, Marco; Orsatti, Roberto

2018-01-01

The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z′- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance. PMID:29495311

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

PubMed

Fanunza, Elisa; Frau, Aldo; Sgarbanti, Marco; Orsatti, Roberto; Corona, Angela; Tramontano, Enzo

2018-02-24

The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z'- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

Monitoring autophagic flux using p62/SQSTM1 based luciferase reporters in glioma cells.

PubMed

Min, Zhang; Ting, Yao; Mingtao, Gong; Xiaofei, Tang; Dong, Yan; Chenguang, Zhang; Wei, Ding

2018-02-01

Autophagy is a highly dynamic process characterized with the term of autophagic flux. In the present study, we developed a quantifiable luciferase reporter system to measure the capacity as well as the dynamics of autophagic flux. Briefly, a luciferase variant of Luc2p was fused with p62/SQSTM1 or its UBA domain deletion mutant (p62ΔU) and transfected into cells. The expressed Luc2p-p62 fusion protein was primarily degraded via autophagy, while Luc2p-p62ΔU was employed as a normalization control due to its resistance to autophagic degradation. The luciferase activity of the lysates from two parallel populations of glioma cells expressing either Luc2p-p62 or Luc2p-p62ΔU was determined and the ratio of Luc2p-p62ΔU/Luc2p-p62 was used to assay the autophagic flux. By this approach, the induction of autophagy was manifested as an increased Luc2p-p62ΔU/Luc2p-p62 ratio, which could be neutralized by autophagy inhibitors or knockdown of ATG5. The performance of our autophagic flux detection system was comparable to a recently reported GFP-LC3-RFP-LC3ΔG probe. We tested the system in TMZ treated glioma cells, and found that coadministration of chloroquine to attenuate cellular autophagic flux significantly improved the TMZ efficacy by triggering more early apoptosis. Collectively, our luciferase-based autophagic flux assay may serve as a useful alternative yet sensitive method for autophagic flux detection in tumor cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Faster experimental validation of microRNA targets using cold fusion cloning and a dual firefly-Renilla luciferase reporter assay.

PubMed

Alvarez, M Lucrecia

2014-01-01

Different target prediction algorithms have been developed to provide a list of candidate target genes for a given animal microRNAs (miRNAs). However, these computational approaches provide both false-positive and false-negative predictions. Therefore, the target genes of a specific miRNA identified in silico should be experimentally validated. In this chapter, we describe a step-by-step protocol for the experimental validation of a direct miRNA target using a faster Dual Firefly-Renilla Luciferase Reporter Assay. We describe how to construct reporter plasmids using the simple, fast, and highly efficient cold fusion cloning technology, which does not require ligase, phosphatase, or restriction enzymes. In addition, we provide a protocol for co-transfection of reporter plasmids with either miRNA mimics or miRNA inhibitors in human embryonic kidney 293 (HEK293) cells, as well as a description on how to measure Firefly and Renilla luciferase activity using the Dual-Glo Luciferase Assay kit. As an example of the use of this technology, we will validate glucose-6-phosphate dehydrogenase (G6PD) as a direct target of miR-1207-5p.

Synthesis and biological evaluation of 2-benzylamino-4(5)-(6-methylpyridin-2-yl)-5(4)-([1,2,4]triazolo[1,5-a]-pyridin-6-yl)thiazoles as transforming growth factor-β type 1 receptor kinase inhibitors.

PubMed

Krishnaiah, Maddeboina; Jin, Cheng Hua; Sreenu, Domalapally; Subrahmanyam, Vura Bala; Rao, Kota Sudhakar; Son, Do-Hyun; Park, Hyun-Ju; Kim, Seung Won; Sheen, Yhun Yhong; Kim, Dae-Kee

2012-11-01

A series of 2-benzylamino-4(5)-(6-methylpyridin-2-yl)-5(4)-([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazoles 12a-ab, 13a, 13b, and 18a-d has been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The N-(3-fluorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12b) inhibited ALK5 phosphorylation with an IC(50) value of 7.01 nM and showed 61% inhibition at 30 nM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output.

PubMed

Loening, Andreas Markus; Fenn, Timothy David; Wu, Anna M; Gambhir, Sanjiv Sam

2006-09-01

Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational analysis were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output. These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.

The smallest natural high-active luciferase: cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa.

PubMed

Markova, Svetlana V; Larionova, Marina D; Burakova, Ludmila P; Vysotski, Eugene S

2015-01-30

Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 SS bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of ∼3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. Copyright © 2014 Elsevier Inc. All rights reserved.

Functional Geno,ic Analysis of Breast Cancer Cell Tumorigenicity Using a Noval Gene Silencing Resource

DTIC Science & Technology

2006-04-01

Fig. 2B). In addition, luciferase assay on cells co-transfected with constructs expressing firefly and renilla luciferase genes showed a significant...positive cells. (C) BT474 cells were co-transfected with pGL3 plasmid expressing firefly luciferase, pRL plasmid expressing renilla luciferase, and...genes Per1 (A) and Bmal1 (B). BT474 cells were transfected with Per1 (A) and Bmal1 (B) firefly luciferase reporters, pRL plasmid expressing renilla

Epirubicin, Identified Using a Novel Luciferase Reporter Assay for Foxp3 Inhibitors, Inhibits Regulatory T Cell Activity.

PubMed

Kashima, Hajime; Momose, Fumiyasu; Umehara, Hiroshi; Miyoshi, Nao; Ogo, Naohisa; Muraoka, Daisuke; Shiku, Hiroshi; Harada, Naozumi; Asai, Akira

2016-01-01

Forkhead box protein p3 (Foxp3) is crucial to the development and suppressor function of regulatory T cells (Tregs) that have a significant role in tumor-associated immune suppression. Development of small molecule inhibitors of Foxp3 function is therefore considered a promising strategy to enhance anti-tumor immunity. In this study, we developed a novel cell-based assay system in which the NF-κB luciferase reporter signal is suppressed by the co-expressed Foxp3 protein. Using this system, we screened our chemical library consisting of approximately 2,100 compounds and discovered that a cancer chemotherapeutic drug epirubicin restored the Foxp3-inhibited NF-κB activity in a concentration-dependent manner without influencing cell viability. Using immunoprecipitation assay in a Treg-like cell line Karpas-299, we found that epirubicin inhibited the interaction between Foxp3 and p65. In addition, epirubicin inhibited the suppressor function of murine Tregs and thereby improved effector T cell stimulation in vitro. Administration of low dose epirubicin into tumor-bearing mice modulated the function of immune cells at the tumor site and promoted their IFN-γ production without direct cytotoxicity. In summary, we identified the novel action of epirubicin as a Foxp3 inhibitor using a newly established luciferase-based cellular screen. Our work also demonstrated our screen system is useful in accelerating discovery of Foxp3 inhibitors.

Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging

PubMed Central

Hsieh, Ya-Ju; Ke, Chien-Chih; Yeh, Skye Hsin-Hsien; Lin, Chien-Feng; Chen, Fu-Du; Lin, Kang-Ping; Chen, Ran-Chou; Liu, Ren-Shyan

2014-01-01

Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications. PMID:24809057

Role of RGM coreceptors in bone morphogenetic protein signaling

PubMed Central

Halbrooks, Peter J; Ding, Ru; Wozney, John M; Bain, Gerard

2007-01-01

Background The repulsive guidance molecule (RGM) proteins, originally discovered for their roles in neuronal development, have been recently identified as co-receptors in the bone morphogenetic protein (BMP) signaling pathway. BMPs are members of the TGFβ superfamily of signaling cytokines, and serve to regulate many aspects of cellular growth and differentiation. Results Here, we investigate whether RGMa, RGMb, and RGMc play required roles in BMP and TGFβ signaling in the mouse myoblast C2C12 cell line. These cells are responsive to BMPs and are frequently used to study BMP/TGFβ signaling pathways. Using siRNA reagents to specifically knock down each RGM protein, we show that the RGM co-receptors are required for significant BMP signaling as reported by two cell-based BMP activity assays: endogenous alkaline phosphatase activity and a luciferase-based BMP reporter assay. Similar cell-based assays using a TGFβ-induced luciferase reporter show that the RGM co-receptors are not required for TGFβ signaling. The binding interaction of each RGM co-receptor to each of BMP2 and BMP12 is observed and quantified, and equilibrium dissociation constants in the low nanomolar range are reported. Conclusion Our results demonstrate that the RGMs play a significant role in BMP signaling and reveal that these molecules cannot functionally compensate for one another. PMID:17615080

Development of a cell-based high throughput luciferase enzyme fragment complementation assay to identify nuclear-factor-e2-related transcription factor 2 activators.

PubMed

Xie, Wensheng; Pao, Christina; Graham, Taylor; Dul, Ed; Lu, Quinn; Sweitzer, Thomas D; Ames, Robert S; Li, Hu

2012-12-01

Nuclear-factor-E2-related transcription factor 2 (Nrf2) regulates a large panel of Phase II genes and plays an important role in cell survival. Nrf2 activation has been shown as preventing cigarette smoke-induced alveolar enlargement in mice. Therefore, activation of the Nrf2 protein by small-molecule activators represents an attractive therapeutic strategy that is used for chronic obstructive pulmonary disease. In this article, we describe a cell-based luciferase enzyme fragment complementation assay that identifies Nrf2 activators. This assay is based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2) and is dependent on the reconstitution of a "split" luciferase. Firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively. BacMam technology was used to deliver the fusion constructs into cells for expression of the tagged proteins. When the BacMam-transduced cells were treated with Nrf2 activators, the Nrf2 protein was stabilized and translocated into the nucleus where it interacted with MafK or RunX2. The interaction of Nrf2 and MafK or RunX2 brought together the two luciferase fragments that form an active luciferase. The assay was developed in a 384-well format and was optimized by titrating the BacMam concentration, transduction time, cell density, and fetal bovine serum concentration. It was further validated with known Nrf2 activators. Our data show that this assay is robust, sensitive, and amenable to high throughput screening of a large compound collection for the identification of novel Nrf2 activators.

A new indicator cell line established to monitor bovine foamy virus infection.

PubMed

Guo, Hong-Yan; Liang, Zhi-Bin; Li, Yue; Tan, Juan; Chen, Qi-Min; Qiao, Wen-Tao

2011-10-01

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.

Split luciferase complementation assay for the analysis of G protein-coupled receptor ligand response in Saccharomyces cerevisiae.

PubMed

Fukutani, Yosuke; Ishii, Jun; Kondo, Akihiko; Ozawa, Takeaki; Matsunami, Hiroaki; Yohda, Masafumi

2017-06-01

The budding yeast Saccharomyces cerevisiae is equipped with G protein-coupled receptors (GPCR). Because the yeast GPCR signaling mechanism is partly similar to that of the mammalian system, S. cerevisiae can be used for a host of mammalian GPCR expression and ligand-mediated activation assays. However, currently available yeast systems require several hours to observe the responses because they depend on the expression of reporter genes. In this study, we attempted to develop a simple GPCR assay system using split luciferase and β-arrestin, which are independent of the endogenous S. cerevisiae GPCR signaling pathways. We applied the split luciferase complementation assay method to S. cerevisiae and found that it can be used to analyze the ligand response of the human somatostatin receptor in S. cerevisiae. On the contrary, the response of the pheromone receptor Ste2 was not observed by the assay. Thus, the split luciferase complementation should be free from the effect of the endogenous GPCR signaling. Biotechnol. Bioeng. 2017;114: 1354-1361. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Regulation of AR and (beta)-Catenin Signaling by Pin 1 in Prostate Cancer

DTIC Science & Technology

2006-10-01

hrs. cells were harvested for Luciferase reporter Assay. C, CV-1 cells were transfected with Renilla (2.5ng), G5-Luc (10ng), pBIND-LBD (50ng), pACT-AR...stimulation, cells were cultured in 5 to 10% CDS-FBS. Firefly and internal control Renilla lucif- erase activities were determined using a dual luciferase...reporter assay kit (Pro- mega, Madison, WI), and Renilla activities were not consistently affected by any of the cotransfected vectors. The firefly

Renilla luciferase reporter assay to study 3'UTR-driven posttranscriptional regulations of OPRM1.

PubMed

Vincelli, Gabriele; Bedini, Andrea

2015-01-01

The regulation of MOR expression at the level of mRNA is relevant for its role in pain transmission and in other functions involving opioid receptors. Recently, the role of the 3'UTR in the posttranscriptional regulation of MOR expression has been highlighted. Here we describe a Renilla luciferase reporter assay for the study of the effect of any selective treatment on the 3'UTR-dependent regulation of OPRM1 in a model of glial cells.

NanoLuc reporter for dual luciferase imaging in living animals.

PubMed

Stacer, Amanda C; Nyati, Shyam; Moudgil, Pranav; Iyengar, Rahul; Luker, Kathryn E; Rehemtulla, Alnawaz; Luker, Gary D

2013-10-01

Bioluminescence imaging is widely used for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on the high signal to background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events, as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, adenosine triphosphate–independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc [NL]) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although the detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in transforming growth factor β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as a new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development.

NanoLuc Reporter for Dual Luciferase Imaging in Living Animals

PubMed Central

Stacer, Amanda C.; Nyati, Shyam; Moudgil, Pranav; Iyengar, Rahul; Luker, Kathryn E.; Rehemtulla, Alnawaz; Luker, Gary D.

2014-01-01

Bioluminescence imaging is utilized widely for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on high signal-to-background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, ATP-independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc, NL) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in TGF-β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development. PMID:24371848

Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites

PubMed Central

2015-01-01

New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z′ factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z′ factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing d-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level. PMID:25102353

Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors

PubMed Central

Sadikot, Takrima; Swink, Megan; Eskew, Jeffery D.; Brown, Douglas; Zhao, Huiping; Kusuma, Bhaskar R.; Rajewski, Roger A.; Blagg, Brian S. J.; Matts, Robert L.; Holzbeierlein, Jeffrey M.

2013-01-01

Abstract The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu. PMID:24127661

Evaluation of luciferase and GFP-expressing Nipah viruses for rapid quantitative antiviral screening.

PubMed

Lo, Michael K; Nichol, Stuart T; Spiropoulou, Christina F

2014-06-01

Nipah virus (NiV) outbreaks have occurred in Malaysia, India, and Bangladesh, and the virus continues to cause annual outbreaks of fatal human encephalitis in Bangladesh due to spillover from its bat host reservoir. Due to its high pathogenicity, its potential use for bio/agro-terrorism, and to the current lack of approved therapeutics, NiV is designated as an overlap select agent requiring biosafety level-4 containment. Although the development of therapeutic monoclonal antibodies and soluble protein subunit vaccines have shown great promise, the paucity of effective antiviral drugs against NiV merits further exploration of compound libraries using rapid quantitative antiviral assays. As a proof-of-concept study, we evaluated the use of fluorescent and luminescent reporter NiVs for antiviral screening. We constructed and rescued NiVs expressing either Renilla luciferase or green fluorescent protein, and characterized their reporter signal kinetics in different cell types as well as in the presence of several inhibitors. The 50% effective concentrations (EC50s) derived for inhibitors against both reporter viruses are within range of EC50s derived from virus yield-based dose-response assays against wild-type NiV (within 1Log10), thus demonstrating that both reporter NiVs can serve as robust antiviral screening tools. Utilizing these live NiV-based reporter assays requires modest instrumentation, and circumvents the time and labor-intensive steps associated with cytopathic effect or viral antigen-based assays. These reporter NiVs will not only facilitate antiviral screening, but also the study of host cell components that influence the virus life cycle. Published by Elsevier B.V.

A real-time bioluminescent HTS method for measuring protein kinase activity influenced neither by ATP concentration nor by luciferase inhibition.

PubMed

Lundin, Arne; Eriksson, Jonas

2008-08-01

The firefly luciferin-luciferase reaction has been used to set up an assay for protein kinase based on measuring ATP consumption rate as the first-order rate constant for the kinase reaction. The assay obviates the problems encountered with previous bioluminescent protein kinase assays such as interference with the luciferase reaction from library compounds, nonlinear standard curves, and limited dynamic ranges. In the assay described in the present paper luciferase and luciferin are present during the entire kinase reaction, and the light emission can be measured continuously. In an HTS situation the light emission is measured only twice, i.e., initially and after a predetermined time. After a fivefold reduction of the ATP concentration a Z' value of 0.96 was obtained. Light emission data from samples with kinase are normalized with light emission data from blanks without kinase. First-order rate constants for the kinase reaction calculated from normalized light emission are not affected by a moderate degree of inactivation of luciferase and luciferin during the measuring time. The constants have the same value at all ATP concentrations much lower than the K(m) of the luciferase and the kinase. These factors make the assay very robust and influenced neither by ATP concentration nor by luciferase inhibition. The measuring time depends on the kinase activity and can be varied from minutes to more than 8 h provided the kinase is stable and the evaporation of water from the wells is acceptable. The assay is linear with respect to kinase activity over three orders of magnitude. The new reagents also allowed us to determine K(m) values for ATP and for Kemptide.

Characterization of BRCA2 Transcriptional Regulation

DTIC Science & Technology

2001-12-01

tig of BRCA2 promoter construct and 0.1 and we verify the role of USF in regulation of basal activity of jig of pRL-TK Renilla luciferase vector...Promega) with 4 1 l of Fugene-6 the promoter. was used for each transfection. The pRL-TK Renilla luciferase activity was used to control for transfection...pCMV-CREB, pCMV-Myc, BRCA2 Reporter Constructs-A BAC clone (B489G) containing the 5’ and pCMV-Max. Firefly luciferase and Renilla luciferase assays

Molecular Cloning of Secreted Luciferases from Marine Planktonic Copepods.

PubMed

Takenaka, Yasuhiro; Ikeo, Kazuho; Shigeri, Yasushi

2016-01-01

Secreted luciferases isolated from copepod crustaceans are frequently used for nondisruptive reporter-gene assays, such as the continuous, automated and/or high-throughput monitoring of gene expression in living cells. All known copepod luciferases share highly conserved amino acid residues in two similar, repeated domains in the sequence. The similarity in the domains are ideal nature for designing PCR primers to amplify cDNA fragments of unidentified copepod luciferases from bioluminescent copepod crustaceans. Here, we introduce how to establish a cDNA encoding novel copepod luciferases from a copepod specimen by PCR with degenerated primers.

Sensitive dual color in vivo bioluminescence imaging using a new red codon optimized firefly luciferase and a green click beetle luciferase.

PubMed

Mezzanotte, Laura; Que, Ivo; Kaijzel, Eric; Branchini, Bruce; Roda, Aldo; Löwik, Clemens

2011-04-22

Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color-coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99. Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate. We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time.

Sensitive Dual Color In Vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase

PubMed Central

Mezzanotte, Laura; Que, Ivo; Kaijzel, Eric; Branchini, Bruce; Roda, Aldo; Löwik, Clemens

2011-01-01

Background Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color-coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99. Principal Findings Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate. Significance We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time. PMID:21544210

A simple heterogeneous one-step assay for screening estrogenic compounds.

PubMed

Huovinen, Tuomas; Rytkönen, Kalle; Lamminmäki, Urpo; Pellinen, Teijo

2013-01-01

Estrogen receptor (ER) modulators are a serious health issue but estrogenic compounds, especially antagonists of ER function, are widely screened for in search of novel therapeutics against hormonal diseases such as the breast cancer. Here we report a novel and a simple bioassay for estrogenic and anti-estrogenic compounds based on ligand-dependent recruitment of ER co-activator steroid receptor co-activator 1 (SRC-1) to purified Renilla luciferase-tagged ERα. In this assay, in vivo-biotinylated (E. coli) SRC-1, purified Renilla luciferase-ERα, and the analyte sample are mixed and incubated for 2 h in a streptavidin-coated microtiter wells, and after one washing step, luminescence is measured with a simple instrument. The assay does not require chemical labeling of the components and shows good sensitivity (25 pM E(2)) and wide dynamic range of more than four orders of magnitude.

Substrate Cooperativity in Marine Luciferases

PubMed Central

Tzertzinis, George; Schildkraut, Ezra; Schildkraut, Ira

2012-01-01

Marine luciferases are increasingly used as reporters to study gene regulation. These luciferases have utility in bioluminescent assay development, although little has been reported on their catalytic properties in response to substrate concentration. Here, we report that the two marine luciferases from the copepods, Gaussia princeps (GLuc) and Metridia longa (MLuc) were found, surprisingly, to produce light in a cooperative manner with respect to their luciferin substrate concentration; as the substrate concentration was decreased 10 fold the rate of light production decreased 1000 fold. This positive cooperative effect is likely a result of allostery between the two proposed catalytic domains found in Gaussia and Metridia. In contrast, the marine luciferases from Renilla reniformis (RLuc) and Cypridina noctiluca (CLuc) demonstrate a linear relationship between the concentration of their respective luciferin and the rate of light produced. The consequences of these enzyme responses are discussed. PMID:22768230

R/L, a double reporter mouse line that expresses luciferase gene upon Cre-mediated excision, followed by inactivation of mRFP expression.

PubMed

Jia, Junshuang; Lin, Xiaolin; Lin, Xia; Lin, Taoyan; Chen, Bangzhu; Hao, Weichao; Cheng, Yushuang; Liu, Yu; Dian, Meijuan; Yao, Kaitai; Xiao, Dong; Gu, Weiwang

2016-10-01

The Cre/loxP system has become an important tool for the conditional gene knockout and conditional gene expression in genetically engineered mice. The applications of this system depend on transgenic reporter mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. To develop a sensitive assay for monitoring Cre-mediated DNA excisions in mice, we generated Cre-mediated excision reporter mice, designated R/L mice (R/L: mRFP(monomeric red fluorescent protein)/luciferase), express mRFP throughout embryonic development and adult stages, while Cre-mediated excision deletes a loxP-flanked mRFP reporter gene and STOP sequence, thereby activating the expression of the second reporter gene luciferase, as assayed by in vivo and ex vivo bioluminescence imaging. After germ line deletion of the floxed mRFP and STOP sequence in R/L mice by EIIa-Cre mice, the resulting luciferase transgenic mice in which the loxP-mRFP-STOP-loxP cassette is excised from all cells express luciferase in all tissues and organs examined. The expression of luciferase transgene was activated in liver of RL/Alb-Cre double transgenic mice and in brain of RL/Nestin-Cre double transgenic mice when R/L reporter mice were mated with Alb-Cre mice and Nestin-Cre mice, respectively. Our findings reveal that the double reporter R/L mouse line is able to indicate the occurrence of Cre-mediated excision from early embryonic to adult lineages. Taken together, these findings demonstrate that the R/L mice serve as a sensitive reporter for Cre-mediated DNA excision both in living animals and in organs, tissues, and cells following necropsy.

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

DTIC Science & Technology

2013-11-01

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

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

DTIC Science & Technology

2011-09-01

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

High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.

PubMed

Ausseil, Frederic; Samson, Arnaud; Aussagues, Yannick; Vandenberghe, Isabelle; Creancier, Laurent; Pouny, Isabelle; Kruczynski, Anna; Massiot, Georges; Bailly, Christian

2007-02-01

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

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.

Analyzing the promoters of two CYP9A genes in the silkworm Bombyx mori by dual-luciferase reporter assay.

PubMed

Zhao, Si-Si; Zhao, Guo-Dong; Di, Tian-Yuan; Ding, Hua; Wan, Xiao-Ling; Li, Bing; Chen, Yu-Hua; Xu, Ya-Xiang; Shen, Wei-De; Wei, Zheng-Guo

2013-02-01

Cytochrome P450s (CYPs) are widespread proteins that interact with exogenous chemicals from the diet or the environment. CYP9A subfamily genes are important in the silkworm Bombyx mori. We previously reported transcriptional levels of two CYP9A genes in different tissues and their responses to sodium fluoride (NaF). In this study, promoter truncation analysis using a dual-luciferase reporter assay in B. mori ovary cells (BmN) showed that the regions -1,496 to -1,102 bp for CYP9A19, and -1,630 to -1,210 bp for CYP9A22 were essential for basal transcriptional activity. Sequence analysis of these regions revealed several transcriptional regulatory elements but no typical promoter elements. Promoter activities were regulated after NaF induction and with an obvious dose effect. Although the dual-luciferase assay has been widely used to determine the activity of a given promoter in cell lines, problems with it still exist. Our results indicate that both plasmid size and construct protocols affect the experimental results.

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

Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC.

PubMed

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

2010-12-05

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. Copyright © 2010 Elsevier Inc. All rights reserved.

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

PubMed

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(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. Copyright © 2012 Elsevier Inc. All rights reserved.

miRNA-1297 induces cell proliferation by targeting phosphatase and tensin homolog in testicular germ cell tumor cells.

PubMed

Yang, Nian-Qin; Zhang, Jian; Tang, Qun-Ye; Guo, Jian-Ming; Wang, Guo-Min

2014-01-01

To investigate the role of miR-1297 and the tumor suppressor gene PTEN in cell proliferation of testicular germ cell tumors (TGCT). MTT assays were used to test the effect of miR-1297 on proliferation of the NCCIT testicular germ cell tumor cell line. In NCCIT cells, the expression of PTEN was assessed by Western blotting further. In order to confirm target association between miR-1297 and 3'-UTR of PTEN, a luciferase reporter activity assay was employed. Moreover, roles of PTEN in proliferation of NCCIT cells were evaluated by transfection of PTEN siRNA. Proliferation of NCCIT cells was promoted by miR-1297 in a concentration-dependent manner. In addition, miR-1297 could bind to the 3'-UTR of PTEN based on luciferase reporter activity assay, and reduced expression of PTEN at protein level was found. Proliferation of NCCIT cells was significantly enhanced after knockdown of PTEN by siRNA. miR-1297 as a potential oncogene could induce cell proliferation by targeting PTEN in NCCIT cells.

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

4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5(3)-(6-methylpyridin-2-yl)imidazole and -pyrazole derivatives as potent and selective inhibitors of transforming growth factor-β type I receptor kinase.

PubMed

Jin, Cheng Hua; Krishnaiah, Maddeboina; Sreenu, Domalapally; Subrahmanyam, Vura Bala; Park, Hyun-Ju; Park, So-Jung; Sheen, Yhun Yhong; Kim, Dae-Kee

2014-05-01

A series of 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5(3)-(6-methylpyridin-2-yl)imidazoles and -pyrazoles 14a-c, 15a-c, 16a, 16b, 19a-d, 21a, and 21b has been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. Among them, the pyrazole derivative 21b inhibited ALK5 phosphorylation with an IC50 value of 0.018 μM and showed 95% inhibition at 0.03 μM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct. The 21b showed a high selectivity index of 284 against p38α MAP kinase. The binding pose of 21b generated by docking analysis reveals that it fits well into the ATP binding cavity of ALK5 by forming several hydrogen bond interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

DTIC Science & Technology

2012-09-01

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

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

PubMed Central

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

2008-01-01

Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence (λmax = 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, E. 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 posttranslational 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. PMID:18595733

Bacterial expression and re-engineering of Gaussia princeps luciferase and its use as a reporter protein.

PubMed

Wu, Nan; Rathnayaka, Tharangani; Kuroda, Yutaka

2015-10-01

Bioluminescence, the generation of visible light in a living organism, is widely observed in nature, and a large variety of bioluminescent proteins have been discovered and characterized. Luciferase is a generic term for bioluminescent enzymes that catalyze the emission of light through the oxidization of a luciferin (also a generic term). Luciferase are not necessarily evolutionary related and do not share sequence or structural similarities. Some luciferases, such as those from fireflies and Renilla, have been thoroughly characterized and are being used in a wide range of applications in bio-imaging. Gaussia luciferase (GLuc) from the marine copepod Gaussia princeps is the smallest known luciferase, and it is attracting much attention as a potential reporter protein. GLuc identification is relatively recent, and its structure and its biophysical properties remain to be fully characterized. Here, we review the bacterial production of natively folded GLuc with special emphasis on its disulfide bond formation and the re-engineering of its bioluminescence properties. We also compare the bioluminescent properties under a strictly controlled in vitro condition of selected GLuc's variants using extensively purified proteins with native disulfide bonds. Furthermore, we discuss and predict the domain structure and location of the catalytic core based on literature and on bioinformatics analysis. Finally, we review some examples of GLuc's emerging use in biomolecular imaging and biochemical assay systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Use of a special Brazilian red-light emitting railroad worm Luciferase in bioassays of NEK7 protein Kinase and Creatine Kinase.

PubMed

Marina Perez, Arina; Aquino, Bruno; Viviani, Vadim; Kobarg, Jörg

2017-07-19

Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP. Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP. In this work we used, after several optimization reactions, creatine kinase isoforms as well as NEK7 protein kinase in the absence or presence of ATP analogous inhibitors  to validate this new luminescence method. With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.

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

PubMed

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

2017-10-01

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

Restoration of Wild-Type Activity to Mutant p53 in Prostate Cancer: A Novel Therapeutic Approach

DTIC Science & Technology

2006-01-01

B) Cells were transfected with 1 mg of the indicated luciferase reporter constructs and 50 ng of pRL- Renilla . 24 hrs post transfections p53 was...induced by removal of tetracyline. Cells were lysed and assayed for luciferase and Renilla activities 24 hrs after induction of p53. The indicated

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.

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

PubMed

Bain, Peter A; Kumar, Anu

2018-05-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Manning, Gillian E., E-mail: gmann017@uottawa.ca; Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3; Farmahin, Reza, E-mail: mfarm070@uottawa.ca

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 usedmore » 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 relative potency values.« less

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

PubMed

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

2016-03-01

Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the "Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk" (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519 ng/mL for LUCIMU, 814 ng/mL for LUCRH, and 320 ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low positive titers needs to be further evaluated. Copyright © 2016 Elsevier B.V. All rights reserved.

Site-directed mutagenesis of firefly luciferase: implication of conserved residue(s) in bioluminescence emission spectra among firefly luciferases.

PubMed

Tafreshi, Narges Kh; Sadeghizadeh, Majid; Emamzadeh, Rahman; Ranjbar, Bijan; Naderi-Manesh, Hossein; Hosseinkhani, Saman

2008-05-15

The bioluminescence colours of firefly luciferases are determined by assay conditions and luciferase structure. Owing to red light having lower energy than green light and being less absorbed by biological tissues, red-emitting luciferases have been considered as useful reporters in imaging technology. A set of red-emitting mutants of Lampyris turkestanicus (Iranian firefly) luciferase has been made by site-directed mutagenesis. Among different beetle luciferases, those from Phrixothrix (railroad worm) emit either green or red bioluminescence colours naturally. By substitution of three specific amino acids using site-specific mutagenesis in a green-emitting luciferase (from L. turkestanicus), the colour of emitted light was changed to red concomitant with decreasing decay rate. Different specific mutations (H245N, S284T and H431Y) led to changes in the bioluminescence colour. Meanwhile, the luciferase reaction took place with relative retention of its basic kinetic properties such as K(m) and relative activity. Structural comparison of the native and mutant luciferases using intrinsic fluorescence, far-UV CD spectra and homology modelling revealed a significant conformational change in mutant forms. A change in the colour of emitted light indicates the critical role of these conserved residues in bioluminescence colour determination among firefly luciferases. Relatively high specific activity and emission of red light might make these mutants suitable as reporters for the study of gene expression and bioluminescence imaging.

Extracts of Fruits and Vegetables Activate the Antioxidant Response Element in IMR-32 Cells.

PubMed

Orena, Stephen; Owen, Jennifer; Jin, Fuxia; Fabian, Morgan; Gillitt, Nicholas D; Zeisel, Steven H

2015-09-01

The biological effects of antioxidant nutrients are mediated in part by activation of antioxidant response elements (AREs) on genes for enzymes involved in endogenous pathways that prevent free radical damage. Traditional approaches for identifying antioxidant molecules in foods, such as total phenolic compound (TP) content or oxygen radical absorption capacity (ORAC), do not measure capacity to activate AREs. The goal of this study was to develop an assay to assess the ARE activation capacity of fruit and vegetable extracts and determine whether such capacity was predicted by TP content and/or ORAC activity. Fruits and vegetables were homogenized, extracted with acidified ethanol, lyophilized, and resuspended in growth medium. Human IMR-32 neuroblastoma cells, transfected with an ARE-firefly luciferase reporter, were exposed to extracts for 5 h. Firefly luciferase was normalized to constitutively expressed Renilla luciferase with tertiary butylhydroquinone (tBHQ) as a positive control. TP content and ORAC activity were measured for each extract. Relations between TPs and ORAC and ARE activity were determined. A total of 107 of 134 extracts tested significantly activated the ARE-luciferase reporter from 1.2- to 58-fold above that of the solvent control (P < 0.05) in human IMR-32 cells. ARE activity, TP content, and ORAC ranked higher in peels than in associated flesh. Despite this relation, ARE activity did not correlate with TP content (Spearman ρ = 0.05, P = 0.57) and only modestly but negatively correlated with ORAC (Spearman ρ = -0.24, P < 0.01). Many extracts activated the ARE more than predicted by the TP content or ORAC. The ARE reporter assay identified many active fruit and vegetable extracts in human IMR-32 cells. There are components of fruits and vegetables that activate the ARE but are not phenolic compounds and are low in ORAC. The ARE-luciferase reporter assay is likely a better predictor of the antioxidant benefits of fruits and vegetables than TP or ORAC. © 2015 American Society for Nutrition.

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. Copyright © 2013 Elsevier Inc. All rights reserved.

Renilla Luciferase-LC3 Based Reporter Assay for Measuring Autophagic Flux.

PubMed

Farkas, T; Jäättelä, M

2017-01-01

Macroautophagy (autophagy) is a dynamic intracellular degradation pathway. Monitoring the flux through the autophagy pathway is experimentally challenging but obviously a prerequisite for the proper investigation of the process. Here, we present an indirect autophagy flux assay based on monitoring the degradation of an autophagosome-associated fusion protein Rluc-LC3 by luminescence detection. The method is suitable for screening purposes with a high number of parallel samples and can be used for measurements in cell lysates as well as in living cells. The Rluc-LC3 assay has proven useful for the identification of genes, miRNAs, and small molecules that regulate autophagy flux in mammalian cells. © 2017 Elsevier Inc. All rights reserved.

False responses of Renilla luciferase reporter control to nuclear receptor TR4.

PubMed

Zhang, Dongyun; Atlasi, Sam S; Patel, Krishna K; Zhuang, Zihao; Heaney, Anthony P

2017-06-01

Renilla luciferase reporter is a widely used internal control in dual luciferase reporter assay system, where its transcription is driven by a constitutively active promoter. However, the authenticity of the Renilla luciferase response in some experimental settings has recently been questioned. Testicular receptor 4 (TR4, also known as NR2C2) belongs to the subfamily 2 of nuclear receptors. TR4 binds to a direct repeat regulatory element in the promoter of a variety of target genes and plays a key role in tumorigenesis, lipoprotein regulation, and central nervous system development. In our experimental system using murine pituitary corticotroph tumor AtT20 cells to investigate TR4 actions on POMC transcription, we found that overexpression of TR4 resulted in reduced Renilla luciferase expression whereas knockdown TR4 increased Renilla luciferase expression. The TR4 inhibitory effect was mediated by the TR4 DNA-binding domain and behaved similarly to the GR and its agonist, Dexamethasone. We further demonstrated that the chimeric intron, commonly present in various Renilla plasmid backbones such as pRL-Null, pRL-SV40, and pRL-TK, was responsible for TR4's inhibitory effect. The results suggest that an intron-free Renilla luciferase reporter may provide a satisfactory internal control for TR4 at certain dose range. Our findings advocate caution on the use of Renilla luciferase as an internal control in TR4-directed studies to avoid misleading data interpretation.

Regulatory single nucleotide polymorphisms (rSNPs) at the promoters 1A and 1B of the human APC gene.

PubMed

Matveeva, Marina Yu; Kashina, Elena V; Reshetnikov, Vasily V; Bryzgalov, Leonid O; Antontseva, Elena V; Bondar, Natalia P; Merkulova, Tatiana I

2016-12-22

Germline mutations in the coding sequence of the tumour suppressor APC gene give rise to familial adenomatous polyposis (which leads to colorectal cancer) and are associated with many other oncopathologies. The loss of APC function because of deletion of putative promoter 1A or 1B also results in the development of colorectal cancer. Since the regions of promoters 1A and 1B contain many single nucleotide polymorphisms (SNPs), the aim of this study was to perform functional analysis of some of these SNPs by means of an electrophoretic mobility shift assay (EMSA) and a luciferase reporter assay. First, it was shown that both putative promoters of APC (1A and 1B) drive transcription in an in vitro reporter experiment. From eleven randomly selected SNPs of promoter 1A and four SNPs of promoter 1B, nine and two respectively showed differential patterns of binding of nuclear proteins to oligonucleotide probes corresponding to alternative alleles. The luciferase reporter assay showed that among the six SNPs tested, the rs75612255 C allele and rs113017087 C allele in promoter 1A as well as the rs138386816 T allele and rs115658307 T allele in promoter 1B significantly increased luciferase activity in the human erythromyeloblastoid leukaemia cell line K562. In human colorectal cancer HCT-116 cells, none of the substitutions under study had any effect, with the exception of minor allele G of rs79896135 in promoter 1B. This allele significantly decreased the luciferase reporter's activity CONCLUSION: Our results indicate that many SNPs in APC promoters 1A and 1B are functionally relevant and that allele G of rs79896135 may be associated with the predisposition to colorectal cancer.

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.

MicroRNA-145 Inhibits Cell Migration and Invasion and Regulates Epithelial-Mesenchymal Transition (EMT) by Targeting Connective Tissue Growth Factor (CTGF) in Esophageal Squamous Cell Carcinoma.

PubMed

Han, Qiang; Zhang, Hua-Yong; Zhong, Bei-Long; Wang, Xiao-Jing; Zhang, Bing; Chen, Hua

2016-10-23

BACKGROUND This study investigated the mechanism of miR-145 in targeting connective tissue growth factor (CTGF), which affects the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of ESCC cells. MATERIAL AND METHODS A total of 50 ESCC tissues and their corresponding normal adjacent esophageal tissue samples were collected. Then, miR-145 expression in both ESCC clinical specimens and cell lines was detected using quantitative real-time PCR. CTGF protein was detected using immunohistochemistry. Dual luciferase reporter gene assay was employed to assess the effect of miR-145 on the 3'UTR luciferase activity of CTGF. Eca109 cells were transfected with miR-145 mimics and CTGF siRNA, respectively, and changes in cellular proliferation, migration, and invasion were detected via MTT assay, wound-healing assay, and Transwell assay, respectively. Western blotting assay was used to detect the expression of marker genes related to EMT. RESULTS MiR-145 was significantly down-regulated in ESCC tissues and cell lines compared with normal tissues and cell lines (P<0.05). We found significantly more positively expressed CTGF protein in ESCC tissues was than in normal adjacent esophageal tissues (P<0.01). Dual luciferase reporter gene assay showed that miR-145 can specifically bind with the 3'UTR of CTGF and significantly inhibit the luciferase activity by 55% (P<0.01). Up-regulation of miR-145 or down-regulation of CTGF can suppress the proliferation, migration, invasion, and EMT process of ESCC cells. CONCLUSIONS MiR-145 was significantly down-regulated in ESCC tissues and cell lines, while the protein expression of CTGF exhibited the opposite trend. MiR-145 inhibited the proliferation, migration, invasiveness, and the EMT process of ESCC cells through targeted regulation of CTGF expression.

Discovery and structural optimization of 4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-ones as RORc inverse agonists.

PubMed

Wu, Xi-Shan; Wang, Rui; Xing, Yan-Li; Xue, Xiao-Qian; Zhang, Yan; Lu, Yong-Zhi; Song, Yu; Luo, Xiao-Yu; Wu, Chun; Zhou, Yu-Lai; Jiang, Jian-Qin; Xu, Yong

2016-11-01

Retinoic acid receptor-related orphan nuclear receptors (RORs) are orphan nuclear receptors that show constitutive activity in the absence of ligands. Among 3 subtypes of RORs, RORc is a promising therapeutic target for the treatment of Th17-mediated autoimmune diseases. Here, we report novel RORc inverse agonists discovered through structure-based drug design. Based on the structure of compound 8, a previously described agonist of RORa, a series of 4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives were designed and synthesized. The interaction between the compounds and RORc was detected at molecular level using AlphaScreen assay. The compounds were further examined in 293T cells transfected with RORc and luciferase reporter gene. Thermal stability shift assay was used to evaluate the effects of the compounds on protein stability. A total of 27 derivatives were designed and synthesized. Among them, the compound 22b was identified as the most potent RORc inverse agonist. Its IC 50 values were 2.39 μmol/L in AlphaScreen assay, and 0.82 μmol/L in inhibition of the cell-based luciferase reporter activity. Furthermore, the compound 22b displayed a 120-fold selectivity for RORc over other nuclear receptors. Moreover, a molecular docking study showed that the structure-activity relationship was consistent with the binding mode of compound 22b in RORc. 4-(4-(Benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives are promising candidates for the treatment of Th17-mediated autoimmune diseases, such as rheumatoid arthritis, psoriasis, and multiple sclerosis.

Utilization of RNA polymerase I promoter and terminator sequences to develop a DNA transfection system for the study of hepatitis C virus internal ribosomal entry site-dependent translation.

PubMed

Oem, Jae-Ku; Xiang, Zhonghua; Zhou, Yan; Babiuk, Lorne A; Liu, Qiang

2007-09-01

Hepatitis C virus (HCV) causes severe liver diseases in a large population worldwide. HCV protein translation is controlled by an internal ribosomal entry site (IRES) within the 5'-untranslated region (UTR). HCV IRES-dependent translation is critical for HCV-associated pathogenesis. To develop a plasmid DNA transfection system by using RNA polymerase I promoter and terminator sequences for studying HCV IRES-dependent translation. A gene cassette containing HCV 5'-UTR, Renilla luciferase reporter gene, and HCV 3'-UTR was inserted between RNA polymerase I promoter and terminator sequences. HCV IRES-directed translation was determined by luciferase assay after transfection. Transfection of the RNA polymerase I-HCV IRES plasmid into human hepatoma Huh-7 and HepG2 cells resulted in luciferase gene expression. Deletion of the IIIf domain in HCV IRES dramatically reduced luciferase activity. Our results indicated that the plasmid vector system-based on RNA polymerase I promoter and terminator sequences represents an effective approach for the study of HCV IRES-dependent translation.

A quantitative assay for mitochondrial fusion using Renilla luciferase complementation

PubMed Central

Huang, Huiyan; Choi, Seok-Yong; Frohman, Michael A.

2010-01-01

Mitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Large mitochondria produce energy more efficiently, whereas small mitochondria translocate better to subcellular sites where local production of ATP is acutely required. Mitochondrial fusion is currently assayed by fusing together cells expressing GFP or RFP in their mitochondria and then scoring the frequency of cells with yellow mitochondria (representing fused green and red mitochondria). However, this assay is labor-intensive and only semi-quantitative. We describe here a reporter system consisting of split fragments of Renilla luciferase and YFP fused to mitochondrial matrix-targeting sequences and to leucine zippers to trigger dimerization. The assay enables fusion to be quantitated both visually for individual cells and on a population level using chemiluminescence, laying the foundation for high throughput small molecule and RNAi screens for modulators of mitochondrial fusion. We use the assay to examine cytoskeletal roles in fusion progression. PMID:20488258

DEVELOPMENT AND CHARACTERIZATION OF A CELL LINE THAT STABLY EXPRESSES AN ESTROGEN-RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ESTROGEN RECEPTOR AGONIST AND ANTAGONISTS

EPA Science Inventory

Screening for endocrine disrupting chemicals (EDCs) that act as estrogens or antiestrogens relies on the use of in vitro binding and gene expression assays coupled with short-term diagnostic in vivo assays. Although binding assays are useful to identify chemicals that are competi...

Mechanical control of Renilla luciferase.

PubMed

Tseng, Chiao-Yu; Zocchi, Giovanni

2013-08-14

We report experiments where the activity of the enzyme luciferase from Renilla reniformis is controlled through a DNA spring attached to the enzyme. In the wake of previous work on kinases, these results establish that mechanical stress applied through the DNA springs is indeed a general method for the artificial control of enzymes, and for the quantitative study of mechano-chemical coupling in these molecules. We also show proof of concept of the luciferase construct as a sensitive molecular probe, detecting a specific DNA target sequence in an easy, one-step, homogeneous assay, as well as SNP detection without melting curve analysis.

FIREFLY LUCIFERASE ATP ASSAY DEVELOPMENT FOR MONITORING BACTERIAL CONCENTRATIONS IN WATER SUPPLIES

EPA Science Inventory

This research program was initiated to develop a rapid, automatable system for measuring total viable microorganisms in potable drinking water supplies using the firefly luciferase ATP assay. The assay was adapted to an automatable flow system that provided comparable sensitivity...

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

PubMed

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

2017-10-01

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

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics.

PubMed

Sun, Kaiwen; Zheng, Yuyu; Zhu, Ziqiang

2017-11-20

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

A functional haplotype of NFKB1 influence susceptibility to oral cancer: a population-based and in vitro study.

PubMed

Chen, Fa; Liu, Fengqiong; Yan, Lingjun; Lin, Lisong; Qiu, Yu; Wang, Jing; Wu, Junfeng; Bao, Xiaodan; Hu, Zhijian; Cai, Lin; He, Baochang

2018-05-01

Genetic variations of NF-κB and its inhibitor IκB genes and their biological mechanism in oral cancer were not well recognized. The purpose of this study was to evaluate the associations of polymorphisms in NFKB1 and NFKBIA with oral cancer susceptibility, and further explore their potential mechanism in vitro. First, the polymorphisms of NFKB1 and NFKBIA were genotyped through iPLEX Sequenom MassARRAY platform in a case-control study with 425 oral cancer patients and 485 healthy controls. Then, the function was explored by a luciferase reporter assay and an electrophoretic mobility shift assay (EMSA) in human tongue squamous cell carcinoma cell lines. The results indicated that NFKB1 rs28362491 Del/Del and rs72696119 G/G genotypes were associated with the risk of oral cancer, with a strong linkage disequilibrium (D' = 0.991, r 2  = 0.971). Moreover, DG haplotype of NFKB1 also showed a significant increased risk (OR = 1.25, 95% CI: 1.02-1.53, P = 0.030). Dual-luciferase reporter assays further revealed that the plasmids with DG or IG or DC haplotype transfected with Tca-8113 cells or CAL-27 cells had a lower luciferase expression than that with IC haplotype. EMSA demonstrated that 4-bp ATTG deletion in the promoter of NFKB1 abolished the binding site of transcription factor. Our preliminary findings suggest that the haplotype of rs28362491 and rs72696119 in NFKB1 could act as a novel genetic marker to predict oral cancer risk in the southeast of China, but much more extensive researches still need to be conducted. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

A novel cell-based assay for inhibitory anti-muscarinic type 3 receptor antibodies in primary Sjögren's syndrome.

PubMed

Bastian, Isabell; Gordon, Tom P; Jackson, Michael W

2015-12-01

Inhibitory autoantibodies acting at the muscarinic acetylcholine receptor type 3 (M3R) are postulated to mediate autonomic dysfunction, including decreased salivary and lacrimal gland output and extra-glandular manifestations, in patients with primary Sjögren's syndrome. However, the contention that anti-M3R antibodies are pathogenic in patients remains untested, due to a lack of assays both sophisticated enough to detect inhibitory anti-M3R antibodies yet suitable for screening large patient cohorts. In the current study, we have established a cell-based bioassay of M3R activity, based on dual transfection of the M3R and a luciferase reporter gene. The bioassay is capable of capturing real-time agonist-mediated signalling of the M3R, which is inhibited specifically by patient IgG that have previously been demonstrated to have anti-M3R activity. The assay can be run in multi-well culture plates, and analysed using simple luminescence readers. As such, the new bioassay incorporating M3R-mediated luciferase transduction is the first assay adaptable to common diagnostic platforms that is capable of determining the presence in patient serum of functionally active anti-M3R autoantibodies. The new bioassay should prove useful for large cohort screening studies aiming to correlate the presence in patients of inhibitory anti-M3R antibodies with symptoms of both glandular and extra-glandular autonomic dysfunction. Copyright © 2015 Elsevier B.V. All rights reserved.

A scalable assessment of Plasmodium falciparum transmission in the standard membrane-feeding assay, using transgenic parasites expressing green fluorescent protein-luciferase.

PubMed

Stone, Will J R; Churcher, Thomas S; Graumans, Wouter; van Gemert, Geert-Jan; Vos, Martijn W; Lanke, Kjerstin H W; van de Vegte-Bolmer, Marga G; Siebelink-Stoter, Rianne; Dechering, Koen J; Vaughan, Ashley M; Camargo, Nelly; Kappe, Stefan H I; Sauerwein, Robert W; Bousema, Teun

2014-11-01

The development of drugs and vaccines to reduce malaria transmission is an important part of eradication plans. The transmission-reducing activity (TRA) of these agents is currently determined in the standard membrane-feeding assay (SMFA), based on subjective microscopy-based readouts and with limitations in upscaling and throughput. Using a Plasmodium falciparum strain expressing the firefly luciferase protein, we present a luminescence-based approach to SMFA evaluation that eliminates the requirement for mosquito dissections in favor of a simple approach in which whole mosquitoes are homogenized and examined directly for luciferase activity. Analysis of 6860 Anopheles stephensi mosquitoes across 68 experimental feeds shows that the luminescence assay was as sensitive as microscopy for infection detection. The mean luminescence intensity of individual and pooled mosquitoes accurately quantifies mean oocyst intensity and generates comparable TRA estimates. The luminescence assay presented here could increase SMFA throughput so that 10-30 experimental feeds could be evaluated in a single 96-well plate. This new method of assessing Plasmodium infection and transmission intensity could expedite the screening of novel drug compounds, vaccine candidates, and sera from malaria-exposed individuals for TRA. Luminescence-based estimates of oocyst intensity in individual mosquitoes should be interpreted with caution. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Discovery and structural optimization of 4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-ones as RORc inverse agonists

PubMed Central

Wu, Xi-shan; Wang, Rui; Xing, Yan-li; Xue, Xiao-qian; Zhang, Yan; Lu, Yong-zhi; Song, Yu; Luo, Xiao-yu; Wu, Chun; Zhou, Yu-lai; Jiang, Jian-qin; Xu, Yong

2016-01-01

Aim: Retinoic acid receptor-related orphan nuclear receptors (RORs) are orphan nuclear receptors that show constitutive activity in the absence of ligands. Among 3 subtypes of RORs, RORc is a promising therapeutic target for the treatment of Th17-mediated autoimmune diseases. Here, we report novel RORc inverse agonists discovered through structure-based drug design. Methods: Based on the structure of compound 8, a previously described agonist of RORa, a series of 4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives were designed and synthesized. The interaction between the compounds and RORc was detected at molecular level using AlphaScreen assay. The compounds were further examined in 293T cells transfected with RORc and luciferase reporter gene. Thermal stability shift assay was used to evaluate the effects of the compounds on protein stability. Results: A total of 27 derivatives were designed and synthesized. Among them, the compound 22b was identified as the most potent RORc inverse agonist. Its IC50 values were 2.39 μmol/L in AlphaScreen assay, and 0.82 μmol/L in inhibition of the cell-based luciferase reporter activity. Furthermore, the compound 22b displayed a 120-fold selectivity for RORc over other nuclear receptors. Moreover, a molecular docking study showed that the structure-activity relationship was consistent with the binding mode of compound 22b in RORc. Conclusion: 4-(4-(Benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives are promising candidates for the treatment of Th17-mediated autoimmune diseases, such as rheumatoid arthritis, psoriasis, and multiple sclerosis. PMID:27374490

A quantitative assay for mitochondrial fusion using Renilla luciferase complementation.

PubMed

Huang, Huiyan; Choi, Seok-Yong; Frohman, Michael A

2010-08-01

Mitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Large mitochondria produce energy more efficiently, whereas small mitochondria translocate better to subcellular sites where local production of ATP is acutely required. Mitochondrial fusion is currently assayed by fusing together cells expressing GFP or RFP in their mitochondria and then scoring the frequency of cells with yellow mitochondria (representing fused green and red mitochondria). However, this assay is labor-intensive and only semi-quantitative. We describe here a reporter system consisting of split fragments of Renilla luciferase and YFP fused to mitochondrial matrix-targeting sequences and to leucine zippers to trigger dimerization. The assay enables fusion to be quantitated both visually for individual cells and on a population level using chemiluminescence, laying the foundation for high throughput small molecule and RNAi screens for modulators of mitochondrial fusion. We use the assay to examine cytoskeletal roles in fusion progression. (c) 2010 Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.

Resveratrol inhibits proteinase-activated receptor-2-induced release of soluble vascular endothelial growth factor receptor-1 from human endothelial cells

PubMed Central

Al-Ani, Bahjat

2013-01-01

We recently reported that (i) activation of the proinflammatory receptor, proteinase-activated receptor-2 (PAR-2) caused the release of an important biomarker in preeclampsia, soluble vascular endothelial growth factor receptor-1 (sVEGFR-1, also known as sFlt-1) from human umbilical vein endothelial cells (HUVECs), and (ii) that the anti-oxidant and anti-inflammatory agent, resveratrol, is capable of inhibiting the proinflammatory cytokine-induced sVEGFR-1 release from human placenta. Based on these findings and because PAR-2 is upregulated by proinflammatory cytokines, we sought to determine whether resveratrol can inhibit PAR-2-induced sVEGFR-1 release. PAR-2 expressing cells, HUVECs and human embryonic kidney cells (HEK-293) transfected with a human VEGFR-1 promoter-luciferase reporter construct were incubated with PAR-2-activating peptide and/or resveratrol. Cell supernatants were assayed for sVEGFR-1 by enzyme-linked immunosorbent assay (ELISA), and VEGFR-1 promoter-luciferase assay was performed on the harvested cell lysates. Preincubation of HEK-293 cells with resveratrol significantly inhibited PAR-2-induced VEGFR-1 promoter activity without affecting cell viability as assessed by MTT assay. The addition of resveratrol also blocked PAR-2-mediated sVEGFR-1 release from HUVECs. The present study demonstrates that resveratrol suppressed both VEGFR-1 promoter activity and sVEGFR-1 protein release induced by PAR-2 activation, which further endorses our recent findings of a potential therapeutic role for resveratrol in preeclampsia. PMID:26933402

A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang

2010-01-22

To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potentialmore » entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.« less

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Using a Split Luciferase Assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins

PubMed Central

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

2015-01-01

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. PMID:26022509

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

PubMed

Gagnon, David; Archambault, Jacques

2015-01-01

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

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.

Targeting androgen receptor and JunD interaction for prevention of prostate cancer progression.

PubMed

Mehraein-Ghomi, Farideh; Kegel, Stacy J; Church, Dawn R; Schmidt, Joseph S; Reuter, Quentin R; Saphner, Elizabeth L; Basu, Hirak S; Wilding, George

2014-05-01

Multiple studies show that reactive oxygen species (ROS) play a major role in prostate cancer (PCa) development and progression. Previously, we reported an induction of Spermidine/Spermine N(1) -Acetyl Transferase (SSAT) by androgen-activated androgen receptor (AR)-JunD protein complex that leads to over-production of ROS in PCa cells. In our current research, we identify small molecules that specifically block AR-JunD in this ROS-generating metabolic pathway. A high throughput assay based on Gaussia Luciferase reconstitution was used to identify inhibitors of the AR-JunD interaction. Selected hits were further screened using a fluorescence polarization competitor assay to eliminate those that bind to the AR Ligand Binding Domain (LBD), in order to identify molecules that specifically target events downstream to androgen activation of AR. Eleven molecules were selected for studies on their efficacy against ROS generation and growth of cultured human PCa cells by DCFH dye-oxidation assay and DNA fluorescence assay, respectively. In situ Proximity Ligation Assay (PLA), SSAT promoter-luciferase reporter assay, and western blotting of apoptosis and cell cycle markers were used to study mechanism of action of the lead compound. Selected lead compound GWARJD10 with EC(50) 10 μM against ROS production was shown to block AR-JunD interaction in situ as well as block androgen-induced SSAT gene expression at IC(50) 5 μM. This compound had no effect on apoptosis markers, but reduced cyclin D1 protein level. Inhibitor of AR-JunD interaction, GWARJD10 shows promise for prevention of progression of PCa at an early stage of the disease by blocking growth and ROS production. © 2014 Wiley Periodicals, Inc.

Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems.

PubMed

Díaz-Triviño, Sara; Long, Yuchen; Scheres, Ben; Blilou, Ikram

2017-01-01

In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6).Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR-SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.

ATPase inhibitor based luciferase assay for prolonged and enhanced ATP pool measurement as an efficient fish freshness indicator.

PubMed

Ranjan, Rajeev; Priyanka, B S; Thakur, M S

2014-07-01

The nucleotide degradation pathway in somatic cells leads to the accumulation of products such as hypoxanthine and inosine, which are commonly used as fish and meat freshness indicators. Assays based on these molecules cannot differentiate the postmortem time over a short period of time (5-10 h). Further, quantification of these degradation products is cumbersome, costly and time-consuming. For the proposed assay, optimal concentrations of 30 and 2 mM, respectively, for the ATPase inhibitors sodium orthovanadate and EDTA were found. Further, it was observed that a firefly luciferase based assay could enhance the sensitivity levels up to 165-fold at 30 °C. In addition, it was observed that the sensitivity for ATP assay was enhanced up to 60-fold even after 12 h. The limit of detection for the ATP assay was 1 pM, unlike other conventional methods, which are sensitive only up to micromolar levels. Moreover, as little as 0.044 g fish fillet was required for the assay, and no time-consuming sample preparation was necessary. Luminescence of prolonged duration was observed in harvested fish kept at -20 °C in comparison with fish kept at 4 and 30 °C, which reflects the shelf life of fish preserved at lower temperatures.

Antiandrogenic activities of diesel exhaust particle extracts in PC3/AR human prostate carcinoma cells.

PubMed

Kizu, Ryoichi; Okamura, Kazumasa; Toriba, Akira; Mizokami, Atsushi; Burnstein, Kerry L; Klinge, Carolyn M; Hayakawa, Kazuichi

2003-12-01

We collected diesel exhaust particles (DEPs) emitted from three diesel-engine vehicles--a car, a bus, and a truck--in daily use, and prepared DEP extracts (DEPEs), designated as EC, EB, or ET, respectively. The androgenic and antiandrogenic effects of the DEPE samples were examined by a luciferase reporter assay in human prostate carcinoma PC3/AR cells transiently transfected with a prostate specific antigen gene promoter-driven luciferase expression vector pGLPSA5.8. PC3/AR is a subline of human prostate carcinoma PC3 transformed to stably express wild-type human androgen receptor (AR). While DEPE samples did not exhibit any androgenic effect, they exerted antiandrogenic effect, inhibiting dihydrotestosterone (10 pM) -induced luciferase activity by 24 to 52% at an extract concentration of 10 microg/ml. The antiandrogenic effect was greater in the following order: ET > EB > EC. Co-treatment of PC3/AR cells with SKF-525A, a nonselective inhibitor of cytochrome P450 (CYP) enzymes, enhanced the antiandrogenic effect, indicating that the antiandrogenic effect is caused by intact species of DEPE constituents. The antiandrogenic effect of DEPE samples was reversed by alpha-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist. The antiandrogenic activity of a DEPE sample correlated with its AhR agonist activity assayed in PC3/AR cells transiently transfected with CYP1A1 gene promoter-driven luciferase expression vector pLUC1A1. Equimolar mixtures of ten polycyclic aromatic hydrocarbons (PAHs) having four or more rings, structures found in the DEPEs, showed significant antiandrogenic effects and AhR agonist activity at concentrations equivalent to those found in DEPE samples. Further, DEPE samples elicited only antiandrogenic effects in recombinant yeast cells, which express beta-galactosidase in response to androgen. A competitive AR binding assay showed that AR-binding constituents exist in DEPE samples, indicating that greater part of AR-binding constituents in DEPEs are AR antagonists. All these findings show that DEPE samples exhibit significant antiandrogenic effect in cell-based transcription assay and that this effect is due in part to the constituents with AhR agonist activity including PAHs and to the constituents with AR antagonist activity.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allegra, Danilo; Cooperation Unit 'Mechanisms of Leukemogenesis', B061, DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg; Mertens, Daniel, E-mail: daniel.mertens@uniklinik-ulm.de

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 microRNAmore » 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.« less

A Rapid and Quantitative Recombinase Activity Assay

USDA-ARS?s Scientific Manuscript database

We present here a comparison between the recombinase systems FLP-FRT and Cre-loxP. A transient excision based dual luciferase expression assay is used for its rapid and repeatable nature. The detection system was designed within an intron to remove the remaining recombinase recognition site and no...

Quantitative and Functional Requirements for Bioluminescent Cancer Models.

PubMed

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

2016-01-01

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

Bioluminescence resonance energy transfer (BRET) imaging of protein–protein interactions within deep tissues of living subjects

PubMed Central

Dragulescu-Andrasi, Anca; Chan, Carmel T.; Massoud, Tarik F.; Gambhir, Sanjiv S.

2011-01-01

Identifying protein–protein interactions (PPIs) is essential for understanding various disease mechanisms and developing new therapeutic approaches. Current methods for assaying cellular intermolecular interactions are mainly used for cells in culture and have limited use for the noninvasive assessment of small animal disease models. Here, we describe red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) that allow for assaying PPIs both in cell culture and deep tissues of small animals. These BRET systems consist of the recently developed Renilla reniformis luciferase (RLuc) variants RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. In addition to the native coelenterazine luciferase substrate, we used the synthetic derivative coelenterazine-v, which further red-shifts the emission maxima of Renilla luciferases by 35 nm. We show the use of these BRET systems for ratiometric imaging of both cells in culture and deep-tissue small animal tumor models and validate their applicability for studying PPIs in mice in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. These red light-emitting BRET systems have great potential for investigating PPIs in the context of drug screening and target validation applications. PMID:21730157

miR-424-5p promotes proliferation of gastric cancer by targeting Smad3 through TGF-β signaling pathway

PubMed Central

Wang, Linjun; Zhang, Lei; Xu, Zekuan

2016-01-01

MiRNAs have been reported to regulate gene expression and be associated with cancer progression. Recently, miR-424-5p was reported to play important role in a variety of tumors. However, the role and molecular mechanisms of miR-424-5p in GC (gastric cancer) remains largely unknown. In this study, we aimed to explore the role of miR-424-5p in GC. QRT-PCR was used to determine the expression levels of miR-424-5p and Smad3. CCK8 assay, plate clone assay and cell cycle assay were used to measure the effects of miR-424-5p on GC cell proliferation. Luciferase reporter assay and western blotting were used to prove that Smad3 was one of the direct targets of miR-424-5p. Tumorigenesis assay was used to investigate the role of miR-424-5p in tumor growth of GC cells in vivo. We found that miR-424-5p was up-regulated in GC tissues and cells. Over-expression of miR-424-5p could promote the proliferation of GC cells. In addition, luciferase reporter assay and western blotting assay revealed that Smad3 was a direct target of miR-424-5p. Over-expression of Smad3 could partially reverse the effects of miR-424-5p on GC cell proliferation. Our study further revealed that miR-424-5p could inhibit TGF-β signaling pathway by Smad3. PMID:27655675

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. © 2013.

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. Copyright © 2013 Elsevier B.V. All rights reserved.

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

LabKey Server NAb: A tool for analyzing, visualizing and sharing results from neutralizing antibody assays

PubMed Central

2011-01-01

Background Multiple types of assays allow sensitive detection of virus-specific neutralizing antibodies. For example, the extent of antibody neutralization of HIV-1, SIV and SHIV can be measured in the TZM-bl cell line through the degree of luciferase reporter gene expression after infection. In the past, neutralization curves and titers for this standard assay have been calculated using an Excel macro. Updating all instances of such a macro with new techniques can be unwieldy and introduce non-uniformity across multi-lab teams. Using Excel also poses challenges in centrally storing, sharing and associating raw data files and results. Results We present LabKey Server's NAb tool for organizing, analyzing and securely sharing data, files and results for neutralizing antibody (NAb) assays, including the luciferase-based TZM-bl NAb assay. The customizable tool supports high-throughput experiments and includes a graphical plate template designer, allowing researchers to quickly adapt calculations to new plate layouts. The tool calculates the percent neutralization for each serum dilution based on luminescence measurements, fits a range of neutralization curves to titration results and uses these curves to estimate the neutralizing antibody titers for benchmark dilutions. Results, curve visualizations and raw data files are stored in a database and shared through a secure, web-based interface. NAb results can be integrated with other data sources based on sample identifiers. It is simple to make results public after publication by updating folder security settings. Conclusions Standardized tools for analyzing, archiving and sharing assay results can improve the reproducibility, comparability and reliability of results obtained across many labs. LabKey Server and its NAb tool are freely available as open source software at http://www.labkey.com under the Apache 2.0 license. Many members of the HIV research community can also access the LabKey Server NAb tool without installing the software by using the Atlas Science Portal (https://atlas.scharp.org). Atlas is an installation of LabKey Server. PMID:21619655

Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging.

PubMed

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

2017-01-01

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

Quantitative and Dynamic Imaging of ATM Kinase Activity.

PubMed

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

2017-01-01

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

Direct Downregulation of B-Cell Translocation Gene 3 by microRNA-93 Is Required for Desensitizing Esophageal Cancer to Radiotherapy.

PubMed

Cui, Hujun; Zhang, Shengqiang; Zhou, Hongbo; Guo, Ling

2017-08-01

Esophageal squamous carcinoma (ESC) is one of the most fatal malignancies worldwide with increasing occurrences yet poor outcome. MicroRNAs were reported to play roles in ESC. We aimed to understand how miRNAs affect the radiotherapy resistance of ESC. MicroRNA assays, real-time PCR, and Western blot were performed for expression analysis of miR-93 and BTG3. Luciferase activity assay was conducted with mutated B-cell translocation gene 3 (BTG3) 3'-UTR sequence in the 3' end of luciferase sequence with miR-93 inhibitor. ESC cells were treated with irradiation (IR) and clonogenic assay was utilized to detect the cell viability. Human ESC xenograft mouse model was established and subjected to target IR treatment followed by tumor size analysis. MiR-93 was decreased and BTG3 was increased in ESC cells, with negative correlation of their expression in ESC tissues. MiR-93 directly targeted BTG3 3'-UTR by luciferase activity assay. Either miR-93 inhibition or BTG3 overexpression decreased radiation resistance. Furthermore, miR-93 inhibition suppressed radiation resistance through BTG3. Direct downregulation of BTG3 by miR-93 is able to render ESC resistant to radiotherapy, and both BTG3 and miR-93 may potentially serve as clinical markers for ESC and contribute to the treatment of ESC.

Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium tuberculosis Growth in Human Macrophages.

PubMed

Sorrentino, Flavia; Gonzalez del Rio, Ruben; Zheng, Xingji; Presa Matilla, Jesus; Torres Gomez, Pedro; Martinez Hoyos, Maria; Perez Herran, Maria Esther; Mendoza Losana, Alfonso; Av-Gay, Yossef

2016-01-01

Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Estrogenic effects of herbal medicines from Costa Rica used for the management of menopausal symptoms.

PubMed

Doyle, Brian J; Frasor, Jonna; Bellows, Lauren E; Locklear, Tracie D; Perez, Alice; Gomez-Laurito, Jorge; Mahady, Gail B

2009-01-01

Outcomes from the Women's Health Initiative have demonstrated adverse effects associated with hormone therapy and have prioritized the need to develop new alternative treatments for the management of menopause and osteoporosis. To this end, we have been investigating natural herbal medicines used by Costa Rican women to manage menopausal symptoms. Seventeen plant species were collected and extracted in Costa Rica. To establish possible mechanisms of action and to determine their potential future use for menopause or osteoporosis, we investigated the estrogenic activities of the herbal extracts in an estrogen-reporter gene estrogen receptor (ER) beta-Chemically Activated Luciferase Expression assay in U2-OS cells and in reporter and endogenous gene assays in MCF-7 cells. Six of the plant extracts bound to the ERs. Four of the six extracts stimulated reporter gene expression in the ER-beta-Chemically Activated Luciferase Expression assay. All six extracts modulated expression of endogenous genes in MCF-7 cells, with four extracts acting as estrogen agonists and two extracts, Pimenta dioica and Smilax domingensis, acting as partial agonist/antagonists by enhancing estradiol-stimulated pS2 mRNA expression but reducing estradiol-stimulated PR and PTGES mRNA expression. Both P. dioica and S. domingensis induced a 2ERE-luciferase reporter gene in transient transfected MCF-7 cells, which was inhibited by the ER antagonist ICI 182,780. This work presents a plausible mechanism of action for many of the herbal medicines used by Costa Rican women to treat menopausal symptoms. However, it further suggests that studies of safety and efficacy are needed before these herbs should be used as alternative therapies to hormone therapy.

Biological characterization of non-steroidal progestins from botanicals used for women’s health

PubMed Central

Toh, MF; Sohn, J; Chen, SN; Yao, P; Bolton, JL; Burdette, JE

2012-01-01

Progesterone plays a central role in women’s reproductive health. Synthetic progestins, such as medroxyprogesterone acetate (MPA) are often used in hormone replacement therapy (HRT), oral contraceptives, and for the treatment of endometriosis and infertility. Although MPA is clinically effective, it also promiscuously binds to androgen and glucocorticoid receptors (AR/GR) leading to many undesirable side effects including cardiovascular diseases and breast cancers. Therefore, identifying alternative progestins is clinically significant. The purpose of this study was to biologically characterize non-steroidal progestins from botanicals by investigating their interaction and activation of progesterone receptor (PR). Eight botanicals commonly used to alleviate menopausal symptoms were investigated to determine if they contain progestins using a progesterone responsive element (PRE) luciferase reporter assay and a PR polarization competitive binding assay. Red clover extract stimulated PRE-luciferase and bound to PR. A library of purified compounds previously isolated from red clover was screened using the luciferase reporter assay. Kaempferol identified in red clover and a structurally similar flavonoid, apigenin, bound to PR and induced progestegenic activity and P4 regulated genes in breast epithelial cells and human endometrial stromal cells (HESC). Kaempferol and apigenin demonstrated higher progestegenic potency in the HESC compared to breast epithelial cells. Furthermore, phytoprogestins were able to activate P4 signaling in breast epithelial cells without downregulating PR expression. These data suggest that botanical extracts used for women’s health may contain compounds capable of activating progesterone receptor signaling. PMID:22484153

Core - shell upconversion nanoparticle - semiconductor heterostructures for photodynamic therapy

NASA Astrophysics Data System (ADS)

Dou, Qing Qing; Rengaramchandran, Adith; Selvan, Subramanian Tamil; Paulmurugan, Ramasamy; Zhang, Yong

2015-02-01

Core-shell nanoparticles (CSNPs) with diverse chemical compositions have been attracting greater attention in recent years. However, it has been a challenge to develop CSNPs with different crystal structures due to the lattice mismatch of the nanocrystals. Here we report a rational design of core-shell heterostructure consisting of NaYF4:Yb,Tm upconversion nanoparticle (UCN) as the core and ZnO semiconductor as the shell for potential application in photodynamic therapy (PDT). The core-shell architecture (confirmed by TEM and STEM) enables for improving the loading efficiency of photosensitizer (ZnO) as the semiconductor is directly coated on the UCN core. Importantly, UCN acts as a transducer to sensitize ZnO and trigger the generation of cytotoxic reactive oxygen species (ROS) to induce cancer cell death. We also present a firefly luciferase (FLuc) reporter gene based molecular biosensor (ARE-FLuc) to measure the antioxidant signaling response activated in cells during the release of ROS in response to the exposure of CSNPs under 980 nm NIR light. The breast cancer cells (MDA-MB-231 and 4T1) exposed to CSNPs showed significant release of ROS as measured by aminophenyl fluorescein (APF) and ARE-FLuc luciferase assays, and ~45% cancer cell death as measured by MTT assay, when illuminated with 980 nm NIR light.

Bacteriophage-based assays for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a meta-analysis.

PubMed

Pai, Madhukar; Kalantri, Shriprakash; Pascopella, Lisa; Riley, Lee W; Reingold, Arthur L

2005-10-01

To summarize, using meta-analysis, the accuracy of bacteriophage-based assays for the detection of rifampicin resistance in Mycobacterium tuberculosis. By searching multiple databases and sources we identified a total of 21 studies eligible for meta-analysis. Of these, 14 studies used phage amplification assays (including eight studies on the commercial FASTPlaque-TB kits), and seven used luciferase reporter phage (LRP) assays. Sensitivity, specificity, and agreement between phage assay and reference standard (e.g. agar proportion method or BACTEC 460) results were the main outcomes of interest. When performed on culture isolates (N=19 studies), phage assays appear to have relatively high sensitivity and specificity. Eleven of 19 (58%) studies reported sensitivity and specificity estimates > or =95%, and 13 of 19 (68%) studies reported > or =95% agreement with reference standard results. Specificity estimates were slightly lower and more variable than sensitivity; 5 of 19 (26%) studies reported specificity <90%. Only two studies performed phage assays directly on sputum specimens; although one study reported sensitivity and specificity of 100 and 99%, respectively, another reported sensitivity of 86% and specificity of 73%. Current evidence is largely restricted to the use of phage assays for the detection of rifampicin resistance in culture isolates. When used on culture isolates, these assays appear to have high sensitivity, but variable and slightly lower specificity. In contrast, evidence is lacking on the accuracy of these assays when they are directly applied to sputum specimens. If phage-based assays can be directly used on clinical specimens and if they are shown to have high accuracy, they have the potential to improve the diagnosis of MDR-TB. However, before phage assays can be successfully used in routine practice, several concerns have to be addressed, including unexplained false positives in some studies, potential for contamination and indeterminate results.

A novel reporter gene assay for recombinant human erythropoietin (rHuEPO) pharmaceutical products.

PubMed

Yang, Yushuai; Zhou, Yong; Yu, Lei; Li, Xiang; Shi, Xinchang; Qin, Xi; Rao, Chunming; Wang, Junzhi

2014-11-01

Accurate determination of in vitro biological activity of therapeutic erythropoietin is essential in quality control of recombinant human erythropoietin (rHuEPO) pharmaceutical products. However, most of currently-used methods leave much to be desired so that a simpler, quicker and more accurate method is urgently needed. The bioassay described here utilizes a sub clone of UT-7/epo cell line stably transfected with luciferase gene under the control of sis inducible element and interferon γ-activated sequence element promoter. Active erythropoietin could induce the expression of luciferase by signaling through the erythropoietin receptor and the dose-response curve showed good linearity, yielding a coefficient of determination of 0.99 or higher. The optimized assay was simpler with the operation completed within 24h and more sensitive with EC50 being 0.077IU/mL. The accuracy estimates ranged from 81.7% to 102.4%, and both intra-assay and inter-assay precision was below 15.0%. The robustness of the assay was demonstrated by no effect of passage levels of the cells on the performance of the assay (p values: 0.772 for sample 1 and 0.943 for sample 2). Besides, Bland-Altman analysis showed a high consistency of the new assay with in vivo reticulocyte assay in results. These results suggested that the new reporter gene assay can be a viable supplement to the traditional reticulocyte assay and employed in potency determination of rHuEPO pharmaceutical products. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

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

PubMed

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

2017-12-01

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

A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

PubMed Central

Guo, Hou-Fu; Cho, Eun Jeong; Devkota, Ashwini K.; Chen, Yulong; Russell, William; Phillips, George N.; Yamauchi, Mitsuo; Dalby, Kevin; Kurie, Jonathan M.

2017-01-01

Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated previously using E coli– or insect-based expression systems was either insoluble or enzymatically unstable, and LH2 enzymatic activity assays have measured radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems. PMID:28216326

"In Vitro" Synthesis and Activity of Reporter Proteins in an "Escherichia coli" S30 Extract System: An Undergraduate Experiment

ERIC Educational Resources Information Center

Higgins, Pamela J.

2005-01-01

This undergraduate laboratory experiment integrates multiple techniques ("in vitro" synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (beta-galactosidase and luciferase) within an "Escherichia coli" S30 transcription/translation extract. Comparison of the data suggests…

Detection of potential (anti)progestagenic endocrine disruptors using a recombinant human progesterone receptor binding and transactivation assay.

PubMed

Viswanath, Gunda; Halder, Sujata; Divya, Gunda; Majumder, Chandrajeet B; Roy, Partha

2008-11-25

The present work describes the identification of (anti)progestin endocrine disrupting chemicals (EDC) using a two step screening system. In the first step a competitive binding assay was developed using recombinant human progesterone receptor (hPR). The tested chemicals were of various classes like insecticides, their metabolites, industrial chemicals and waste water treatment plant (WWTP) effluents. All the tested chemicals demonstrated a high affinity binding for hPR. The average IC50 values of the test chemicals were within the range of 1-25microM. In the second step of screening, a mammalian cell-based hPR transactivation assay was developed where HEK 293 cells were co-transfected with hPR and luciferase reporter gene under the control of progesterone-response element. Stimulation of the cells with progesterone resulted in about 25-fold up regulation of luciferase activity, with EC50 value of 4nM. Potent anti-progesterone, RU486, significantly inhibited progesterone-induced transactivation and non-progestagenic steroids failed to transactivate hPR till 1microM concentrations. The chemicals showing high binding affinities in competitive binding assays were then tested in transactivation assay and all of them were found to be anti-progestative except WWTP effluents. Transactivation assays using extracted water samples from five different WWTP effluents showed that it was rich in progestative compounds. The levels of induction caused by these effluents were in the range of 15-25% of induction by progesterone and they represented about 6ng/l equivalent progesterone activities. In conclusion, we demonstrated that this two step assay provides an efficient screening tool for the detection of (anti)progestative EDC in various samples.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Lei; Sasai, Ken; Akagi, Tsuyoshi

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 bymore » 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.« less

Antiosteoporotic compounds from seeds of Cuscuta chinensis.

PubMed

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

2011-05-17

The seeds of Cuscuta chinensis (Tu-Si-Zi, TSZ) have long been used for the treatment of osteoporosis in China and some Asian countries. The compounds in TSZ responsible for the antiosteoporotic activity are still poorly understood. The present study was designed to investigate the osteogenic compounds in TSZ, and to evaluate their antiosteoporotic effects in osteoblastic cells. Osteoblast-like UMR-106 cells were used for bioactivity-guided isolation of the active compounds. The activity of alkaline phosphatase (ALP) in UMR-106 cells was measured by p-nitrophenyl sodium phosphate assay. The proliferation of UMR-106 cells was assayed by Alamar-Blue method. Estrogenic activity of the extracts and isolated compounds was evaluated by activation of estrogen response element (ERE) luciferase reporter expression in HeLa cells co-transfected with human estrogen receptor subtypes (ERα or ERβ) expression vectors and 5×ERE luciferase reporter plasmid. Antiestrogenic activity of the extracts and isolated compounds were evaluated by activation of activator protein-1 (AP-1) luciferase reporter expression in HeLa cells co-transfected with human estrogen receptor subtypes (ERα or ERβ) expression vectors and 6×AP-1 luciferase reporter plasmid. ALP-guided fractionation led to the isolation of five known flavonoids, quercetin, kaempferol, isorhamnetin, hyperoside and astragalin from the crude ethanolic extract of TSZ. Further study showed that kaempferol and hyperoside significantly increased the ALP activity in UMR-106 cells. Astragalin promoted the proliferation of UMR-106 cells whereas other compounds had no such effect. The isolated compounds showed estrogenic activity but quercetin, kaempferol and isorhamnetin showed more potent ERβ agonist activity. However, compared with their ER agonist activity, only quercetin and kaempferol showed potent ER antagonist activity by activating ERα/β-mediated AP-1 reporter expression. Our findings validated the clinical use of TSZ in the treatment of osteoporosis, and demonstrated that kaempferol and hyperoside are the active compounds in TSZ for the osteogenic effect. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Comparing the effects of tetrabromobisphenol-A, bisphenol A, and their potential replacement alternatives, TBBPA-bis(2,3-dibromopropyl ether) and bisphenol S, on cell viability and messenger ribonucleic acid expression in chicken embryonic hepatocytes.

PubMed

Ma, Melissa; Crump, Doug; Farmahin, Reza; Kennedy, Sean W

2015-02-01

A market for alternative brominated flame retardants (BFRs) has emerged recently due to the phase out of persistent and inherently toxic BFRs. Several of these replacement compounds have been detected in environmental matrices, including wild birds. A chicken embryonic hepatocyte (CEH) assay was utilized to assess the effects of the BFR, tetrabromobisphenol-A (TBBPA), and its replacement alternative, tetrabromobisphenol A bis(2,3-dibromopropyl ether [TBBPA-DBPE]) on cell viability and messenger ribonucleic acid (mRNA) expression. Bisphenol A (BPA) and 1 of its replacement alternatives, bisphenol S (BPS), were also screened for effects. Both TBBPA and BPA decreased CEH viability with calculated median lethal concentration (LC50) values of 40.6 μM and 61.7 μM, respectively. However, the replacement alternatives, TBBPA-DBPE and BPS, did not affect cell viability (up to 300 μM). Effects on mRNA expression were determined using an Avian ToxChip polymerse chain reaction (PCR) array and a real-time (RT)-PCR assay for the estrogen-responsive genes, apolipoproteinII (ApoII) and vitellogenin (Vtg). A luciferase reporter gene assay was used to assess dioxin-like effects. Tetrabromobisphenol-A altered mRNA levels of 4 genes from multiple toxicity pathways and increased luciferase activity in the luciferase reporter gene assay, whereas its alternative, TBBPA-DBPE, only altered 1 gene on the array, Cyp1a4, and increased luciferase activity. At 300 μM, a concentration that decreased cell viability for TBBPA and BPA, the BPA replacement, BPS, altered the greatest number of transcripts, including both ApoII and Vtg. Bisphenol A exposure did not alter any genes on the array but did up-regulate Vtg at 10 μM. Characterization of the potential toxicological and molecular-level effects of these compounds will ideally be useful to chemical regulators tasked with assessing the risk of new and existing chemicals. © 2014 SETAC.

Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase.

PubMed

Zhou, Min; Kitagawa, Yoshinori; Yamaguchi, Mayu; Uchiyama, Chika; Itoh, Masae; Gotoh, Bin

2013-01-01

Human metapneumovirus (HMPV) is a common cause of respiratory diseases in persons of all ages. Because of its slow replication and weak cytopathic effect in cultured cells, conventional neutralization assays for HMPV require around one week for completion. The purpose of this study is to establish a rapid neutralization assay based on a recombinant virus expressing Renilla luciferase (Rluc). A recombinant HMPV expressing both Rluc and green fluorescent protein (GFP) was created by reverse genetics method. Two-fold serial dilutions of human 23 sera were made in a 96-well plate and incubated with 50 pfu/well of the recombinant virus at 4°C for 1 h. The mixtures were then transferred to LLC-MK2 cells in a 96-well plate, incubated for 2 h, and replaced with trypsin-free fresh media. After incubation at 32°C for 24 h, the cells were lysed and measured for Rluc activity. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity. The novel assay could be completed within 24 h and eliminated the requirement of trypsin supporting multistep replication in cultured cells, as well as laborious processes including the plaque assay with immunostaining. Neutralization titers correlated well with those determined by a GFP-based assay previously developed. The neutralization assay based on Rluc activity is the fastest and the most straightforward of all previous assays, and may be available for high throughput screening of neutralizing antibodies. Copyright © 2012 Elsevier B.V. All rights reserved.

Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition

PubMed Central

Hatzios, Stavroula K.; Ringgaard, Simon; Davis, Brigid M.; Waldor, Matthew K.

2012-01-01

The luciferase protein fragment complementation assay is a powerful tool for studying protein-protein interactions. Two inactive fragments of luciferase are genetically fused to interacting proteins, and when these two proteins interact, the luciferase fragments can reversibly associate and reconstitute enzyme activity. Though this technology has been used extensively in live eukaryotic cells, split luciferase complementation has not yet been applied to studies of dynamic protein-protein interactions in live bacteria. As proof of concept and to develop a new tool for studies of bacterial chemotaxis, fragments of Renilla luciferase (Rluc) were fused to the chemotaxis-associated response regulator CheY3 and its phosphatase CheZ in the enteric pathogen Vibrio cholerae. Luciferase activity was dependent on the presence of both CheY3 and CheZ fusion proteins, demonstrating the specificity of the assay. Furthermore, enzyme activity was markedly reduced in V. cholerae chemotaxis mutants, suggesting that this approach can measure defects in chemotactic signaling. However, attempts to measure changes in dynamic CheY3-CheZ interactions in response to various chemoeffectors were undermined by nonspecific inhibition of the full-length luciferase. These observations reveal an unexpected limitation of split Rluc complementation that may have implications for existing data and highlight the need for great caution when evaluating small molecule effects on dynamic protein-protein interactions using the split luciferase technology. PMID:22905225

Studies of dynamic protein-protein interactions in bacteria using Renilla luciferase complementation are undermined by nonspecific enzyme inhibition.

PubMed

Hatzios, Stavroula K; Ringgaard, Simon; Davis, Brigid M; Waldor, Matthew K

2012-01-01

The luciferase protein fragment complementation assay is a powerful tool for studying protein-protein interactions. Two inactive fragments of luciferase are genetically fused to interacting proteins, and when these two proteins interact, the luciferase fragments can reversibly associate and reconstitute enzyme activity. Though this technology has been used extensively in live eukaryotic cells, split luciferase complementation has not yet been applied to studies of dynamic protein-protein interactions in live bacteria. As proof of concept and to develop a new tool for studies of bacterial chemotaxis, fragments of Renilla luciferase (Rluc) were fused to the chemotaxis-associated response regulator CheY3 and its phosphatase CheZ in the enteric pathogen Vibrio cholerae. Luciferase activity was dependent on the presence of both CheY3 and CheZ fusion proteins, demonstrating the specificity of the assay. Furthermore, enzyme activity was markedly reduced in V. cholerae chemotaxis mutants, suggesting that this approach can measure defects in chemotactic signaling. However, attempts to measure changes in dynamic CheY3-CheZ interactions in response to various chemoeffectors were undermined by nonspecific inhibition of the full-length luciferase. These observations reveal an unexpected limitation of split Rluc complementation that may have implications for existing data and highlight the need for great caution when evaluating small molecule effects on dynamic protein-protein interactions using the split luciferase technology.

Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells.

PubMed

Wan, Qingwen; Okashah, Najeah; Inoue, Asuka; Nehmé, Rony; Carpenter, Byron; Tate, Christopher G; Lambert, Nevin A

2018-05-11

G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase-mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype-biased ligands. © 2018 Wan et al.

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

PubMed

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

2016-08-19

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

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.

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.

Basic and Applied Aspects of Color Tuning of Bioluminescence Systems

NASA Astrophysics Data System (ADS)

Ohmiya, Yoshihiro

2005-09-01

V. Viviani et al. [Biochemistry 38 (1999) 8271] were the first to succeed in cloning the red-emitting enzyme from the South American railroad worm, which is the only bioluminescent organism known to emit a red-colored light. The application of red bioluminescence has been our goal because the transmittance of longer-wavelength light is superior to that of the other colors for visualization of biological functions in living cells. Now, different color luciferases, which emit with wavelength maxima ranging from 400 to 630 nm, are available and are being used. For example, based on different color luciferases, Nakajima et al. developed a tricolor reporter in vitro assay system based on these different color luciferases in which the expression of three genes can be monitored simultaneously. On the other hand, bioluminescence resonance energy transfer (BRET) is a natural phenomenon caused by the intermolecular interaction between a bioluminescent protein and a fluorophore on a second protein, resulting in the light from the bioluminescence reaction having the spectrum of the fluorophore. Otsuji et al. [Anal. Biochem. 329 (2004) 230] showed that the change in the efficiency of energy transfer in intramolecular BRET can quantify cellular functions in living cells. In this review, I introduce the basic mechanisms of color tuning in bioluminescent systems and new applications based on color tuning in the life sciences.

FcUni-RLuc: an engineered Renilla luciferase with Fc binding ability and light emission activity.

PubMed

Farzannia, A; Roghanian, R; Zarkesh-Esfahani, S H; Nazari, M; Emamzadeh, R

2015-03-07

A novel and advanced Fc-binding probe – FcUni-RLuc namely – has been produced and functionally assayed for labelling IgGs. The Fc antibody binding sequence – HWRGWV – was fused to Renilla luciferase, and the purified probe was employed for bioluminescence enzyme-linked immunoabsorbance assay of Her2 positive cells.

Homogeneous bioluminescence competitive binding assay for folate based on a coupled glucose-6-phosphate dehydrogenase--bacterial luciferase enzyme system.

PubMed

Huang, W; Feltus, A; Witkowski, A; Daunert, S

1996-05-01

A homogeneous bioluminescence competitive binding assay for folate was developed by using a coupled enzyme system of glucose-6-phosphate dehydrogenase (G6PDH) and bacterial luciferase. A highly substituted G6PDH-folate conjugate was prepared by employing an N-hydroxysuccinimide/carbodiimide method. Folate binding protein inhibits the activity of the conjugate. In the presence of folate, there is a competition between folate and the G6PDH-folate conjugate for the binding site of the folate binding protein, and the activity of the conjugate is recovered. Thus, the concentration of folate can be related to the activity of the G6PDH-folate conjugate, which is directly related to the bioluminescence produced by the coupled enzyme reaction. Using this assay, dose-response curves with a detection limit of 2.5 x 10(-8) M folate were obtained, which is an improvement of an order of magnitude with respect to an assay that monitors G6PDH activity spectrophotometrically. The assay was validated using vitamin tablets and a cell culture medium.

Dual-Color Click Beetle Luciferase Heteroprotein Fragment Complementation Assays

PubMed Central

Villalobos, Victor; Naik, Snehal; Bruinsma, Monique; Dothager, Robin S.; Pan, Mei-Hsiu; Samrakandi, Mustapha; Moss, Britney; Elhammali, Adnan; Piwnica-Worms, David

2010-01-01

Summary Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically-relevant time scales. Herein we describe a novel set of reversible, multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discreet pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to β-TrCP, an E3-ligase common to the regulation of both β-catenin and IκBα, GSK3β was identified as a novel candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells. PMID:20851351

Functional artificial luciferases as an optical readout for bioassays.

PubMed

Kim, Sung Bae; Izumi, Hiroshi

2014-06-13

This study elucidates functional artificial luciferases (ALucs) wholly synthesized for bioassays and molecular imaging. The ALucs bearing epitopes were newly created by amending the sequences of our previously reported ALucs in light of a multi-sequence alignment and hydrophobicity search. The synthesized ALucs are survived in live cells and stable in culture media for 25 days after secretion. The epitopes in ALucs are exposed during the secretion process and indeed valid for column purification and immunological assays. The ALucs exerted a 9400-times stronger optical intensity with a coelenterazine derivative (CTZ i), when compared with Renilla reniformis luciferase 8.6-535. A supersecondary structure of ALuc30 was predicted with respect to the X-ray crystallographic information of the coelenterazine-binding protein (CBP). The structure revealed that ALuc30 has a room for accommodating the iodide of CTZ i. This study guides on how to create functional artificial luciferases and predicts the structural details with the current bioinformatics technologies. Copyright © 2014 Elsevier Inc. All rights reserved.

Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

PubMed Central

2014-01-01

Background The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. Results A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. Conclusions A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome. PMID:24987490

MiR-300 Serves as Potential Biomarker to Screen Knee Osteoarthritis Patients by Targeting TNFα.

PubMed

Zhou, Zihao; Tian, Fengde; An, Ning; Zhang, Yao; Wang, Changcheng; Guo, Lin

2018-04-01

This study mainly explored the serum level of miR-300 with the risk of knee OA, thereby evaluating their diagnostic ability for treatment of knee osteoarthritis (KOA) patients. In the current study, we evaluated the level of TNFα in KOA patients and HCs. The serum was used to quantify the level of TNF-α by way of a sandwich enzyme-linked immunosorbent assay. Dual luciferase reporter assay was carried out to identify the possible target gene of miR-300. In line with previous studies, our data showed that serum TNFα level was increased along with K/L grades and WOMAC scoring, suggesting TNFα induced inflammatory responses correlating with the severity of KOA. We also showed that serum miR-300 level was increased with the severity of KOA according to X ray examination and K/L grades. Furthermore, we showed that serum miR-300 level positively correlated with K/L grades, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain scoring and WOMAC function scoring. Bioinformatic predictions showed a conserved binding site of miR-300 in the 3'UTR of IκBα. We then carried out a dual luciferase reporter assay and found miR-300 significantly suppressed pmirGLO-IκBα-3'UTR luciferase activity. Serum miR-300 is increased with the severity of KOA according to X-ray examination and K/L grades, thereby reflecting the severity of KOA and the degree of cartilage damage. Therefore, it could be used as a potential biomarker to screen KOA patients from healthy controls.

In vivo testing of Renilla luciferase substrate analogs in an orthotopic murine model of human glioblastoma.

PubMed

Otto-Duessel, Maya; Khankaldyyan, Vazgen; Gonzalez-Gomez, Ignacio; Jensen, Michael C; Laug, Walter E; Rosol, Michael

2006-01-01

In vivo bioluminescent imaging using cells expressing Renilla luciferase is becoming increasingly common. Hindrances to the more widespread use of Renilla luciferase are the high autoluminescence of its natural substrate, coelenterazine, in plasma, the relatively high absorbance by tissue of the light emitted by the enzyme-substrate reaction; rapid clearance of the substrate; and significant cost. These factors, save for the cost, which has its own limiting effect on use, can combine to reduce the sensitivity of in vivo assays utilizing this reporter system, and methods of increasing light output or decreasing autoluminescence could be of great benefit. A number of analogs of coelenterazine are being investigated may accomplish one or both of these goals. In this study that we report on the testing of two new substrate analogs, EnduRen and ViViren, manufactured by Promega Corporation, in an orthotopic murine model of human glioblastoma expressing Renilla luciferase. We have tested these analogs in this cell line both in vitro and in vivo, and find that the substrate viviren results in significantly greater light output than the natural substrate or the other analog EnduRen. This new substrate could be valuable for studies where greater sensitivity is important.

Efficient production of glycosylated Cypridina luciferase using plant cells.

PubMed

Mitani, Yasuo; Oshima, Yoshimi; Mitsuda, Nobutaka; Tomioka, Azusa; Sukegawa, Masako; Fujita, Mika; Kaji, Hiroyuki; Ohmiya, Yoshihiro

2017-05-01

Cypridina noctiluca luciferase has been utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays. Some of these applications require a large amount of purified luciferase. However, conventional protein expression systems are not capable of producing sufficient quantities of protein with a high quality and purity without laborious and costly purification processes. To improve the productivity and expand the breadth of possibilities for Cypridina luciferase applications, we employed a variety of secretion expression systems, including yeast, mammalian cells, and silk worms. In this study, we established a simple production procedure using plant cell cultures. The plant cell culture BY-2 efficiently secreted luciferase, which was easily purified using a simple one-step ion-exchange chromatography method. The production yield was 20-30 mg of luciferase per liter of culture medium, and its Km for the luciferin (0.45 μM) was similar to that of the native protein. Additionally, we characterized its glycosylation pattern and confirmed that the two potential N-glycosylation sites were modified with plant-type oligosaccharide chains. Interestingly, the oligosaccharide chains could be trimmed without any detectable decrease in recombinant protein activity. Therefore, the results of our study indicate that this method offers a more cost-effective production method for Cypridina luciferase than conventional methods. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2017-01-01

Background A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. Methods The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3′end of the reporter gene and the VP2 start sequence to allow co-translational ‘cleavage’ of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones. Results Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing. Conclusion NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication. PMID:29379384

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP.

PubMed

Henderson, Mark J; Wires, Emily S; Trychta, Kathleen A; Yan, Xiaokang; Harvey, Brandon K

2015-09-06

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

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...

Simultaneous monitoring of independent gene expression patterns in two types of cocultured fibroblasts with different color-emitting luciferases

PubMed Central

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

2008-01-01

Background Luciferase assay systems enable the real-time monitoring of gene expression in living cells. We have developed a dual-color luciferase assay system in which the expression of multiple genes can be tracked simultaneously using green- and red-emitting beetle luciferases. We have applied the system to monitoring independent gene expressions in two types of cocultured fibroblasts in real time. Results Two Rat-1 cell lines were established that stably express either green- or red-emitting luciferases under the control of the mBmal1 promoter, a canonical clock gene. We cocultured these cell lines, and gene expression profiles in both were monitored simultaneously. The circadian rhythms of these cell lines are independent, oscillating following their intrinsic circadian phases, even when cocultured. Furthermore, the independent rhythms were synchronized by medium change as an external stimulus. Conclusion Using this system, we successfully monitored independent gene expression patterns in two lines of cocultured fibroblasts. PMID:18416852

Identification of two novel LRP5 mutations in families with familial exudative vitreoretinopathy

PubMed Central

Fei, Ping; Zhang, Qi; Huang, Luling; Xu, Yu; Zhu, Xiong; Tai, Zhengfu; Gong, Bo; Ma, Shi; Yao, Quanyao; Li, Jing; Zhao, Peiquan

2014-01-01

Purpose To investigate the clinical features and disease-causing mutations in two Chinese families with familial exudative vitreoretinopathy (FEVR). Methods Clinical data and genomic DNA were collected for patients with FEVR. The coding exons and adjacent intronic regions of FZD4, LRP5, TSPAN12, and NDP were amplified with PCR, and the resulting amplicons were analyzed with Sanger sequencing. Wild-type and mutant LRP5 proteins were assayed for the Norrin/β-catenin pathway by luciferase reporter assays. Results Two novel heterozygous mutations in the LRP5 gene were identified in two relatives—p.A422T and p.L540P. Typical FEVR fundus change and mild reduced bone mineral density (BMD) was found in the two patients and the affected parent. In the luciferase studies, both p.A422T and p.L540P mutants displayed a significant reduction of the luciferase activity in SuperTopFlash (STF) cells in response to Norrin (87% reduction for p.A422T and 97% reduction for p.L540P). Both patients had an additional LRP5 sequence change (p.Q816P in Patient 1 from the unaffected mother and p.T852M in Patient 2 verified as a new mutation). Luciferase assay showed no reduction for p.Q816P and 94.9% reduction for the new mutation p.T852M, suggesting that p.Q816P may be not pathogenic and p.T852M may be pathogenic. Conclusions Our findings demonstrated two new novel LRP5 mutations in Chinese patients with FEVR and mild reduced BMD. They emphasize the complexity of FEVR mutations and phenotypes. PMID:24715757

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.

Generation of a Gaussia luciferase-expressing endotheliotropic cytomegalovirus for screening approaches and mutant analyses.

PubMed

Falk, Jessica J; Laib Sampaio, Kerstin; Stegmann, Cora; Lieber, Diana; Kropff, Barbara; Mach, Michael; Sinzger, Christian

2016-09-01

For many questions in human cytomegalovirus (HCMV) research, assays are desired that allow robust and fast quantification of infection efficiencies under high-throughput conditions. The secreted Gaussia luciferase has been demonstrated as a suitable reporter in the context of a fibroblast-adapted HCMV strain, which however is greatly restricted in the number of cell types to which it can be applied. We inserted the Gaussia luciferase expression cassette into the BAC-cloned virus strain TB40-BAC4, which displays the natural broad cell tropism of HCMV and hence allows application to screening approaches in a variety of cell types including fibroblasts, epithelial, and endothelial cells. Here, we applied the reporter virus TB40-BAC4-IE-GLuc to identify mouse hybridoma clones that preferentially neutralize infection of endothelial cells. In addition, as the Gaussia luciferase is secreted into culture supernatants from infected cells it allows kinetic analyses in living cultures. This can speed up and facilitate phenotypic characterization of BAC-cloned mutants. For example, we analyzed a UL74 stop-mutant of TB40-BAC4-IE-GLuc immediately after reconstitution in transfected cultures and found the increase of luciferase delayed and reduced as compared to wild type. Phenotypic monitoring directly in transfected cultures can minimize the risk of compensating mutations that might occur with extended passaging. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of a Novel Type of HIV-1 Particle Assembly Inhibitor Using a Quantitative Luciferase-Vpr Packaging-Based Assay

PubMed Central

Errazuriz, Elisabeth; Coric, Pascale; Souquet, Florence; Turcaud, Serge; Boulanger, Pierre; Bouaziz, Serge; Hong, Saw See

2011-01-01

The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity. PMID:22073298

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction

PubMed Central

Parashurama, Natesh; Ahn, Byeong-Cheol; Ziv, Keren; Ito, Ken; Paulmurugan, Ramasamy; Willmann, Jürgen K.; Chung, Jaehoon; Ikeno, Fumiaki; Swanson, Julia C.; Merk, Denis R.; Lyons, Jennifer K.; Yerushalmi, David; Teramoto, Tomohiko; Kosuge, Hisanori; Dao, Catherine N.; Ray, Pritha; Patel, Manishkumar; Chang, Ya-fang; Mahmoudi, Morteza; Cohen, Jeff Eric; Goldstone, Andrew Brooks; Habte, Frezghi; Bhaumik, Srabani; Yaghoubi, Shahriar; Robbins, Robert C.; Dash, Rajesh; Yang, Phillip C.; Brinton, Todd J.; Yock, Paul G.; McConnell, Michael V.

2016-01-01

Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow–derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. © RSNA, 2016 Online supplemental material is available for this article. PMID:27308957

A variant in a cis-regulatory element enhances claudin-14 expression and is associated with pediatric-onset hypercalciuria and kidney stones.

PubMed

Ure, Megan E; Heydari, Emma; Pan, Wanling; Ramesh, Ajay; Rehman, Sabah; Morgan, Catherine; Pinsk, Maury; Erickson, Robin; Herrmann, Johannes M; Dimke, Henrik; Cordat, Emmanuelle; Lemaire, Mathieu; Walter, Michael; Alexander, R Todd

2017-06-01

The greatest risk factor for kidney stones is hypercalciuria, the etiology of which is largely unknown. A recent genome-wide association study (GWAS) linked hypercalciuria and kidney stones to a claudin-14 (CLDN14) risk haplotype. However, the underlying molecular mechanism was not delineated. Recently, renal CLDN14 expression was found to increase in response to increased plasma calcium, thereby inducing calciuria. We hypothesized therefore that some children with hypercalciuria and kidney stones harbor a CLDN14 variant that inappropriately increases gene expression. To test this hypothesis, we sequenced the CLDN14 risk haplotype in a cohort of children with idiopathic hypercalciuria and kidney stones. An intronic SNP was more frequent in affected children. Dual luciferase and cell-based assays demonstrated increased reporter or CLDN14 expression when this polymorphism was introduced. In silico studies predicted the SNP introduced a novel insulinoma-associated 1 (INSM1) transcription factor binding site. Consistent with this, repeating the dual luciferase assay in the presence of INSM1 further increased reporter expression. Our data suggest that children with the INSM1 binding site within the CLDN14 risk haplotype have a higher likelihood of hypercalciuria and kidney stones. Enhanced CLDN14 expression may play a role in the pathophysiology of their hypercalciuria. © 2017 Wiley Periodicals, Inc.

Analysis of JC virus DNA replication using a quantitative and high-throughput assay

PubMed Central

Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

2015-01-01

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. PMID:25155200

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

PubMed

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

2015-01-01

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

Enhancing the response of CALUX and CAFLUX cell bioassays for quantitative detection of dioxin-like compounds

PubMed Central

ZHAO, Bin; BASTON, David S.; KHAN, Elaine; SORRENTINO, Claudio; DENISON, Michael S.

2011-01-01

Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications. Over the past 10 years, reporter gene assays have been widely accepted and used for analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like compounds in various types of matrices, such as biological, environmental, food and feed samples, given that high-resolution instrumental analysis techniques are impractical for large-scale screening analysis. The most sensitive cell-based reporter gene bioassay systems developed are the mechanism-based CALUX (Chemically Activated Luciferase Expression) and CAFLUX (Chemically Activated Fluorescent Expression) bioassays, which utilize recombinant cell lines containing stably transfected dioxin (AhR)-responsive firefly luciferase or enhanced green fluorescent protein (EGFP) reporter genes, respectively. While the current CALUX and CAFLUX bioassays are very sensitive, increasing their lower limit of sensitivity, magnitude of response and dynamic range for chemical detection would significantly increase their utility, particularly for those samples that contain low levels of dioxin-like HAHs (i.e., serum). In this study, we report that the addition of modulators of cell signaling pathways or modification of cell culture conditions results in significant improvement in the magnitude and overall responsiveness of the existing CALUX and CAFLUX cell bioassays. PMID:21394221

Modeling Ebola Virus Genome Replication and Transcription with Minigenome Systems.

PubMed

Cressey, Tessa; Brauburger, Kristina; Mühlberger, Elke

2017-01-01

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

Rapid assessment of antibody-induced ricin neutralization by employing a novel functional cell-based assay.

PubMed

Gal, Yoav; Alcalay, Ron; Sabo, Tamar; Noy-Porat, Tal; Epstein, Eyal; Kronman, Chanoch; Mazor, Ohad

2015-09-01

Ricin is one of the most potent and lethal toxins known against which there is no available antidote. Currently, the most promising countermeasures against the toxin are based on neutralizing antibodies elicited by active vaccination or administered passively. A cell-based assay is widely applied for the primary screening and evaluation of anti-ricin antibodies, yet such assays are usually time-consuming (18-72 h). Here, we report of a novel assay to monitor ricin activity, based on HeLa cells that stably express the rapidly-degraded ubiquitin-luciferase (Ub-FL, half-life of 2 min). Ricin-induced arrest of protein synthesis could be quantified within 3 to 6h post intoxication (IC90 of 300 and 100 ng/ml, respectively). Furthermore, by stabilizing the intracellular levels of Ub-FL in the last hour of the assay, a 3-fold increase in the assay sensitivity was attained. We applied this assay to monitor the efficacy of a ricin holotoxin-based vaccine by measuring the formation of neutralizing antibodies throughout the immunization course. The potency of anti-ricin monoclonal antibodies (directed to either subunit of the toxin) could also be easily and accurately measured in this assay format. Owing to its simplicity, this assay may be implemented for high-throughput screening of ricin-neutralizing antibodies and for identification of small-molecule inhibitors of the toxin, as well as other ribosome-inactivating toxins. Copyright © 2015 Elsevier B.V. All rights reserved.

A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome.

PubMed

Omachi, Kohei; Kamura, Misato; Teramoto, Keisuke; Kojima, Haruka; Yokota, Tsubasa; Kaseda, Shota; Kuwazuru, Jun; Fukuda, Ryosuke; Koyama, Kosuke; Matsuyama, Shingo; Motomura, Keishi; Shuto, Tsuyoshi; Suico, Mary Ann; Kai, Hirofumi

2018-05-17

Alport syndrome is a hereditary glomerular disease caused by mutation in type IV collagen α3-α5 chains (α3-α5(IV)), which disrupts trimerization, leading to glomerular basement membrane degeneration. Correcting the trimerization of α3/α4/α5 chain is a feasible therapeutic approach, but is hindered by lack of information on the regulation of intracellular α(IV) chain and the absence of high-throughput screening (HTS) platforms to assess α345(IV) trimer formation. Here, we developed sets of split NanoLuc-fusion α345(IV) proteins to monitor α345(IV) trimerization of wild-type and clinically associated mutant α5(IV). The α345(IV) trimer assay, which satisfied the acceptance criteria for HTS, enabled the characterization of intracellular- and secretion-dependent defects of mutant α5(IV). Small interfering RNA-based and chemical screening targeting the ER identified several chemical chaperones that have potential to promote α345(IV) trimer formation. This split luciferase-based trimer formation assay is a functional HTS platform that realizes the feasibility of targeting α345(IV) trimers to treat Alport syndrome. Copyright © 2018 Elsevier Ltd. All rights reserved.

Multiplexing a high-throughput liability assay to leverage efficiencies.

PubMed

Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

2009-06-01

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

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.

Fluoro-luminometric real-time measurement of bacterial viability and killing.

PubMed

Lehtinen, Janne; Virta, Marko; Lilius, Esa Matti

2003-10-01

The viability and killing of Escherichia coli was measured on a real-time basis using a fluoro-luminometric device, which allows successive measurements of fluorescence and bioluminescence without user intervention. Bacteria were made fluorescent and bioluminescent by expression of gfp and insect luciferase (lucFF) genes. The green fluorescent protein (GFP) is a highly fluorescent, extremely stable protein, which accumulates in cells during growth, and therefore the measured fluorescence signal was proportional to the total number of cells. The luciferase reaction is dependent of ATP produced by living cells, so that the bioluminescence level was a direct measure of the viable cells. In contrast to the bacterial luciferase, the insect luciferase uses a water-soluble and nonvolatile substrate, which makes automated multi-well microplate assay possible. For the validation of the assay, the proportion of living and dead cell populations was experimentally modified by incubating E. coli cells in the presence of various ethanol concentrations. Bacterial viability and killing measured by a fluoro-luminometric assay correlated fairly well with the reference methods: conventional plate counting, optical density measurement and various flow cytometric analyses. The real-time assay described here allows following the changes in bacterial cultures and assessing the bactericidal and other effects of various chemical, immunological and physical agents simultaneously in large numbers of samples.

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo.

PubMed

Stefan, E; Aquin, S; Berger, N; Landry, C R; Nyfeler, B; Bouvier, M; Michnick, S W

2007-10-23

The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Galpha(s) protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive beta-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.

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.

miR-379 Inhibits Cell Proliferation, Invasion, and Migration of Vascular Smooth Muscle Cells by Targeting Insulin-Like Factor-1.

PubMed

Li, Kai; Wang, Yong; Zhang, Anji; Liu, Baixue; Jia, Li

2017-01-01

MicroRNAs are small non-coding RNAs that play important roles in vascular smooth muscle cell (VSMC) function. This study investigated the role of miR-379 on proliferation, invasion, and migration of VSMCs and explored underlying mechanisms thereof. MicroRNA, mRNA, and protein levels were determined by quantitative real-time PCR and western blot. The proliferative, invasive, and migratory abilities of VSMCs were measured by CCK-8, invasion, and wound healing assay, respectively. Luciferase reporter assay was used to confirm the target of miR-379. Platelet-derived growth factor-bb was found to promote cell proliferation and suppress miR-379 expression in VSMCs. Functional assays demonstrated that miR-379 inhibited cell proliferation, cell invasion, and migration. Flow cytometry results further showed that miR-379 induced apoptosis in VSMCs. TargetScan analysis and luciferase report assay confirmed that insulin-like growth factor-1 (IGF-1) 3'UTR is a direct target of miR-379, and mRNA and protein levels of miR-379 and IGF-1 were inversely correlated. Rescue experiments showed that enforced expression of IGF-1 sufficiently overcomes the inhibitory effect of miR-379 on cell proliferation, invasion, and migration in VSMCs. Our results suggest that miR-379 plays an important role in regulating VSMCs proliferation, invasion, and migration by targeting IGF-1.

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

PubMed

Chiu, Jen-Hwey; Chang, Chun-Ju; Wu, Jing-Chong; Liu, Hui-Ju; Wen, Che-Sheng; Hsu, Chung-Hua; Chen, Jiun-Liang; Tseng, Ling-Ming; Chen, Wei-Shone; Shyr, Yi-Ming

2014-01-01

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

Aerosol-delivered programmed cell death 4 enhanced apoptosis, controlled cell cycle and suppressed AP-1 activity in the lungs of AP-1 luciferase reporter mice.

PubMed

Hwang, S-K; Jin, H; Kwon, J T; Chang, S-H; Kim, T H; Cho, C-S; Lee, K H; Young, M R; Colburn, N H; Beck, G R; Yang, H-S; Cho, M-H

2007-09-01

The long-term survival of lung cancer patients treated with conventional therapies remains poor and therefore the need for novel approaches remains high. This has led to the re-emergence of aerosol delivery as a therapeutic intervention. In this study, glucosylated polyethylenimine (GPEI) was used as carrier to investigate programmed cell death 4 (PDCD4) and PDCD4 mutant (D418A), an eIF4A-binding mutant, on PDCD4-related signaling and activator protein-1 (AP-1) activity in the lungs of AP-1 luciferase reporter mice. After confirming the efficiency of GPEI as a carrier in lungs, the effects of aerosol-delivered PDCD4 were investigated in AP-1 luciferase reporter mice. Aerosol delivery of GPEI/PDCD4 through a nose-only inhalation facilitated the apoptosis of lungs whereas aerosol PDCD4 mutant did not. Also, such aerosol delivery regulated proteins relevant to cell-cycle control and suppressed AP-1 activity. Results obtained by western blot analysis, immunohistochemistry, luciferase assay and deoxynucleotidyl-transferase-mediated nick end labeling study suggest that combined actions such as facilitating apoptosis, controlling cell cycle and suppression of AP-1 activity by PDCD4 may provide useful tool for designing lung tumor prevention and treatment by which PDCD4 functions as a transformation suppressor in the future.

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

PubMed

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

2018-01-01

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

Extraction and quantification of adenosine triphosphate in mammalian tissues and cells.

PubMed

Chida, Junji; Kido, Hiroshi

2014-01-01

Adenosine 5'-triphosphate (ATP) is the "energy currency" of organisms and plays central roles in bioenergetics, whereby its level is used to evaluate cell viability, proliferation, death, and energy transmission. In this chapter, we describe an improved and efficient method for extraction of ATP from tissues and cells using phenol-based reagents. The chaotropic extraction reagents reported so far co-precipitate ATP with insoluble proteins during extraction and with salts during neutralization. In comparison, the phenol-based reagents extract ATP well without the risks of co-precipitation. The extracted ATP can be quantified by the luciferase assay or high-performance liquid chromatography.

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone.

PubMed

Smirnova, Daria V; Samsonova, Jeanne V; Ugarova, Natalia N

2016-01-01

The sensitive BRET system for the homogeneous immunoassay of a low-molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)-the "red" mutant with λmax.em = 590 nm (RedLuc) and the "green" mutant with λmax.em = 550 nm (GreenLuc)-were tested as the donors. The water-soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase-progesterone (Luc-Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF-Ab) were developed. Both conjugates retained their functional properties, had high antigen-antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL(-1) . © 2015 The American Society of Photobiology.

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

PubMed

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

2017-08-01

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. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2011-01-01

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

Quantitative bioluminescent detection of bacteria

NASA Technical Reports Server (NTRS)

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

1976-01-01

Phosphoflavins in sample are measured using photobacterial luciferase assay technique for flavin mononucleotide (FMN). Boiling perchloric acid is used to rupture cells to free bound flavin and to hydrolyze flavin adenine dinucleotide to FMN. Base-stabilized water solution of sodium borohydride is used as reactant.

A Luciferase-fragment Complementation Assay to Detect Lipid Droplet-associated Protein-Protein Interactions*

PubMed Central

Kolkhof, Petra; Werthebach, Michael; van de Venn, Anna; Poschmann, Gereon; Chen, Lili; Welte, Michael; Stühler, Kai; Beller, Mathias

2017-01-01

A critical challenge for all organisms is to carefully control the amount of lipids they store. An important node for this regulation is the protein coat present at the surface of lipid droplets (LDs), the intracellular organelles dedicated to lipid storage. Only limited aspects of this regulation are understood so far. For the probably best characterized case, the regulation of lipolysis in mammals, some of the major protein players have been identified, and it has been established that this process crucially depends on an orchestrated set of protein-protein interactions. Proteomic analysis has revealed that LDs are associated with dozens, if not hundreds, of different proteins, most of them poorly characterized, with even fewer data regarding which of them might physically interact. To comprehensively understand the mechanism of lipid storage regulation, it will likely be essential to define the interactome of LD-associated proteins. Previous studies of such interactions were hampered by technical limitations. Therefore, we have developed a split-luciferase based protein-protein interaction assay and test for interactions among 47 proteins from Drosophila and from mouse. We confirmed previously described interactions and identified many new ones. In 1561 complementation tests, we assayed for interactions among 487 protein pairs of which 92 (19%) resulted in a successful luciferase complementation. These results suggest that a prominent fraction of the LD-associated proteome participates in protein-protein interactions. In targeted experiments, we analyzed the two proteins Jabba and CG9186 in greater detail. Jabba mediates the sequestration of histones to LDs. We successfully applied our split luciferase complementation assay to learn more about this function as we were e.g. able to map the interaction between Jabba and histones. For CG9186, expression levels affect the positioning of LDs. Here, we reveal the ubiquitination of CG9186, and link this posttranslational modification to LD cluster induction. PMID:27956707

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. © 2013.

Dual-Color Luciferase Complementation for Chemokine Receptor Signaling.

PubMed

Luker, Kathryn E; Luker, Gary D

2016-01-01

Chemokine receptors may share common ligands, setting up potential competition for ligand binding, and association of activated receptors with downstream signaling molecules such as β-arrestin. Determining the "winner" of competition for shared effector molecules is essential for understanding integrated functions of chemokine receptor signaling in normal physiology, disease, and response to therapy. We describe a dual-color click beetle luciferase complementation assay for cell-based analysis of interactions of two different chemokine receptors, CXCR4 and ACKR3, with the intracellular scaffolding protein β-arrestin 2. This assay provides real-time quantification of receptor activation and signaling in response to chemokine CXCL12. More broadly, this general imaging strategy can be applied to quantify interactions of any set of two proteins that interact with a common binding partner. © 2016 Elsevier Inc. All rights reserved.

Characterization of cis-acting elements required for autorepression of the equine herpesvirus 1 IE gene

PubMed Central

Kim, Seongman; Dai, Gan; O’Callaghan, Dennis J.; Kim, Seong Kee

2012-01-01

The immediate-early protein (IEP), the major regulatory protein encoded by the IE gene of equine herpesvirus 1 (EHV-1), plays a crucial role as both transcription activator and repressor during a productive lytic infection. To investigate the mechanism by which the EHV-1 IEP inhibits its own promoter, IE promoter-luciferase reporter plasmids containing wild-type and mutant IEP-binding site (IEBS) were constructed and used for luciferase reporter assays. The IEP inhibited transcription from its own promoter in the presence of a consensus IEBS (5’-ATCGT-3’) located near the transcription initiation site but did not inhibit when the consensus sequence was deleted. To determine whether the distance between the TATA box and the IEBS affects transcriptional repression, the IEBS was displaced from the original site by the insertion of synthetic DNA sequences. Luciferase reporter assays revealed that the IEP is able to repress its own promoter when the IEBS is located within 26-bp from the TATA box. We also found that the proper orientation and position of the IEBS were required for the repression by the IEP. Interestingly, the level of repression was significantly reduced when a consensus TATA sequence was deleted from the promoter region, indicating that the IEP efficiently inhibits its own promoter in a TATA box-dependent manner. Taken together, these results suggest that the EHV-1 IEP delicately modulates autoregulation of its gene through the consensus IEBS that is near the transcription initiation site and the TATA box. PMID:22265772

Characterization of cis-acting elements required for autorepression of the equine herpesvirus 1 IE gene.

PubMed

Kim, Seongman; Dai, Gan; O'Callaghan, Dennis J; Kim, Seong Kee

2012-04-01

The immediate-early protein (IEP), the major regulatory protein encoded by the IE gene of equine herpesvirus 1 (EHV-1), plays a crucial role as both transcription activator and repressor during a productive lytic infection. To investigate the mechanism by which the EHV-1 IEP inhibits its own promoter, IE promoter-luciferase reporter plasmids containing wild-type and mutant IEP-binding site (IEBS) were constructed and used for luciferase reporter assays. The IEP inhibited transcription from its own promoter in the presence of a consensus IEBS (5'-ATCGT-3') located near the transcription initiation site but did not inhibit when the consensus sequence was deleted. To determine whether the distance between the TATA box and the IEBS affects transcriptional repression, the IEBS was displaced from the original site by the insertion of synthetic DNA sequences. Luciferase reporter assays revealed that the IEP is able to repress its own promoter when the IEBS is located within 26-bp from the TATA box. We also found that the proper orientation and position of the IEBS were required for the repression by the IEP. Interestingly, the level of repression was significantly reduced when a consensus TATA sequence was deleted from the promoter region, indicating that the IEP efficiently inhibits its own promoter in a TATA box-dependent manner. Taken together, these results suggest that the EHV-1 IEP delicately modulates autoregulation of its gene through the consensus IEBS that is near the transcription initiation site and the TATA box. Copyright © 2012. Published by Elsevier B.V.

Data on regulation of the gene for the adipocyte-enriched micropeptide Adig/Smaf1 by qPCR analysis and luciferase reporter assay.

PubMed

Ren, Gang; Cairl, Nicholas; Kim, Ji Young; Smas, Cynthia M

2016-12-01

This article describes qPCR analysis for the Adig/Smaf1 gene in multiple in vitro adipocyte differentiation models including white and brown adipogenesis, cell lines and primary cultures. The article also contains qPCR data for transcript levels of Adig/Smaf1 in a wide panel of murine tissues. Expression of Adig/Smaf1 transcript in white and brown adipose tissue in fasted and refed mice is reported and also data for Adig/Smaf1 transcript expression in genetically obese ob/ob mice. Data on the effects of siRNA-mediated knockdown of Srebp1c on Adig/Smaf1 transcript levels in 3T3-L1 adipocytes are shown. Luciferase reporter assays provide data for regulation of an ~ 2 kb fragment of the 5' flanking region of Adig/Smaf1 gene by PPARγ/RXRα. This data is related to a research article describing Adig/Smaf1 protein expression, "Expression, regulation and functional assessment of the 80 amino acid Small Adipocyte Factor 1 (Smaf1) protein in adipocytes" (G. Ren, P. Eskandari, S. Wang, C.M. Smas, 2016) [1].

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 Central

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. PMID:25326916

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

Profiling the immunotoxicity of chemicals based on in vitro evaluation by a combination of the Multi-ImmunoTox assay and the IL-8 Luc assay.

PubMed

Kimura, Yutaka; Fujimura, Chizu; Ito, Yumiko; Takahashi, Toshiya; Terui, Hitoshi; Aiba, Setsuya

2018-06-01

We established a luciferase reporter assay system, the Multi-ImmunoTox Assay (MITA), which can evaluate the effects of chemicals on the promoter activities of four cytokines: IL-2, IFN-γ, IL-1β, and IL-8. We previously reported that MITA correctly reflected the change in mRNA of human whole-blood cells treated with dexamethasone, cyclosporine, FK506, or several other immunosuppressive drugs. In this study, we combined MITA with the IL-8 Luc assay to detect skin sensitization chemicals (OECD 442E) (modified MITA: mMITA) and established a data set of 60 chemicals examined by mMITA. Using the mMITA results, chemicals can be classified based on the lowest observed effect level (LOEL) of chemicals in suppressing or augmenting the promoter activities of the four cytokines. Moreover, we demonstrated that K-means clustering and hierarchical clustering of the 60 chemicals based on the LOEL for their effects on IL-2 and IL-8 promoter activities and the judgment by the IL-8 Luc assay resulted in the same 6-cluster solution: cluster 1 with preferential suppression of IL-8, cluster 2 with suppression of IL-2 and a positive IL-8 Luc assay result, cluster 3 with suppression of both IL-2 and IL-8, cluster 4 with no effects on IL-2 or IL-8 and a negative IL-8 Luc assay result, cluster 5 with suppression of both IL-2 and IL-8 and a negative IL-8 Luc assay result, and cluster 6 with preferential suppression of IL-8. These data suggest that mMITA is a promising novel high-throughput approach for detecting unrecognized immunological effects of chemicals and for profiling their immunotoxic effects.

Long noncoding RNA ANCR suppresses bone formation of periodontal ligament stem cells via sponging miRNA-758.

PubMed

Peng, Wei; Deng, Wei; Zhang, Jing; Pei, Gengwang; Rong, Qiong; Zhu, Shuangxi

2018-06-22

Long noncoding RNAs (lncRNAs) were proposed to be important regulators influencing various differentiation processes. Yet, the molecular mechanisms of lncRNAs governing osteogenic differentiation of Periodontal Ligament Stem Cells (PDLSCs) remain unclear. Here, PDLSCs were isolated from normal periodontal ligament of human (PDL) whereas P-PDLSCs were isolated from periodontitis affected PDL. Quantitative real-time PCR (qRT-PCR) was performed to examine the relative expression level of lncRNA-ANCR and of Osterix (OSX), Alkaline Phosphatase (ALP) as well as Runt-related transcription factor 2 (RUNX2) in PDLSCs. Gain- and loss-of- function experiments was performed to study the role of lncRNA-ANCR. Alizarin Red staining was used to evaluate the function of lncRNA-ANCR and miRNA-758 on osteogenic differentiation. In addition, via dual luciferase reporter assay and RNA immunoprecipitation the microRNA sponge potential of lncRNA-ANCR was assessed. A luciferase reporter assay identified the correlation between miR-758 and Notch2. Our results showed that the expression of ALP, RUNX2 and OSX were increased whereas lncRNA-ANCR was decreased during the process of differentiation in PDLSCs. Overexpression of lncRNA-ANCR decreased the expression of ALP, RUNX2 and OSX as confirmed by Alizarin red staining. Overexpression of lncRNA-ANCR resulted in reduction of the miR-758 expression level. Furthermore, RNA immunoprecipitation proved that lncRNA-ANCR targets miR-758 directly. The results of dual luciferase reporter assay also demonstrated that miR-758 regulated Notch2 expression by targeting 3'-UTR of Notch2. In conclusion, the novel pathway lncRNA-ANCR/miR-758/Notch2 plays an important role in the process of regulating osteogenic differentiation of PDLSCs. Copyright © 2018 Elsevier Inc. All rights reserved.

Nicotine suppresses bone sialoprotein gene expression.

PubMed

Nakayama, Y; Mezawa, M; Araki, S; Sasaki, Y; Wang, S; Han, J; Li, X; Takai, H; Ogata, Y

2009-10-01

Tobacco smoking is a risk factor for periodontitis and osteoporosis. Nicotine is a major component of tobacco, and has been reported to inhibit proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. The purpose of this study was to determine the effects of nicotine on bone metabolism. We used rat osteobast-like UMR106 and ROS 17/2.8 cells and rat stromal bone marrow RBMC-D8 cells. To determine the molecular basis of the transcriptional regulation of the BSP gene by nicotine, we conducted Northern hybridization, transient transfection analyses with chimeric constructs of the BSP gene promoter linked to a luciferase reporter gene and gel mobility shift assays. Nicotine (250 microg/mL) decreased the BSP mRNA levels at 12 and 24 h in UMR106 and ROS 17/2.8 cells. From transient transfection assays using various sized BSP promoter-luciferase constructs, nicotine decreased the luciferase activities of the construct, including the promoter sequence nucleotides -116 to +60, in UMR106 and RBMC-D8 cells. Nicotine decreased the nuclear protein binding to the cAMP response element (CRE), fibroblast growth factor 2 response element (FRE) and homeodomain protein-binding site (HOX) at 12 and 24 h. This study indicates that nicotine suppresses BSP transcription mediated through CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene.

Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay: a "quantitative code".

PubMed

Vaghi, Valentina; Polacchini, Alessio; Baj, Gabriele; Pinheiro, Vera L M; Vicario, Annalisa; Tongiorgi, Enrico

2014-10-03

The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5'- and 3'UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5'- and 3'UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5'UTR or a 3'UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5'UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3'UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent "a quantitative code" for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression, purification and luminescence properties of coelenterazine-utilizing luciferases from Renilla, Oplophorus and Gaussia: comparison of substrate specificity for C2-modified coelenterazines.

PubMed

Inouye, Satoshi; Sahara-Miura, Yuiko; Sato, Jun-ichi; Iimori, Rie; Yoshida, Suguru; Hosoya, Takamitsu

2013-03-01

The cold-induced expression system in Escherichia coli is useful and we have applied this system to prepare the coelenterazine-utilizing luciferases including Renilla luciferase (RLase), a red-shifted variant of Renilla luciferase (RLase-547), the catalytic domain of Oplophorus luciferase (19kOLase) and Gaussia luciferase (GLase). The luminescence properties of the purified luciferases were characterized by using 10 kinds of C2-modified coelenterazine analogues as a substrate. The order of the maximal luminescence intensity for native coelenterazine was GLase (100%)>RLase (8.0%)>RLase-547 (0.73%)>19kOLase (0.09%) under our assay conditions. The substrate specificities of coelenterazine-utilizing luciferases for the C2-modified analogues showed significant differences, but the emission peaks catalyzed by coelenterazine-utilizing luciferases were not affected by the C2-substituted coelenterazine. These results suggest that the catalytic environment for the oxygenation process of coelenterazine and the excited species of coelenteramide might be different among coelenterazine-utilizing luciferases. Copyright © 2012 Elsevier Inc. All rights reserved.

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

PubMed

Deng, Zhongyuan; Zhang, Shen; Gu, Shaohua; Ni, Xinzhi; Zeng, Wenxian; Li, Xianchun

2018-01-17

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Manning, Gillian E., E-mail: gmann017@uottawa.ca; Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3; Mundy, Lukas J., E-mail: lukas.mundy@ec.gc.ca

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, inmore » 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 CYP1A4/5 mRNA expression.« less

Optimization and Validation of the TZM-bl Assay for Standardized Assessments of Neutralizing Antibodies Against HIV-1

PubMed Central

Sarzotti-Kelsoe, Marcella; Bailer, Robert T; Turk, Ellen; Lin, Chen-li; Bilska, Miroslawa; Greene, Kelli M.; Gao, Hongmei; Todd, Christopher A.; Ozaki, Daniel A.; Seaman, Michael S.; Mascola, John R.; Montefiori, David C.

2014-01-01

The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of preclinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format. PMID:24291345

Automatic instrument for chemical processing to detect microorganism in biological samples by measuring light reactions

NASA Technical Reports Server (NTRS)

Kelbaugh, B. N.; Picciolo, G. L.; Chappelle, E. W.; Colburn, M. E. (Inventor)

1973-01-01

An automated apparatus is reported for sequentially assaying urine samples for the presence of bacterial adenosine triphosphate (ATP) that comprises a rotary table which carries a plurality of sample containing vials and automatically dispenses fluid reagents into the vials preparatory to injecting a light producing luciferase-luciferin mixture into the samples. The device automatically measures the light produced in each urine sample by a bioluminescence reaction of the free bacterial adenosine triphosphate with the luciferase-luciferin mixture. The light measured is proportional to the concentration of bacterial adenosine triphosphate which, in turn, is proportional to the number of bacteria present in the respective urine sample.

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

PubMed

Brennan, Jennifer C; Tillitt, Donald E

2018-03-01

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

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

USGS Publications Warehouse

Brennan, Jennifer; Tillitt, Donald E.

2018-01-01

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

Bioactivity screening and mass spectrometric confirmation for the detection of PPARδ agonists that increase type 1 muscle fibres.

PubMed

Bovee, Toine F H; Blokland, Marco; Kersten, Sander; Hamers, Astrid R M; Heskamp, Henri H; Essers, Martien L; Nielen, Michel W F; van Ginkel, Leendert A

2014-01-01

Sensitive and robust bioassays able to detect nuclear receptor activation are very useful for veterinary and doping control, pharmaceutical industry and environmental scientists. Here, we used bioassays based on human leukemic monocyte lymphoma U937 and human liver hepatocellular carcinoma HepG2 cell lines to detect the ligand-induced activation of the peroxisome proliferator-activated receptor delta (PPARδ). Exposure of U937 cells to the PPARδ agonist GW501516 resulted in a marked increase in mRNA expression of the PPARδ target gene Angptl4 which was quantified by qRT-PCR analysis. Exposure of HepG2 cells transiently transfected with a PPARδ expression plasmid and a PPAR-response element-driven luciferase reporter plasmid to PPARδ agonists GW501516, GW610742 and L-165041 resulted in clear dose-response curves. Although the qRT-PCR resulted in higher fold inductions, the luciferase assay with transfected HepG2 cells is cheaper and quicker and about ten times more sensitive to GW501516 compared to analysis of Angptl4 mRNA expression in U937 cells by qRT-PCR. The HepG2-based luciferase assay was therefore used to screen GW501516-spiked supplements and feed and water samples. After liquid extraction and clean-up by solid phase extraction using a weak anion exchange column, extracts were screened in the HepG2 bioassay followed by confirmation with a newly developed UPLC-MS/MS method, using two transitions for each compound, i.e., for GW501516, 454.07>188.15 (collision energy (CE) 46 V) and 454.07>257.08 (CE 30 V); for GW610742, 472.07>206.2 (CE 48 V) and 472.07>275.08 (CE 30 V); and for L-165041, 401.2>193.15 (CE 26 V) and 401.2>343.2 (CE 20 V).

Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis.

PubMed

Lin, Shin-Jen; Ho, Hsin-Chiu; Lee, Yi-Fen; Liu, Ning-Chun; Liu, Su; Li, Gonghui; Shyr, Chih-Rong; Chang, Chawnshang

2012-06-07

Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

The hURAT1 rs559946 polymorphism and the incidence of gout in Han Chinese men.

PubMed

Li, C; Yu, Q; Han, L; Wang, C; Chu, N; Liu, S

2014-01-01

Our previous study identified rs559946, a human urate transporter 1 (hURAT1) single nucleotide polymorphism (SNP), as being significantly associated with risk of primary hyperuricaemia (HUA) in a Han Chinese population. In the current study we aimed to identify the genetic effects of rs559946 on gout susceptibility in Han Chinese men. A total of 335 patients with gout and 376 healthy controls were recruited for a case-control association study. To examine the functional effect of rs559946, we performed luciferase reporter assays and an electrophoretic mobility shift assay (EMSA). rs559946 was found to be significantly associated with gout susceptibility (p = 0.004), with T-allele carriers showing a decreased risk of gout [odds ratio (OR) 0.70, 95% confidence interval (CI) 0.55-0.89]. Multiple linear regression analysis identified a significant association between rs559946 genotypes and tophi. Luciferase reporter assays show increased transcriptional activity of the hURAT1 promoter with the C allele of rs559946. EMSA detected binding of nuclear proteins to both the T and C alleles, although increased binding was observed with the T allele. Cold competition assays suggest that rs559946 may bind within a glucocorticoid receptor (GR) binding motif. Our study suggests that the rs559946 polymorphism is associated with increased HUA risk and may also contribute to gout development in Han Chinese men. The T to C substitution within rs559946 increased the transcriptional activity, and potentially increases gout susceptibility.

Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments.

PubMed

Yanik, Mert; Ponnam, Surya Prakash Goud; Wimmer, Tobias; Trimborn, Lennart; Müller, Carina; Gambert, Isabel; Ginsberg, Johanna; Janise, Annabella; Domicke, Janina; Wende, Wolfgang; Lorenz, Birgit; Stieger, Knut

2018-06-01

Common genome-editing strategies are either based on non-homologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomology-mediated end joining [MMEJ]) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ after CRISPR/Cas9-mediated cleavage in cell culture. Following successful proof of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by microhomologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse retinitis pigmentosa GTPase regulator (RPGR)-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by mhs. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Monitoring Intracellular pH Change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells.

PubMed

Zhang, Yunfei; Robertson, J Brian; Xie, Qiguang; Johnson, Carl Hirschie

2016-01-01

"pHlash" is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characterization of pHlash, and also in vivo assays including in yeast cells and in HeLa cells using pHlash as a cytoplasmic pH indicator.

Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function.

PubMed

Alberti, Michael O; Jones, Jennifer J; Miglietta, Riccardo; Ding, Haitao; Bakshi, Rakesh K; Edmonds, Tara G; Kappes, John C; Ochsenbauer, Christina

2015-12-01

We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a "ribosome skipping" T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4(+) T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, "6ATRi"] demonstrated Nef expression and function similar to parental "nonreporter" virus. In a previously validated (nef-independent) T cell-based NAb neutralization assay, LucR.6ATRi reporter virus performed indistinguishably from LucR.T2A reporter virus. In summary, reporter viruses comprising the "6ATRi" element promise to augment HIV-1 vaccine and transmission research approaches requiring a sensitive reporter readout combined with wild-type Nef function.

Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function

PubMed Central

Alberti, Michael O.; Jones, Jennifer J.; Miglietta, Riccardo; Ding, Haitao; Bakshi, Rakesh K.; Edmonds, Tara G.; Kappes, John C.

2015-01-01

Abstract We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a “ribosome skipping” T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4+ T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, “6ATRi”] demonstrated Nef expression and function similar to parental “nonreporter” virus. In a previously validated (nef-independent) T cell-based NAb neutralization assay, LucR.6ATRi reporter virus performed indistinguishably from LucR.T2A reporter virus. In summary, reporter viruses comprising the “6ATRi” element promise to augment HIV-1 vaccine and transmission research approaches requiring a sensitive reporter readout combined with wild-type Nef function. PMID:26101895

NanoLuc: A Small Luciferase is Brightening up the Field of Bioluminescence

PubMed Central

Cai, Weibo

2016-01-01

The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a non-ideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a non-biased manner, allowing the audience to adopt their own views of this novel system. PMID:27045664

Bromodomain and Extra-terminal (BET) Protein Inhibitors Suppress Chondrocyte Differentiation and Restrain Bone Growth.

PubMed

Niu, Ningning; Shao, Rui; Yan, Guang; Zou, Weiguo

2016-12-23

Small molecule inhibitors for bromodomain and extra-terminal (BET) proteins have recently emerged as potential therapeutic agents in clinical trials for various cancers. However, to date, it is unknown whether these inhibitors have side effects on bone structures. Here, we report that inhibition of BET bromodomain proteins may suppress chondrocyte differentiation and restrain bone growth. We generated a luciferase reporter system using the chondrogenic cell line ATDC5 in which the luciferase gene was driven by the promoter of Col2a1, an elementary collagen of the chondrocyte. The Col2a1-luciferase ATDC5 system was used for rapidly screening both activators and repressors of human collagen Col2a1 gene expression, and we found that BET bromodomain inhibitors reduce the Col2a1-luciferase. Consistent with the luciferase assay, BET inhibitors decrease the expression of Col2a1 Furthermore, we constructed a zebrafish line in which the enhanced green fluorescent protein (EGFP) expression was driven by col2a1 promoter. The transgenic (col2a1-EGFP) zebrafish line demonstrated that BET inhibitors I-BET151 and (+)-JQ1 may affect EGFP expression in zebrafish. Furthermore, we found that I-BET151 and (+)-JQ1 may affect chondrocyte differentiation in vitro and inhibit zebrafish growth in vivo Mechanistic analysis revealed that BET inhibitors influenced the depletion of RNA polymerase II from the Col2a1 promoter. Collectively, these results suggest that BET bromodomain inhibition may have side effects on skeletal bone structures. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolf, Frank; Li Wenrong; Li Fang

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 systemsmore » 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.« less

Imaging CXCL12-CXCR4 Signaling and Inhibition in Ovarian Cancer

DTIC Science & Technology

2013-10-01

arrestin 2 and reconstitution of luciferase enzymes to produce light (see attached manuscript from PLoS One). Fig 2. CXCL12-dependent activation...With the luciferase complementa- tion system, interactions between CXCR4 and b-arrestin 2 reconstitute active click beetle luciferase to produce light ...dimensional cell culture experiments We plated 1.56104 HeyA8-CXCR4-CBRN/Ar-CBC cells per well in black wall 96 well plates one day before assays. We

Re-engineering of Bacterial Luciferase; For New Aspects of Bioluminescence.

PubMed

Kim, Da-Som; Choi, Jeong-Ran; Ko, Jeong-Ae; Kim, Kangmin

2018-01-01

Bacterial luminescence is the end-product of biochemical reactions catalyzed by the luciferase enzyme. Nowadays, this fascinating phenomenon has been widely used as reporter and/or sensors to detect a variety of biological and environmental processes. The enhancement or diversification of the luciferase activities will increase the versatility of bacterial luminescence. Here, to establish the strategy for luciferase engineering, we summarized the identity and relevant roles of key amino acid residues modulating luciferase in Vibrio harveyi, a model luminous bacterium. The current opinions on crystal structures and the critical amino acid residues involved in the substrate binding sites and unstructured loop have been delineated. Based on these, the potential target residues and/or parameters for enzyme engineering were also suggested in limited scale. In conclusion, even though the accurate knowledge on the bacterial luciferase is yet to be reported, the structure-guided site-directed mutagenesis approaches targeting the regulatory amino acids will provide a useful platform to re-engineer the bacterial luciferase in the future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

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.

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

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.

Caged ATP - an internal calibration method for ATP bioluminescence assays.

PubMed

Calvert, R M; Hopkins, H C; Reilly, M J; Forsythe, S J

2000-03-01

ATP bioluminescence, based on the firefly luciferase system, is used for the rapid determination of hygienic practices in the food industry. This study has demonstrated the use of caged ATP as an internal ATP standard and quantified the effects of industrial cleansing solutions, alcoholic beverages and pH on firefly luciferase activity. The light signal was quenched 6-47% by five cleansing solutions at standard working concentrations. Ethanol at 1% (v/v) inhibited bioluminescence by 15% (w/v) whereas concentrations above 4% enhanced the light output. The light signal was quenched by 20-25% at pH values below pH 4 and above pH 10.

1001 lights: luciferins, luciferases, their mechanisms of action and applications in chemical analysis, biology and medicine.

PubMed

Kaskova, Zinaida M; Tsarkova, Aleksandra S; Yampolsky, Ilia V

2016-10-24

Bioluminescence (BL) is a spectacular phenomenon involving light emission by live organisms. It is caused by the oxidation of a small organic molecule, luciferin, with molecular oxygen, which is catalysed by the enzyme luciferase. In nature, there are approximately 30 different BL systems, of which only 9 have been studied to various degrees in terms of their reaction mechanisms. A vast range of in vitro and in vivo analytical techniques have been developed based on BL, including tests for different analytes, immunoassays, gene expression assays, drug screening, bioimaging of live organisms, cancer studies, the investigation of infectious diseases and environmental monitoring. This review aims to cover the major existing applications for bioluminescence in the context of the diversity of luciferases and their substrates, luciferins. Particularly, the properties and applications of d-luciferin, coelenterazine, bacterial, Cypridina and dinoflagellate luciferins and their analogues along with their corresponding luciferases are described. Finally, four other rarely studied bioluminescent systems (those of limpet Latia, earthworms Diplocardia and Fridericia and higher fungi), which are promising for future use, are also discussed.

Prediction of luciferase inhibitors by the high-performance MIEC-GBDT approach based on interaction energetic patterns.

PubMed

Chen, Fu; Sun, Huiyong; Liu, Hui; Li, Dan; Li, Youyong; Hou, Tingjun

2017-04-12

High-throughput screening (HTS) is widely applied in many fields ranging from drug discovery to clinical diagnostics and toxicity assessment. Firefly luciferase is commonly used as a reporter to monitor the effect of chemical compounds on the activity of a specific target or pathway in HTS. However, the false positive rate of luciferase-based HTS is relatively high because many artifacts or promiscuous compounds that have direct interaction with the luciferase reporter enzyme are usually identified as active compounds (hits). Therefore, it is necessary to develop a rapid screening method to identify these compounds that can inhibit the luciferase activity directly. In this study, a virtual screening (VS) classification model called MIEC-GBDT (MIEC: Molecular Interaction Energy Components; GBDT: Gradient Boosting Decision Tree) was developed to distinguish luciferase inhibitors from non-inhibitors. The MIECs calculated by Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) free energy decomposition were used to energetically characterize the binding pattern of each small molecule at the active site of luciferase, and then the GBDT algorithm was employed to construct the classifiers based on MIECs. The predictions to the test set show that the optimized MIEC-GBDT model outperformed molecular docking and MM/GBSA rescoring. The best MIEC-GBDT model based on the MIECs with the energy terms of ΔG ele , ΔG vdW , ΔG GB , and ΔG SA achieves the prediction accuracies of 87.2% and 90.3% for the inhibitors and non-inhibitors in the test sets, respectively. Moreover, the energetic analysis of the vital residues suggests that the energetic contributions of the vital residues to the binding of inhibitors are quite different from those to the binding of non-inhibitors. These results suggest that the MIEC-GBDT model is reliable and can be used as a powerful tool to identify potential interference compounds in luciferase-based HTS experiments.

A reporter gene assay for the detection of phytoestrogens in traditional Chinese medicine.

PubMed

Miller-Martini, D M; Chan, R Y; Ip, N Y; Sheu, S J; Wong, Y H

2001-09-01

Bupleurum & Peony Formula (Jia Wei Xiao Yao San) is a herbal formula which possesses a clinical history for the treatment of menopausal syndrome and menstrual irregularity. The present investigation reports the ability to monitor the formula's phytoestrogen content that will allow for the implementation of a standardization protocol that is based on a quantifiable biological response. Utilizing an oestrogen-sensitive chimeric receptor/reporter gene element which has been stably transfected into HeLa cells, the botanical formula was shown to induce the expression of the reporter gene, luciferase, in a dose dependent manner. Pretreatment of the HeLa cells with the botanical formula produced a 5-fold increase in bioluminescence compared with the control. Additionally, our studies showed that the response of the cells, when challenged by the botanical formula, was oestrogen specific. Pretreatment of the cells with tamoxifen effectively blocked the activation of the chimeric oestrogen receptor by the botanical formula. The cell line provides a sensitive assay that can easily detect the presence of phytoestrogens in complex botanical formulas. Copyright 2001 John Wiley & Sons, Ltd.

Modification of the hTERT promoter by heat shock elements enhances the efficiency and specificity of cancer targeted gene therapy.

PubMed

Wang, Xiaolong; Zhou, PeiHua; Sun, XueJun; Wei, GuangBing; Zhang, Li; Wang, Hui; Yao, JianFeng; Jia, PengBo; Zheng, JianBao

2016-05-01

One of the current challenges facing cancer gene therapy is the tumour-specific targeting of therapeutic genes. Effective targeting in gene therapy requires accurate spatial and temporal control of gene expression. To develop a sufficient and accurate tumour-targeting method for cancer gene therapy, we have investigated the use of hyperthermia to control the expression of a transgene under the control of the human telomerase reverse transcriptase (hTERT) promoter and eight heat shock elements (8HSEs). Luciferase reporters were constructed by inserting eight HSEs and the hTERT promoter (8HSEs-hTERTp) upstream of the pGL4.20 vector luciferase gene. The luciferase activity of the hTERT promoter and 8HSEs-hTERT promoter were then compared in the presence and absence of heat. The differences in luciferase activity were analysed using dual luciferase assays in SW480 (high hTERT expression), MKN28 and MRC-5 cells (low hTERT expression). The luciferase activity of the Hsp70B promoter was also compared to the 8HSEs-hTERT promoter in the above listed cell lines. Lentiviral vector and heat-induced expression of EGFP expression under the control of the 8HSEs-hTERT promoter in cultured cells and mouse tumour xenografts was measured by reverse transcription polymerase (RT-PCR), Western blot and immunofluorescence assays. hTERT promoter activity was higher in SW480 cells than in MKN28 or MRC-5 cells. At 43 °C, the luciferase activity of the 8HSEs-hTERT promoter was significantly increased in SW480 cells, but not in MKN28 or MRC-5 cells. Importantly, the differences in luciferase activity were much more obvious in both high (SW480) and low (MKN28 and MRC-5) hTERT expressing cells when the activity of the 8HSEs-hTERT promoter was compared to the Hsp70B promoter. Moreover, under the control of 8HSEs-hTERT promoter in vitro and in vivo, EGFP expression was obviously increased by heat treatment in SW480 cells but not in MKN28 or MRC-5 cells, nor was expression increased under normal temperature conditions. The hTERT promoter is a potentially powerful tumour-specific promoter and gene therapy tool for cancer treatment. Incorporating heat-inducible therapeutic elements (8HSEs) into the hTERT promoter may enhance the efficiency and specificity of cancer targeting gene therapy under hyperthermic clinical conditions.

MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer.

PubMed

Liu, Minxia; Zhou, Kecheng; Cao, Yi

2016-09-26

MicroRNAs (miRNAs) have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC) and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D) and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfection were used to investigate interaction between the miRNA and target gene. miR-944 was significantly down-regulated in NSCLC and had many putative targets. Moreover, the forced expression of miR-944 significantly inhibited the proliferation of NSCLC cells in vitro. By integrating mRNA expression data and miR-944-target prediction, we disclosed that EPHA7 was a potential target of miR-944, which was further verified by luciferase reporter assay and microRNA transfection. Our data indicated that miR-944 targets EPHA7 in NSCLC and regulates NSCLC cell proliferation, which may offer a new mechanism underlying the development and progression of NSCLC.

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

PubMed Central

Millet, Jean Kaoru; Whittaker, Gary R.

2016-01-01

Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay. PMID:28018942

Synthesis and evaluation of (+)-decursin derivatives as inhibitors of the Wnt/β-catenin pathway.

PubMed

Lee, Jee-Hyun; Kim, Min-Ah; Park, Seoyoung; Cho, Soo-Hyun; Yun, Eunju; O, Yu-Seok; Kim, Jiseon; Goo, Ja-Il; Yun, Mi-Young; Choi, Yongseok; Oh, Sangtaek; Song, Gyu-Yong

2016-08-01

We synthesized (+)-decursin derivatives substituted with cinnamoyl- and phenyl propionyl groups originating from (+)-CGK062 and screened them using a cell-based assay to detect relative luciferase reporter activity. Of this series, compound 8b, in which a 3-acetoxy cinnamoyl group was introduced, most potently inhibited (97.0%) the Wnt/β-catenin pathway. Specifically, compound 8b dose-dependently inhibited Wnt3a-induced expression of the β-catenin response transcription (CRT) and increased β-catenin degradation in HEK293 reporter cells. Furthermore, compound 8b suppressed expression of the downstream β-catenin target genes cyclin D1 and c-myc and suppressed PC3 cell growth in a concentration-dependent manner. Copyright © 2016. Published by Elsevier Ltd.

Renilla luciferase-based quantitation of Potato virus A infection initiated with Agrobacterium infiltration of N. benthamiana leaves.

PubMed

Eskelin, K; Suntio, T; Hyvärinen, S; Hafren, A; Mäkinen, K

2010-03-01

A quantitation method based on the sensitive detection of Renilla luciferase (Rluc) activity was developed and optimized for Potato virus A (PVA; genus Potyviridae) gene expression. This system is based on infections initiated by Agrobacterium infiltration and subsequent detection of the translation of PVA::Rluc RNA, which is enhanced by viral replication, first within the cells infected initially and later by translation and replication within new cells after spread of the virus. Firefly luciferase (Fluc) was used as an internal control to normalize the Rluc activity. An approximately 10-fold difference in the Rluc/Fluc activity ratio between a movement-deficient and a replication-deficient mutant was observed starting from 48h post Agrobacterium infiltration (h.p.i.). The Rluc activity derived from wild type (wt) PVA increased significantly between 48 and 72h.p.i. and the Rluc/Fluc activity deviated clearly from that of the mutant viruses. Quantitation of the Rluc and Fluc mRNAs by semi-quantitative RT-PCR indicated that increases and decreases in the Renillareniformis luciferase (rluc) mRNA levels coincided with changes in Rluc activity. However, a subtle increase in the mRNA level led to pronounced changes in Rluc activity. PVA CP accumulation was quantitated by enzyme-linked immunosorbent assay. The increase in Rluc activity correlated closely with virus accumulation. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Shikonin, an ingredient of Lithospermum erythrorhizon, down-regulates the expression of steroid sulfatase genes in breast cancer cells.

PubMed

Zhang, Yi; Qian, Rui-Qin; Li, Ping-Ping

2009-10-18

Steroid sulfatase (STS) has an important role in regulating the biosynthesis of estrogen within breast tumors. We aimed to investigate whether shikonin, an ingredient of Lithospermum erythrorhizon, could modulate STS expression in breast cancer cells. By MTT assay, shikonin inhibited the cell proliferation of breast cancer cells MCF-7 and SK-BR-3. Moreover, by semi-quantitative/quantitative reverse transcription polymerase chain reaction and dual-luciferase reporter based bioluminescent measurements, the mRNA and enzymatic activity levels of STS were decreased after shikonin treatment. Concluding, shikonin could act as a selective estrogen enzyme modulator by down-regulating the STS expression.

Dexamethasone Enhances 1α,25-Dihydroxyvitamin D3 Effects by Increasing Vitamin D Receptor Transcription*

PubMed Central

Hidalgo, Alejandro A.; Deeb, Kristin K.; Pike, J. Wesley; Johnson, Candace S.; Trump, Donald L.

2011-01-01

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner. PMID:21868377

Searching for Synergistic Bronchodilators and Novel Therapeutic Regimens for Chronic Lung Diseases from a Traditional Chinese Medicine, Qingfei Xiaoyan Wan

PubMed Central

Hou, Yuanyuan; Cheng, Binfeng; Zhou, Mengge; Fang, Runping; Jiang, Min; Hou, Wenbin; Bai, Gang

2014-01-01

Classical Chinese pharmacopeias describe numerous excellent herbal formulations, and each prescription is an outstanding pool of effective compounds for drug discovery. Clarifying the bioactivity of the combined mechanisms of the ingredients in complex traditional Chinese medicine formulas is challenging. A classical formula known as Qingfei Xiaoyan Wan, used clinically as a treatment for prevalent chronic lung disease, was investigated in this work. A mutually enhanced bioactivity-guided ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) characterization system was proposed, coupled with a dual-luciferase reporter assay for β2AR-agonist cofactor screening. Arctiin, arctigenin, descurainoside and descurainolide B, four lignin compounds that showed synergistic bronchodilation effects with ephedrine, were revealed. The synergistic mechanism of arctigenin with the β2ARagonist involved with the reduction of free Ca2+ was clarified by a dual-luciferase reporter assay for intracellular calcium and the Ca2+ indicator fluo-4/AM to monitor changes in the fluorescence. The relaxant and contractile responses of airway smooth muscle are regulated by crosstalk between the intracellular cAMP and calcium signaling pathways. Our data indicated the non-selective βAR agonist ephedrine as the principal bronchodilator of the formula, whereas the lignin ingredients served as adjuvant ingredients. A greater understanding of the mechanisms governing the control of these pathways, based on conventional wisdom, could lead to the identification of novel therapeutic targets or new agents for the treatment of asthma and COPD. PMID:25397687

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamaji, Takashi; Lopez, David; Pellegrini, Matteo

Upon fertilization Chlamydomonas reinhardtii zygotes undergo a program of differentiation into a diploid zygospore that is accompanied by transcription of hundreds of zygote-specific genes. We identified a distinct sequence motif we term a zygotic response element (ZYRE) that is highly enriched in promoter regions of C. reinhardtii early zygotic genes. A luciferase reporter assay was used to show that native ZYRE motifs within the promoter of zygotic gene ZYS3 or intron of zygotic gene DMT4 are necessary for zygotic induction. A synthetic luciferase reporter with a minimal promoter was used to show that ZYRE motifs introduced upstream are sufficient tomore » confer zygotic upregulation, and that ZYRE-controlled zygotic transcription is dependent on the homeodomain transcription factor GSP1. Furthermore, we predict that ZYRE motifs will correspond to binding sites for the homeodomain proteins GSP1-GSM1 that heterodimerize and activate zygotic gene expression in early zygotes.« less

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

Development of a luciferase based viral inhibition assay to evaluate vaccine induced CD8 T-cell responses

PubMed Central

Naarding, Marloes A.; Fernandez-Fernandez, Natalia; Kappes, John C.; Hayes, Peter; Ahmed, Tina; Icyuz, Mert; Edmonds, Tara G.; Bergin, Philip; Anzala, Omu; Hanke, Tomas; Clark, Lorna; Cox, Josephine H.; Cormier, Emmanuel; Ochsenbauer, Christina; Gilmour, Jill

2014-01-01

Emergence of SIV and HIV specific CD8 T cells has been shown to correlate with control of in vivo replication. Poor correlation between IFN-γ ELISPOT responses and in vivo control of the virus has triggered the development of more relevant assays to assess functional HIV-1 specific CD8 T-cell responses for the evaluation and prioritization of new HIV-1 vaccine candidates. We previously established a viral inhibition assay (VIA) that measures the ability of vaccine-induced CD8 T-cell responses to inhibit viral replication in autologous CD4 T cells. In this assay, viral replication is determined by measuring p24 in the culture supernatant. Here we describe the development of a novel VIA, referred to as IMC LucR VIA that exploits replication-competent HIV-1 infectious molecular clones (IMCs) in which the complete proviral genome is strain-specific and which express the Renilla luciferase (LucR) gene to determine viral growth and inhibition. The introduction of the luciferase readout does provide significant improvement of the read out time. In addition to switching to the LucR read out, changes made to the overall protocol resulted in the miniaturization of the assay from a 48 to a 96-well plate format, which preserved sample and allowed for the introduction of replicates. The overall assay time was reduced from 13 to 8 days. The assay has a high degree of specificity, and the previously observed non-specific background inhibition in cells from HIV-1 negative volunteers has been reduced dramatically. Importantly, we observed an increase in positive responses, indicating an improvement in sensitivity compared to the original VIA. Currently, only a limited number of “whole-genome” IMC-LucR viruses are available and our efforts will focus on expanding the panel to better evaluate anti-viral breadth. Overall, we believe the IMC LucR VIA provides a platform to assess functional CD8 T-cell responses in large-scale clinical trial testing, which will enhance the ability to select the most promising HIV-1 vaccine candidates capable of controlling HIV-1 replication in vivo. PMID:24291126

MiR-137 inhibited cell proliferation and migration of vascular smooth muscle cells via targeting IGFBP-5 and modulating the mTOR/STAT3 signaling

PubMed Central

Li, Kai; Huang, Wei; Zhang, Xiaoqing

2017-01-01

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to the development of cardiovascular diseases. Studies have shown the great impact of microRNAs (miRNAs) on the cell proliferation of VSMCs. This study examined the effects of miR-137 on the cell proliferation and migration of VSMCs and also explored the underlying molecular mechanisms. The mRNA and protein expression levels were determined by qRT-PCR and western blot assays, respectively. The CCK-8 assay, wound healing assay and transwell migration assay were performed to measure cell proliferation and migration of VSMCs. The miR-137-targeted 3’untranslated region of insulin-like growth factor-binding protein-5 (IGFBP-5) was confirmed by luciferase reporter assay. Platelet-derived growth factor-bb (PDGF-bb) treatment enhanced cell proliferation and suppressed the expression of miR-137 in VSMCs. The gain-of-function and loss-of-function assays showed that overexpression of miR-137 suppressed the cell proliferation and migration, and also inhibited the expression of matrix genes of VSMCs; down-regulation of miR-137 had the opposite effects on VSMCs. Bioinformatics analysis and luciferase report assay results showed that IGFBP-5 was a direct target of miR-137, and miR-137 overexpression suppressed the IGFBP-5 expression and down-regulation of miR-137 increased the IGFBP-5 expression in VSMCs. PDGF-bb treatment also increased the IGFBP-5 mRNA expression. In addition, enforced expression of IGFBP-5 reversed the inhibitory effects of miR-137 on cell proliferation and migration of VSMCs. More importantly, overexpression of miR-137 also suppressed the activity of mTOR/STAT3 signaling in VSMCs. Taken together, the results suggest that miR-137 may suppress cell proliferation and migration of VSMCs via targeting IGFBP-5 and modulating mTOR/STAT3 signaling pathway. PMID:29016699

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...

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...

The Effects of Statistical Multiplicity of Infection on Virus Quantification and Infectivity Assays.

PubMed

Mistry, Bhaven A; D'Orsogna, Maria R; Chou, Tom

2018-06-19

Many biological assays are employed in virology to quantify parameters of interest. Two such classes of assays, virus quantification assays (VQAs) and infectivity assays (IAs), aim to estimate the number of viruses present in a solution and the ability of a viral strain to successfully infect a host cell, respectively. VQAs operate at extremely dilute concentrations, and results can be subject to stochastic variability in virus-cell interactions. At the other extreme, high viral-particle concentrations are used in IAs, resulting in large numbers of viruses infecting each cell, enough for measurable change in total transcription activity. Furthermore, host cells can be infected at any concentration regime by multiple particles, resulting in a statistical multiplicity of infection and yielding potentially significant variability in the assay signal and parameter estimates. We develop probabilistic models for statistical multiplicity of infection at low and high viral-particle-concentration limits and apply them to the plaque (VQA), endpoint dilution (VQA), and luciferase reporter (IA) assays. A web-based tool implementing our models and analysis is also developed and presented. We test our proposed new methods for inferring experimental parameters from data using numerical simulations and show improvement on existing procedures in all limits. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

An interferon-beta promoter reporter assay for high throughput identification of compounds against multiple RNA viruses.

PubMed

Guo, Fang; Zhao, Xuesen; Gill, Tina; Zhou, Yan; Campagna, Matthew; Wang, Lijuan; Liu, Fei; Zhang, Pinghu; DiPaolo, Laura; Du, Yanming; Xu, Xiaodong; Jiang, Dong; Wei, Lai; Cuconati, Andrea; Block, Timothy M; Guo, Ju-Tao; Chang, Jinhong

2014-07-01

Virus infection of host cells is sensed by innate pattern recognition receptors (PRRs) and induces production of type I interferons (IFNs) and other inflammatory cytokines. These cytokines orchestrate the elimination of the viruses but are occasionally detrimental to the hosts. The outcomes and pathogenesis of viral infection are largely determined by the specific interaction between the viruses and their host cells. Therefore, compounds that either inhibit viral infection or modulate virus-induced cytokine response should be considered as candidates for managing virus infection. The aim of the study was to identify compounds in both categories, using a single cell-based assay. Our screening platform is a HEK293 cell-based reporter assay where the expression of a firefly luciferase is under the control of a human IFN-β promoter. We have demonstrated that infection of the reporter cell line with a panel of RNA viruses activated the reporter gene expression that correlates quantitatively with the levels of virus replication and progeny virus production, and could be inhibited in a dose-dependent manner by known antiviral compound or inhibitors of PRR signal transduction pathways. Using Dengue virus as an example, a pilot screening of a small molecule library consisting of 26,900 compounds proved the concept that the IFN-β promoter reporter assay can serve as a convenient high throughput screening platform for simultaneous discovery of antiviral and innate immune response modulating compounds. A representative antiviral compound from the pilot screening, 1-(6-ethoxybenzo[d]thiazol-2-yl)-3-(3-methoxyphenyl) urea, was demonstrated to specifically inhibit several viruses belonging to the family of flaviviridae. Copyright © 2014 Elsevier B.V. All rights reserved.

Optimization and validation of a high throughput method for detecting neutralizing antibodies against human papillomavirus (HPV) based on pseudovirons.

PubMed

Nie, Jianhui; Huang, Weijin; Wu, Xueling; Wang, Youchun

2014-09-01

The pseudoviron-based neutralization assay is accepted as the gold standard to evaluate the functional humoral immune response against HPV. The goal of this study was to develop and optimize a human papillomavirus (HPV) neutralization assay using HPV pseudovirons with Gaussia luciferase (Gluc) as the reporter gene. For this purpose, high-titers Gluc pseudovirons were generated by cotransfecting 293TT cells with HPV structural genes and Gluc expressing plasmids. Six types of neutralizing monoclonal antibodies, vaccines immunized serum samples and WHO international antibody standard were used to validate the new developed assay. The ideal circumstances of the assay were identified for cell counts (30,000/well for 96-well plate), pseudoviron inoculating size (100 times RLU above background) and incubation time (72 hr). The sensitivity of the Gluc assay was comparable to secreted alkaline phosphatase (SEAP) assay and higher than the green florescent protein (GFP) assay. The non-specific background for different types of sample was significantly different (rabbit sera > human sera > mouse sera, P < 0.01). The non-specific neutralization effects were not attributed to IgG antibody. The cutoff value for this assay was determined as 50% inhibition at a dilution of 1:40. Without requirements of sample dilution and different incubation times at different temperature before processing, the detection time was shortened from more than 90 min to less than 5 min for a 96-well plate compared with the SEAP-based assay. With the advantages of short detection time and easy-to-use procedure, the newly developed assay is more suitable for large sero-epidemiological studies or clinical trials and more amenable to automation. © 2014 Wiley Periodicals, Inc.

Fabrication of a New Lineage of Artificial Luciferases from Natural Luciferase Pools.

PubMed

Kim, Sung Bae; Nishihara, Ryo; Citterio, Daniel; Suzuki, Koji

2017-09-11

The fabrication of artificial luciferases (ALucs) with unique optical properties has a fundamental impact on bioassays and molecular imaging. In this study, we developed a new lineage of ALucs with unique substrate preferences by extracting consensus amino acids from the alignment of 25 copepod luciferase sequences available in natural luciferase pools. The primary sequence was first created with a sequence logo generator resulting in a total of 11 sibling sequences. Phylogenetic analysis shows that the newly fabricated ALucs form an independent branch, genetically isolated from the natural luciferases, and from a prior series of ALucs produced by our laboratory using a smaller basis set. The new lineage of ALucs were strongly luminescent in living mammalian cells with specific substrate selectivity to native coelenterazine. A single-residue-level comparison of the C-terminal sequences of new ALucs reveals that some amino acids in the C-terminal ends are greatly influential on the optical intensities but limited in the color variance. The success of this approach guides on how to engineer and functionalize marine luciferases for bioluminescence imaging and assays.

Tamoxifen Dependent Interaction Between the Estrogen Receptor and a Novel P21 Activated Kinase

DTIC Science & Technology

2002-06-01

binding domain at the C Cv1 cells were cotransfected with ARE4-Luc reporter (100 teric G prti Tse bindin dominat the C ng), pRL-SV40 ( Renilla control...48 h and assayed for luciferase and Renilla Three of the four residues defining a putative het- activity. erotrimeric G protein binding motif were...nases. Taken together, these results demonstrated expression of the control Renilla reporter regulated by that the PAK6 CRIB domain was functional with re

Cardenolide glycosides from Elaeodendron australe var. integrifolium.

PubMed

Butler, Mark S; Towerzey, Leanne; Pham, Ngoc B; Hyde, Edward; Wadi, Sao Khemar; Guymer, Gordon P; Quinn, Ronald J

2014-02-01

Extracts from dried leaf and stems of Elaeodendron australe var. integrifolium (Celastraceae) collected in South East Queensland, Australia, were active in an assay that measured Ca(2+) driven expression of IL-2/luciferase designed to identify inhibitors of the ICRAC channel. Bioassay-guided isolation using C18 and polyamide column chromatography, HPLC (Phenyl and C18) and centrifugal partition chromatography (CPC) led to the isolation of digitoxigenin (1) and three cardenolide glycosides, glucoside 2, quinovoside 3 and the new natural product xyloside 4, as the active components with low nM activity in the reporter assay. Copyright © 2013 Elsevier Ltd. All rights reserved.

A functional polymorphism of the TNF-{alpha} gene that is associated with type 2 DM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Susa, Shinji; Daimon, Makoto; Sakabe, Jun-Ichi

2008-05-09

To examine the association of the tumor necrosis factor-{alpha} (TNF-{alpha}) gene region with type 2 diabetes (DM), 11 single-nucleotide polymorphisms (SNPs) of the region were analyzed. The initial study using a sample set (148 cases vs. 227 controls) showed a significant association of the SNP IVS1G + 123A of the TNF-{alpha} gene with DM (p = 0.0056). Multiple logistic regression analysis using an enlarged sample set (225 vs. 716) revealed the significant association of the SNP with DM independently of any clinical traits examined (OR: 1.49, p = 0.014). The functional relevance of the SNP were examined by the electrophoreticmore » mobility shift assays using nuclear extracts from the U937 and NIH3T3 cells and luciferase assays in these cells with Simian virus 40 promoter- and TNF-{alpha} promoter-reporter gene constructs. The functional analyses showed that YY1 transcription factor bound allele-specifically to the SNP region and, the IVS1 + 123A allele had an increase in luciferase expression compared with the G allele.« less

Coordinated regulation of IFITM1, 2 and 3 genes by an IFN-responsive enhancer through long-range chromatin interactions.

PubMed

Li, Ping; Shi, Ming-Lei; Shen, Wen-Long; Zhang, Zhang; Xie, De-Jian; Zhang, Xiang-Yuan; He, Chao; Zhang, Yan; Zhao, Zhi-Hu

2017-08-01

Interferon-induced transmembrane protein (IFITM) 1, 2 and 3 genes encode a family of interferon (IFN)-induced transmembrane proteins that block entry of a broad spectrum of pathogens. However, the transcriptional regulation of these genes, especially whether there exist any enhancers and their roles during the IFN induction process remain elusive. Here, through public data mining, episomal luciferase reporter assay and in vivo CRISPR-Cas9 genome editing, we identified an IFN-responsive enhancer located 35kb upstream of IFITM3 gene promoter upregulating the IFN-induced expression of IFITM1, 2 and 3 genes. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and luciferase reporter assay demonstrated that signal transducers and activators of transcription (STAT) 1 bound to the enhancer with the treatment of IFN and was indispensable for the enhancer activity. Furthermore, using chromosome conformation capture technique, we revealed that the IFITM1, 2 and 3 genes physically clustered together and constitutively looped to the distal enhancer through long-range interactions in both HEK293 and A549 cells, providing structural basis for coordinated regulation of IFITM1, 2 and 3 by the enhancer. Finally, we showed that in vivo truncation of the enhancer impaired IFN-induced resistance to influenza A virus (IAV) infection. These findings expand our understanding of the mechanisms underlying the transcriptional regulation of IFITM1, 2 and 3 expression and its ability to mediate IFN signaling. Copyright © 2017 Elsevier B.V. All rights reserved.

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. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Ciculating miRNA-21 as a Biomarker Predicts Polycystic Ovary Syndrome (PCOS) in Patients.

PubMed

Jiang, Liyan; Li, Wei; Wu, Minmin; Cao, Sifan

2015-01-01

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, hyperinsulinemia, and infertility. In PCOS, abnormal regulation of relevant genes is required for follicular development. By binding to the 3' untranslated region (3'URT), microRNAs (miRNAs) are widely involved in posttranscriptional gene regulation. However, few studies have been conducted on circulating miRNA expression in PCOS. This study aims to describe altered expression of circulating miR-21 in PCOS. The expression of serum miRNAs of PCOS patients were explored using the TaqMan Low Density Array followed by individual quantitative reverse transcription polymerase chain reaction assays. The protein level of LATS1 was determined using Western blot. To validate whether miR-21 targeted LATS1, the luciferase assay was applied. In comparison with normal subjects, the circulating level of miRNA-21 was significantly enhanced in PCOS patients. In PCOS patients, the expression levels of MST1/2, LATS1/2, TAZ were much lower than the control subjects. Luciferase reporter assay revealed that LATS1 was a downstream target of miR-21. In comparison with normal subjects, serum miR-21 is obviously increased in PCOS patients. Through targeting LATS1, miR-21 could prompt PCOS progression and could act as a novel non-invasive biomarker for diagnosis of PCOS.

Hormone treatment enhances WT1 activation of Renilla luciferase constructs in LNCaP cells.

PubMed

Hanson, Julie; Reese, Jennifer; Gorman, Jacquelyn; Cash, Jennifer; Fraizer, Gail

2007-01-01

The zinc finger transcription factor, WT1, regulates many growth control genes, repressing or activating transcription depending on the gene and cell type. Based on earlier analyses of the effect of WT1 on androgen responsive genes, we hypothesized that there may be an interaction between the androgen signaling pathway and WT1, such that the commonly used Renilla luciferase control vectors were activated in LNCaP prostate cancer cells. Using cotransfection assays we tested the effects of WT1 and/or the androgen analog, R1881, on two Renilla luciferase vectors, pRL-SV40 and the promoter-less pRL-null. To determine whether the zinc finger DNA binding domain was required, the zinc finger mutant DDS-WT1 (R394W) was tested; but it had no significant effect on the Renilla luciferase vectors. To determine whether the androgen signaling pathway was required, WT1 was co-transfected with Renilla vectors in cells with varied hormone responsiveness. The WT1 effect on pRL-null varied from no significant effect in 293 and PC3 cells to very strong enhancement in LNCaP cells treated with 5 nM R1881. Overall, these results suggest that hormone enhanced WT1 mediated activation of Renilla luciferase and that these interactions require an intact WT1 zinc finger DNA binding domain.

Enhanced red-emitting railroad worm luciferase for bioassays and bioimaging

PubMed Central

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°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. PMID:19866487

Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice.

PubMed

Bhaumik, S; Lewis, X Z; Gambhir, S S

2004-01-01

We have recently demonstrated that Renilla luciferase (Rluc) is a promising bioluminescence reporter gene that can be used for noninvasive optical imaging of reporter gene expression in living mice, with the aid of a cooled charged couple device (CCD) camera. In the current study, we explore the expression of a novel synthetic Renilla luciferase reporter gene (hRluc) in living mice, which has previously been reported to be a more sensitive reporter than native Rluc in mammalian cells. We explore the strategies of simultaneous imaging of both Renilla luciferase enzyme (RL) and synthetic Renilla luciferase enzyme (hRL):coelenterazine (substrate for RL/hRL) in the same living mouse. We also demonstrate that hRL:coelenterazine can yield a higher signal when compared to Firefly luciferase enzyme (FL): D-Luciferin, both in cell culture studies and when imaged from cells at the surface and from lungs of living mice. These studies demonstrate that hRluc should be a useful primary reporter gene with high sensitivity when used alone or in conjunction with other bioluminescence reporter genes for imaging in living rodents. (c) 2004 Society of Photo-Optical Instrumentation Engineers.

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.

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

PubMed

Kassar, Telissa C; Magalhães, Tereza; S, José V J; Carvalho, Amanda G O; Silva, Andréa N M R DA; Queiroz, Sabrina R A; Bertani, Giovani R; Gil, Laura H V G

2017-01-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Junwei; Zhang, Huan; Fang, Liurong

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 luciferasemore » 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.« less

Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells.

PubMed

Pathak, Bhakti R; Breed, Ananya A; Deshmukh, Priyanka; Mahale, Smita D

2018-07-01

Cysteine-rich secretory protein 3 (CRISP3) is one of the most upregulated genes in prostate cancer. Androgen receptor (AR) plays an important role not only in initial stages of prostate cancer development but also in the advanced stage of castration-resistant prostate cancer (CRPC). Role of AR in regulation of CRISP3 expression is not yet known. In order to understand the regulation of CRISP3 expression, various overlapping fragments of CRISP3 promoter were cloned in pGL3 luciferase reporter vector. All constructs were transiently and stably transfected in PC3 (CRISP3 negative) and LNCaP (CRISP3 positive) cell lines and promoter activity was measured by luciferase assay. Promoter activity of LNCaP stable clones was significantly higher than PC3 stable clones. Further in CRISP3 negative PC3 and RWPE-1 cells, CRISP3 promoter was shown to be silenced by histone deacetylation. Treatment of LNCaP cells with DHT resulted in increase in levels of CRISP3 transcript and protein. AR dependency of CRISP3 promoter was also evaluated in LNCaP stable clones by luciferase assay. To provide molecular evidence of epigenetic regulation of CRISP3 promoter and its response to DHT, ChIP PCR was performed in PC3 and LNCaP cells. Our results demonstrate that CRISP3 expression in prostate cancer cells is androgen dependent and in AR positive cells, CRISP3 promoter is epigenetically regulated by AR. Copyright © 2018 Elsevier Ltd. All rights reserved.

Monitoring nanoparticle-mediated cellular hyperthermia with a high-sensitivity biosensor

PubMed Central

Mukherjee, Amarnath; Castanares, Mark; Hedayati, Mohammad; Wabler, Michele; Trock, Bruce; Kulkarni, Prakash; Rodriguez, Ronald; Getzenberg, Robert H; DeWeese, Theodore L; Ivkov, Robert; Lupold, Shawn E

2014-01-01

Aim To develop and apply a heat-responsive and secreted reporter assay for comparing cellular response to nanoparticle (NP)- and macroscopic-mediated sublethal hyperthermia. Materials & methods Reporter cells were heated by water bath (macroscopic heating) or iron oxide NPs activated by alternating magnetic fields (nanoscopic heating). Cellular responses to these thermal stresses were measured in the conditioned media by secreted luciferase assay. Results & conclusion Reporter activity was responsive to macroscopic and nanoparticle heating and activity correlated with measured macroscopic thermal dose. Significant cellular responses were observed with NP heating under doses that were insufficient to measurably change the temperature of the system. Under these conditions, the reporter response correlated with proximity to cells loaded with heated nanoparticles. These results suggest that NP and macroscopic hyperthermia may be distinctive under conditions of mild hyperthermia. PMID:24547783

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

PubMed

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

1997-03-14

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

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.

Measuring antiviral activity of benzimidazole molecules that alter IRES RNA structure with an infectious hepatitis C virus chimera expressing Renilla luciferase.

PubMed

Liu, Shuanghu; Nelson, Cassie A; Xiao, Li; Lu, Ling; Seth, Punit P; Davis, Darrell R; Hagedorn, Curt H

2011-01-01

Major progress has been made in developing infectious HCV cell culture systems and these systems have been useful in identifying novel HCV antivirals. However, more rapid and sensitive assays using infectious cell based HCV systems would facilitate the development of additional antivirals, including small molecules directed at unique targets such as the HCV RNA internal ribosomal entry site (IRES). We have found that the V3 region (28 aa) of NS5A of HCV JFH1 can be deleted from the genome with only modest effects on the titer of infectious virus produced in cell culture. Moreover, the V3 region can be replaced with the Renilla reniformis luciferase (Rluc) gene resulting in an infectious virus that stably expresses an NS5A-Rluc fusion protein. Infected cells cultured in 96-well plates provided a robust luciferase signal that accurately reflected the production of infectious virus. This infectious HCV reporter system was used to test the activity of three benzimidazole compounds that bind the HCV RNA IRES. Compounds in this chemical class of small molecules bind and alter the IRES RNA structure at low to sub-micromolar concentrations and interfere with viral replication. The current study shows that these compounds inhibit HCV replication in an infectious HCV cell culture system, defines their IC(50) in this system, and provides a platform for the rapid testing of next generation inhibitors. Copyright © 2010 Elsevier B.V. All rights reserved.

Measuring Antiviral Activity of Benzimidazole Molecules that Alter IRES RNA Structure with an Infectious Hepatitis C Virus Chimera Expressing Renilla Luciferase

PubMed Central

Liu, Shuanghu; Nelson, Cassie A.; Xiao, Li; Lu, Ling; Seth, Punit P.; Davis, Darrell R.; Hagedorn, Curt H.

2010-01-01

Major progress has been made in developing infectious HCV cell culture systems and these systems have been useful in identifying novel HCV antivirals. However, more rapid and sensitive assays using infectious cell based HCV systems would facilitate the development of additional antivirals, including small molecules directed at unique targets such as the HCV RNA internal ribosomal entry site (IRES). We have found that the V3 region (28 aa) of NS5A of HCV JFH1 can be deleted from the genome with only modest effects on the titer of infectious virus produced in cell culture. Moreover, the V3 region can be replaced with the Renilla reniformis luciferase (Rluc) gene resulting in an infectious virus that stably expresses an NS5A-Rluc fusion protein. Infected cells cultured in 96-well plates provided a robust luciferase signal that accurately reflected the production of infectious virus. This infectious HCV reporter system was used to test the activity of three benzimidazole compounds that bind the HCV RNA IRES. Compounds in this chemical class of small molecules bind and alter the IRES RNA structure at low to sub-micromolar concentrations and interfere with viral replication. The current study shows that these compounds inhibit HCV replication in an infectious HCV cell culture system, defines their IC50 in this system, and provides a platform for the rapid testing of next generation inhibitors. PMID:21075143

[Comparison of TTR and CMV promoters in vivo and in vitro via a secreted luciferase reporter system].

PubMed

Luo, Shun-Tao; Tian, Wen-Hong; Wang, Gang; Dong, Xiao-Yan; Yang, Li; Wu, Xiao-Bing

2009-11-01

GLuc (Gaussia luciferase) is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLuc. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then, 4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/c mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5 microl tail tip blood since 2 h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLuc driven by CMV promoter was 50-300 times higher than that of GLuc driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PCMV's. Thus, it is more suitable for maintaining longer expression of target genes in liver.

A quantitative and high-throughput assay of human papillomavirus DNA replication.

PubMed

Gagnon, David; Fradet-Turcotte, Amélie; Archambault, Jacques

2015-01-01

Replication of the human papillomavirus (HPV) double-stranded DNA genome is accomplished by the two viral proteins E1 and E2 in concert with host DNA replication factors. HPV DNA replication is an established model of eukaryotic DNA replication and a potential target for antiviral therapy. Assays to measure the transient replication of HPV DNA in transfected cells have been developed, which rely on a plasmid carrying the viral origin of DNA replication (ori) together with expression vectors for E1 and E2. Replication of the ori-plasmid is typically measured by Southern blotting or PCR analysis of newly replicated DNA (i.e., DpnI digested DNA) several days post-transfection. Although extremely valuable, these assays have been difficult to perform in a high-throughput and quantitative manner. Here, we describe a modified version of the transient DNA replication assay that circumvents these limitations by incorporating a firefly luciferase expression cassette in cis of the ori. Replication of this ori-plasmid by E1 and E2 results in increased levels of firefly luciferase activity that can be accurately quantified and normalized to those of Renilla luciferase expressed from a control plasmid, thus obviating the need for DNA extraction, digestion, and analysis. We provide a detailed protocol for performing the HPV type 31 DNA replication assay in a 96-well plate format suitable for small-molecule screening and EC50 determinations. The quantitative and high-throughput nature of the assay should greatly facilitate the study of HPV DNA replication and the identification of inhibitors thereof.

Editor's Highlight: Structure-Based Investigation on the Binding and Activation of Typical Pesticides With Thyroid Receptor.

PubMed

Xiang, Dandan; Han, Jian; Yao, Tingting; Wang, Qiangwei; Zhou, Bingsheng; Mohamed, Abou Donia; Zhu, Guonian

2017-12-01

A broad range of pesticides have been reported to interfere with the normal function of the thyroid endocrine system. However, the precise mechanism(s) of action has not yet been thoroughly elucidated. In this study, 21 pesticides were assessed for their binding interactions and the potential to disrupt thyroid homeostasis. In the GH3 luciferase reporter gene assays, 5 of the pesticides tested had agonistic effects in the order of procymidone > imidacloprid > mancozeb > fluroxypyr > atrazine. 11 pesticides inhibited luciferase activity of T3 to varying degrees, demonstrating their antagonistic activity. And there are 4 pesticides showed mixed effects when treated with different concentrations. Surface plasmon resonance (SPR) biosensor technique was used to directly measure the binding interactions of these pesticides to the human thyroid hormone receptor (hTR). 13 pesticides were observed to bind directly with TR, with a KD ranging from 4.80E-08 M to 9.44E-07 M. The association and disassociation of the hTR/pesticide complex revealed 2 distinctive binding modes between the agonists and antagonists. At the same time, a different binding mode was displayed by the pesticides showed mix agonist and antagonist activity. In addition, the molecular docking simulation analyses indicated that the interaction energy calculated by CDOCKER for the agonists and antagonists correlated well with the KD values measured by the surface plasmon resonance assay. These results help to explain the differences of the TR activities of these tested pesticides. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cyp2a5 Promoter-based Gene Reporter Assay: A Novel Design of Cell-based Bioassay for Toxicity Prediction.

PubMed

Abu-Bakar, A'edah; Hu, Hao; Lang, Matti A

2018-05-22

The murine cytochrome P450 2a5 (Cyp2a5) gene is regulated by complex interactions of various stress-activated transcription factors (TFs). Elevated Cyp2a5 transcription under chemical-induced stress conditions is achieved by interplay between the various TFs-including as aryl hydrocarbon receptor (AhR) and nuclear factor (erythroid-derived 2)-like 2 wild-type (Nrf2)-at the "stress-responding" cluster of response elements on the Cyp2a5 promoter, as well as through mRNA stabilisation mediated by interaction of the stress-activated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with the 3'UTR of the CYP2A5 mRNA. We design a unique toxicity pathway-based reporter assay to include regulatory regions from both the 5' and the 3' untranslated regions of Cyp2a5 in a luciferase reporter plasmid to reflect in vivo responses to chemical insult. Human breast cancer, MCF-7 cells were stably transfected with pGL4.38-Cyp2a5_Wt3k (wildtype) or mutants-pGL4.38-Cyp2a5-StREMut and pGL4.38-Cyp2a5-XREMut-reporter gene to monitor chemical-induced cellular response mediated by AhR and Nrf2 signalling. The recombinant cells were treated with representative of AhR agonist, polycyclic aromatic hydrocarbons, brominated flame retardant, fluorosurfactant, aromatic organic compound and metal, to determine sensitivity of the Cyp2a5 promoter-based gene reporter assays to chemical insults by measuring the LC 50 and EC 50 of the respective chemicals. The three assays are sensitive to sub-lethal cellular responses of chemicals, which is an ideal feature for toxicity pathway-based bioassay for toxicity prediction. The wildtype reporter responded well to chemicals that activate cross-talk between the AhR and Nrf2, whilst the mutant reporters effectively gauge cellular response driven by either Nrf2/StRE or AhR/XRE signalling. Thus, the three gene reporter assays could be used tandemly to determine the predominant toxicity pathway of a given compound. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

PubMed Central

Alpert, Michael D.; Heyer, Lisa N.; Williams, David E. J.; Harvey, Jackson D.; Greenough, Thomas; Allhorn, Maria

2012-01-01

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4+ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays. PMID:22933282

MicroRNA-9 promotes the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1.

PubMed

Liu, D-Z; Chang, B; Li, X-D; Zhang, Q-H; Zou, Y-H

2017-09-01

The objective of the study was to investigate the role of microRNA-9 (miR-9) targeting forkhead box O1 (FOXO1) in the proliferation, migration, and invasion of breast cancer cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expressions of miR-9 and FOXO1 mRNA in breast cancer tissues, normal breast tissues, breast cancer cell lines, and normal breast epithelial cells. After the up-regulation of miR-9 expression, qRT-PCR and Western blotting were used to determine the expression of FOXO1. The luciferase reporter gene assay was used to validate the target gene. The CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay were used to investigate the changes in the proliferation, migration, and invasion of breast cancer cells, respectively. MicroRNA-9 expression was significantly up-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P < 0.05). FOXO1 mRNA and protein expressions were substantially down-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P < 0.05). There can be a negative correlation between miR-9 and FOXO1 mRNA in breast cancer. Luciferase reporter gene assay indicated that miR-9 can down-regulate FOXO1 expression at a post-transcriptional level through binding specifically to FOXO1 3'UTR. The results of CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay revealed that the inhibition of miR-9 can suppress MCF7 cell proliferation, migration, and invasion. Additionally, the expression of miR-9 increased significantly whilst that of FOXO1 decreased substantially as the disease progressed (P < 0.05). Our study provides evidence that miR-9 can promote the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1.

Cloning the uteroglobin gene promoter from the relic volcano rabbit (Romerolagus diazi) reveals an ancient estrogen-response element.

PubMed

Acosta-MontesdeOca, Adriana; Zariñán, Teresa; Macías, Héctor; Pérez-Solís, Marco A; Ulloa-Aguirre, Alfredo; Gutiérrez-Sagal, Rubén

2012-05-01

To gain further insight on the estrogen-dependent transcriptional regulation of the uteroglobin (UG) gene, we cloned the 5'-flanking region of the UG gene from the phylogenetically ancient volcano rabbit (Romerolagus diazi; Rd). The cloned region spans 812 base pairs (bp; -812/-1) and contains a noncanonical TATA box (TACA). The translation start site is 48 bp downstream from the putative transcription initiation site (AGA), and is preceded by a consensus Kozak box. Comparison of the Rd-UG gene with that previously isolated from rabbits (Oryctolagus cuniculus) showed 93% in sequence identity as well as a number of conserved cis-acting elements, including the estrogen-response element (ERE; -265/-251), which differs from the consensus by two nucleotides. In MCF-7 cells, 17β-estradiol (E(2)) induced transcription of a luciferase reporter driven by the Rd-UG promoter in a similar manner as in an equivalent rabbit UG reporter; the Rd-UG promoter was 30% more responsive to E(2) than the rabbit promoter. Mutagenesis studies on the Rd-ERE confirmed this cis-element as a target of E(2) as two luciferase mutant reporters of the Rd-promoter, one with the rabbit and the other with the consensus ERE, were more responsive to the hormone than the wild-type reporter. Gel shift and super-shift assays showed that estrogen receptor-α indeed binds to the imperfect palindromic sequence of the Rd-ERE. Copyright © 2012 Wiley Periodicals, Inc.

MicroRNA-138 inhibits proliferation of cervical cancer cells by targeting c-Met.

PubMed

Li, B; Yang, X-X; Wang, D; Ji, H-K

2016-01-01

MicroRNAs (miRNAs) function as important post-transcriptional regulators involved in a wide range of biological behaviors. MicroRNA-138 (miR-138) has been shown to play a critical role in tumor pathogenesis, the present study aimed to investigate the role of miR-138 in cervical cancer. CCK-8 assay was performed to measure the viabilities of cancer cells. Quantitative real-time PCR (qRT-PCR) and western blot were used to detect the mRNA and protein expression, respectively. Moreover, the miRNA target genes were validated with luciferase activity assay. In the current study, we found that the expression of miR-138 was significantly down-regulated in cervical cancer tissues compared to the adjacent non-cancer tissues. CCK-8 assay showed that over-expression of miR-138 suppressed the proliferation of four cervical cancer cell lines including HeLa, SiHa, C33A and CaSki. By contrast, down-regulation of miR-138 promoted the growth of cervical cancer cells. In addition, increased expression of miR-138 led to a reduction in c-Met expression, whereas inhibition of miR-138 enhanced c-Met levels in cervical cancer cells. The luciferase reporter assay showed that c-Met was a direct target of miR-138 in cervical cancer cells. These findings demonstrated that miR-138 inhibited cervical cancer cells proliferation via c-Met, providing a novel target for the molecular treatment of cervical cancer.

New Horizons on Molecular Pharmacology Applied to Drug Discovery: When Resonance Overcomes Radioligand Binding.

PubMed

Pernomian, Larissa; Gomes, Mayara Santos; Moreira, Josimar Dornelas; da Silva, Carlos Henrique Tomich de Paula; Rosa, Joaquin Maria Campos; Cardoso, Cristina Ribeiro de Barros

2017-01-01

One of the cornerstones of rational drug development is the measurement of molecular parameters derived from ligand-receptor interaction, which guides therapeutic windows definition. Over the last decades, radioligand binding has provided valuable contributions in this field as key method for such purposes. However, its limitations spurred the development of more exquisite techniques for determining such parameters. For instance, safety risks related to radioactivity waste, expensive and controlled disposal of radioisotopes, radiotracer separation-dependence for affinity analysis, and one-site mathematical models-based fitting of data make radioligand binding a suboptimal approach in providing measures of actual affinity conformations from ligands and G proteincoupled receptors (GPCR). Current advances on high-throughput screening (HTS) assays have markedly extended the options of sparing sensitive ways for monitoring ligand affinity. The advent of the novel bioluminescent donor NanoLuc luciferase (Nluc), engineered from Oplophorus gracilirostris luciferase, allowed fitting bioluminescence resonance energy transfer (BRET) for monitoring ligand binding. Such novel approach named Nluc-based BRET (NanoBRET) binding assay consists of a real-time homogeneous proximity assay that overcomes radioligand binding limitations but ensures the quality in affinity measurements. Here, we cover the main advantages of NanoBRET protocol and the undesirable drawbacks of radioligand binding as molecular methods that span pharmacological toolbox applied to Drug Discovery. Also, we provide a novel perspective for the application of NanoBRET technology in affinity assays for multiple-state binding mechanisms involving oligomerization and/or functional biased selectivity. This new angle was proposed based on specific biophysical criteria required for the real-time homogeneity assigned to the proximity NanoBRET protocol. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Dynamic monitoring of p53 translocation to mitochondria for the analysis of specific inhibitors using luciferase-fragment complementation.

PubMed

Noda, Natsumi; Awais, Raheela; Sutton, Robert; Awais, Muhammad; Ozawa, Takeaki

2017-12-01

Intracellular protein translocation plays a pivotal role in regulating complex biological processes, including cell death. The tumor suppressor p53 is a transcription factor activated by DNA damage and oxidative stress that also translocates from the cytosol into the mitochondrial matrix to facilitate necrotic cell death. However, specific inhibitors of p53 mitochondrial translocation are largely unknown. To explore the inhibitors of p53, we developed a bioluminescent probe to monitor p53 translocation from cytosol to mitochondria using luciferase fragment complementation assays. The probe is composed of a novel pair of luciferase fragments, the N-terminus of green click beetle luciferase CBG68 (CBGN) and multiple-complement luciferase fragment (McLuc1). The combination of luciferase fragments showed significant luminescence intensity and high signal-to-background ratio. When the p53 connected with McLuc1 translocates from cytosol into mitochondrial matrix, CBGN in mitochondrial matrix enables to complement with McLuc1, resulting in the restoration of the luminescence. The luminescence intensity was significantly increased under hydrogen peroxide-induced oxidative stress following the complementation of CBGN and McLuc1. Pifithrin-μ, a selective inhibitor of p53 mitochondrial translocation, prevented the mitochondrial translocation of the p53 probe in a concentration-dependent manner. Furthermore, the high luminescence intensity made it easier to visualize the p53 translocation at a single cell level under a bioluminescence microscope. This p53 mitochondrial translocation assay is a new tool for high-throughput screening to identify novel p53 inhibitors, which could be developed as drugs to treat diseases in which necrotic cell death is a major contributor. © 2017 Wiley Periodicals, Inc.

Functional elements in the minimal promoter of the human proton-coupled folate transporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stark, Michal; Gonen, Nitzan; Assaraf, Yehuda G., E-mail: assaraf@tx.technion.ac.il

2009-10-09

The proton-coupled folate transporter (PCFT) is the dominant intestinal folate transporter, however, its promoter has yet to be revealed. Hence, we here cloned a 3.1 kb fragment upstream to the first ATG of the human PCFT gene and generated sequential deletion constructs evaluated in luciferase reporter assay. This analysis mapped the minimal promoter to 157 bp upstream to the first ATG. Crucial GC-box sites were identified within the minimal promoter and in its close vicinity which substantially contribute to promoter activity, as their disruption resulted in 94% loss of luciferase activity. We also identified upstream enhancer elements including YY1 andmore » AP1 which, although distantly located, prominently transactivated the minimal promoter, as their inactivation resulted in 50% decrease in reporter activity. This is the first functional identification of the minimal PCFT promoter harboring crucial GC-box elements that markedly contribute to its transcriptional activation via putative interaction with distal YY1 and AP1 enhancer elements.« less

Identification and characterization of a cis-regulatory element for zygotic gene expression in Chlamydomonas reinhardtii

DOE PAGES

Hamaji, Takashi; Lopez, David; Pellegrini, Matteo; ...

2016-03-26

Upon fertilization Chlamydomonas reinhardtii zygotes undergo a program of differentiation into a diploid zygospore that is accompanied by transcription of hundreds of zygote-specific genes. We identified a distinct sequence motif we term a zygotic response element (ZYRE) that is highly enriched in promoter regions of C. reinhardtii early zygotic genes. A luciferase reporter assay was used to show that native ZYRE motifs within the promoter of zygotic gene ZYS3 or intron of zygotic gene DMT4 are necessary for zygotic induction. A synthetic luciferase reporter with a minimal promoter was used to show that ZYRE motifs introduced upstream are sufficient tomore » confer zygotic upregulation, and that ZYRE-controlled zygotic transcription is dependent on the homeodomain transcription factor GSP1. Furthermore, we predict that ZYRE motifs will correspond to binding sites for the homeodomain proteins GSP1-GSM1 that heterodimerize and activate zygotic gene expression in early zygotes.« less

An ultrasensitive NanoLuc-based luminescence system for monitoring Plasmodium berghei throughout its life cycle.

PubMed

De Niz, Mariana; Stanway, Rebecca R; Wacker, Rahel; Keller, Derya; Heussler, Volker T

2016-04-21

Bioluminescence imaging is widely used for cell-based assays and animal imaging studies, both in biomedical research and drug development. Its main advantages include its high-throughput applicability, affordability, high sensitivity, operational simplicity, and quantitative outputs. In malaria research, bioluminescence has been used for drug discovery in vivo and in vitro, exploring host-pathogen interactions, and studying multiple aspects of Plasmodium biology. While the number of fluorescent proteins available for imaging has undergone a great expansion over the last two decades, enabling simultaneous visualization of multiple molecular and cellular events, expansion of available luciferases has lagged behind. The most widely used bioluminescent probe in malaria research is the Photinus pyralis firefly luciferase, followed by the more recently introduced Click-beetle and Renilla luciferases. Ultra-sensitive imaging of Plasmodium at low parasite densities has not been previously achieved. With the purpose of overcoming these challenges, a Plasmodium berghei line expressing the novel ultra-bright luciferase enzyme NanoLuc, called PbNLuc has been generated, and is presented in this work. NanoLuc shows at least 150 times brighter signal than firefly luciferase in vitro, allowing single parasite detection in mosquito, liver, and sexual and asexual blood stages. As a proof-of-concept, the PbNLuc parasites were used to image parasite development in the mosquito, liver and blood stages of infection, and to specifically explore parasite liver stage egress, and pre-patency period in vivo. PbNLuc is a suitable parasite line for sensitive imaging of the entire Plasmodium life cycle. Its sensitivity makes it a promising line to be used as a reference for drug candidate testing, as well as the characterization of mutant parasites to explore the function of parasite proteins, host-parasite interactions, and the better understanding of Plasmodium biology. Since the substrate requirements of NanoLuc are different from those of firefly luciferase, dual bioluminescence imaging for the simultaneous characterization of two lines, or two separate biological processes, is possible, as demonstrated in this work.

Analysis of JC virus DNA replication using a quantitative and high-throughput assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Jong; Phelan, Paul J.; Chhum, Panharith

2014-11-15

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCVmore » DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.« less

Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

PubMed

Kopechek, Jonathan A; Carson, Andrew R; McTiernan, Charles F; Chen, Xucai; Klein, Edwin C; Villanueva, Flordeliza S

2016-01-01

RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.

Aurantiamide acetate from baphicacanthus cusia root exhibits anti-inflammatory and anti-viral effects via inhibition of the NF-κB signaling pathway in Influenza A virus-infected cells.

PubMed

Zhou, Beixian; Yang, Zifeng; Feng, Qitong; Liang, Xiaoli; Li, Jing; Zanin, Mark; Jiang, Zhihong; Zhong, Nanshan

2017-03-06

Baphicacanthus cusia root also names "Nan Ban Lan Gen" has been traditionally used to prevent and treat influenza A virus infections. Here, we identified a peptide derivative, aurantiamide acetate (compound E17), as an active compound in extracts of B. cusia root. Although studies have shown that aurantiamide acetate possesses antioxidant and anti-inflammatory properties, the effects and mechanism by which it functions as an anti-viral or as an anti-inflammatory during influenza virus infection are poorly defined. Here we investigated the anti-viral activity and possible mechanism of compound E17 against influenza virus infection. The anti-viral activity of compound E17 against Influenza A virus (IAV) was determined using the cytopathic effect (CPE) inhibition assay. Viruses were titrated on Madin-Darby canine kidney (MDCK) cells by plaque assays. Ribonucleoprotein (RNP) luciferase reporter assay was further conducted to investigate the effect of compound E17 on the activity of the viral polymerase complex. HEK293T cells with a stably transfected NF-κB luciferase reporter plasmid were employed to examine the activity of compound E17 on NF-κB activation. Activation of the host signaling pathway induced by IAV infection in the absence or presence of compound E17 was assessed by western blotting. The effect of compound E17 on IAV-induced expression of pro-inflammatory cytokines was measured by real-time quantitative PCR and Luminex assays. Compound E17 exerted an inhibitory effect on IAV replication in MDCK cells but had no effect on avian IAV and influenza B virus. Treatment with compound E17 resulted in a reduction of RNP activity and virus titers. Compound E17 treatment inhibited the transcriptional activity of NF-κB in a NF-κB luciferase reporter stable HEK293 cell after stimulation with TNF-α. Furthermore, compound E17 blocked the activation of the NF-κB signaling pathway and decreased mRNA expression levels of pro-inflammatory genes in infected cells. Compound E17 also suppressed the production of IL-6, TNF-α, IL-8, IP-10 and RANTES from IAV-infected lung epithelial (A549) cells. These results indicate that compound E17 isolated from B. cusia root has potent anti-viral and anti-inflammatory effects on IAV-infected cells via inhibition of the NF-κB pathway. Therefore, compound E17 could be a potential therapeutic agent for the treatment of influenza. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

MiR-320 inhibits the growth of glioma cells through downregulating PBX3.

PubMed

Pan, Cuicui; Gao, Hua; Zheng, Ni; Gao, Qi; Si, Yuanquan; Zhao, Yueran

2017-09-21

MiR-320 is downregulated in multiple cancers, including glioma and acts as tumor suppressor through inhibiting tumor cells proliferation and inducing apoptosis. PBX3 (Pre-B cell leukemia homeobox 3), a putative target gene of miR-320, has been reported to be upregulated in various tumors and promote tumor cell growth through regulating MAKP/ERK pathway. This study aimed to verify whether miR-320 influences glioma cells growth through regulating PBX3. Twenty-four human glioma and paired adjacent nontumorous tissues were collected for determination of miR-320 and PBX3 expression using RT-qPCR and western blot assays. Luciferase reporter assay was performed to verify the interaction between miR-320 and its targeting sequence in the 3' UTR of PBX3 in glioma cells U87 and U251. Increased miR-320 level in U87 and U251 cells was achieved through miR-320 mimic transfection and the effect of which on glioma cells growth, proliferation, cell cycle, apoptosis and activation of Raf-1/MAPK pathway was determined using MTT, colony formation, flow cytometry and western blot assays. PBX3 knockdown was performed using shPBX3 and the influence on MAPK pathway activation was evaluated. MiR-320 downregulation and PBX3 upregulation was found in glioma tissues. Luciferase reporter assays identified miR-320 directly blinds to the 3' UTR of PBX3 in glioma cells. MiR-320 mimic transfection suppressed glioma cells proliferation, and induced cell cycle arrest and apoptosis. Both miR-320 overexpression and PBX3 knockdown inhibited Raf-1/MAPK activation. MiR-320 may suppress glioma cells growth and induced apoptosis through the PBX3/Raf-1/MAPK axis, and miR-320 oligonucleotides may be a potential cancer therapeutic for glioma.

Effect of brain- and tumor-derived connective tissue growth factor on glioma invasion.

PubMed

Edwards, Lincoln A; Woolard, Kevin; Son, Myung Jin; Li, Aiguo; Lee, Jeongwu; Ene, Chibawanye; Mantey, Samuel A; Maric, Dragan; Song, Hua; Belova, Galina; Jensen, Robert T; Zhang, Wei; Fine, Howard A

2011-08-03

Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets. Highly infiltrative patient-derived glioma tumor-initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin β1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided. Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1-TrkA receptor complexes and nuclear factor kappa B (NF-κB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF(200 ng/mL): 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2, P < .001). NF-κB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF(200 ng/mL) + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most infiltrative glioma cells in situ and that the surrounding reactive astrocytes secrete CTGF. A CTGF-rich microenvironment facilitates CTGF-ITGB1-TrkA complex activation in TIC/TSCs, thereby increasing the invasiveness of malignant gliomas.

Novel functional Renilla luciferase mutant provides long-term serum stability and high luminescence activity.

PubMed

Song, Woo Chul; Sung, Hye-Jin; Park, Kyung Soo; Choi, Jeong-Woo; Cho, Je-Yeol; Um, Soong Ho

2013-10-01

Fluorescent and luminescent chemical probes are essential in recent medical diagnostics. However, the use of these probes in vivo has raised concerns due to their low sensitivity, background signal interference, and non-biocompatibility. Therefore, biological chromophores have received much attention as new alternatives. In particular, luciferase, a class of oxidative enzyme with bioluminescence, has emerged as a promising fluorophore due to its improved biocompatibility. However, the enzyme usually possesses weaker luminescence and stability relative to its chemically-based competitors. Here, we report a novel functional mutant luciferase with both enhanced luminescence and long-term serum stability. For the preparation of the modified Renilla luciferase, a new bacterial subcloning design was established. The luciferase coding DNA sequence was redesigned so that mutant luciferase could be easily expressed in an Escherichia coli system. The mutant Renilla luciferase, which we called "m-Rluc," demonstrated characteristic enzymatic functions and showed a 5.6-fold increase in luminescence activity. In addition, the enzyme's physiological stability remained >80% for more than 5days, in contrast to conventional luciferase, termed "hrluc," which disappeared within a few hours. We suggest that this novel biological luciferase probe may be a great tool for both in vitro and in vivo medical diagnostics. Copyright © 2013 Elsevier Inc. All rights reserved.

Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches

PubMed Central

Ekland, Eric H.; Schneider, Jessica; Fidock, David A.

2011-01-01

Malarial parasites have evolved resistance to all previously used therapies, and recent evidence suggests emerging resistance to the first-line artemisinins. To identify antimalarials with novel mechanisms of action, we have developed a high-throughput screen targeting the apicoplast organelle of Plasmodium falciparum. Antibiotics known to interfere with this organelle, such as azithromycin, exhibit an unusual phenotype whereby the progeny of drug-treated parasites die. Our screen exploits this phenomenon by assaying for “delayed death” compounds that exhibit a higher potency after two cycles of intraerythrocytic development compared to one. We report a primary assay employing parasites with an integrated copy of a firefly luciferase reporter gene and a secondary flow cytometry-based assay using a nucleic acid stain paired with a mitochondrial vital dye. Screening of the U.S. National Institutes of Health Clinical Collection identified known and novel antimalarials including kitasamycin. This inexpensive macrolide, used for agricultural applications, exhibited an in vitro IC50 in the 50 nM range, comparable to the 30 nM activity of our control drug, azithromycin. Imaging and pharmacologic studies confirmed kitasamycin action against the apicoplast, and in vivo activity was observed in a murine malaria model. These assays provide the foundation for high-throughput campaigns to identify novel chemotypes for combination therapies to treat multidrug-resistant malaria.—Ekland, E. H., Schneider, J., Fidock, D. A. Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches. PMID:21746861

Regulatory motifs for CREB-binding protein and Nfe2l2 transcription factors in the upstream enhancer of the mitochondrial uncoupling protein 1 gene.

PubMed

Rim, Jong S; Kozak, Leslie P

2002-09-13

Thermogenesis against cold exposure in mammals occurs in brown adipose tissue (BAT) through mitochondrial uncoupling protein (UCP1). Expression of the Ucp1 gene is unique in brown adipocytes and is regulated tightly. The 5'-flanking region of the mouse Ucp1 gene contains cis-acting elements including PPRE, TRE, and four half-site cAMP-responsive elements (CRE) with BAT-specific enhancer elements. In the course of analyzing how these half-site CREs are involved in Ucp1 expression, we found that a DNA regulatory element for NF-E2 overlaps CRE2. Electrophoretic mobility shift assay and competition assays with the CRE2 element indicates that nuclear proteins from BAT, inguinal fat, and retroperitoneal fat tissue interact with the CRE2 motif (CGTCA) in a specific manner. A supershift assay using an antibody against the CRE-binding protein (CREB) shows specific affinity to the complex from CRE2 and nuclear extract of BAT. Additionally, Western blot analysis for phospho-CREB/ATF1 shows an increase in phosphorylation of CREB/ATF1 in HIB-1B cells after norepinephrine treatment. Transient transfection assay using luciferase reporter constructs also indicates that the two half-site CREs are involved in transcriptional regulation of Ucp1 in response to norepinephrine and cAMP. We also show that a second DNA regulatory element for NF-E2 is located upstream of the CRE2 region. This element, which is found in a similar location in the 5'-flanking region of the human and rodent Ucp1 genes, shows specific binding to rat and human NF-E2 by electrophoretic mobility shift assay with nuclear extracts from brown fat. Co-transfections with an Nfe2l2 expression vector and a luciferase reporter construct of the Ucp1 enhancer region provide additional evidence that Nfe2l2 is involved in the regulation of Ucp1 by cAMP-mediated signaling.

C/EBPβ Promotes STAT3 Expression and Affects Cell Apoptosis and Proliferation in Porcine Ovarian Granulosa Cells.

PubMed

Yuan, Xiaolong; Zhou, Xiaofeng; He, Yingting; Zhong, Yuyi; Zhang, Ailing; Zhang, Zhe; Zhang, Hao; Li, Jiaqi

2018-06-13

Previous studies suggest that signal transducer and activator of transcription 3 (STAT3) and CCAAT/enhancer binding protein beta (C/EBPβ) play an essential role in ovarian granulosa cells (GCs) for mammalian follicular development. Several C/EBPβ putative binding sites were previously predicted on the STAT3 promoter in mammals. However, the molecular regulation of C/EBPβ on STAT3 and their effects on cell proliferation and apoptosis remain virtually unexplored in GCs. Using porcine GCs as a model, the 5′-deletion, luciferase report assay, mutation, chromatin immunoprecipitation, Annexin-V/PI staining and EdU assays were applied to investigate the molecular mechanism for C/EBPβ regulating the expression of STAT3 and their effects on the cell proliferation and apoptosis ability. We found that over and interfering with the expression of C/EBPβ significantly increased and decreased the messenger RNA (mRNA) and protein levels of STAT3 , respectively. The dual luciferase reporter assay showed that C/EBPβ directly bound at −1397/−1387 of STAT3 to positively regulate the mRNA and protein expressions of STAT3 . Both C/EBPβ and STAT3 were observed to inhibit cell apoptosis and promote cell proliferation. Furthermore, C/EBPβ might enhance the antiapoptotic and pro-proliferative effects of STAT3 . These results would be of great insight in further exploring the molecular mechanism of C/EBPβ and STAT3 on the function of GCs and the development of ovarian follicles in mammals.

Synthesis, biological evaluation and molecular modeling of GW 501516 analogues.

PubMed

Ciocoiu, Calin C; Ravna, Aina W; Sylte, Ingebrigt; Hansen, Trond Vidar

2010-11-01

Eleven analogues of GW 501516 (1) were prepared and subjected to biological testing in a semi-high throughput human skeletal muscle cell assay. The assay testing indicated that all analogues elicited oxidation of oleic acid. Among the most potent agonists, 2e (2-{2-ethyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)thiazol-5-yl)methylthio]phenoxy}-2-methylpropanoic acid), was also subjected to a luciferase-based transfection assay, which showed that this compound is a potent agonist against PPARδ and a moderate agonist against PPARα. Docking of compound 2e into PPARδ revealed that it occupied the agonist binding site and exhibited key hydrogen bonding interactions with His323, His449, and Tyr473.

miR-494 represses HOXA10 expression and inhibits cell proliferation in oral cancer.

PubMed

Libório-Kimura, Tatiana N; Jung, Hyun Min; Chan, Edward K L

2015-02-01

miR-494 was identified as a candidate of the most significantly underexpressed microRNAs (miRNAs) in our oral cancer screen. The aim of this study was to validate whether miR-494 has a functional role in oral cancer. Quantitative miRNA analyses were performed on oral tumor RNA and oral cancer cell lines. HOXA10 was selected for further analysis based on bioinformatics analysis of miR-494 targets and a previous report of overexpression of HOXA10 in oral cancer. Transient transfection of miRNA-mimic and inhibitor were performed in SCC-25 (tongue), CAL 27 (tongue), and FaDu (pharynx) cancer cells and regulation of HOXA10 by miR-494 was investigated. Dual luciferase assay was used to verify the interaction between miR-494 and HOXA10 in reporter cells. The effect of miR-494 on cell proliferation was examined. Our data showed that miR-494 was underexpressed whereas HOXA10 was overexpressed in oral cancer compared to normal tissues. An inverse correlation between miR-494 and HOXA10 was observed in the human tissues (p<0.05). Transient transfection of miR-494 in all cancer cell lines significantly reduced the expression of HOXA10 mRNA. The luciferase reporter that contains the 3'UTR of HOXA10 showed a significantly reduced luciferase activity by miR-494 indicating a direct interaction between HOXA10 and miR-494. Significant reduction in cell proliferation was demonstrated in tongue cancer cells transfected with miR-494. miR-494 repressed the expression of HOXA10 and also reduced the proliferation of oral cancer cells. These data give more evidence of the role of miR-494 as a tumor suppressor miRNA in oral cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

A minimal murine Msx-1 gene promoter. Organization of its cis-regulatory motifs and their role in transcriptional activation in cells in culture and in transgenic mice.

PubMed

Takahashi, T; Guron, C; Shetty, S; Matsui, H; Raghow, R

1997-09-05

To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from -1282 to +106 base pairs (bp)) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C2C12 cells, and the expression of luciferase was assessed in transient expression assays. The functional consequences of the sequential 5' deletions of the promotor revealed that multiple positive and negative regulatory elements participate in regulating transcription of the Msx-1 gene. Surprisingly, however, the optimal expression of Msx-1 promoter in either NIH3T3 or C2C12 cells required only 165 bp of the upstream sequence to warrant detailed examination of its structure. Therefore, the functional consequences of site-specific deletions and point mutations of the cis-acting elements of the minimal Msx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with the cis-acting elements of the minimal promoter were also studied by gel electrophoretic mobility shift assays and DNase I footprinting. Combined analyses of the minimal promoter by DNase I footprinting, electrophoretic mobility shift assays, and super shift assays with specific antibodies revealed that 5'-flanking regions from -161 to -154 and from -26 to -13 of the Msx-1 promoter contains an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1), respectively. Additionally, we observed that the promoter activation was seriously hampered if the proximal E box was removed or mutated, and the promoter activity was eliminated completely if the proximal Sp1 site was similarly altered. Absolute dependence of the Msx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient Drosophila cell line cotransfected with Msx-1-luciferase and an Sp1 expression vector pPacSp1. The transgenic mice embryos containing -165/106-bp Msx-1 promoter-LacZ DNA in their genomes abundantly expressed beta-galactosidase in maxillae and mandibles and in the cellular primordia involved in the formation of the meninges and the bones of the skull. Thus, the truncated murine Msx-1 promoter can target expression of a heterologous gene in the craniofacial tissues of transgenic embryos known for high level of expression of the endogenous Msx-1 gene and found to be severely defective in the Msx-1 knock-out mice.

Diversity in Genetic In Vivo Methods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

PubMed Central

Stynen, Bram; Tournu, Hélène; Tavernier, Jan

2012-01-01

Summary: The yeast two-hybrid system pioneered the field of in vivo protein-protein interaction methods and undisputedly gave rise to a palette of ingenious techniques that are constantly pushing further the limits of the original method. Sensitivity and selectivity have improved because of various technical tricks and experimental designs. Here we present an exhaustive overview of the genetic approaches available to study in vivo binary protein interactions, based on two-hybrid and protein fragment complementation assays. These methods have been engineered and employed successfully in microorganisms such as Saccharomyces cerevisiae and Escherichia coli, but also in higher eukaryotes. From single binary pairwise interactions to whole-genome interactome mapping, the self-reassembly concept has been employed widely. Innovative studies report the use of proteins such as ubiquitin, dihydrofolate reductase, and adenylate cyclase as reconstituted reporters. Protein fragment complementation assays have extended the possibilities in protein-protein interaction studies, with technologies that enable spatial and temporal analyses of protein complexes. In addition, one-hybrid and three-hybrid systems have broadened the types of interactions that can be studied and the findings that can be obtained. Applications of these technologies are discussed, together with the advantages and limitations of the available assays. PMID:22688816

The split Renilla luciferase complementation assay is useful for identifying the interaction of Epstein-Barr virus protein kinase BGLF4 and a heat shock protein Hsp90.

PubMed

Wang, J; Guo, W; Long, C; Zhou, H; Wang, H; Sun, X

2016-03-01

Protein-protein interactions can regulate different cellular processes, such as transcription, translation, and oncogenic transformation. The split Renilla luciferase complementation assay (SRLCA) is one of the techniques that detect protein-protein interactions. The SRLCA is based on the complementation of the LN and LC non-functional halves of Renilla luciferase fused to possibly interacting proteins which after interaction form a functional enzyme and emit luminescence. The BGLF4 of Epstein-Barr virus (EBV) is a viral protein kinase that is expressed during the early and late stages of lytic cycles, which can regulate multiple cellular and viral substrates to optimize the DNA replication environment. The heat shock protein Hsp90 is a molecular chaperone that maintains the integrity of structure and function of various interacting proteins, which can form a complex with BGLF4 and stabilize its expression in cells. The interaction between BGLF4 and Hsp90 could be specifically detected through the SRLCA. The region of aa 250-295 of BGLF4 is essential for the BGLF4/Hsp90 interaction and the mutation of Phe-254, Leu-266, and Leu-267 can disrupt this interaction. These results suggest that the SRLCA can specifically detect the BGLF4/Hsp90 interaction and provide a reference to develop inhibitors that disrupt the BGLF4/Hsp90 interaction.

Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro.

PubMed

Jung, Song-yi; Willard, Scott T

2014-01-30

The porcine oocyte maturation in vivo occurs within the ovarian follicle and is regulated by the interactions between oocytes and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. Therefore, the antral follicle is an essential microenvironment for efficient oocyte maturation and its developmental competence. Quantitative bioluminescence imaging of firefly luciferase reporter genes in an intact antral follicle would allow investigation of changes in cellular and molecular events and in the context of the whole follicles. In this study, we investigate factors influencing bioluminescence measurements as a first step towards developing a new bioluminescence imaging system for intact antral follicles. We analyzed the time course of bioluminescence emitted from transfected living intact follicles using a cationic lipid mediated gene transfer method with increasing doses (1-3 μg) of firefly luciferase reporter gene (pGL4). In addition, a standard luciferase assay was used to confirm the luciferase expression in granulosa cells in the transfected intact antral follicles. Finally, the dose effects of substrate, D-luciferin, were determined for optimal quantitative bioluminescence imaging of intact porcine antral follicles in vitro. The level of luciferase activity of follicles with 3 μg pGL4 was significantly (P < 0.05) greater than the 1 μg and 2 μg groups at 1 min after D-luciferin injection. The bioluminescence intensity of transfected follicles reached a peak at 1 min, and then it was significantly (P < 0.05) reduced within 2 min after injection of D-luciferin; with the level of bioluminescence emission remained constant from 2.5 to 10 min. The bioluminescence emission was maximal with 300 μg of D-luciferin. The results of this study suggested that the investigation of factors influencing bioluminescence measurements is a critical step toward developing a new bioluminescence imaging model. This study is the first to demonstrate that reporter genes can be transferred to intact granulosa cells with a lipid-mediated gene transfer method within intact follicles in vitro, and the level of transgene expression can be assessed by bioluminescence imaging in living intact antral follicles.

Synergistic cooperation of Sall4 and Cyclin D1 in transcriptional repression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boehm, Johann; Kaiser, Frank J.; Borozdin, Wiktor

2007-05-11

Loss of function mutations in SALL4 cause Okihiro syndrome, an autosomal dominant disorder characterised by radial ray malformations associated with Duane anomaly. In zebrafish and mouse Sall4 interacts with TBX5 during limb and heart development and plays a crucial role for embryonic stem (ES) cell pluripotency. Here we report the nuclear interaction of murine Sall4 with Cyclin D1, one of the main regulators of G{sub 1} to S phase transition in cell cycle, verified by yeast two-hybrid assay, co-immunoprecipitation and intracellular co-localisation. Furthermore, using luciferase reporter gene assays we demonstrate that Sall4 operates as a transcriptional repressor located to heterochromatinmore » and that this activity is modulated by Cyclin D1.« less

The human insulin receptor mRNA contains a functional internal ribosome entry segment

PubMed Central

Spriggs, Keith A.; Cobbold, Laura C.; Ridley, Simon H.; Coldwell, Mark; Bottley, Andrew; Bushell, Martin; Willis, Anne E.; Siddle, Kenneth

2009-01-01

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5′-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective. PMID:19654240

Hu antigen R (HuR) multimerization contributes to glioma disease progression.

PubMed

Filippova, Natalia; Yang, Xiuhua; Ananthan, Subramaniam; Sorochinsky, Anastasia; Hackney, James R; Gentry, Zachery; Bae, Sejong; King, Peter; Nabors, L Burt

2017-10-13

Among primary brain cancers, gliomas are the most deadly and most refractory to current treatment modalities. Previous reports overwhelmingly support the role of the RNA-binding protein Hu antigen R (HuR) as a positive regulator of glioma disease progression. HuR expression is consistently elevated in tumor tissues, and a cytoplasmic localization appears essential for HuR-dependent oncogenic transformation. Here, we report HuR aggregation (multimerization) in glioma and the analysis of this tumor-specific HuR protein multimerization in clinical brain tumor samples. Using a split luciferase assay, a bioluminescence resonance energy transfer technique, and site-directed mutagenesis, we examined the domains involved in HuR multimerization. Results obtained with the combination of the split HuR luciferase assay with the bioluminescence resonance energy transfer technique suggested that multiple (at least three) HuR molecules come together during HuR multimerization in glioma cells. Using these data, we developed a model of HuR multimerization in glioma cells. We also demonstrate that exposing glioma cells to the HuR inhibitor tanshinone group compound 15,16-dihydrotanshinone-I or to the newly identified compound 5 disrupts HuR multimerization modules and reduces tumor cell survival and proliferation. In summary, our findings provide new insights into HuR multimerization in glioma and highlight possible pharmacological approaches for targeting HuR domains involved in cancer cell-specific multimerization.

Mutations in the Promoter Region of the Aldolase B Gene that cause Hereditary Fructose Intolerance

PubMed Central

Coffee, Erin M.; Tolan, Dean R.

2010-01-01

SUMMARY Hereditary fructose intolerance (HFI) is a potentially fatal inherited metabolic disease caused by a deficiency of aldolase B activity in the liver and kidney. Over 40 disease-causing mutations are known in the protein-coding region of ALDOB. Mutations upstream of the protein-coding portion of ALDOB are reported here for the first time. DNA sequence analysis of 61 HFI patients revealed single base mutations in the promoter, intronic enhancer, and the first exon, which is entirely untranslated. One mutation, g.–132G>A, is located within the promoter at an evolutionarily conserved nucleotide within a transcription factor-binding site. A second mutation, IVS1+1G>C, is at the donor splice site of the first exon. In vitro electrophoretic mobility shift assays show a decrease in nuclear extract-protein binding at the g.–132G>A mutant site. The promoter mutation results in decreased transcription using luciferase reporter plasmids. Analysis of cDNA from cells transfected with plasmids harboring the IVS1+1G>C mutation results in aberrant splicing leading to complete retention of the first intron (~ 5 kb). The IVS1+1G>C splicing mutation results in loss of luciferase activity from a reporter plasmid. These novel mutations in ALDOB represent 2% of alleles in American HFI patients, with IVS1+1G>C representing a significantly higher allele frequency (6%) among HFI patients of Hispanic and African-American ethnicity. PMID:20882353

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

PubMed Central

Baston, David S.; Denison, Michael S.

2011-01-01

The chemically activated luciferase expression (CALUX) system is a mechanistically based recombinant luciferase reporter gene cell bioassay used in combination with chemical extraction and clean-up methods for the detection and relative quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like halogenated aromatic hydrocarbons in a wide variety of sample matrices. While sample extracts containing complex mixtures of chemicals can produce a variety of distinct concentration-dependent luciferase induction responses in CALUX cells, these effects are produced through a common mechanism of action (i.e. the Ah receptor (AhR)) allowing normalization of results and sample potency determination. Here we describe the diversity in CALUX response to PCDD/Fs from sediment and soil extracts and not only report the occurrence of superinduction of the CALUX bioassay, but we describe a mechanistically based approach for normalization of superinduction data that results in a more accurate estimation of the relative potency of such sample extracts. PMID:21238730

The human tartrate-resistant acid phosphatase (TRAP): involvement of the hemin responsive elements (HRE) in transcriptional regulation.

PubMed

Fleckenstein, E C; Dirks, W G; Drexler, H G

2000-02-01

The biochemical properties and protein structure of the tartrate-resistant acid phosphatase (TRAP), an iron-containing lysosomal glycoprotein in cells of the mononuclear phagocyte system, are well known. In contrast, little is known about the physiology and genic structure of this unique enzyme. In some diseases, like hairy cell leukemia, Gaucher's disease and osteoclastoma, cytochemically detected TRAP expression is used as a disease-associated marker. In order to begin to elucidate the regulation of this gene we generated different deletion constructs of the TRAP 5'-flanking region, placed them upstream of the luciferase reporter gene and assayed them for their ability to direct luciferase expression in human 293 cells. Treatment of these cells with the iron-modulating reagents transferrin and hemin causes opposite effects on the TRAP promoter activity. Two regulatory GAGGC tandem repeat sequences (the hemin responsive elements, HRE) within the 5'-flanking region of the human TRAP gene were identified. Studies with specific HRE-deletion constructs of the human TRAP 5'-flanking region upstream of the luciferase reporter gene document the functionality of these HRE-sequences which are apparently responsible for mediating transcriptional inhibition upon exposure to hemin. In addition to the previously published functional characterization of the murine TRAP HRE motifs, these results provide the first description of a new iron/hemin-responsive transcriptional regulation in the human TRAP gene.

In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs

PubMed Central

Röck, Ruth; Bachmann, Verena; Bhang, Hyo-eun C; Malleshaiah, Mohan; Raffeiner, Philipp; Mayrhofer, Johanna E; Tschaikner, Philipp M; Bister, Klaus; Aanstad, Pia; Pomper, Martin G; Michnick, Stephen W; Stefan, Eduard

2015-01-01

Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems. PMID:26099953

Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model.

PubMed

Liu, Yu; Song, Ge; Shao, Xiao-Xia; Liu, Ya-Li; Guo, Zhan-Yun

2015-02-01

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence to date. In the present work, we developed NanoLuc as a sensitive bioluminescent reporter to measure quantitatively the internalization of cell membrane receptors, based on the pH dependence of the reporter activity. The G protein-coupled receptor RXFP3, the cognate receptor of relaxin-3/INSL7, was used as a model receptor. We first generated stable HEK293T cells that inducibly coexpressed a C-terminally NanoLuc-tagged human RXFP3 and a C-terminally enhanced green fluorescent protein (EGFP)-tagged human RXFP3. The C-terminal EGFP-tag and NanoLuc-tag had no detrimental effects on the ligand-binding potency and intracellular trafficking of RXFP3. Based on the fluorescence of the tagged EGFP reporter, the ligand-induced RXFP3 internalization was visualized directly under a fluorescence microscope. Based on the bioluminescence of the tagged NanoLuc reporter, the ligand-induced RXFP3 internalization was measured quantitatively by a convenient bioluminescent assay. Coexpression of an EGFP-tagged inactive [E141R]RXFP3 had no detrimental effect on the ligand-binding potency and ligand-induced internalization of the NanoLuc-tagged wild-type RXFP3, suggesting that the mutant RXFP3 and wild-type RXFP3 worked independently. The present bioluminescent internalization assay could be extended to other G protein-coupled receptors and other cell membrane receptors to study ligand-receptor and receptor-receptor interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1.

PubMed

Guo, Xiaodong; Yu, Ling; Zhang, Zhengpei; Dai, Guo; Gao, Tian; Guo, Weichun

2017-01-01

Evidence is accumulating to link cancer stem cells to the pathogenesis and progression of osteosarcoma. The aim of this study is to investigate the role of miR-335 in osteosarcoma stem cells. Tumor spheroid culture and flow cytometry were applied to screen out osteosarcoma stem cells. Real-time quantitative PCR was used to detect the expression level of miR-335 in MG63, U2OS and 143B osteosarcoma stem cells. The relationship of miR-335 expression with osteosarcoma stem cells was then analyzed. Transwell assay and transplantation assay were performed to elucidate biological effects of miR-335 on cell invasion and vivo tumor formation. Western Blot and luciferase assays were executed to investigate the regulation of POU5F1 by miR-335. The expression of miR-335 in osteosarcoma stem cells was lower than their differentiated counterparts. Cells expressing miR-335 possessed decreased stem cell-like properties. Gain or loss of function assays were applied to find that miR-335 antagonist promoted stem cell-like properties as well as invasion. Luciferase report and transfection assay showed that POU5F1 was downregulated by miR-335. Pre-miR-335 resulted in tumor enhanced sensitivity to traditional chemotherapy, whereas anti-miR-335 promoted chemoresistance. Finally, the inhibitory effect of miR-335 on in vivo tumor formation showed that combination of pre-miR-335 with cisplatin further reduced the tumor size, and miR-335 brought down the sphere formation capacity induced by cisplatin. The current study demonstrates that miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1, and miR-335 could target CSCs to synergize with traditional chemotherapeutic agents to overcome osteosarcoma.

Long noncoding RNA GAPLINC promotes gastric cancer cell proliferation by acting as a molecular sponge of miR-378 to modulate MAPK1 expression.

PubMed

Diao, Lingyun; Wang, Shengying; Sun, Zhiguang

2018-01-01

Dysregulated long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play key roles in the development of human cancers. LncRNA GAPLINC has been reported to be increased in gastric cancer (GC) tissues. Real-time PCR assays were used to measure expressions of GAPLINC, miR-378, and MAPK1 mRNA. Western blot assays were employed to examine MAPK1 protein expression. Cell proliferation and cell cycle were measured by CCK-8 and propidium iodide-detection assays, respectively. The interaction between GAPLINC and miR-378 was confirmed by site-directed mutagenesis and luciferase assays. Luciferase assays were also used to study whether GAPLINC was able to act as a molecular sponge of miR-378 to modulate MAPK1 expression. The lncRNA GAPLINC expression was upregulated and positively correlated with MAPK1 expression in gastric cancer tissues and cells. Additionally, lncRNA GAPLINC promoted the expression of MAPK1 and the enhancement of GC cell proliferation and cell cycle progression by LncRNA GAPLINC was dependent on MAPK1 in vitro and in vivo. Consequently, we found that miR-378 expression was inversely correlated with GAPLINC expression in GC tissues and cells. miR-378 could directly bind to GAPLINC and decreased GAPLINC expression, thus reducing MAPK1 expression. Furthermore, overexpression of miR-378 inhibited MAPK1 expression, cell proliferation, and cell cycle progression of gastric cancer cells, while these effects were abrogated by upregulating lncRNA GAPLINC expression. Taken together, lncRNA GAPLINC promotes gastric cancer cell proliferation by acting as a molecular sponge of miR-378 to modulate MAPK1 expression.

Disposable luciferase-based microfluidic chip for rapid assay of water pollution.

PubMed

Denisov, Ivan; Lukyanenko, Kirill; Yakimov, Anton; Kukhtevich, Igor; Esimbekova, Elena; Belobrov, Peter

2018-06-21

In the present study, we demonstrate the use of a disposable luciferase-based microfluidic bioassay chip for environmental monitoring and methods for fabrication. The designed microfluidic system includes a chamber with immobilized enzymes of bioluminescent bacteria Photobacterium leiognathi and Vibrio fischeri and their substrates, which dissolve after the introduction of the water sample and thus activate bioluminescent reactions. Limits of detection for copper (II) sulfate, 1,3-dihydroxybenzene and 1,4-benzoquinone for the proposed microfluidic biosensor measured 3 μM, 15 mM, and 2 μM respectively, and these values are higher or close to the level of conventional environmental biosensors based on lyophilized bacteria. Approaches for entrapment of enzymes on poly(methyl methacrylate) (PMMA) plates using a gelatin scaffold and solvent bonding of PMMA chip plates under room temperature were suggested. The proposed microfluidic system may be used with some available luminometers and future portable luminescence readers. © 2018 John Wiley & Sons, Ltd.

Comparison of bioluminescent kinase assays using substrate depletion and product formation.

PubMed

Tanega, Cordelle; Shen, Min; Mott, Bryan T; Thomas, Craig J; MacArthur, Ryan; Inglese, James; Auld, Douglas S

2009-12-01

Assays for ATPases have been enabled for high-throughput screening (HTS) by employing firefly luciferase to detect the remaining ATP in the assay. However, for any enzyme assay, measurement of product formation is a more sensitive assay design. Recently, technologies that allow detection of the ADP product from ATPase reactions have been described using fluorescent methods of detection. We describe here the characterization of a bioluminescent assay that employs firefly luciferase in a coupled-enzyme assay format to enable detection of ADP levels from ATPase assays (ADP-Glo, Promega Corp.). We determined the performance of the ADP-Glo assay in 1,536-well microtiter plates using the protein kinase Clk4 and a 1,352 member kinase focused combinatorial library. The ADP-Glo assay was compared to the Clk4 assay performed using a bioluminescence ATP-depletion format (Kinase-Glo, Promega Corp). We performed this analysis using quantitative HTS (qHTS) where we determined potency values for all library members and identified approximately 300 compounds with potencies ranging from as low as 50 nM to >10 microM, yielding a robust dataset for the comparison. Both assay formats showed high performance (Z'-factors approximately 0.9) and showed a similar potency distribution for the actives. We conclude that the bioluminescence ADP detection assay system is a viable generic alternative to the widely used ATP-depletion assay for ATPases and discuss the advantages and disadvantages of both approaches.

A Novel Method for Determining the Level of Viable Disseminated Prostate Cancer Cells

DTIC Science & Technology

2012-10-01

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...need for multiple replicates of plates in time course studies. Recently a naturally secreted luciferase was identified and cloned from the marine ...well solid white flat bottom polystyrene microplates (Corning, Cat#3917, Lowell, MA). After 24 hours, conditioned media was harvested and remaining

High-throughput Titration of Luciferase-expressing Recombinant Viruses

PubMed Central

Garcia, Vanessa; Krishnan, Ramya; Davis, Colin; Batenchuk, Cory; Le Boeuf, Fabrice; Abdelbary, Hesham; Diallo, Jean-Simon

2014-01-01

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay. PMID:25285536

Dual regulation of skin sensitizer-induced HMOX1 expression by Bach1 and Nrf2: Comparison to regulation of the AKR1C2-ARE element in the KeratinoSens cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Emter, Roger; Natsch, Andreas, E-mail: andreas.natsch@givaudan.com

2015-11-01

Heme oxygenase (decycling) 1 (HMOX1) is the most consistently found genetic marker induced by skin sensitizers. HMOX1 is often referred to as typical gene regulated by nuclear factor erythroid 2-related factor 2 (Nrf2), however, it is also regulated by other DNA-binding factors, including BTB and CNC homolog 1 (Bach1). The KeratinoSens™ assay is the first validated in vitro assay for sensitizers that measures gene induction. It is based on luciferase expression regulated by the antioxidant response element (ARE) of the aldoketoreductase 1C2 (AKR1C2) gene. Luciferase upregulation is dependent on Nrf2, while HMOX1 upregulation is only partially Nrf2-dependent. Thus, sensitizer-dependent activationmore » of HMOX1 may integrate multiple signals thereby providing additional information. We constructed reporter cell lines containing the full HMOX1 regulatory region or the HMOX1-ARE sequence and compared them with the construct containing the AKR1C2-ARE sequence. Induction of the AKR1C2-ARE depends on Nrf2, but not on the repressor Bach1. Results obtained with HMOX1-ARE and the full HMOX1 promoter indicate that, within the HMOX1 promoter, the HMOX1-ARE is sufficient to explain the induction by sensitizers and that (i) inhibiting Bach1 leads to strong basal expression, (ii) fold-induction by sensitizers above this level is reduced in the absence of Bach1 and (iii) these constructs are less dependent on Nrf2 as compared to the AKR1C2-ARE. Nevertheless, congruent dose response curves for luciferase activity were obtained with all constructs. Thus, while sensitizer-induced HMOX1 activation is dependent on Nrf2 and Bach1, all constructs give identical information for the in vitro prediction of the sensitization potential. - Highlights: • HMOX1 is a key genetic marker up-regulated by skin sensitizers. • HMOX1-, but not AKR1C2-upregulation, is dependent on both Nrf2 and Bach1. • AKR1C2 and HMOX1-dependent reporter constructs yield congruent dose response curves. • Combining both constructs offers no advantage over either construct used alone.« less

MicroRNA-1231 exerts a tumor suppressor role through regulating the EGFR/PI3K/AKT axis in glioma.

PubMed

Zhang, Jiale; Zhang, Jie; Qiu, Wenjin; Zhang, Jian; Li, Yangyang; Kong, Enjun; Lu, Ailin; Xu, Jia; Lu, Xiaoming

2018-05-17

MicroRNAs (miRNAs) have been shown to be involved in the initiation and progression of glioma. However, the underlying molecular mechanisms are still unclear. We performed microarray analysis to evaluate miRNA expression levels in 158 glioma tissue samples, and examined miR-1231 levels in glioma samples and healthy brain tissues using qRT-PCR. In vitro analyses were performed using miR-1231 mimics, inhibitors, and siRNA targeting EGFR. We used flow cytometry, CCK-8 assays, and colony formation assays to examine glioma proliferation and cell cycle analysis. A dual luciferase reporter assay was performed to examine miR-1231 regulation of EGFR, and the effect of upregulated miR-1231 was investigated in a subcutaneous GBM model. We found that miR-1231 expression was decreased in human glioma tissues and negatively correlated with EGFR levels. Moreover, the downregulation of miR-1231 negatively correlated with the clinical stage of human glioma patients. miR-1231 overexpression dramatically downregulated glioma cell proliferation, and suppressed tumor growth in a nude mouse model. Bioinformatics prediction and a luciferase assay confirmed EGFR as a direct target of miR-1231. EGFR overexpression abrogated the suppressive effect of miR-1231 on the PI3K/AKT pathway and G1 arrest. Taken together, these results demonstrated that EGFR is a direct target of miR-1231. Our findings suggest that the miR-1231/EGFR axis may be a helpful future diagnostic target for malignant glioma.

Suppression of microRNA-629 enhances sensitivity of cervical cancer cells to 1'S-1'-acetoxychavicol acetate via regulating RSU1.

PubMed

Phuah, Neoh Hun; Azmi, Mohamad Nurul; Awang, Khalijah; Nagoor, Noor Hasima

2017-01-01

Cervical cancer is the fourth most frequent malignancy affecting women worldwide, but drug resistance and toxicities remain a major challenge in chemotherapy. The use of natural compounds is promising because they are less toxic and able to target multiple signaling pathways. The 1'S-1'-acetoxychavicol acetate (ACA), a natural compound isolated from wild ginger Alpinia conchigera , induced cytotoxicity on various cancer cells including cervical cancer. MicroRNAs (miRNAs) are short noncoding RNAs that regulate numerous biological processes, such as apoptosis and chemosensitivity. Past studies reported that miR-629 is upregulated in many cancers, and its expression was altered in ACA-treated cervical cancer cells. However, the role of miR-629 in regulating sensitivity toward ACA or other anticancer agents has not been reported. Hence, this study aims to investigate the role of miR-629 in regulating response toward ACA on cervical cancer cells. The miR-629 expression following transfection with miR-629 hairpin inhibitor and hairpin inhibitor negative control was measured using quantitative real-time polymerase chain reaction (RT-qPCR). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to investigate sensitivity toward ACA. Apoptosis was detected using Annexin V/propidium iodide and Caspase 3/7 assays. The gene target for miR-629 was identified using miRNA target prediction programs, luciferase reporter assay and Western blots. Gene overexpression studies were performed to evaluate its role in regulating response toward ACA. Transfection with miR-629 hairpin inhibitor downregulated its expression in both cervical cancer cell lines. Suppression of miR-629 increased sensitivity toward ACA by reducing cell proliferation and inducing apoptosis. Luciferase reporter assay confirmed RSU1 as a direct target of miR-629. Overexpression of miR-629 decreased RSU1 protein expression, while inhibition of miR-629 increased RSU1 protein expression. Overexpression of RSU1 augmented antiproliferative and apoptosis-inducing effects of ACA. Our findings showed that combination of ACA with miR-629 and RSU1 may provide a potential strategy in treating cervical cancer.

Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound

PubMed Central

McTiernan, Charles F.; Chen, Xucai; Klein, Edwin C.; Villanueva, Flordeliza S.

2016-01-01

RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease. PMID:27471848

Suppression of survivin promoter activity by YM155 involves disruption of Sp1-DNA interaction in the survivin core promoter

PubMed Central

Cheng, Qiuying; Ling, Xiang; Haller, Andrew; Nakahara, Takahito; Yamanaka, Kentaro; Kita, Aya; Koutoku, Hiroshi; Takeuchi, Masahiro; Brattain, Michael G; Li, Fengzhi

2012-01-01

YM155, a novel survivin suppressant, shows potent antitumor activity against various human cancers and is currently in phase II clinical trials. In this study, we investigated whether YM155 selectively inhibits survivin transcription. We hypothesize that inhibition of survivin transcription plays a role in YM155-mediated survivin inhibition. We found that YM155 inhibited survivin promoter activity, while it showed minimal inhibitory effect on four control gene promoters in transfection and luciferase activity assay experiments, indicating its selectivity. Transfection of various survivin promoter-luciferase constructs followed by luciferase assays revealed that the survivin core promoter (269 bp) plays a major role in YM155-mediated inhibitory effects. However, flow cytometry analysis indicated that inhibition of survivin promoter activity by YM155 is cell cycle-independent without G1 cell arrests. Electrophoretic mobility shift assays (EMSA) identified that YM155 abrogates nuclear proteins binding to the region of -149 to -71, in which Sp1 is a major candidate, and that YM155 treatment induces Sp1 re-subcellular localization without inhibiting its expression. Forced expression of Sp1 neutralized YM155-mediated downregulation of survivin promoter activity. Consistently, mutation of the identified Sp1 sites in the oligonucleotide probe diminished DNA-protein interactions in EMSA experiments, and mutation of the Sp1 sites in the survivin promoter-luciferase construct diminished survivin promoter activity. These findings indicate that YM155 inhibition of survivin expression is at least in part through its inhibition of survivin transcription by disruption of Sp1 interaction with the region of -149 to -71 in the survivin core promoter. PMID:22773958

Transcriptional regulation of α1H T-type calcium channel under hypoxia

PubMed Central

Sellak, Hassan; Zhou, Chun; Liu, Bainan; Chen, Hairu; Lincoln, Thomas M.

2014-01-01

The low-voltage-activated T-type Ca2+ channels play an important role in mediating the cellular responses to altered oxygen tension. Among three T-type channel isoforms, α1G, α1H, and α1I, only α1H was found to be upregulated under hypoxia. However, mechanisms underlying such hypoxia-dependent isoform-specific gene regulation remain incompletely understood. We, therefore, studied the hypoxia-dependent transcriptional regulation of α1G and α1H gene promoters with the aim to identify the functional hypoxia-response elements (HREs). In rat pulmonary artery smooth muscle cells (PASMCs) and pheochromocytoma (PC12) cells after hypoxia (3% O2) exposure, we observed a prominent increase in α1H mRNA at 12 h along with a significant rise in α1H-mediated T-type current at 24 and 48 h. We then cloned two promoter fragments from the 5′-flanking regions of rat α1G and α1H gene, 2,000 and 3,076 bp, respectively, and inserted these fragments into a luciferase reporter vector. Transient transfection of PASMCs and PC12 cells with these recombinant constructs and subsequent luciferase assay revealed a significant increase in luciferase activity from the reporter containing the α1H, but not α1G, promoter fragment under hypoxia. Using serial deletion and point mutation analysis strategies, we identified a functional HRE at site −1,173cacgc−1,169 within the α1H promoter region. Furthermore, an electrophoretic mobility shift assay using this site as a DNA probe demonstrated an increased binding activity to nuclear protein extracts from the cells after hypoxia exposure. Taken together, these findings indicate that hypoxia-induced α1H upregulation involves binding of hypoxia-inducible factor to an HRE within the α1H promoter region. PMID:25099734

Alpha-synuclein mRNA isoform formation and translation affected by polymorphism in the human SNCA 3'UTR.

PubMed

Barrie, Elizabeth S; Lee, Sung-Ha; Frater, John T; Kataki, Maria; Scharre, Douglas W; Sadee, Wolfgang

2018-05-06

Multiple variants in SNCA, encoding alpha-synuclein, a main component of Lewy bodies, are implicated in Parkinson's disease. We searched for cis-acting SNCA variants using allelic mRNA ratios in human brain tissues. In a SNCA 3'UTR (2,520 bp) luciferase reporter gene assay, translation in SH-SY5Y cells in the presence of the rs17016074 G/A alleles was measured. To assess clinical impact, we queried neurocognitive genome-wide association studies. Allelic ratios deviated up to twofold, measured at a marker SNP in the middle of a long 3' untranslated region (3'UTR), but not at a marker at its start, suggesting regulation of 3'UTR processing. 3'UTR SNP rs17016074 G/A, minor allele frequency (MAF) <1% in Caucasians, 13% in Africans, strongly associates with large allelic mRNA expression imbalance (AEI), resulting in reduced expression of long 3'UTR isoforms. A second 3'UTR SNP (rs356165) associates with moderate AEI and enhances SNCA mRNA expression. The rs17016074 A allele reduces overall 3'UTR expression in luciferase reporter gene assays but supports more efficient translation, resolving previous contradictory results. We failed to detect significant genome-wide associations for rs17016074, possibly a result of low MAF in Caucasians or its absence from most genotyping panels. In the "Genome Wide Association Study of Yoruba in Nigeria," rs356165 was associated with reduced memory performance. Here, we identify two cis-acting regulatory variants affecting SNCA mRNA expression, measured by allelic ratios in the 3'UTR. The rs17016074 minor A allele is associated with higher expression of luciferase protein activity. Resolving the genetic influence of SNCA polymorphisms requires study of the interactions between multiple regulatory variants with distinct frequencies among populations. © 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

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

PubMed

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

2014-01-24

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

Developing Breast Cancer Program at Xavier; Genomic and Proteomic Analysis of Signaling Pathways Involved in Xenohormone and MEK5 Regulation of Breast

DTIC Science & Technology

2008-05-01

will build a core of human talent that will address scientific problems such as drug resistance and the effect of environmental agents on breast...normal for Xavier University after the continuing effects of Hurricane Katrina. Xavier University of Louisiana did an amazing recovery from the...an increase in expression of ERβ . To determine the effects of MEK5 on ER signaling we used an ERE-luciferase reporter assay. Parental MCF-7 cells

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

PubMed

Nazari, Mahboobeh; Hosseinkhani, Saman; Hassani, Leila

2013-02-01

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

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

PubMed

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

2016-12-23

Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples ( n =5) and cervical intraepithelial neoplasia tissue samples ( n =5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P <0.05). The MTT assay revealed that the cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group ( P <0.05), and the cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group ( P <0.05) but higher than the miR-143 mimic group significantly ( P <0.05). The expression levels of K-ras protein in the miR-143 mimic group, the negative control group and the miR-143 mimic+ K-ras group were lowest, moderate, and highest, respectively (115.27±34.08, 521.36±41.89, and 706.52±89.44, all P <0.05). In the tissue samples, the miR-143 expression in the cervical cancer group was significantly lower than that of the cervical intraepithelial neoplasia group (0.32±0.06 vs. 0.93±0.17, P <0.05); whereas the K-ras protein expression in the cervical cancer group was significantly higher than that in the cervical intraepithelial neoplasia group (584.39±72.34 vs. 114.23±25.82, P <0.05). Conclusions: In vitro, miR-143 can inhibit the proliferative activity of HeLa cells through targeted regulating the expression of K-ras gene. In human cervical cancer tissues of a small sample set, the expression of miR-143 is downregulated, and the expression of K-ras is upregulated.

A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger

PubMed Central

Schäpe, Paul; Müller-Hagen, Dirk; Ouedraogo, Jean-Paul; Heiderich, Caroline; Jedamzick, Johanna; van den Hondel, Cees A.; Ram, Arthur F.; Meyer, Vera

2016-01-01

Understanding the genetic, molecular and evolutionary basis of cysteine-stabilized antifungal proteins (AFPs) from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In the current study, a transcriptome meta-analysis of the Aspergillus niger γ-core protein AnAFP was performed to explore co-expressed genes and pathways, based on independent expression profiling microarrays covering 155 distinct cultivation conditions. This analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we demonstrated that the anafp promoter is active in highly vacuolated compartments and foraging hyphal cells during carbon starvation with CreA and FlbA, but not BrlA, as most likely regulators of anafp. A co-expression network analysis supported by luciferase-based reporter assays uncovered that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators. The transcriptomic resources available for A. niger provide unparalleled resources to investigate the function of proteins. Our work illustrates how transcriptomic meta-analyses can lead to hypotheses regarding protein function and predict a role for AnAFP during slow growth, allorecognition, asexual development and nutrient recycling of A. niger and propose that it interacts with the autophagic machinery to enable these processes. PMID:27835655

A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger.

PubMed

Paege, Norman; Jung, Sascha; Schäpe, Paul; Müller-Hagen, Dirk; Ouedraogo, Jean-Paul; Heiderich, Caroline; Jedamzick, Johanna; Nitsche, Benjamin M; van den Hondel, Cees A; Ram, Arthur F; Meyer, Vera

2016-01-01

Understanding the genetic, molecular and evolutionary basis of cysteine-stabilized antifungal proteins (AFPs) from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In the current study, a transcriptome meta-analysis of the Aspergillus niger γ-core protein AnAFP was performed to explore co-expressed genes and pathways, based on independent expression profiling microarrays covering 155 distinct cultivation conditions. This analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we demonstrated that the anafp promoter is active in highly vacuolated compartments and foraging hyphal cells during carbon starvation with CreA and FlbA, but not BrlA, as most likely regulators of anafp. A co-expression network analysis supported by luciferase-based reporter assays uncovered that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators. The transcriptomic resources available for A. niger provide unparalleled resources to investigate the function of proteins. Our work illustrates how transcriptomic meta-analyses can lead to hypotheses regarding protein function and predict a role for AnAFP during slow growth, allorecognition, asexual development and nutrient recycling of A. niger and propose that it interacts with the autophagic machinery to enable these processes.

Inhibition of Th17 Cell Differentiation as a Treatment for Multiple Sclerosis

DTIC Science & Technology

2013-10-01

luciferase reporter construct into the cells. This reporter construct allows for both measurement of the transfection efficiency by Renilla luciferase...miR326 (delivered either by lentivirus or cotransfection) should result in reduced Firefly luminescence, with no change in Renilla luminescence. The...using Lipofectamine. After 48 hours Dual Glo substrate was added to the cells and luciferase activity and Renilla Luciferase activity were measured

In vivo levels of diadenosine tetraphosphate and adenosine tetraphospho-guanosine in Physarum polycephalum during the cell cycle and oxidative stress.

PubMed Central

Garrison, P N; Mathis, S A; Barnes, L D

1986-01-01

Cellular levels of diadenosine tetraphosphate (Ap4A) and adenosine tetraphospho-guanosine (Ap4G) were specifically measured during the cell cycle of Physarum polycephalum by a high-pressure liquid chromatographic method. Ap4A was also measured indirectly by a coupled phosphodiesterase-luciferase assay. No cell cycle-specific changes in either Ap4A or Ap4G were detected in experiments involving different methods of assay, different strains of P. polycephalum, or different methods of fixation of macroplasmodia. Our results on Ap4A are in contrast with those reported previously (C. Weinmann-Dorsch, G. Pierron, R. Wick, H. Sauer, and F. Grummt, Exp. Cell Res. 155:171-177, 1984). Weinmann-Dorsch et al. reported an 8- to 30-fold increase in Ap4A in early S phase in P. polycephalum, as measured by the phosphodiesterase-luciferase assay. We also measured levels of Ap4A, Ap4G, and ATP in macroplasmodia treated with 0.1 mM dinitrophenol. Ap4A and Ap4G transiently increased three- to sevenfold after 1 h and then decreased concomitantly with an 80% decrease in the level of ATP after 2 h in the presence of dinitrophenol. These results do not support the hypothesis that Ap4A is a positive pleiotypic activator that modulates DNA replication, but they are consistent with the hypothesis proposed for procaryotes that Ap4A and Ap4G are signal nucleotides or alarmones of oxidative stress (B.R. Bochner, P.C. Lee, S.W. Wilson, C.W. Cutler, and B.N. Ames, Cell 37:225-232, 1984). PMID:3785160

MicroRNA-124 inhibits the proliferation of C6 glioma cells by targeting Smad4.

PubMed

Zhang, Zechuan; Gong, Qiaoyun; Li, Meiying; Xu, Jinying; Zheng, Yangyang; Ge, Pengfei; Chi, Guangfan

2017-10-01

MicroRNA-124 (miR-124) has been shown to be downregulated in glioma; however, its biological functions in glioma are not yet fully understood. The aim of this study was to examine the Smad4‑dependent effects of miR‑124 on C6 glioma cell proliferation. In this study, the level of miR‑124 was found to be enhanced in C6 cells upon transfection with miR‑124 mimics, and the mechanisms of action of miR‑124 in C6 cells were investigated by reverse transcriptase-quantitative polymerase chain reaction, MTT assay, western blot analysis and luciferase reporter assays in vitro. The results revealed that miR‑124 expression was significantly lower in the C6 cells than in either normal rat brain tissue or astrocytes. Upon the overexpression of miR‑124, the proliferation of the C6 cells decreased and Smad4 expression was significantly suppressed. Smad4 was identified as a direct target of miR‑124 through luciferase reporter assays. Furthermore, miR‑124 was found to modulate signal transducer and activator of transcription 3 (Stat3) by downregulating Smad4 expression. Using small interfering RNA targeting Smad4 mRNA, we also confirmed that miR‑124 downregulated c‑Myc by modulating Smad4 expression. In addition, caspase‑3 expression was induced by miR‑124 overexpression, but not via Smad4 downregulation. On the whole, our results demonstrate that miR‑124 upregulation inhibits the growth of C6 glioma cells by targeting Smad4 directly. These findings may be clinically useful for the development of therapeutic strategies directed toward miR‑124 function in patients with glioma.

[miR-497 suppresses proliferation of human cervical carcinoma HeLa cells by targeting cyclin E1].

PubMed

Han, Jiming; Huo, Manpeng; Mu, Mingtao; Liu, Junjun; Zhang, Jing

2014-06-01

To evaluate the effect of miR-497 on proliferation of human cervical carcinoma HeLa cells and target relationship between miR-497 and cyclin E1 (CCNE1). Pre-miR-497 sequences were synthesized and cloned into pcDNATM6.2-GW to construct recombinant plasmid pcDNATM6.2-GW-pre-miR-497 and identified by real-time quantitative PCR (qRT-PCR). In addition, sequences of the wild-type CCNE1 (WT-CCNE1) and mutant CCNE1 (MT-CCNE1) were respectively cloned into pmirGLO vectors. MTT assay was used to explore the impact of miR-497 on the proliferation of HeLa cells. Furthermore, the target effect of miR-497 on the CCNE1 was identified by dual-luciferase reporter assay system, qRT-PCR and Western blotting. The recombinant plasmids pcDNATM6.2-GW-pre-miR-497 and pmirGLO-WT-CCNE1, pmirGLO-MT-CCNE1 were successfully constructed, and the miR-497 expression level in HeLa cells transfected with pre-miR-497 was significantly higher than that in the neg-miR group (P<0.05). MTT assay showed that miR-497 could significantly inhibit the proliferation of HeLa cells (P<0.05). A remarkable reduction of luciferase activities of WT-CCNE1 reporter was observed in HeLa cells with pre-miR-497 transfection (P<0.01), and the mRNA and protein expression levels of CCNE1 were down-regulated in HeLa cells transfected with pre-miR-497 (P<0.05). Over-expressed miR-497 in HeLa cells could suppress cell proliferation by targeting CCNE1.

Renilla luciferase- Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals.

PubMed

Wang, Y; Yu, Y A; Shabahang, S; Wang, G; Szalay, A A

2002-10-01

Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the "humanized" GFP (gfp) from Aequorea. A plasmid containing the fusion gene construct was successfully transformed into, and expressed in, mammalian cells. The transformed cells exhibited both Renilla luciferase activity in the presence of coelenterazine and GFP fluorescence upon excitation with UV light. Spectrofluorometry of cells containing the Ruc-GFP fusion protein, in the absence of wavelengths capable of exciting GFP fluorescence but in the presence of the luciferase substrate, coelenterazine, showed an emission spectrum with two peaks at 475 nm and 508 nm. These two peaks correspond to the emission maximum of Renilla luciferase at 475 nm and that of GFP at 508 nm. The peak at 508 nm generated in the presence of coelenterazine alone (without UV excitation) is the result of intramolecular energy transfer from Renilla luciferase to Aequorea GFP. Southern analysis of genomic DNA purified from transformed Chinese hamster ovary (CHO) cells and fluorescence in situ hybridization (FISH) to metaphase chromosomes confirmed the integration of the ruc-gfp fusion gene on a single chromosome. The bifunctional Ruc-GFP fusion protein allows the detection of gene expression at the single-cell level based on green fluorescence, and in a group of cells based on luminescence emission. Furthermore, animal experiments revealed that light emission from the Ruc-GFP fusion protein can be detected externally in the organs or tissues of live animals bearing the gene construct.

Secreted Gaussia princeps luciferase as a reporter of Escherichia coli replication in a mouse tissue cage model of infection.

PubMed

Liu, Mingyu; Blinn, Christina; McLeod, Sarah M; Wiseman, John W; Newman, Joseph V; Fisher, Stewart L; Walkup, Grant K

2014-01-01

Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.

MiR-593 mediates curcumin-induced radiosensitization of nasopharyngeal carcinoma cells via MDR1.

PubMed

Fan, Haoning; Shao, Meng; Huang, Shaohui; Liu, Ying; Liu, Jie; Wang, Zhiyuan; Diao, Jianxin; Liu, Yuanliang; Tong, L I; Fan, Qin

2016-06-01

Curcumin (Cur) exhibits radiosensitization effects to a variety of malignant tumors. The present study investigates the radiosensitizing effect of Cur on nasopharyngeal carcinoma (NPC) cells and whether its mechanism is associated with microRNA-593 (miR-593) and multidrug resistance gene 1 (MDR1). A clonogenic assay was performed to measure the radiosensitizing effect. The expression of miR-593 and MDR1 was analyzed by quantitative polymerase chain reaction (qPCR) or western blot assay. A transplanted tumor model was established to identify the radiosensitizing effect in vivo . A luciferase-based reporter was constructed to evaluate the effect of direct binding of miR-593 to the putative target site on the 3' UTR of MDR1. The clonogenic assay showed that Cur enhanced the radiosensitivity of cells. Cur (100 mg/kg) combined with 4 Gy irradiation inhibited the growth of a transplanted tumor model in vivo , resulting in the higher inhibition ratio compared with the radiotherapy-alone group. These results demonstrated that Cur had a radiosensitizing effect on NPC cells in vivo and in vitro ; Cur-mediated upregulation of miR-593 resulted in reduced MDR1 expression, which may promote radiosensitivity of NPC cells.

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

PubMed

Dionisi, Mauro; Alexander, Stephen P H; Bennett, Andrew J

2012-05-14

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

FOXD3 inhibits SCN2A gene transcription in intractable epilepsy cell models.

PubMed

Xiang, Jun; Wen, Fang; Zhang, Lingyun; Zhou, Yu

2018-04-01

The expression of sodium voltage-gated channel alpha subunit 2 (SCN2A) is closely related to the development of epilepsy. This study investigated regulatory element of the SCN2A gene involved in epilepsy. An intractable epilepsy cell model was constructed using hippocampal primary neurons and the SH-SY5Y cell line. SCN2A protein and gene expression in cells as well as the level of lactic acid dehydrogenase (LDH) in the cell culture supernatants was detected. Potential regulatory factors of SCN2A and its upstream regulatory elements were identified using the dual-luciferase reporter assay. Finally, the role of the hypothetical transcription factor in epilepsy was examined by using its small interfering RNA (siRNA). Results found that levels of LDH and expression of the hypothetical transcription factor, Forkhead box D3 (FOXD3), was both increased in the model cells, whereas that of SCN2A was decreased. The results of dual-luciferase reporter assays revealed that an upstream region of SCN2A gene spanning from nucleotides -1617 to -1470 was a transcription factor binding region and a trans-acting factor role of FOXD3 was identified in the core region (GGCAAAATTAT). Then the FOXD3 binding site was further verified by the chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA). After SH-SY5Y cells were transfected with FOXD3 siRNA, the release of LDH into culture supernatants and the LDH expression levels in cells were significantly decreased. SCN2A expression in model cells was increased by knockdown of FOXD3. Therefore, this study demonstrated that FOXD3 is a trans-acting factor of SCN2A, and this mechanism may play a role in cell injury after epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

Contribution of Priority PAHs and POPs to Ah Receptor-Mediated Activities in Sediment Samples from the River Elbe Estuary, Germany

PubMed Central

Otte, Jens C.; Keiter, Steffen; Faßbender, Christopher; Higley, Eric B.; Rocha, Paula Suares; Brinkmann, Markus; Wahrendorf, Dierk-Steffen; Manz, Werner; Wetzel, Markus A.; Braunbeck, Thomas; Giesy, John P.; Hecker, Markus; Hollert, Henner

2013-01-01

The estuary of the River Elbe between Hamburg and the North Sea (Germany) is a sink for contaminated sediment and suspended particulate matter (SPM). One major concern is the effect of human activities on the hydrodynamics, particularly the intensive dredging activities in this area that may result in remobilization of sediment-bound pollutants. The aim of this study was to identify pollutants contributing to the toxicological risk associated with re-suspension of sediments in the Elbe Estuary by use of an effect-directed analysis that combines chemical and biological analyses in with specific fractionation techniques. Sediments were collected from sites along the Elbe Estuary and a site from a small harbor basin of the Elbe Estuary that is known to be polluted. The sixteen priority EPA-PAHs were quantified in organic extracts of sediments. In addition, dioxin equivalents of sediments were investigated by use of the 7-ethoxyresorufin O-deethylase assay with RTL-W1 cells and the Ah receptor-mediated luciferase transactivation assay with H4IIE-luc cells. Quantification of the 16 priority PAHs revealed that sediments were moderately contaminated at all of the sites in the Elbe River Estuary (<0.02–0.906 µg/g dw). Sediments contained relatively small concentrations of dioxin equivalents (Bio-TEQ) with concentrations ranging from 15.5 to 322 pg/g dw, which were significantly correlated with dioxin equivalents calculated based on toxicity reference values and concentrations of PAH. The concentration of Bio-TEQ at the reference site exceeded 200,000 pg/g dw. In a potency balance the 16 PAHs explained between 47 and 118% of the Bio-TEQ in the luciferase assay, which can be explained by the constant input of PAHs bound to SPM from the upper course of the Elbe River into its estuary. Successful identification of a significant portion of dioxin-like activity to priority PAHs in complex environmental samples such as sediments has rarely been reported. PMID:24146763

Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

PubMed Central

Gandelman, Olga A.; Church, Vicki L.; Moore, Cathy A.; Kiddle, Guy; Carne, Christopher A.; Parmar, Surendra; Jalal, Hamid; Tisi, Laurence C.; Murray, James A. H.

2010-01-01

Background The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. Principal Findings Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. Conclusions The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings. PMID:21152399

Reactivation of Embryonic Nodal Signaling is Associated with Tumor Progression and Promotes the Growth of Prostate Cancer Cells

PubMed Central

Lawrence, Mitchell G.; Margaryan, Naira V.; Loessner, Daniela; Collins, Angus; Kerr, Kris M.; Turner, Megan; Seftor, Elisabeth A.; Stephens, Carson R.; Lai, John; BioResource, APC; Postovit, Lynne-Marie; Clements, Judith A.; Hendrix, Mary J.C.

2011-01-01

Background Nodal is a member of the Transforming Growth Factor β (TGFβ) superfamily that directs embryonic patterning and promotes the plasticity and tumorigenicity of tumor cells, but its role in the prostate is unknown. The goal of this study was to characterize the expression and function of Nodal in prostate cancer and determine whether, like other TGFβ ligands, it modulates androgen receptor (AR) activity. Methods Nodal expression was investigated using immunohistochemistry of tissue microarrays and Western blots of prostate cell lines. The functional role of Nodal was examined using Matrigel and soft agar growth assays. Cross-talk between Nodal and AR signaling was assessed with luciferase reporter assays and expression of endogenous androgen regulated genes. Results Significantly increased Nodal expression was observed in cancer compared with benign prostate specimens. Nodal was only expressed by DU145 and PC3 cells. All cell lines expressed Nodal’s co-receptor, Cripto-1, but lacked Lefty, a critical negative regulator of Nodal signaling. Recombinant human Nodal triggered downstream Smad2 phosphorylation in DU145 and LNCaP cells, and stable transfection of pre-pro-Nodal enhanced the growth of LNCaP cells in Matrigel and soft agar. Finally, Nodal attenuated AR signaling, reducing the activity of a PSA promoter construct in luciferase assays and down-regulating the endogenous expression of androgen regulated genes. Conclusions An aberrant Nodal signaling pathway is re-expressed and functionally active in prostate cancer cells. PMID:21656830

MicroRNA-17-5p contributes to osteoarthritis progression by binding p62/SQSTM1.

PubMed

Li, Huihui; Miao, Daoyi; Zhu, Qi; Huang, Jianghua; Lu, Guangxian; Xu, Weiguo

2018-02-01

Autophagy has been reported to be widely involved in the pathogenesis of osteoarthritis (OA). Increasing evidence suggested the important role of microRNAs (miRs) in the progression of OA. However, the functional role of miR-17-5p in OA development has remained to be fully elucidated. First, a mouse model of OA was established and the relative level of miR-17-5p was determined using PCR. Safranin O-fast green staining was applied to determine cartilage degeneration. TargetScan software and a dual luciferase reporter assay were applied to determine potential target genes of miR-17-5P. Autophagy measurement was performed using green fluorescent protein-microtubule-associated protein 1 light chain 3 (LC3) dot analysis. The results demonstrated that the relative expression of miR-17-5p was significantly decreased in OA model mice. In addition, the level of miR-17-5p was decreased in SW1353 human chondrosarcoma cells treated with interleukin-1β. Furthermore, autophagy was found to be suppressed in the knee joints of experimental OA model mice. The dual luciferase reporter assay confirmed that p62/sequestosome 1 was a target gene of miR-17-5p. Of note, miR-17-5p inhibitor-induced reduction of LC3 dots was markedly reversed by knockdown of p62 in SW1353 cells. In conclusion, decreased miR-17-5p expression in chondrocytes induced autophagy mainly through suppressing the expression of p62, thereby contributing to OA progression.

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

PubMed

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

2015-01-01

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

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

PubMed Central

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

2015-01-01

Stra8 encodes stimulated by retinoic acid gene 8, a protein that is important for initiation of meiosis in mammals and birds. This study was aimed at identifying the active control area of chicken STRA8 gene core promoter, to screen optimum inducers of the STRA8 gene, thus to enhance the differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells. Fragments of chicken STRA8 gene promoter were cloned into fluorescent reporter plasmids and transfected into DF-1 cells. Then Dual-Luciferase® Reporter Assay System was used to identify the activity of the STRA8 gene under different inducers. Our studies showed that the promoter fragment −1055 bp to +54 bp of Suqin chicken Stra8 revealed the strongest activity. The dual-luciferase® reporter showed that Tamibarotene (Am80) and TrichostatinA (TSA) could significantly enhance STRA8 transcription. The in vitro inductive culture of chicken ESCs demonstrated that spermatogonial stem cells (SSC)-like cells appeared and Integrinβ1 protein was expressed on day 10, indicating that Am80 and TSA can promote ESCs differentiation into SSCs via regulation of Stra8. PMID:26606052

A homogeneous nucleic acid hybridization assay based on strand displacement.

PubMed Central

Vary, C P

1987-01-01

A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal strand. Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form. The single-strand specificity of polynucleotide phosphorylase (EC 2.7.7.8) allows discrimination between double-helical and single-stranded forms of the RNA signal strand. As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate. The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40). Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement. Images PMID:3309890

Inhibitors of SOD1 Interaction as an Approach to Slow the Progressive Spread of ALS Symptoms

DTIC Science & Technology

2016-07-01

luciferase enzyme can be split into 2 halves. These 2 halves can be forced to reconstitute an active enzyme if they are brought together by some...force. In our assay, this force is the normal interaction that occurs when 2 individual SOD1 proteins come together to form a normal active enzyme ...Using recombinant DNA, we create fusion proteins of SOD1 and each half of the luciferase enzyme . In the past year, we have characterized and optimized

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

PubMed

Baston, David S; Denison, Michael S

2011-02-15

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

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

USDA-ARS?s Scientific Manuscript database

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...

A panel of Trypanosoma brucei strains tagged with blue and red-shifted luciferases for bioluminescent imaging in murine infection models.

PubMed

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

2014-08-01

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. 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. 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 kinetics of their substrate D-luciferin.

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 kinetics of their substrate D-luciferin. PMID:25144573

Effect of Interleukin-18 Gene Polymorphisms on Sensitization to Wheat Flour in Bakery Workers

PubMed Central

Kim, Seung-Hyun; Hur, Gyu-Young; Jin, Hyun Jung; Choi, Hyunna

2012-01-01

Lower respiratory symptoms in bakery workers may be induced by wheat flour and endotoxins. We hypothesized that endotoxins from wheat flour may stimulate innate immunity and that interleukin-18 (IL-18) gene polymorphisms may affect their regulatory role in innate immune responses to endotoxins. To investigate the genetic contribution of IL-18 to sensitization to wheat flour, we performed a genetic association study of IL-18 in Korean bakery workers. A total of 373 bakery workers undertook a questionnaire regarding work-related symptoms. Skin prick tests with common and occupational allergens were performed and specific antibodies to wheat flour were measured by ELISA. Three polymorphisms of the IL-18 gene (-607A/C, -137G/C, 8674C/G) were genotyped, and the functional effects of the polymorphisms were analyzed using the luciferase reporter assay. Genotypes of -137G/C (GC or CC) and haplotype ht3 [ACC] showed a significant association with the rate of sensitization to wheat flour. Luciferase activity assay indicated ht3 [AC] as a low transcript haplotype. In conclusion, the regulatory role of IL-18 in lipopolysaccharide-induced responses in bakery workers may be affected by this polymorphism, thus contributing to the development of sensitization to wheat flour and work-related respiratory symptoms. PMID:22468101

Optical imaging of tumor cells in hollow fibers: evaluation of the antitumor activities of anticancer drugs and target validation.

PubMed

Zhang, Guo-Jun; Chen, Tsing-Bau; Bednar, Bohumil; Connolly, Brett M; Hargreaves, Richard; Sur, Cyrille; Williams, David L

2007-08-01

The in vivo hollow fiber assay, in which semipermeable hollow fibers filled with tumor cells, are implanted into animals, was originally developed to screen for anticancer compounds before assessment in more complex tumor models. To enhance screening and evaluation of anticancer drugs, we have applied optical imaging technology to this assay. To demonstrate that tumor cells inside hollow fibers can communicate with the host mice, we have used fluorescence imaging in vivo and CD31 immunostaining ex vivo to show that angiogenesis occurs around cell-filled hollow fibers by 2 weeks after subcutaneous implantation. Bioluminescence imaging has been used to follow the number of luciferase-expressing tumor cells within implanted hollow fibers; proliferation of those cells was found to be significantly inhibited by docetaxel or irinotecan. We also used bioluminescence imaging of hollow fibers to monitor the nuclear factor kappaB (NFkappaB) pathway in vivo; NFkappaB activation by lipopolysaccharide and tumor necrosis factor-alpha was evaluated in tumor cell lines genetically engineered to express luciferase controlled by an NFkappaB-responsive element. These results demonstrate that optical imaging of hollow fibers containing reporter tumor cells can be used for the rapid and accurate evaluation of antitumor activities of anticancer drugs and for measurement of molecular pathways.

Long non-coding RNA HOTTIP promotes prostate cancer cells proliferation and migration by sponging miR-216a-5p.

PubMed

Yang, Bin; Gao, Ge; Wang, Zhixin; Sun, Daju; Wei, Xin; Ma, Yanan; Ding, Youpeng

2018-06-08

Long non-coding RNAs (lncRNAs) are a class of ncRNAs with > 200 nucleotides in length that regulate gene expression. The HOXA transcript at the distal tip (HOTTIP) lncRNA plays an important role in carcinogenesis, however, the underlying role of HOTTIP in prostate cancer (PCa) remain unknown. The aim of the present study was to evaluate the expression and function of HOTTIP in PCa. In the present study, we analyzed HOTTIP expression levels of 86 PCa patients in tumor and adjacent normal tissue by real-time quantitative PCR. Knockdown or overexpression of HOTTIP was performed to explore its roles in cell proliferation, migration, invasion, and cell cycle. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOTTIP and miR-216a-5p in PCa cells. Our results found that HOTTIP was up-regulated in human primary PCa tissues with lymph node metastasis. Knockdown of HOTTIP inhibited PCa cell proliferation, migration and invasion. Overexpression of HOTTIP promoted cell proliferation, migration and invasion of PCa cells. Bioinformatics online programs predicted that HOTTIP sponge miR-216a-5p at 3'-UTR with complementary binding sites, which was validated using luciferase reporter assay. HOTTIP could negatively regulate the expression of miR-216a-5p in PCa cells. Above all, knockdown of HOTTIP could represent a rational therapeutic strategy for PCa. ©2018 The Author(s).

Optimization of a cAMP response element signal pathway reporter system.

PubMed

Shan, Qiang; Storm, Daniel R

2010-08-15

A sensitive cAMP response element (CRE) reporter system is essential for studying the cAMP/protein kinase A/cAMP response element binding protein signal pathway. Here we have tested a few CRE promoters and found one with high sensitivity to external stimuli. Using this optimal CRE promoter and the enhanced green fluorescent protein as the reporter, we have established a CRE reporter cell line. This cell line can be used to study the signal pathway by fluorescent microscope, fluorescence-activated cell analysis and luciferase assay. This cell line's sensitivity to forskolin, using the technique of fluorescence-activated cell sorting, was increased to approximately seven times that of its parental HEK 293 cell line, which is currently the most commonly used cell line in the field for the signal pathway study. Therefore, this newly created cell line is potentially useful for studying the signal pathway's modulators, which generally have weaker effect than its mediators. Our research has also established a general procedure for optimizing transcription-based reporter cell lines, which might be useful in performing the same task when studying many other transcription-based signal pathways. (c) 2010 Elsevier B.V. All rights reserved.

Effect of Brain- and Tumor-Derived Connective Tissue Growth Factor on Glioma Invasion

PubMed Central

Edwards, Lincoln A.; Woolard, Kevin; Son, Myung Jin; Li, Aiguo; Lee, Jeongwu; Ene, Chibawanye; Mantey, Samuel A.; Maric, Dragan; Song, Hua; Belova, Galina; Jensen, Robert T.; Zhang, Wei

2011-01-01

Background Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets. Methods Highly infiltrative patient-derived glioma tumor–initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin β1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided. Results Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1–TrkA receptor complexes and nuclear factor kappa B (NF-κB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF200 ng/mL: 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2, P < .001). NF-κB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF200 ng/mL + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most infiltrative glioma cells in situ and that the surrounding reactive astrocytes secrete CTGF. Conclusion A CTGF-rich microenvironment facilitates CTGF–ITGB1–TrkA complex activation in TIC/TSCs, thereby increasing the invasiveness of malignant gliomas. PMID:21771732

In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography

PubMed Central

Genevois, Coralie; Loiseau, Hugues; Couillaud, Franck

2016-01-01

Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT®). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors. PMID:27809256

In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography.

PubMed

Genevois, Coralie; Loiseau, Hugues; Couillaud, Franck

2016-10-31

Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT ® ). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors.

Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research.

PubMed

Siciliano, Giulia; Alano, Pietro

2015-01-01

The unicellular protozoan parasites of the genus Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have been essential to unveil mechanisms of parasite gene expression and to develop in vivo imaging approaches in mouse malaria models. Mainly the human malaria parasite Plasmodium falciparum and the rodent parasite P. berghei have been engineered to express bioluminescent reporters in almost all the developmental stages of the parasite along its complex life cycle between the insect and the vertebrate hosts. Plasmodium lines expressing conventional and improved luciferase reporters are now gaining a central role to develop cell based assays in the much needed search of new antimalarial drugs and to open innovative approaches for both fundamental and applied research in malaria.

Effect of cadmium on lake water bacteria as determined by the luciferase assay of adenosine triphosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seyfried, P.L.; Horgan, C.B.L.

1981-10-01

A firefly luciferase assay of bacterial adenosine triphosphate (ATP) was developed to measure the toxic effects of cadmium ions on aquatic organisms. Toxicity was monitored using intracellular (I/C) ATP (in micrograms per litre) as well as plate counts (colony-forming units per millilitre). The bacteria, which belonged mainly to the families Enterobacteriaceae and Pseudomonadaceae, exhibited varying degrees of resistance to up to 100 ppm cadmium when grown in a glucose-salts medium at pH 6.8. Among the organisms tested, cadmium resistance decreased in the following order: Pseudomonas vesicularis > P. aeruginosa > Enterobacter sp. > P. fluorescens > Chromobacter sp. > Serratiamore » sp. A rise in the pH of the growth medium from 5 to 7 resulted in increased toxicity of cadmium.« less

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

Luminogenic cytochrome P450 assays.

PubMed

Cali, James J; Ma, Dongping; Sobol, Mary; Simpson, Daniel J; Frackman, Susan; Good, Troy D; Daily, William J; Liu, David

2006-08-01

Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

Dopamine D2-like receptors (DRD2 and DRD4) in chickens: Tissue distribution, functional analysis, and their involvement in dopamine inhibition of pituitary prolactin expression.

PubMed

Lv, Can; Mo, Chunheng; Liu, Haikun; Wu, Chao; Li, Zhengyang; Li, Juan; Wang, Yajun

2018-04-20

Dopamine (DA) D2-like (and D1-like) receptors are suggested to mediate the dopamine actions in the anterior pituitary and/or CNS of birds. However, the information regarding the structure, functionality, and expression of avian D2-like receptors have not been fully characterized. In this study, we cloned two D2-like receptors (cDRD2, cDRD4) from chicken brain using RACE PCR. The cloned cDRD4 is a 378-amino acid receptor, which shows 57% amino acid (a.a.) identity with mouse DRD4. As in mammals, two cDRD2 isoforms, cDRD2L (long isoform, 437 a.a.) and cDRD2S (short isoform, 408 a.a.), which differ in their third intracellular loop, were identified in chickens. Using cell-based luciferase reporter assays or Western blot, we demonstrated that cDRD4, cDRD2L and cDRD2S could be activated by dopamine and quinpirole (a D2-like receptor agonist) dose-dependently, and their activation inhibits cAMP signaling pathway and stimulates MAPK/ERK signaling cascade, indicating that they are functional receptors capable of mediating dopamine actions. Quantitative real-time PCR revealed that cDRD2 and cDRD4 are widely expressed in chicken tissues with abundant expression noted in anterior pituitary, and their expressions are likely controlled by their promoters near exon 1, as demonstrated by dual-luciferase reporter assays in DF-1 cells. In accordance with cDRD2/cDRD4 expression in the pituitary, DA or quinpirole could partially inhibit vasoactive intestinal peptide-induced prolactin expression in cultured chick pituitary cells. Together, our data proves the functionality of DRD2 and DRD4 in birds and aids to uncover the conserved roles of DA/D2-like receptor system in vertebrates, such as its action on the pituitary. Copyright © 2018. Published by Elsevier B.V.

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

PubMed

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

2015-10-01

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

Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins

PubMed Central

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. PMID:24842154

Essiac? and Flor-Essence? herbal tonics stimulate the in vitro growth of human breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kulp, K S; Montgomery, J L; McLimans, B

People diagnosed with cancer often self-administer complementary and alternative medicines (CAMs) to supplement their conventional treatments, improve health, or prevent recurrence. Flor-Essence{reg_sign} and Essiac{reg_sign} Herbal Tonics are commercially available complex mixtures of herbal extracts sold as dietary supplements and used by cancer patients based on anecdotal evidence that they can treat or prevent disease. In this study, we evaluated Flor-Essence{reg_sign} and Essiac{reg_sign} for their effects on the growth of human tumor cells in culture. The effect of Flor-Essence{reg_sign} and Essiac{reg_sign} herbal tonics on cell proliferation was tested in MCF-7, MDA-MB-436, MDA-MB-231, and T47D cancer cells isolated from human breast tumors.more » Estrogen receptor (ER) dependent activation of a luciferase reporter construct was tested in MCF-7 cells. Specific binding to the ER was tested using an ICI 182,780 competition assay. Flor-Essence{reg_sign} and Essiac{reg_sign} herbal tonics at 1%, 2%, 4% and 8% stimulated cell proliferation relative to untreated controls and activated ER dependent luciferase activity in MCF-7 cells. A 10{sup -7} M concentration of ICI 870,780 inhibited the induction of ER dependent luciferase activity by Flor-Essence{reg_sign} and Essiac{reg_sign}, but did not affect cell proliferation. Flor-Essence{reg_sign} and Essiac{reg_sign} Herbal Tonics can stimulate the growth of human breast cancer cells through ER mediated as well as ER independent mechanisms of action. Cancer patients and health care providers can use this information to make informed decisions about the use of these CAMs.« less

Artemin protects cells and proteins against oxidative and salt stress.

PubMed

Takalloo, Zeinab; Sajedi, Reza H; Hosseinkhani, Saman; Moazzenzade, Taghi

2017-02-01

Artemin is an abundant thermostable protein in Artemia encysted embryos under environmental stresses. It is confirmed that high regulatory expression of artemin is relevant to stress resistance in this crustacean. Here, the protective role of artemin from Artemia urmiana has been investigated on survival of bacterial cells under salt and oxidative shocks. Also, for continuous monitoring of the effect of artemin in prevention of proteins aggregation/inactivation, co-expression of artemin and luciferase (as an intracellular reporter) in bacterial cells was performed. According to the results, residual activity of luciferase in artemin expressing E. coli cells exposing to different concentrations of H 2 O 2 and NaCl was significantly higher than non-expressing cells. The luciferase activity was rapidly lost in control cells under salt treatments while in co-transformed cells, the activity was considerably retained at higher salt concentrations. Also, analysis from cell viability assays showed that artemin-expressing cells exhibited more resistance to both stress conditions. In the present study, we document for the first time that artemin can protect proteins and bacterial cells against oxidative and salt stress conditions. These results can declare the resistance property of this crustacean against harsh environmental conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Stalk-dependent and Stalk-independent Signaling by the Adhesion G Protein-coupled Receptors GPR56 (ADGRG1) and BAI1 (ADGRB1).

PubMed

Kishore, Ayush; Purcell, Ryan H; Nassiri-Toosi, Zahra; Hall, Randy A

2016-02-12

The adhesion G protein-coupled receptors (aGPCRs) are a large yet poorly understood family of seven-transmembrane proteins. A defining characteristic of the aGPCR family is the conserved GAIN domain, which has autoproteolytic activity and can cleave the receptors near the first transmembrane domain. Several aGPCRs, including ADGRB1 (BAI1 or B1) and ADGRG1 (GPR56 or G1), have been found to exhibit significantly increased constitutive activity when truncated to mimic GAIN domain cleavage (ΔNT). Recent reports have suggested that the new N-terminal stalk, which is revealed by GAIN domain cleavage, can directly activate aGPCRs as a tethered agonist. We tested this hypothesis in studies on two distinct aGPCRs, B1 and G1, by engineering mutant receptors lacking the entire NT including the stalk (B1- and G1-SL, with "SL" indicating "stalkless"). These receptors were evaluated in a battery of signaling assays and compared with full-length wild-type and cleavage-mimicking (ΔNT) forms of the two receptors. We found that B1-SL, in multiple assays, exhibited robust signaling activity, suggesting that the membrane-proximal stalk region is not necessary for its activation. For G1, however, the results were mixed, with the SL mutant exhibiting robust activity in several signaling assays (including TGFα shedding, activation of NFAT luciferase, and β-arrestin recruitment) but reduced activity relative to ΔNT in a distinct assay (activation of SRF luciferase). These data support a model in which the activation of certain pathways downstream of aGPCRs is stalk-dependent, whereas signaling to other pathways is stalk-independent. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

MicroRNA-203 Induces Apoptosis by Targeting Bmi-1 in YD-38 Oral Cancer Cells.

PubMed

Kim, Jae-Sung; Choi, Dae Woo; Kim, Chun Sung; Yu, Sun-Kyoung; Kim, Heung-Joong; Go, Dae-San; Lee, Seul Ah; Moon, Sung Min; Kim, Su Gwan; Chun, Hong Sung; Kim, Jeongsun; Kim, Jong-Keun; Kim, DO Kyung

2018-06-01

MicroRNAs (miRNAs) are closely associated with a number of cellular processes, including cell development, differentiation, proliferation, carcinogenesis, and apoptosis. The aim of the present study was to elucidate the molecular mechanisms underlying the tumor suppressor activity of miRNA-203 (miR-203) in YD-38 human oral cancer cells. Polymerase chain reaction analysis, MTT assay, DNA fragmentation assay, fluorescence-activated cell-sorting analysis, gene array, immunoblotting, and luciferase assay were carried out in YD-38 cells. miR-203 expression was significantly down-regulated in YD-38 cells compared to expression levels in normal human oral keratinocytes. miR-203 decreased the viability of YD-38 cells in a time- and dose-dependent manner. In addition, over-expression of miR-203 significantly increased not only DNA segmentation, but also the apoptotic population of YD-38 cells. These results indicate that miR-203 overexpression induces apoptosis in YD-38 cells. Target gene array analysis revealed that the expression of the polycomb complex protein gene Bmi-1, a representative oncogene, was significantly down-regulated by miR-203 in YD-38 cells. Moreover, both mRNA and protein levels of Bmi-1 were significantly reduced in YD-38 cells transfected with miR-203. These results indicate that Bmi-1 is a target gene of miR-203. A luciferase reporter assay confirmed that miR-203 suppressed Bmi-1 expression by directly targeting the 3'-untranslated region. miR-203 induces apoptosis in YD-38 cells by directly targeting Bmi-1, which suggests its possible application as an anti-cancer therapeutic. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

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

PubMed

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

2017-08-01

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

MicroRNA-143 suppresses gastric cancer cell growth and induces apoptosis by targeting COX-2

PubMed Central

Wu, Xiao-Li; Cheng, Bin; Li, Pei-Yuan; Huang, Huan-Jun; Zhao, Qiu; Dan, Zi-Li; Tian, De-An; Zhang, Peng

2013-01-01

AIM: To investigate the function of microRNA-143 (miR-143) in gastric cancer and explore the target genes of miR-143. METHODS: A quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to evaluate miR-143 expression in gastric cancer cell lines. After transfecting gastric cancer cells with miR-143-5p and miR-143-3p precursors, Alamar blue and apoptosis assays were used to measure the respective proliferation and apoptosis rates. Cyclooxygenase-2 (COX-2) expression was determined by real-time RT-PCR and Western blot assays after miR-143 transfection. Reporter plasmids were constructed, and a luciferase reporter assay was used to identify the miR-143 binding site on COX-2. RESULTS: Both miR-143-5p and miR-143-3p were significantly downregulated in multiple gastric cancer cell lines. Forced miR-143-5p and miR-143-3p expression in gastric cancer cells produced a profound cytotoxic effect. MiR-145-5p transfection into gastric cancer cells resulted in a greater growth inhibitory effect (61.23% ± 3.16% vs 46.58% ± 4.28%, P < 0.05 in the MKN-1 cell line) and a higher apoptosis rate (28.74% ± 1.93% vs 22.13% ± 3.31%, P < 0.05 in the MKN-1 cell line) than miR-143-3p transfection. Further analysis indicated that COX-2 expression was potently suppressed by miR-143-5p but not by miR-143-3p. The activity of a luciferase reporter construct that contained the 3’-untranslated region (UTR) of COX-2 was downregulated by miR-143-5p (43.6% ± 4.86%, P < 0.01) but not by miR-143-3p. A mutation in the miR-145-5p binding site completely ablated the regulatory effect on luciferase activity, which suggests that there is a direct miR-145-5p binding site in the 3’-UTR of COX-2. CONCLUSION: Both miR-143-5p and miR-143-3p function as anti-oncomirs in gastric cancer. However, miR-143-5p alone directly targets COX-2, and it exhibits a stronger tumor suppressive effect than miR-143-3p. PMID:24616567

MiR-130a inhibition protects rat cardiac myocytes from hypoxia-triggered apoptosis by targeting Smad4.

PubMed

Li, Yuanshi; Du, Yingrong; Cao, Junxian; Gao, Qianping; Li, Hongjuan; Chen, Yangjun; Lu, Nihong

2018-02-05

Cardiomyocyte death facilitates the pathological process underlying ischemic heart diseases, such as myocardial infarction. Emerging evidence suggests that microRNAs play a critical role in the pathological process underlying myocardial infarction by regulating cardiomyocyte apoptosis. However, the relevance of miR-130a in regulating cardiomyocyte apoptosis and the mechanism of regulation is still uncertain. This study aimed to explore the regulatory effect of miR-130a on hypoxic cardiomyocyte apoptosis. The expression of miR-130a was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell survival was determined by the MTT assay. The lactate dehydrogenase (LDH) assay was performed to determine the severity of hypoxia-induced cell injury. Apoptosis was assessed via caspase-3 analysis. Protein expression level was determined by Western blotting. The genes targeted by miR-130a were predicted using bioinformatics and were validated via the dual-luciferase reporter assay. We found that miR-130a expression was greatly increased in hypoxic cardiac myocytes, and that the downregulation of miR-130a effectively shielded cardiac myocytes from hypoxia-triggered apoptosis. The results of our bioinformatic analysis predicted the Smad4 gene to be the target of miR-130a. This finding was validated through the Western blot assay, dual-luciferase reporter gene assay, and qRT-PCR. MiR-130a inhibition significantly promoted the activation of Smad4 in hypoxic cardiomyocytes. Interestingly, knockdown of Smad4 markedly reversed the protective effects induced by miR-130a inhibition. Moreover, we found that the inhibition of miR-130a promoted the activation of TGF-β signaling. Blocking Smad4 signaling significantly abrogated the protective effects of miR-130a inhibition. Overall, these findings indicate that inhibition of miR-130a, which targets the Smad4 gene, shields cardiac myocytes from hypoxic apoptosis. This study offers a novel perspective of the molecular basis of hypoxia-induced cardiomyocyte apoptosis and suggests a possible drug target for the treatment of myocardial infarction.

Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro

PubMed Central

2014-01-01

Background The porcine oocyte maturation in vivo occurs within the ovarian follicle and is regulated by the interactions between oocytes and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. Therefore, the antral follicle is an essential microenvironment for efficient oocyte maturation and its developmental competence. Quantitative bioluminescence imaging of firefly luciferase reporter genes in an intact antral follicle would allow investigation of changes in cellular and molecular events and in the context of the whole follicles. In this study, we investigate factors influencing bioluminescence measurements as a first step towards developing a new bioluminescence imaging system for intact antral follicles. Methods We analyzed the time course of bioluminescence emitted from transfected living intact follicles using a cationic lipid mediated gene transfer method with increasing doses (1-3 μg) of firefly luciferase reporter gene (pGL4). In addition, a standard luciferase assay was used to confirm the luciferase expression in granulosa cells in the transfected intact antral follicles. Finally, the dose effects of substrate, D-luciferin, were determined for optimal quantitative bioluminescence imaging of intact porcine antral follicles in vitro. Results The level of luciferase activity of follicles with 3 μg pGL4 was significantly (P < 0.05) greater than the 1 μg and 2 μg groups at 1 min after D-luciferin injection. The bioluminescence intensity of transfected follicles reached a peak at 1 min, and then it was significantly (P < 0.05) reduced within 2 min after injection of D-luciferin; with the level of bioluminescence emission remained constant from 2.5 to 10 min. The bioluminescence emission was maximal with 300 μg of D-luciferin. Conclusions The results of this study suggested that the investigation of factors influencing bioluminescence measurements is a critical step toward developing a new bioluminescence imaging model. This study is the first to demonstrate that reporter genes can be transferred to intact granulosa cells with a lipid-mediated gene transfer method within intact follicles in vitro, and the level of transgene expression can be assessed by bioluminescence imaging in living intact antral follicles. PMID:24479789

Development of a novel genetically modified bioluminescent-bacteria-based assay for detection of fluoroquinolones in animal-derived foods.

PubMed

Cheng, Guyue; Dong, Xiaobing; Wang, Yulian; Peng, Dapeng; Wang, Xu; Hao, Haihong; Xie, Shuyu; Qu, Wei; Liu, Zhenli; Yuan, Zonghui

2014-12-01

Fluoroquinolones (FQNs) are broad-spectrum antibacterial agents widely used in animal husbandry and aquaculture. The residues and antimicrobial resistance of such antibiotics are a major public health concern. To realize multianalyte detection of FQN residues, a genetically modified bacterium, Escherichia coli pK12 harboring plasmid pRecAlux3, was constructed in this study to develop a bioluminescent-bacteria-based assay for the detection of FQNs in animal-derived foods. This assay was based on the principle of induction of an SOS response by FQNs via inducing the recA-promoter-fused luciferase reporter gene existing on the plasmid pRecAlux3. E. coli pK12 was able to recognize 11 FQNs: difloxacin, enrofloxacin, ciprofloxacin, sarafloxacin, norfloxacin, danofloxacin, ofloxacin, pefloxacin, lomefloxacin, marbofloxacin, and orbifloxacin. This method could be applied to 11 edible tissues, including milk, fish muscle, and the muscles, livers, and kidneys of cattle, chickens, and pigs, with a very simple and rapid sample extraction procedure using only phosphate-buffered saline. The limits of detection of the FQNs were between 12.5 and 100 μg kg(-1), all of which were lower than the maximum residue limits. Most of the recoveries of the FQNs were in the range from 60 to 120 %, and the interassay coefficients of variation were less than 30 %. This method, confirmed by high-performance liquid chromatography, is reliable and can be used as both a screening test and a semiquantitative assay, when the identity of a single type of FQN is known.

Functional Characterization of Promoter Variants of the Adiponectin Gene Complemented by Epidemiological Data

PubMed Central

Laumen, Helmut; Saningong, Akuma D.; Heid, Iris M.; Hess, Jochen; Herder, Christian; Claussnitzer, Melina; Baumert, Jens; Lamina, Claudia; Rathmann, Wolfgang; Sedlmeier, Eva-Maria; Klopp, Norman; Thorand, Barbara; Wichmann, H.-Erich; Illig, Thomas; Hauner, Hans

2009-01-01

OBJECTIVE Adiponectin (APM1, ACDC) is an adipocyte-derived protein with downregulated expression in obesity and insulin-resistant states. Several potentially regulatory single nucleotide polymorphisms (SNPs) within the APM1 gene promoter region have been associated with circulating adiponectin levels. None of them have been functionally characterized in adiponectin-expressing cells. Hence, we investigated three SNPs (rs16861194, rs17300539, and rs266729) for their influence on adiponectin promoter activity and their association with circulating adiponectin levels. RESEARCH DESIGN AND METHODS Basal and rosiglitazone-induced promoter activity of different SNP combinations (haplotypes) was analyzed in 3T3-L1 adipocytes using luciferase reporter gene assays and DNA binding studies comparing all possible APM1 haplotypes. This functional approach was complemented with analysis of epidemiological population-based data of 1,692 participants of the MONICA/KORA S123 cohort and 696 participants from the KORA S4 cohort for SNP and haplotype association with circulating adiponectin levels. RESULTS Major to minor allele replacements of the three SNPs revealed significant effects on promoter activity in luciferase assays. Particularly, a minor variant in rs16861194 resulted in reduced basal and rosiglitazone-induced promoter activity and hypoadiponectinemia in the epidemiological datasets. The haplotype with the minor allele in all three SNPs showed a complete loss of promoter activity, and no subject carried this haplotype in either of the epidemiological samples (combined P value for statistically significant difference from a random sample was 0.006). CONCLUSIONS Our results clearly demonstrate that promoter variants associated with hypoadiponectinemia in humans substantially affect adiponectin promoter activity in adipocytes. Our combination of functional experiments with epidemiological data overcomes the drawback of each approach alone. PMID:19074982

A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.

PubMed

Burbelo, Peter D; Goldman, Radoslav; Mattson, Thomas L

2005-08-18

Assays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data. We developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20-85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically. This technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications.

A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins

PubMed Central

Burbelo, Peter D; Goldman, Radoslav; Mattson, Thomas L

2005-01-01

Background Assays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data. Results We developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20–85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically. Conclusion This technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications. PMID:16109166

Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice.

PubMed

Iyer, Meera; Berenji, Manijeh; Templeton, Nancy S; Gambhir, Sanjiv S

2002-10-01

Gene therapy involves the safe and effective delivery of one or more genes of interest to target cells in vivo. The advantages of using nonviral delivery systems include ease of preparation, low toxicity, and weak immunogenicity. Nonviral delivery methods, when combined with a noninvasive, clinically applicable imaging assay, will greatly aid in the optimization of gene therapy approaches for cancer. We demonstrate cationic lipid-mediated noninvasive monitoring of reporter gene expression of firefly (Photinus pyralis) luciferase (fl) and a mutant herpes simplex virus type I thymidine kinase (HSV1-sr39tk, tk) in living mice using a cooled charge coupled device (CCD) camera and positron emission tomography (PET), respectively. We observe a high level of fl and tk reporter gene expression predominantly in the lungs after a single injection of the extruded DOTAP:cholesterol DNA liposome complexes by way of the tail vein, seen to be time- and dose-dependent. We observe a good correlation between the in vivo bioluminescent signal and the ex vivo firefly luciferase enzyme (FL) activity in different organs. We further demonstrate the feasibility of noninvasively imaging both optical and PET reporter gene expression in the same animal using the CCD camera and microPET, respectively.

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cruz, P.G.; Auld, D.S.; Schultz, P.J.

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 ofmore » 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.« less

A rapid and reagent-free bioassay for the detection of dioxin-like compounds and other aryl hydrocarbon receptor (AhR) agonists using autobioluminescent yeast.

PubMed

Xu, Tingting; Young, Anna; Marr, Enolia; Sayler, Gary; Ripp, Steven; Close, Dan

2018-02-01

An autonomously bioluminescent Saccharomyces cerevisiae BLYAhS bioreporter was developed in this study for the simple and rapid detection of dioxin-like compounds (DLCs) and aryl hydrocarbon receptor (AhR) agonists. This recombinant yeast reporter was based on a synthetic bacterial luciferase reporter gene cassette (lux) that can produce the luciferase as well as the enzymes capable of self-synthesizing the requisite substrates for bioluminescent production from endogenous cellular metabolites. As a result, bioluminescent signal production is generated continuously and autonomously without cell lysis or exogenous reagent addition. By linking the expression of the autobioluminescent lux reporter cassette to AhR activation via the use of a dioxin-responsive promoter, the S. cerevisiae BLYAhS bioreporter emitted a bioluminescent signal in response to DLC exposure in a dose-responsive manner. The model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), could be detected within 4 h with a half maximal effective concentration (EC 50 ) of ~ 8.1 nM and a lower detection limit of 500 pM. The autobioluminescent response of BLYAhS to other AhR agonists, including 2,3,7,8-tetrachlorodibenzofuran (TCDF), polychlorinated bisphenyl congener 126 (PCB-126) and 169 (PCB-169), 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD), benzo[a]pyrene (BaP), and β-naphthoflavone (bNF), were also characterized in this study. The non-destructive and reagent-free nature of the BLYAhS reporter assay facilitated near-continuous, automated signal acquisition without additional hands-on effort and cost, providing a simple and cost-effective method for rapid DLC detection.

Visualising Androgen Receptor Activity in Male and Female Mice

PubMed Central

Dart, D. Alwyn; Waxman, Jonathan; Aboagye, Eric O.; Bevan, Charlotte L.

2013-01-01

Androgens, required for normal development and fertility of males and females, have vital roles in the reproductive tract, brain, cardiovascular system, smooth muscle and bone. Androgens function via the androgen receptor (AR), a ligand-dependent transcription factor. To assay and localise AR activity in vivo we generated the transgenic “ARE-Luc” mouse, expressing a luciferase reporter gene under the control of activated endogenous AR. In vivo imaging of androgen-mediated luciferase activity revealed several strongly expressing tissues in the male mouse as expected and also in certain female tissues. In males the testes, prostate, seminal vesicles and bone marrow all showed high AR activity. In females, strong activity was seen in the ovaries, uterus, omentum tissue and mammary glands. In both sexes AR expression and activity was also found in salivary glands, the eye (and associated glands), adipose tissue, spleen and, notably, regions of the brain. Luciferase protein expression was found in the same cell layers as androgen receptor expression. Additionally, mouse AR expression and activity correlated well with AR expression in human tissues. The anti-androgen bicalutamide reduced luciferase signal in all tissues. Our model demonstrates that androgens can act in these tissues directly via AR, rather than exclusively via androgen aromatisation to estrogens and activation of the estrogen receptor. Additionally, it visually demonstrates the fundamental importance of AR signalling outside the normal role in the reproductive organs. This model represents an important tool for physiological and developmental analysis of androgen signalling, and for characterization of known and novel androgenic or antiandrogenic compounds. PMID:23940781

Molecular cloning and identification of the transcriptional regulatory domain of the goat neurokinin B gene TAC3.

PubMed

Suetomi, Yuta; Matsuda, Fuko; Uenoyama, Yoshihisa; Maeda, Kei-ichiro; Tsukamura, Hiroko; Ohkura, Satoshi

2013-10-01

Neurokinin B (NKB), encoded by TAC3, is thought to be an important accelerator of pulsatile gonadotropin-releasing hormone release. This study aimed to clarify the transcriptional regulatory mechanism of goat TAC3. First, we determined the full-length mRNA sequence of goat TAC3 from the hypothalamus to be 820 b, including a 381 b coding region, with the putative transcription start site located 143-b upstream of the start codon. The deduced amino acid sequence of NKB, which is produced from preproNKB, was completely conserved among goat, cattle, and human. Next, we cloned 5'-upstream region of goat TAC3 up to 3400 b from the translation initiation site, and this region was highly homologous with cattle TAC3 (89%). We used this goat TAC3 5'-upstream region to perform luciferase assays. We created a luciferase reporter vector containing DNA constructs from -2706, -1837, -834, -335, or -197 to +166 bp (the putative transcription start site was designated as +1) of goat TAC3 and these were transiently transfected into mouse hypothalamus-derived N7 cells and human neuroblastoma-derived SK-N-AS cells. The luciferase activity gradually increased with the deletion of the 5'-upstream region, suggesting that the transcriptional suppressive region is located between -2706 and -336 bp and that the core promoter exists downstream of -197 bp. Estradiol treatment did not lead to significant suppression of luciferase activity of any constructs, suggesting the existence of other factor(s) that regulate goat TAC3 transcription.

miR-1182 inhibits growth and mediates the chemosensitivity of bladder cancer by targeting hTERT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Jun; Dai, Wenbin, E-mail: daiwenbin271@163.com; Song, Jianming

2016-02-05

microRNAs (miRNAs) have been demonstrated to contribute to tumor progression and metastasis and proposed to be key regulators of diverse biological processes. In this study, we report that miR-1182 is deregulated in bladder cancer tissues and cell lines. To characterize the role of miR-1182 in bladder cancer cells, we performed functional assays. The overexpression of miR-1182 significantly inhibits bladder cancer cell proliferation, colony formation, and invasion. Moreover, its up-regulation induced cell cycle arrest and apoptosis and mediated chemosensitivity to cisplatin in bladder cancer. Furthermore, a luciferase reporter assay and a rescue experiment indicated that miR-1182 directly targets hTERT by bindingmore » its 3′UTR. In conclusion, these results demonstrate that miR-1182 acts as a tumor suppressor and may be a potential biomarker for bladder cancer diagnosis and treatment.« less

High-Sensitivity Real-Time Imaging of Dual Protein-Protein Interactions in Living Subjects Using Multicolor Luciferases

PubMed Central

Hida, Naoki; Awais, Muhammad; Takeuchi, Masaki; Ueno, Naoto; Tashiro, Mayuri; Takagi, Chiyo; Singh, Tanuja; Hayashi, Makoto; Ohmiya, Yoshihiro; Ozawa, Takeaki

2009-01-01

Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1–Smad4 and Smad2–Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects. PMID:19536355

Molecular basis of thermostability enhancement of Renilla luciferase at higher temperatures by insertion of a disulfide bridge into the structure.

PubMed

Fanaei Kahrani, Zahra; Emamzadeh, Rahman; Nazari, Mahboobeh; Rasa, Seyed Mohammad Mahdi

2017-02-01

Renilla luciferase (RLuc), also known as Renilla-luciferin 2-monooxygenase, is a light producing enzyme used in many biotechnological applications such as bioreporters. However, its kinetics stability -especially at higher temperatures- is a limiting factor for developing thermostable bioreporters. The aim of this study was to improve the stability of super Renilla luciferase 8 (SRLuc 8) which is a red-emitter variety of RLuc at higher temperatures, by introduction of a disulfide bridge into its structure. In this study, the choice of the proper disulfide bond formation was based on computational methods and enzyme functionality (active site position) which is called geometric-functional method. N45 and A71 at the N-terminal of the enzyme were selected for directed evolution. The engineered luciferase was called C-SRLuc 8 and its activity and stability were assayed. The results indicated that the kinetic stability of C-SRLuc 8 increased significantly at 60°C to 70°C as compared to SRLuc 8; the residual activity of C-SRLuc 8 was approximately 20% after incubation at 65°C for 5min. Moreover, the enzyme activity decreased compared with SRLuc 8. The molecular basis of the structural changes was considered using molecular dynamics simulations and the results indicated that the N45C/A71C crosslink was involved in a hotspot foldon which seemed to be the rate-limiting step of conformational collapse at higher temperatures. The present study may provide an opportunity for the development of the next-generation of thermostable RLuc-based biosensors. Copyright © 2016. Published by Elsevier B.V.

A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain

2016-09-15

The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc.more » The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.« less

Luciferase-tagged wild-type and tropism-deficient mouse cytomegaloviruses reveal early dynamics of host colonization following peripheral challenge.

PubMed

Farrell, Helen; Oliveira, Martha; Macdonald, Kate; Yunis, Joseph; Mach, Michael; Bruce, Kimberley; Stevenson, Philip; Cardin, Rhonda; Davis-Poynter, Nicholas

2016-12-01

Cytomegaloviruses (CMVs) establish persistent, systemic infections and cause disease by maternal-foetal transfer, suggesting that their dissemination is a key target for antiviral intervention. Late clinical presentation has meant that human CMV (HCMV) dissemination is not well understood. Murine CMV (MCMV) provides a tractable model. Whole mouse imaging of virus-expressed luciferase has proved a useful way to track systemic infections. MCMV, in which the abundant lytic gene M78 was luciferase-tagged via a self-cleaving peptide (M78-LUC), allowed serial, unbiased imaging of systemic and peripheral infection without significant virus attenuation. Ex vivo luciferase imaging showed greater sensitivity than plaque assay, and revealed both well-known infection sites (the lungs, lymph nodes, salivary glands, liver, spleen and pancreas) and less explored sites (the bone marrow and upper respiratory tract). We applied luciferase imaging to tracking MCMV lacking M33, a chemokine receptor conserved in HCMV and a proposed anti-viral drug target. M33-deficient M78-LUC colonized normally in peripheral sites and local draining lymph nodes but spread poorly to the salivary gland, suggesting a defect in vascular transport consistent with properties of a chemokine receptor.

Intragenic Locus in Human PIWIL2 Gene Shares Promoter and Enhancer Functions.

PubMed

Skvortsova, Yulia V; Kondratieva, Sofia A; Zinovyeva, Marina V; Nikolaev, Lev G; Azhikina, Tatyana L; Gainetdinov, Ildar V

2016-01-01

Recently, more evidence supporting common nature of promoters and enhancers has been accumulated. In this work, we present data on chromatin modifications and non-polyadenylated transcription characteristic for enhancers as well as results of in vitro luciferase reporter assays suggesting that PIWIL2 alternative promoter in exon 7 also functions as an enhancer for gene PHYHIP located 60Kb upstream. This finding of an intragenic enhancer serving as a promoter for a shorter protein isoform implies broader impact on understanding enhancer-promoter networks in regulation of gene expression.

Development and implementation of an international proficiency testing program for a neutralizing antibody assay for HIV-1 in TZM-bl cells.

PubMed

Todd, Christopher A; Greene, Kelli M; Yu, Xuesong; Ozaki, Daniel A; Gao, Hongmei; Huang, Yunda; Wang, Maggie; Li, Gary; Brown, Ronald; Wood, Blake; D'Souza, M Patricia; Gilbert, Peter; Montefiori, David C; Sarzotti-Kelsoe, Marcella

2012-01-31

Recent advances in assay technology have led to major improvements in how HIV-1 neutralizing antibodies are measured. A luciferase reporter gene assay performed in TZM-bl (JC53bl-13) cells has been optimized and validated. Because this assay has been adopted by multiple laboratories worldwide, an external proficiency testing program was developed to ensure data equivalency across laboratories performing this neutralizing antibody assay for HIV/AIDS vaccine clinical trials. The program was optimized by conducting three independent rounds of testing, with an increased level of stringency from the first to third round. Results from the participating domestic and international laboratories improved each round as factors that contributed to inter-assay variability were identified and minimized. Key contributors to increased agreement were experience among laboratories and standardization of reagents. A statistical qualification rule was developed using a simulation procedure based on the three optimization rounds of testing, where a laboratory qualifies if at least 25 of the 30 ID50 values lie within the acceptance ranges. This ensures no more than a 20% risk that a participating laboratory fails to qualify when it should, as defined by the simulation procedure. Five experienced reference laboratories were identified and tested a series of standardized reagents to derive the acceptance ranges for pass-fail criteria. This Standardized Proficiency Testing Program is the first available for the evaluation and documentation of assay equivalency for laboratories performing HIV-1 neutralizing antibody assays and may provide guidance for the development of future proficiency testing programs for other assay platforms. Copyright © 2011 Elsevier B.V. All rights reserved.

Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis.

PubMed

Loening, Andreas Markus; Fenn, Timothy David; Gambhir, Sanjiv Sam

2007-12-07

Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 A and demonstrate a classic alpha/beta-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity‡

PubMed Central

Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B.; Titus, Louisa; Boden, Scott D.

2010-01-01

The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1ΔSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell-based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post-translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression-based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP-1. Among them, we selected SVAK-3, a compound that showed a dose-dependent potentiation of BMP-2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP-1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP-2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored. PMID:19862690

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

PubMed

Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

2009-12-01

The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell-based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post-translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression-based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP-1. Among them, we selected SVAK-3, a compound that showed a dose-dependent potentiation of BMP-2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP-1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP-2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored.

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.

[Construction and function identification of luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR].

PubMed

Yang, Shuo; Li, Jia-li; Bi, Hui-chang; Zhou, Shou-ning; Liu, Xiao-man; Zeng, Hang; Hu, Bing-fang; Huang, Min

2016-01-01

This study aims to investigate the function of two SNPs (rs8904C > T and rs696G >A) in 3' untranslated region (3'UTR) of NFKBIA gene by constructing luciferase reporter gene. A patient's genomic DNA with rs8904 CC and rs696 GA genotype was used as the PCR template. Full-length 3'UTR of NFKBIA gene was amplified by different primers. After sequencing validation, these fragments were inserted to the luciferase reporter vector, pGL3-promoter to construct recombinant plasmids containing four kinds of haplotypes, pGL3-rs8904C/rs696G, pGL3-rs8904C/rs696A, pGL3-rs8904T/rs696G and pGL3-rs8904T/rs696A. Then these plasmids were transfected into LS174T cells and the luciferase activity was detected. Compared with pGL3-vector transfected cells (negative control), the luciferase activity of the four kinds of recombinant plasmids was significantly decreased (P < 0.001). For rs696G > A, the luciferase activity of the recombinant plasmids containing A allele (pGL3-rs8904C/rs696A and pGL3-rs8904T/rs696A) was about 45.1% (P < 0.05) and 56.1% (P < 0.001) lower than those containing G allele (pGL3-rs8904C/rs696G and pGL3-rs8904T/rs696G), respectively. For rs8904C > T, there were no significant differences in the luciferase activity between the recombinant plasmids containing T allele and those with C allele. Together, the luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR were constructed successfully and rs696G > A could decrease the luciferase activity while rs8904C >T didn't have much effect on the luciferase activity.

Screening Nylon-3 Polymers, a New Class of Cationic Amphiphiles, for siRNA Delivery

PubMed Central

2015-01-01

Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20–65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent. PMID:25437915

Screening nylon-3 polymers, a new class of cationic amphiphiles, for siRNA delivery.

PubMed

Nadithe, Venkatareddy; Liu, Runhui; Killinger, Bryan A; Movassaghian, Sara; Kim, Na Hyung; Moszczynska, Anna B; Masters, Kristyn S; Gellman, Samuel H; Merkel, Olivia M

2015-02-02

Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20-65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent.

Development of a rapid and quantitative method for the analysis of viral entry and release using a NanoLuc luciferase complementation assay.

PubMed

Sasaki, Michihito; Anindita, Paulina D; Phongphaew, Wallaya; Carr, Michael; Kobayashi, Shintaro; Orba, Yasuko; Sawa, Hirofumi

2018-01-02

Subviral particles (SVPs) self-assemble and are released from cells transfected with expression plasmids encoding flavivirus structural proteins. Flavivirus-like particles (VLPs), consisting of flavivirus structural proteins and a subgenomic replicon, can enter cells and cause single-round infections. Neither SVPs or VLPs possess complete viral RNA genomes, therefore are replication-incompetent systems; however, they retain the capacity to fuse and bud from target cells and follow the same maturation process as whole virions. SVPs and VLPs have been previously employed in studies analyzing entry and release steps of viral life cycles. In this study, we have developed quantitative methods for the detection of cellular entry and release of SVPs and VLPs by applying a luciferase complementation assay based on the high affinity interaction between the split NanoLuc luciferase protein, LgBiT and the small peptide, HiBiT. We introduced HiBiT into the structural protein of West Nile virus and generated SVPs and VLPs harboring HiBiT (SVP-HiBiT and VLP-HiBiT, respectively). As SVP-HiBiT emitted strong luminescence upon exposure to LgBiT and its substrate, the nascently budded SVP-HiBiT in the supernatant was readily quantified by luminometry. Similarly, the cellular entry of VLP-HiBiT generated luminescence when VLP-HiBiT was infected into LgBiT-expressing cells. These methods utilizing SVP-HiBiT and VLP-HiBiT will facilitate research into life cycles of flaviviruses, including WNV. Copyright © 2017 Elsevier B.V. All rights reserved.

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

microRNA-497 overexpression decreases proliferation, migration and invasion of human retinoblastoma cells via targeting vascular endothelial growth factor A

PubMed Central

Li, Jianjun; Zhang, Yinghui; Wang, Xiuchao; Zhao, Ruibo

2017-01-01

The expression level and roles of microRNA-497 (miR-497) have been frequently reported in previous studies on cancer. However, its expression, function and associated molecular mechanisms in retinoblastoma remain unknown. In the present study, miR-497 expression levels in human retinoblastoma tissues, normal retinal tissues and retinoblastoma cell lines were determined using reverse transcription-quantitative polymerase chain reaction. In addition, a Cell Counting Kit-8 assay, cell migration assay, cell invasion assay, western blot analysis and Dual-Luciferase reporter assay were used to explore the expression, functions and molecular mechanisms of miR-497 in human retinoblastoma. It was demonstrated that miR-497 was significantly downregulated in retinoblastoma tissues and cell lines compared with normal retinal tissues. Ectopic expression of miR-497 decreased the proliferation, migration and invasion of retinoblastoma cells. Furthermore, VEGFA was verified as a potential direct target of miR-497 in vitro. Taken together, the results indicate that miR-497 functions as a tumor suppressor in the carcinogenesis and progression of retinoblastoma via targeting VEGFA. miR-497 should be investigated as a potential therapeutic target for the treatment of retinoblastoma. PMID:28588740

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

USDA-ARS?s Scientific Manuscript database

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...

Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae

PubMed Central

Krishnamoorthy, Archana

2015-01-01

Luciferase is a useful, noninvasive reporter of gene regulation that can be continuously monitored over long periods of time; however, its use is problematic in fast-growing microbes like bacteria and yeast because rapidly changing cell numbers and metabolic states also influence bioluminescence, thereby confounding the reporter's signal. Here we show that these problems can be overcome in the budding yeast Saccharomyces cerevisiae by simultaneously monitoring bioluminescence from two different colors of beetle luciferase, where one color (green) reports activity of a gene of interest, while a second color (red) is stably expressed and used to continuously normalize green bioluminescence for fluctuations in signal intensity that are unrelated to gene regulation. We use this dual-luciferase strategy in conjunction with a light-inducible promoter system to test whether different phases of yeast respiratory oscillations are more suitable for heterologous protein production than others. By using pulses of light to activate production of a green luciferase while normalizing signal variation to a red luciferase, we show that the early reductive phase of the yeast metabolic cycle produces more luciferase than other phases. PMID:26162874

MiRNA-21 mediates the antiangiogenic activity of metformin through targeting PTEN and SMAD7 expression and PI3K/AKT pathway

PubMed Central

Luo, Mao; Tan, Xiaoyong; Mu, Lin; Luo, Yulin; Li, Rong; Deng, Xin; Chen, Ni; Ren, Meiping; Li, Yongjie; Wang, Liqun; Wu, Jianbo; Wan, Qin

2017-01-01

Metformin, an anti-diabetic drug commonly used for type 2 diabetes therapy, is associated with anti-angiogenic effects in conditions beyond diabetes. miR-21 has been reported to be involved in the process of angiogenesis. However, the precise regulatory mechanisms by which the metformin-induced endothelial suppression and its effects on miR-21-dependent pathways are still unclear. Bioinformatic analysis and identification of miR-21 and its targets and their effects on metformin-induced antiangiogenic activity were assessed using luciferase assays, quantitative real-time PCR, western blots, scratch assays, CCK-8 assays and tubule formation assays. In this study, miR-21 was strikingly downregulated by metformin in a time- and dose-dependent manner. miR-21 directly targeted the 3′-UTR of PTEN and SMAD7, and negatively regulated their expression. Overexpression of miR-21 abrogated the metformin-mediated inhibition of endothelial cells proliferation, migration, tubule formation and the TGF-β-induced AKT, SMAD- and ERK-dependent phosphorylations, and conversely, down-regulation of miR-21 aggravated metformin’s action and revealed significant promotion effects. Our study broadens our understanding of the regulatory mechanism of miR-21 mediating metformin-induced anti-angiogenic effects, providing important implications regarding the design of novel miRNA-based therapeutic strategies against angiogenesis. PMID:28230206

Increased serum miR-300 level serves as a potential biomarker of lipopolysaccharide-induced lung injury by targeting IκBα.

PubMed

Cao, Wei; Dai, Hong; Yang, Shengqing; Liu, Zhijun; Yi Chen, Qian

2017-01-10

MicroRNAs (miRs) are reported to play key roles in various disease models. In this study, the functional role of miR-300 in the regulation of lung injury was explored to assess the feasibility of serum miR-300 as a potential biomarker for lung injury. Firstly, the expression of miR-300 was studied in the serum of 50 lung injury patients and 50 healthy controls. And the expression of miR-300 was also explored in the serum and lung tissues of mouse models. To further explore the possible mechanism in which miR-300 may contribute to lung injury, the target genes of miR-300 were predicted by TargetScan and validated using dual luciferase reporter assay. Moreover, the expression of inflammation factors was studied after transfection of miR-300 mimics and inhibitors into A549 cells. Here, we first identified that the level of miR-300 was significantly upregulated in the blood samples of acute lung injury patients compared with healthy control. Meanwhile, miR-300 was also found to be enhanced in the blood samples and lung tissues of LPS-induced mouse models. Further study showed that miR-300 significantly suppressed the expression of IκBα and luciferase reporter assay showed that IκBα was a target gene of miR-300. More importantly, the levels of inflammatory factors, such as TNFα, COX-2, iNOS, IL-6 and IL8, were significantly upregulated accompanied by overexpression of miR-300 in A549 cells. In summary, enhanced miR-300 expression in the peripheral blood contributed to the lung injury mainly by inhibiting the expression of IκBα.

Curcumin activates human glutathione S-transferase P1 expression through antioxidant response element.

PubMed

Nishinaka, Toru; Ichijo, Yusuke; Ito, Maki; Kimura, Masayoshi; Katsuyama, Masato; Iwata, Kazumi; Miura, Takeshi; Terada, Tomoyuki; Yabe-Nishimura, Chihiro

2007-05-15

Curcumin is a plant-derived diferuloylmethane compound extracted from Curcuma longa, possessing antioxidative and anticarcinogenic properties. Antioxidants and oxidative stress are known to induce the expression of certain classes of detoxification enzymes. Since the upregulation of detoxifying enzymes affects the drug metabolism and cell defense system, it is important to understand the gene regulation by such agents. In this study, we demonstrated that curcumin could induce the expression of human glutathione S-transferase P1 (GSTP1). In HepG2 cells treated with 20muM curcumin, the level of GSTP1 mRNA was significantly increased. In luciferase reporter assays, curcumin augmented the promoter activity of a reporter construct carrying 336bp upstream of the 5'-flanking region of the GSTP1 gene. Mutation analyses revealed that the region including antioxidant response element (ARE), which overlaps AP1 in sequence, was essential to the response to curcumin. While the introduction of a wild-type Nrf2 expression construct augmented the promoter activity of the GSTP1 gene, co-expression of a dominant-negative Nrf2 abolished the responsiveness to curcumin. In addition, curcumin activated the expression of the luciferase gene from a reporter construct carrying multiple ARE consensus sequences but not one with multiple AP1 sites. In a gel mobility shift assay with an oligonucleotide with GSTP1 ARE, an increase in the amount of the binding complex was observed in the nuclear extracts of curcumin-treated HepG2 cells. These results suggested that ARE is the primary sequence for the curcumin-induced transactivation of the GSTP1 gene. The induction of GSTP1 may be one of the mechanisms underlying the multiple actions of curcumin.

Molecular and functional evaluation of a novel HIF inhibitor, benzopyranyl 1,2,3-triazole compound

PubMed Central

Park, Kyunghye; Lee, Hye Eun; Lee, Sun Hee; Lee, Doohyun; Lee, Taeho; Lee, You Mie

2017-01-01

Hypoxia occurs in a variety of pathological events, including the formation of solid tumors. Hypoxia-inducible factor (HIF)-1α is stabilized under hypoxic conditions and is a key molecule in tumor growth and angiogenesis. Seeking to develop novel cancer therapeutics, we investigated small molecules from our in-house chemical libraries to target HIF-1α. We employed a dual-luciferase assay that uses a luciferase (Luc) reporter vector harboring five copies of hypoxia-responsive element (HRE) in the promoter. Under hypoxic conditions that increased Luc reporter activity by four-fold, we screened 144 different compounds, nine of which showed 30–50% inhibition of hypoxia-induced Luc reporter activity. Among these, “Compound 12, a benzopyranyl 1,2,3-triazole” was the most efficient at inhibiting the expression of HIF-1α under hypoxic conditions, reducing its expression by 80%. Under hypoxic conditions, the half maximal IC50 of the compound was 24 nM in HEK-293 human embryonic kidney cells, and 2 nM in A549 human lung carcinoma cells. Under hypoxic conditions, Compound 12 increased hydroxylated HIF-1α levels and HIF-1α ubiquitination, and also dose-dependently decreased HIF-1α target gene expression as well as vascular endothelial growth factor (VEGF) secretion. Furthermore, this compound inhibited VEGF-induced in vitro angiogenesis in human umbilical vein endothelial cells (HUVECs), and in vivo, it inhibited chick chorioallantoic membrane angiogenesis. In allogaft assays, cotreatment with Compound 12 and gefitinib significantly inhibited tumor growth and angiogenesis. Compound 12 can be a novel inhibitor of HIF-1α by accelerating its degradation, and shows much potential as an anti-cancer agent through its ability to suppress tumor growth and angiogenesis. PMID:27999195

Photoaging protective effects of BIOGF1K, a compound-K-rich fraction prepared from Panax ginseng.

PubMed

Hong, Yo Han; Kim, Donghyun; Nam, Gibaeg; Yoo, Sulgi; Han, Sang Yun; Jeong, Seong-Gu; Kim, Eunji; Jeong, Deok; Yoon, Keejung; Kim, Sunggyu; Park, Junseong; Cho, Jae Youl

2018-01-01

BIOGF1K, a compound-K-rich fraction, has been shown to display anti-inflammatory activity. Although Panax ginseng is widely used for the prevention of photoaging events induced by UVB irradiation, the effect of BIOGF1K on photoaging has not yet been examined. In this study, we investigated the effects of BIOGF1K on UVB-induced photoaging events. We analyzed the ability of BIOGF1K to prevent UVB-induced apoptosis, enhance matrix metalloproteinase (MMP) expression, upregulate anti-inflammatory activity, reduce sirtuin 1 expression, and melanin production using reverse transcription-polymerase chain reaction, melanin content assay, tyrosinase assay, and flow cytometry. We also evaluated the effects of BIOGF1K on the activator protein-1 signaling pathway, which plays an important role in photoaging, by immunoblot analysis and luciferase reporter gene assays. Treatment of UVB-irradiated NIH3T3 fibroblasts with BIOGF1K prevented UVB-induced cell death, inhibited apoptosis, suppressed morphological changes, reduced melanin secretion, restored the levels of type I procollagen and sirtuin 1, and prevented mRNA upregulation of MMP-1, MMP-2, and cyclo-oxygenase-2; these effects all occurred in a dose-dependent manner. In addition, BIOGF1K markedly reduced activator-protein-1-mediated luciferase activity and decreased the activity of mitogen-activated protein kinases (extracellular response kinase, p38, and C-Jun N-terminal kinase). Our results strongly suggest that BIOGF1K has anti-photoaging activity and that BIOGF1K could be used in anti-aging cosmeceutical preparations.

[miR-25 promotes cell proliferation by targeting RECK in human cervical carcinoma HeLa cells].

PubMed

Qiu, Gang; Fang, Baoshuan; Xin, Guohong; Wei, Qiang; Yuan, Xiaoye; Wu, Dayong

2015-01-01

To investigate the effect of miR-25 on the proliferation of human cervical carcinoma HeLa cells and its association with reversion-inducing cysteine-rich protein with Kazal motifs (RECK). The recombinant plasmids of pcDNATM6.2-GW-pre-miR-25, pmirGLO-RECK-WT, pmirGLO-RECK-MT and anti-miR-25 were constructed, and their transfection efficiencies into HeLa cells were identified by real-time quantitative PCR (qRT-PCR). The potential proliferation-stimulating function of miR-25 was analyzed by MTT assay in HeLa cells. Furthermore, the target effect of miR-25 on the RECK was determined by dual-luciferase reporter assay system, qRT-PCR and Western blotting. Sequence analysis demonstrated that the recombinant plasmids of pcDNATM6.2-GW-pre-miR-25 and pmirGLO-RECK-WT, pmirGLO-RECK-MT were successfully constructed, and qRT-PCR revealed that the transfection efficiencies of pre-miR-25 and anti-miR-25 were desirable in HeLa cells. MTT assay showed that miR-25 over-expression promoted the proliferation of HeLa cells. In addition, the luciferase activity was significantly reduced in HeLa cells cotransfected with pre-miR-25 and RECK-WT. The qRT-PCR and Western blotting indicated that the expression level of RECK was up-regulated in HeLa cells transfected with anti-miR-25 at the transcriptional and posttranscriptional levels. miR-25 could promote cell proliferation by targeting RECK in HeLa cells.

Low-dose 6-bromoindirubin-3′-oxime induces partial dedifferentiation of endothelial cells to promote increased neovascularization† (R1)

PubMed Central

KOHLER, ERIN E.; BARUAH, JUGAJYOTI; URAO, NORIFUMI; USHIO-FUKAI, MASUKO; FUKAI, TOHRU; CHATTERJEE, ISHITA; WARY, KISHORE K.

2014-01-01

Endothelial cell (EC) dedifferentiation in relation to neovascularization is a poorly understood process. In this report we addressed the role of Wnt signaling in the mechanisms of neovascularization in adult tissues. Here, we show that a low-dose of 6-bromoindirubin-3′-oxime (BIO), a competitive inhibitor of Glycogen Synthase Kinase (GSK)-3β, induced the stabilization of β-catenin and its subsequent direct interaction with the transcription factor NANOG in the nucleus of ECs. This event induced loss of VE-cadherin from the adherens junctions, increased EC proliferation accompanied by asymmetric cell division (ACD), and formed cellular aggregates in a hanging drop assays indicating the acquisition of a dedifferentiated state. In a chromatin immunoprecipitation assay, nuclear NANOG protein bound to the NANOG- and VEGFR2-promoters in ECs, and the addition of BIO activated the NANOG-promoter-luciferase reporter system in a cell-based assay. Consequently, NANOG-knockdown decreased BIO-induced NOTCH-1 expression, thereby decreasing cell proliferation, ACD and neovascularization. In a Matrigel plug assay, BIO induced increased neovascularization, secondary to the presence of VEGF. Moreover, in a mouse model of hind limb ischemia, BIO augmented neovascularization that was coupled with increased expression of NOTCH-1 in ECs and increased smooth muscle α-actin (SMA)+ cell recruitment around the neovessels. Thus, these results show the ability of a low-dose of BIO to augment neovascularization secondary to VEGF, a process that was accompanied by a partial dedifferentiation of ECs via β-catenin and the NANOG signaling pathway. PMID:24496925

Humanized Androgen Receptor Mice: A Genetic Model for Differential Response to Prostate Cancer Therapy

DTIC Science & Technology

2009-07-01

transfected with 4 ng of receptor (12Q, 21Q or 48Q), 400 ng of reporter and 100 ng of promoterless renilla luciferase as an internal control. 24 hrs...The histograms represent average values normalized to renilla from either three (A and B) or two (C) independent trials. Fold activation was...luciferase activities normalized to renilla from three independent trials. * p≤0.02, significant differences based on Anova single factor analysis

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

PubMed Central

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

2013-01-01

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

A Functional Variant rs6435156C > T in BMPR2 is Associated With Increased Risk of Chronic Obstructive Pulmonary Disease (COPD) in Southern Chinese Population.

PubMed

Wang, Jian; Zhang, Chenting; Zhang, Zili; Zheng, Zeguang; Sun, Dejun; Yang, Quan; Hadadi, Cyrus; Li, Defu; Xu, Xiaoming; Xiong, Mingmei; Zhou, Qipeng; Guo, Meihua; Wang, Yingfeng; Tang, Chun; Xu, Guihua; Yang, Kai; Zhong, Nanshan; Lu, Wenju

2016-03-01

Bone morphogenetic protein receptor type 2 (BMPR2) signaling is anti-inflammatory. Decreased BMPR2 expression was seen in lung tissue from chronic obstructive pulmonary disease (COPD) patients. The selected single nucleotide polymorphisms (SNPs) in BMPR2 were genotyped with polymerase chain reaction (PCR) ligase detection reaction. The effects of SNPs on gene expression were analyzed with luciferase assays. The mRNA and protein expression levels of BMPR2 in peripheral blood mononuclear cells (PBMCs) from COPD patients were determined by quantitative PCR and western blotting, respectively. Two SNPs, rs6435156C > T and rs1048829G > T in the 3'-untranslated region (3'UTR) of BMPR2 were selected and genotyped in COPD case and healthy control subjects from southern Chinese population. Both of them were found associated with significantly increased COPD risk (adjusted odds ratio [OR] = 1.58 with 95% confidence interval [CI] = 1.14-2.15, P = 0.0056 for rs6435156C > T; adjusted OR = 1.47 and 95% CI = 1.10-1.97, P = 0.0092 for rs1048829G > T). Older age, cigarette smoking, family history of cancer and COPD were all factors that interacted with rs6435156C > T and rs1048829G > T causing increased COPD risk. Cigarette smokers with rs6435156 (CT + TT) or rs1048829 (GT + TT) were more susceptible to COPD than that with the rs6435156CC or rs1048829GG genotypes. In A549 human alveolar epithelial cells, luciferase reporter assays revealed that introduction of 3'UTR of BMPR2 plasmids carrying rs6435156T allele but not rs1048829T led to lower luciferase activity than the wild-type C or G alleles. Comparing to rs6435156CC, treatment with hsa-miR-20a mimics deceased whereas hsa-miR-20a inhibitor restored the luciferase reporter activity in cells transfected with constructs carrying rs6435156TT. BMPR2 mRNA and protein expressions were significantly lower in PBMCs from COPD smokers than that in non-smokers. COPD patients carrying rs6435156T allele had less BMPR2 expression in PBMCs. This study demonstrated that both rs6435156C > T and rs1048829G > T variants in BMPR2 contributed to increased susceptibility to COPD. The T variants of rs6435156 increased COPD risk likely by binding with hsa-miR-20a, thus leading to downregulated BMPR2 expression in lung epithelial and immune cells.

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

PubMed

Erdmann, Simon E; Dietz, Rune; Sonne, Christian; Bechshøft, Thea Ø; Vorkamp, Katrin; Letcher, Robert J; Long, Manhai; Bonefeld-Jørgensen, Eva C

2013-07-01

The aims of the project were to (i) extract the lipophilic persistent organic pollutants (POPs) from the blood of 99 East Greenland polar bears and assess the combined mixture effect on the estrogen receptor (ER) and the aryl hydrocarbon receptor (AhR) mediated transactivity; (ii) To evaluate whether the receptor transactivities were associated with selected POP markers, and (iii) compare the receptor transactivities in polar bears with earlier studies on Greenlandic Inuit. Lipophilic POPs were extracted using a combination of solid-phase extraction (SPE) and high performance liquid chromatography (HPLC). ER mediated transactivity was determined using the ER luciferase reporter MVLN cell assay. The extracts were tested alone (XER) and together with 17β-estradiol (E2) as a physiological mimic (XERcomp). Dioxins and dioxin-like (DL) compounds were extracted by a combination of SPE and the Supelco Dioxin Prep System®. AhR mediated dioxin-like transactivity was determined using the AhR luciferase reporter Hepa 1.12cR cell assay. Agonistic ER transactivity was elicited by 19% of the samples, and a further increased E2 induced ER response was found for 52%, whereas 17% antagonized the E2 induced ER response. Positive correlations were found in subadult bears between XER and several POP biomarkers. XER and XERcomp correlated positively to each other. A total of 91% of the polar bear blood extracts elicited agonistic AhR transactivity. The AhR-TCDD equivalent (AhR-TEQ) median levels were higher among adult bears compared to subadult bears, but not significantly. Copyright © 2013 Elsevier Ltd. All rights reserved.

PPARγ regulates exocrine pancreas lipase.

PubMed

Danino, Hila; Naor, Ronny Peri-; Fogel, Chen; Ben-Harosh, Yael; Kadir, Rotem; Salem, Hagit; Birk, Ruth

2016-12-01

Pancreatic lipase (triacylglycerol lipase EC 3.1.1.3) is an essential enzyme in hydrolysis of dietary fat. Dietary fat, especially polyunsaturated fatty acids (PUFA), regulate pancreatic lipase (PNLIP); however, the molecular mechanism underlying this regulation is mostly unknown. As PUFA are known to regulate expression of proliferator-activated receptor gamma (PPARγ), and as we identified in-silico putative PPARγ binding sites within the putative PNLIP promoter sequence, we hypothesized that PUFA regulation of PNLIP might be mediated by PPARγ. We used in silico bioinformatics tools, reporter luciferase assay, PPARγ agonists and antagonists, PPARγ overexpression in exocrine pancreas AR42J and primary cells to study PPARγ regulation of PNLIP. Using in silico bioinformatics tools we mapped PPARγ binding sites (PPRE) to the putative promoter region of PNLIP. Reporter luciferase assay in AR42J rat exocrine pancreas acinar cells transfected with various constructs of the putative PNLIP promoter showed that PNLIP transcription is significantly enhanced by PPARγ dose-dependently, reaching maximal levels with multi PPRE sites. This effect was significantly augmented in the presence of PPARγ agonists and reduced by PPARγ antagonists or mutagenesis abrogating PPRE sites. Over-expression of PPARγ significantly elevated PNLIP transcript and protein levels in AR42J cells and in primary pancreas cells. Moreover, PNLIP expression was up-regulated by PPARγ agonists (pioglitazone and 15dPGJ2) and significantly down-regulated by PPARγ antagonists in non-transfected rat exocrine pancreas AR42J cell line cells. PPARγ transcriptionally regulates PNLIP gene expression. This transcript regulation resolves part of the missing link between dietary PUFA direct regulation of PNLIP. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression.

PubMed

Puckette, Michael; Burrage, Thomas; Neilan, John G; Rasmussen, Max

2017-06-12

The Gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. We sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the Gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2A translational interrupter sequence. We produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2A sequence placed either N-terminal or C-terminal to the Gaussia princeps luciferase gene. Two plasmids included novel Gaussia princeps luciferase variants with the position 1 methionine deleted. Placing a foot-and-mouth disease virus 2A translational interrupter sequence on either the N- or C-terminus of the Gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. We also measured the ability of another polycistronic plasmid vector with a 2A-luciferase sequence placed downstream of the foot-and-mouth disease virus P1 and 3C protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. Incorporation of the 2A-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles. We demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs.

Androgen receptor requires JunD as a coactivator to switch on an oxidative stress generation pathway in prostate cancer cells.

PubMed

Mehraein-Ghomi, Farideh; Basu, Hirak S; Church, Dawn R; Hoffmann, F Michael; Wilding, George

2010-06-01

Relatively high oxidative stress levels in the prostate are postulated to be a major factor for prostate carcinogenesis and prostate cancer (CaP) progression. We focused on elucidating metabolic pathways of oxidative stress generation in CaP cells. Previously, we showed that the transcription factor JunD is essential for androgen-induced reactive oxygen species (ROS) production in androgen-dependent human CaP cells. We also recently showed that androgen induces the first and regulatory enzyme spermidine/spermine N1-acetyltransferase (SSAT) in a polyamine catabolic pathway that produces copious amounts of metabolic ROS. Here, we present coimmunoprecipitation and Gaussia luciferase reconstitution assay data that show that JunD forms a complex with androgen-activated androgen receptor (AR) in situ. Our chromatin immunoprecipitation assay data show that JunD binds directly to a specific SSAT promoter sequence only in androgen-treated LNCaP cells. Using a vector containing a luciferase reporter gene connected to the SSAT promoter and a JunD-silenced LNCaP cell line, we show that JunD is essential for androgen-induced SSAT gene expression. The elucidation of JunD-AR complex inducing SSAT expression leading to polyamine oxidation establishes the mechanistic basis of androgen-induced ROS production in CaP cells and opens up a new prostate-specific target for CaP chemopreventive/chemotherapeutic drug development. Copyright 2010 AACR.

Powerful workhorses for antimicrobial peptide expression and characterization.

PubMed

Li, Chun; Blencke, Hans-Matti; Paulsen, Victoria; Haug, Tor; Stensvåg, Klara

2010-01-01

Discovery of antimicrobial peptides (AMP) is to a large extent based on screening of fractions of natural samples in bacterial growth inhibition assays. However, the use of bacteria is not limited to screening for antimicrobial substances. In later steps, bioengineered "bugs" can be applied to both production and characterization of AMPs. Here we describe the idea to use genetically modified Escherichia coli strains for both these purposes. This approach allowed us to investigate SpStrongylocins 1 and 2 from the purple sea urchin Strongylocentrotus purpuratus only based on sequence information from a cDNA library and without previous direct isolation or chemical synthesis of these peptides. The recombinant peptides are proved active against all bacterial strains tested. An assay based on a recombinant E. coli sensor strain expressing insect luciferase, revealed that SpStrongylocins are not interfering with membrane integrity and are therefore likely to have intracellular targets. © 2010 Landes Bioscience

An RNAi-mediated screen identifies novel targets for next-generation antiepileptic drugs based on increased expression of the homeostatic regulator pumilio.

PubMed

Lin, Wei-Hsiang; He, Miaomiao; Fan, Yuen Ngan; Baines, Richard A

2018-05-02

Despite availability of a diverse range of anti-epileptic drugs (AEDs), only about two-thirds of epilepsy patients respond well to drug treatment. Thus, novel targets are required to catalyse the design of next-generation AEDs. Manipulation of neuron firing-rate homoeostasis, through enhancing Pumilio (Pum) activity, has been shown to be potently anticonvulsant in Drosophila. In this study, we performed a genome-wide RNAi screen in S2R + cells, using a luciferase-based dPum activity reporter and identified 1166 genes involved in dPum regulation. Of these genes, we focused on 699 genes that, on knock-down, potentiate dPum activity/expression. Of this subgroup, 101 genes are activity-dependent based on comparison with genes previously identified as activity-dependent by RNA-sequencing. Functional cluster analysis shows these genes are enriched in pathways involved in DNA damage, regulation of cell cycle and proteasomal protein catabolism. To test for anticonvulsant activity, we utilised an RNA-interference approach in vivo. RNAi-mediated knockdown showed that 57/101 genes (61%) are sufficient to significantly reduce seizure duration in the characterized seizure mutant, para bss . We further show that chemical inhibitors of protein products of some of the genes targeted are similarly anticonvulsant. Finally, to establish whether the anticonvulsant activity of identified compounds results from increased dpum transcription, we performed a luciferase-based assay to monitor dpum promoter activity. Third instar larvae exposed to sodium fluoride, gemcitabine, metformin, bestatin, WP1066 or valproic acid all showed increased dpum promoter activity. Thus, this study validates Pum as a favourable target for AED design and, moreover, identifies a number of lead compounds capable of increasing the expression of this homeostatic regulator.

Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization.

PubMed

Okamoto-Uchida, Yoshimi; Nakamura, Ryosuke; Matsuzawa, Yumiko; Soma, Megumi; Kawakami, Hiroshi; Ishii-Watabe, Akiko; Nishimaki-Mogami, Tomoko; Teshima, Reiko; Saito, Yoshiro

2016-01-01

The physicochemical nature of allergen molecules differ from the liquid phase to the solid phase. However, conventional allergy tests are based on the detection of immunoglobulin (Ig)E binding to immobilized allergens. We recently developed an in vitro allergy testing method using a luciferase-reporting humanized rat mast cell line to detect IgE crosslinking-induced luciferase expression (EXiLE test). The aim of the present study was to evaluate the effects of antigen immobilization on the results of different in vitro allergy tests using two anti-ovalbumin (OVA) antibodies (Abs), E-C1 and E-G5, with different properties in the OVA-induced allergic reaction. Both Abs showed clear binding to OVA with an enzyme-linked immunosorbent assay and by BIAcore analysis. However, only E-C1 potentiated EXiLE response for the liquid-phase OVA. On the other hand, OVA immobilized on solid-phase induced EXiLE responses in both E-C1 Ab- and E-G5 Ab-sensitized mast cells. Western blotting of OVA indicated that E-C1 Ab binds both to OVA monomers and dimers, unlike E-G5 Ab, which probably binds only to the OVA dimer. These results suggest that antigen immobilization enhanced IgE crosslinking ability through multimerization of allergen molecules in the solid phase, resulting in an increase in false positives in IgE binding-based conventional in vitro allergy tests. These findings shed light on the physicochemical nature of antigens as an important factor for the development and evaluation of in vitro allergy tests and suggest that mast cell activation-based allergy testing with liquid-phase allergens is a promising strategy to evaluate the physiological interactions of IgE and allergens.

MicroRNA-650 targets ING4 to promote gastric cancer tumorigenicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, XueLi, E-mail: zhangxueli.200010@yahoo.com.cn; Zhu, WeiYing; Zhang, JiFa

2010-04-30

MicroRNAs (miRNAs) are non-coding RNAs that regulate the expression of target mRNAs. Altered expression of specific miRNAs in human gastric cancer progression has been reported; however, the role of miR-650 in gastric cancer is poorly understood. In this study, we show that miR-650 is involved in lymphatic and distant metastasis in human gastric cancer, and we find that ectopic expression of miR-650 promotes tumorigenesis and proliferation of gastric cancer cells. A luciferase reporter assay demonstrates that Inhibitor of Growth 4 (ING4) is a direct target of miR-650. Collectively, our study demonstrates that over-expression of miR-650 in gastric cancer may promotemore » proliferation and growth of cancer cells, at least partially through directly targeting ING4. These findings help clarify the molecular mechanisms involved in gastric carcinogenesis and indicate that miR-650 modulation may be a bona fide miRNA-based treatment of gastric cancer.« less

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.

PubMed

Tuckow, A P; Temeyer, K B

2015-08-01

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 vector of bovine babesiosis and anaplasmosis. Commercial promoters were evaluated for transcriptional activity driving luciferase expression in the tick cell lines. The human phosphoglycerate kinase (PGK) promoter resulted in detectable firefly luciferase activity within 2 days post-transfection of the R. microplus cell line BME26, with maximal activity at 5 days post-transfection. Several other promoters were weaker or inactive in the tick cells, prompting identification and assessment of transcriptional activity of the homologous ribosomal protein L4 (rpL4, GenBank accession no.: KM516205) and elongation factor 1α (EF-1α, GenBank accession no.: KM516204) promoters cloned from R. microplus. Evaluation of luciferase expression driven by various promoters in tick cell culture resulted in selection of the R. microplus rpL4 promoter and the human PGK promoter driving transcription of sequences encoding modified firefly and NanoLuc® luciferases for construction of a dual luciferase reporter system for use in tick cell culture. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Cytotoxicity and genotoxicity of nanosilver in stable GADD45α promoter-driven luciferase reporter HepG2 and A549 cells.

PubMed

Che, Bizhong; Luo, Qiulin; Zhai, Bingzhong; Fan, Guoqiang; Liu, Zhiyong; Cheng, Kaiming; Xin, Lili

2017-09-01

The intense commercial application of silver nanoparticles (AgNPs) has been raising concerns about their potential adverse health effects to human. This study aimed to explore the potency of AgNPs to induce GADD45α gene, an important stress sensor, and its relationships with the cytotoxicity and genotoxicity elicited by AgNPs. Two established HepG2 and A549 cell lines containing the GADD45α promoter-driven luciferase reporter were treated with increasing concentrations of AgNPs for 48 hours. After the treatment, transcriptional activation of GADD45α indicated by luciferase activity, cell viability, cell cycle arrest, and levels of genotoxicity were determined. The uptake and intracellular localization of AgNPs, cellular Ag doses as well as Ag + release were also detected. AgNPs could activate GADD45α gene at the transcriptional level as demonstrated by the dose-dependent increases in luciferase activity in both the reporter cells. The relative luciferase activity was greater than 12× the control level in HepG2-luciferase cells at the highest concentration tested where the cell viability decreased to 17.0% of the control. These results was generally in accordance with the positive responses in cytotoxicity, cell cycle arrest of Sub G1 and G2/M phase, Olive tail moment, micronuclei frequency, and the cellular Ag content. The cytotoxicity and genotoxicity of AgNPs seems to occur mainly via particles uptake and the subsequent liberation of ions inside the cells. And furthermore, the GADD45α promoter-driven luciferase reporter cells, especially the HepG2-luciferase cells, could provide a new and valuable tool for predicting nanomaterials genotoxicity in humans. © 2017 Wiley Periodicals, Inc.

Six1-Eya-Dach Network in Breast Cancer

DTIC Science & Technology

2009-05-01

Ctrl scramble controls. Responsiveness was tested using luciferase activity of the 3TP reporter construct and normalized to renilla luciferase...construct and normalized to renilla luciferase activity. Data points show the mean of two individual clones from two experiments and error bars represent

Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase.

PubMed

Stepanyuk, Galina A; Unch, James; Malikova, Natalia P; Markova, Svetlana V; Lee, John; Vysotski, Eugene S

2010-10-01

It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some "yellow" Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca(2+)-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.

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.

Several synthetic progestins disrupt the glial cell specific-brain aromatase expression in developing zebra fish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cano-Nicolau, Joel

The effects of some progestins on fish reproduction have been recently reported revealing the hazard of this class of steroidal pharmaceuticals. However, their effects at the central nervous system level have been poorly studied until now. Notwithstanding, progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. Herein, we investigated the effects of a large set of synthetic ligands of the nuclear progesterone receptor on the glial-specific expression of the zebrafish brain aromatase (cyp19a1b) using zebrafish mechanism-based assays. Progesterone and 24 progestins were first screened on transgenic cyp19a1b-GFP zebrafish embryos. Wemore » showed that progesterone, dydrogesterone, drospirenone and all the progesterone-derived progestins had no effect on GFP expression. Conversely, all progestins derived from 19-nortesterone induced GFP in a concentration-dependent manner with EC{sub 50} ranging from the low nM range to hundreds nM. The 19-nortestosterone derived progestins levonorgestrel (LNG) and norethindrone (NET) were further tested in a radial glial cell context using U251-MG cells co-transfected with zebrafish ER subtypes (zfERα, zfERβ1 or zfERβ2) and cyp19a1b promoter linked to luciferase. Progesterone had no effect on luciferase activity while NET and LNG induced luciferase activity that was blocked by ICI 182,780. Zebrafish-ERs competition assays showed that NET and LNG were unable to bind to ERs, suggesting that the effects of these compounds on cyp19a1b require metabolic activation prior to elicit estrogenic activity. Overall, we demonstrate that 19-nortestosterone derived progestins elicit estrogenic activity by inducing cyp19a1b expression in radial glial cells. Given the crucial role of radial glial cells and neuro-estrogens in early development of brain, the consequences of exposure of fish to these compounds require further investigation. - Highlights: • P4 + 24 progestins were tested on embryonic brain aromatase expression in zebrafish. • 19 nor-testosterone derivatives induced cyp19a1b expression. • cyp19a1b up-regulation involved functional zfERs. • 19 nor-testosterone derivatives are pro-estrogenic compounds. • Effect of progestins should be further investigated at the brain level.« less

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

PubMed

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

2014-11-01

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

miR-138 suppresses the proliferation, metastasis and autophagy of non-small cell lung cancer by targeting Sirt1.

PubMed

Ye, Zaiting; Fang, Bingmu; Pan, Jiongwei; Zhang, Ning; Huang, Jinwei; Xie, Congying; Lou, Tianzheng; Cao, Zhuo

2017-06-01

The present study determined the role and mechanism of miR-138 in non-small cell lung cancer (NSCLC). In total, 45 freshly resected clinical NSCLC tissues were collected. The expression of miR-138 in tissues and cell lines were determined by real-time quantitative PCR. miR-138 mimics were transfected into A549 and Calu-3 cells in vitro, and then the effects of miR-138 on lung cancer cell proliferation, cell cycle, invasion and metastasis were investigated by CCK-8 assay, Transwell and flow cytometry, respectively. The protein expression of the potential target gene Sirt1 in lung cancer cells were determined by western blot analysis. Dual-luciferase reporter assay was performed to further confirm whether Sirt1 was the target gene of miR-138. The expression of miR-138 was significantly lower in lung cancer tissues and was negatively correlated to the differentiation degree and lymph node metastasis of lung cancer. In vitro experiment results showed that miR-138 inhibited lung cancer cell proliferation, invasion and migration. It was verified that miR-138 could downregulate Sirt1 protein expression, inhibit epithelial-mesenchymal transition (EMT), decrease the activity of AMPK signaling pathway and elevate mTOR phosphorylation level. Dual-luciferase reporter assay demonstrated that miR-138 could directly regulate Sirt1. Downregulation of Sirt1 alone can also cause the same molecular and biological function changes. Western blot analysis and confocal microscopy results indicated that overexpression of miR-138 or interference of Sirt1 expression could inhibit lung cancer cell autophagy activity possibly through AMPK-mTOR signaling pathway. miR-138 plays a tumor suppressor function in lung cancer. It may inhibit the proliferation, invasion and migration of lung cancer through downregulation of Sirt1 expression and activation of cell autophagy. The downregulation of miR-138 is closely related to the development of lung cancer.

MicroRNA hsa-let-7b suppresses the odonto/osteogenic differentiation capacity of stem cells from apical papilla by targeting MMP1.

PubMed

Wang, Yanqiu; Pang, Xiyao; Wu, Jintao; Jin, Lin; Yu, Yan; Gobin, Romila; Yu, Jinhua

2018-01-31

MicroRNA let-7 family acts as the key regulator of the differentiation of mesenchymal stem cells (MSCs). However, the influence of let-7b on biological characteristics of stem cells from apical papilla (SCAPs) is still controversial. In this study, the expression of hsa-let-7b was obviously downregulated during the osteogenic differentiation of SCAPs. SCAPs were then infected with hsa-let-7b or hsa-let-7b inhibitor lentiviruses. The proliferation ability was determined by CCK-8 and flow cytometry. The odonto/osteogenic differentiation capacity was analyzed by alkaline phosphatase (ALP) activity, alizarin red staining, Western blot assay, and real-time RT-PCR. Bioinformatics analysis was used to screen out the target of hsa-let-7b and the target relationship was confirmed by dual luciferase reporter assay. Hsa-let-7b was of no influence on the proliferation of SCAPs. Interferential expression of hsa-let-7b increased the ALP activity as well as the formation of calcified nodules of SCAPs. Moreover, the mRNA levels of osteoblastic markers (ALP, RUNX2, OSX, OPN, and OCN) were upregulated while the protein levels of DSPP, ALP, RUNX2, OSX, OPN, and OCN also increased considerably. Conversely, overexpression of hsa-let-7b inhibited the odonto/osteogenic differentiation capacity of SCAPs. Bioinformatics analysis revealed a putative binding site of hsa-let-7b in the matrix metalloproteinase 1 (MMP1) 3'-untranslated region (3'-UTR). Dual luciferase reporter assay confirmed that hsa-let-7b targets MMP1. The odonto/osteogenic differentiation ability of SCAPs ascended after repression of hsa-let-7b, which was then reversed after co-transfection with siMMP1. Together, hsa-let-7b can suppress the odonto/osteogenic differentiation capacity of SCAPs by targeting MMP1. © 2018 Wiley Periodicals, Inc.

Liver X receptor alpha mediated genistein induction of human dehydroepiandrosterone sulfotransferase (hSULT2A1) in Hep G2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yue; Zhang, Shunfen; Zhou, Tianyan

2013-04-15

Cytosolic sulfotransferases are one of the major families of phase II drug metabolizing enzymes. Sulfotransferase-catalyzed sulfonation regulates hormone activities, metabolizes drugs, detoxifies xenobiotics, and bioactivates carcinogens. Human dehydroepiandrosterone sulfotransferase (hSULT2A1) plays important biological roles by sulfating endogenous hydroxysteroids and exogenous xenobiotics. Genistein, mainly existing in soy food products, is a naturally occurring phytoestrogen with both chemopreventive and chemotherapeutic potential. Our previous studies have shown that genistein significantly induces hSULT2A1 in Hep G2 and Caco-2 cells. In this study, we investigated the roles of liver X receptor (LXRα) in the genistein induction of hSULT2A1. LXRs have been shown to induce expressionmore » of mouse Sult2a9 and hSULT2A1 gene. Our results demonstrate that LXRα mediates the genistein induction of hSULT2A1, supported by Western blot analysis results, hSULT2A1 promoter driven luciferase reporter gene assay results, and mRNA interference results. Chromatin immunoprecipitation (ChIP) assay results demonstrate that genistein increase the recruitment of hLXRα binding to the hSULT2A1 promoter. These results suggest that hLXRα plays an important role in the hSULT2A1 gene regulation. The biological functions of phytoestrogens may partially relate to their induction activity toward hydroxysteroid SULT. - Highlights: ► Liver X receptor α mediated genistein induction of hSULT2A1 in Hep G2 cells. ► LXRα and RXRα dimerization further activated this induction. ► Western blot results agreed well with luciferase reporter gene assay results. ► LXRs gene silencing significantly decreased hSULT2A1 expression. ► ChIP analysis suggested that genistein enhances hLXRα binding to the hSULT2A1 promoter.« less

Post-translational regulation of gene expression using the ATF4 oxygen-dependent degradation domain for hypoxia-specific gene therapy.

PubMed

Cho, Su Hee; Oh, Binna; Kim, Hyun Ah; Park, Jeong Hyun; Lee, Minhyung

2013-11-01

Solid tumors have hypoxic regions in their cores, due to low blood supply levels. Therefore, hypoxia-specific gene regulation systems have been developed for tumor-specific gene therapy. In this study, the oxygen-dependent degradation (ODD) domain on activating transcription factor-4 (ATF4) was evaluated for post-translational regulation of proteins. The ATF4 ODD cDNA was amplified by RT-PCR, and a luciferase plasmid containing the ATF4 ODD domain, pSV-Luc-ATF4-ODD, was constructed. Luciferase expression was induced under hypoxia by the ATF4 ODD domain in transfection assays into N2A neuroblastoma cells, C6 glioblastoma cells, and U87 glioblastoma cells. In the transfection assay with pSV-Luc-ATF4-ODD, RT-PCR results showed that the mRNA level did not change under hypoxia. This suggests that the induction of luciferase under hypoxia was mediated by post-translational regulation. A plasmid expressing thymidine kinase from herpes simplex virus (HSV-tk), pSV-HSVtk-ATF4-ODD, was constructed with the ATF4 ODD cDNA. The transfection assay with pSV-TK-ATF4-ODD showed that the ATF4 ODD domain induced HSV-tk expression under hypoxia and facilitated the death of C6 cells in the presence of ganciclovir (GCV). Furthermore, pSV-HSVtk-ATF4-ODD induced caspase-3 activity in the hypoxic cells. In conclusion, the ATF4 ODD may be useful for hypoxia-specific gene therapy by post-translational regulation of gene expression.

A new firefly luciferase with bimodal spectrum: identification of structural determinants of spectral pH-sensitivity in firefly luciferases.

PubMed

Viviani, Vadim R; Oehlmeyer, T L; Arnoldi, F G C; Brochetto-Braga, M R

2005-01-01

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases.

Prenatal exposure of mice to diethylstilbestrol disrupts T-cell differentiation by regulating Fas/Fas ligand expression through estrogen receptor element and nuclear factor-κB motifs.

PubMed

Singh, Narendra P; Singh, Udai P; Nagarkatti, Prakash S; Nagarkatti, Mitzi

2012-11-01

Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans. In the current study, we investigated the effect of prenatal exposure to DES on thymocyte differentiation involving apoptotic pathways. Prenatal DES exposure caused thymic atrophy, apoptosis, and up-regulation of Fas and Fas ligand (FasL) expression in thymocytes. To examine the mechanism underlying DES-mediated regulation of Fas and FasL, we performed luciferase assays using T cells transfected with luciferase reporter constructs containing full-length Fas or FasL promoters. There was significant luciferase induction in the presence of Fas or FasL promoters after DES exposure. Further analysis demonstrated the presence of several cis-regulatory motifs on both Fas and FasL promoters. When DES-induced transcription factors were analyzed, estrogen receptor element (ERE), nuclear factor κB (NF-κB), nuclear factor of activated T cells (NF-AT), and activator protein-1 motifs on the Fas promoter, as well as ERE, NF-κB, and NF-AT motifs on the FasL promoter, showed binding affinity with the transcription factors. Electrophoretic mobility-shift assays were performed to verify the binding affinity of cis-regulatory motifs of Fas or FasL promoters with transcription factors. There was shift in mobility of probes (ERE or NF-κB2) of both Fas and FasL in the presence of nuclear proteins from DES-treated cells, and the shift was specific to DES because these probes failed to shift their mobility in the presence of nuclear proteins from vehicle-treated cells. Together, the current study demonstrates that prenatal exposure to DES triggers significant alterations in apoptotic molecules expressed on thymocytes, which may affect T-cell differentiation and cause long-term effects on the immune functions.

Prenatal Exposure of Mice to Diethylstilbestrol Disrupts T-Cell Differentiation by Regulating Fas/Fas Ligand Expression through Estrogen Receptor Element and Nuclear Factor-κB Motifs

PubMed Central

Singh, Narendra P.; Singh, Udai P.; Nagarkatti, Prakash S.

2012-01-01

Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans. In the current study, we investigated the effect of prenatal exposure to DES on thymocyte differentiation involving apoptotic pathways. Prenatal DES exposure caused thymic atrophy, apoptosis, and up-regulation of Fas and Fas ligand (FasL) expression in thymocytes. To examine the mechanism underlying DES-mediated regulation of Fas and FasL, we performed luciferase assays using T cells transfected with luciferase reporter constructs containing full-length Fas or FasL promoters. There was significant luciferase induction in the presence of Fas or FasL promoters after DES exposure. Further analysis demonstrated the presence of several cis-regulatory motifs on both Fas and FasL promoters. When DES-induced transcription factors were analyzed, estrogen receptor element (ERE), nuclear factor κB (NF-κB), nuclear factor of activated T cells (NF-AT), and activator protein-1 motifs on the Fas promoter, as well as ERE, NF-κB, and NF-AT motifs on the FasL promoter, showed binding affinity with the transcription factors. Electrophoretic mobility-shift assays were performed to verify the binding affinity of cis-regulatory motifs of Fas or FasL promoters with transcription factors. There was shift in mobility of probes (ERE or NF-κB2) of both Fas and FasL in the presence of nuclear proteins from DES-treated cells, and the shift was specific to DES because these probes failed to shift their mobility in the presence of nuclear proteins from vehicle-treated cells. Together, the current study demonstrates that prenatal exposure to DES triggers significant alterations in apoptotic molecules expressed on thymocytes, which may affect T-cell differentiation and cause long-term effects on the immune functions. PMID:22888145

Reporter Gene-Facilitated Detection of Compounds in Arabidopsis Leaf Extracts that Activate the Karrikin Signaling Pathway.

PubMed

Sun, Yueming K; Flematti, Gavin R; Smith, Steven M; Waters, Mark T

2016-01-01

Karrikins are potent germination stimulants generated by the combustion of plant matter. Treatment of Arabidopsis with karrikins triggers a signaling process that is dependent upon a putative receptor protein KARRIKIN INSENSITIVE 2 (KAI2). KAI2 is a homolog of DWARF 14 (D14), the receptor for endogenous strigolactone hormones. Genetic analyses suggest that KAI2 also perceives endogenous signal(s) that are not strigolactones. Activation of KAI2 by addition of karrikins to Arabidopsis plants induces expression of transcripts including D14-LIKE 2 ( DLK2 ). We constructed the synthetic reporter gene DLK2 : LUC in Arabidopsis , which comprises the firefly luciferase gene ( LUC ) driven by the DLK2 promoter. Here we describe a luminescence-based reporter assay with Arabidopsis seeds to detect chemical signals that can activate the KAI2 signaling pathway. We demonstrate that the DLK2 : LUC assay can selectively and sensitively detect karrikins and a functionally similar synthetic strigolactone analog. Crucially we show that crude extracts from Arabidopsis leaves can also activate DLK2 : LUC in a KAI2-dependent manner. Our work provides the first direct evidence for the existence of endogenous chemical signals that can activate the KAI2-mediated signaling pathway in Arabidopsis . This sensitive reporter system can now be used for the bioassay-guided purification and identification of putative endogenous KAI2 ligands or their precursors, and endogenous compounds that might modulate the KAI2 signaling pathway.

Reporter Gene-Facilitated Detection of Compounds in Arabidopsis Leaf Extracts that Activate the Karrikin Signaling Pathway

PubMed Central

Sun, Yueming K.; Flematti, Gavin R.; Smith, Steven M.; Waters, Mark T.

2016-01-01

Karrikins are potent germination stimulants generated by the combustion of plant matter. Treatment of Arabidopsis with karrikins triggers a signaling process that is dependent upon a putative receptor protein KARRIKIN INSENSITIVE 2 (KAI2). KAI2 is a homolog of DWARF 14 (D14), the receptor for endogenous strigolactone hormones. Genetic analyses suggest that KAI2 also perceives endogenous signal(s) that are not strigolactones. Activation of KAI2 by addition of karrikins to Arabidopsis plants induces expression of transcripts including D14-LIKE 2 (DLK2). We constructed the synthetic reporter gene DLK2:LUC in Arabidopsis, which comprises the firefly luciferase gene (LUC) driven by the DLK2 promoter. Here we describe a luminescence-based reporter assay with Arabidopsis seeds to detect chemical signals that can activate the KAI2 signaling pathway. We demonstrate that the DLK2:LUC assay can selectively and sensitively detect karrikins and a functionally similar synthetic strigolactone analog. Crucially we show that crude extracts from Arabidopsis leaves can also activate DLK2:LUC in a KAI2-dependent manner. Our work provides the first direct evidence for the existence of endogenous chemical signals that can activate the KAI2-mediated signaling pathway in Arabidopsis. This sensitive reporter system can now be used for the bioassay-guided purification and identification of putative endogenous KAI2 ligands or their precursors, and endogenous compounds that might modulate the KAI2 signaling pathway. PMID:27994609

Transcriptional regulation of human MUC4 gene: identification of a novel inhibitory element and its nuclear binding protein.

PubMed

Zhang, Jing-Jing; Zhu, Yi; Zhang, Xiong-Fei; Liang, Wen-Biao; Xie, Kun-Ling; Tao, Jin-Qiu; Peng, Yun-Peng; Xu, Ze-Kuan; Miao, Yi

2013-08-01

The human mucin 4 (MUC4) is aberrantly expressed in pancreatic adenocarcinoma and tumor cell lines, while remaining undetectable in normal pancreas, indicating its important role in pancreatic cancer development. Although its transcriptional regulation has been investigated in considerable detail, some important elements remain unknown. The aim of the present study was to demonstrate the existence of a novel inhibitory element in the MUC4 promoter and characterize some of its binding proteins. By luciferase reporter assay, we located the inhibitory element between nucleotides -2530 and -2521 in the MUC4 promoter using a series of deletion and mutant reporter constructs. Electrophoretic mobility shift assay (EMSA) with Bxpc-3 cell nuclear extracts revealed that one protein or protein complex bind to this element. The proteins binding to this element were purified and identified as Yin Yang 1 (YY1) by mass spectrometry. Supershift assay and chromatin immunoprecipitation (ChIP) assay confirmed that YY1 binds to this element in vitro and in vivo. Moreover, transient YY1 overexpression significantly inhibited MUC4 promoter activity and endogenous MUC4 protein expression. In conclusion, we reported here a novel inhibitory element in the human MUC4 promoter. This provides additional data on MUC4 gene regulation and indicates that YY1 may be a potential target for abnormal MUC4 expression.

The organic solute transporters alpha and beta are induced by hypoxia in human hepatocytes

PubMed Central

Schaffner, Carlos A; Mwinyi, Jessica; Gai, Zhibo; Thasler, Wolfgang E; Eloranta, Jyrki J; Kullak-Ublick, Gerd A

2015-01-01

Background & Aims The organic solute transporters alpha and beta (OSTα-OSTβ) form a heterodimeric transporter located at the basolateral membrane of intestinal epithelial cells and hepatocytes. Liver injury caused by ischaemia-reperfusion, cancer, inflammation or cholestasis can induce a state of hypoxia in hepatocytes. Here, we studied the effect of hypoxia on the expression of OSTα-OSTβ. Methods OSTα-OSTβ expression was measured in Huh7 cells and primary human hepatocytes (PHH) exposed to chenodeoxycholic acid (CDCA), hypoxia or both. OSTα-OSTβ promoter activity was analysed in luciferase reporter gene assays. Binding of hypoxia-inducible factor-1 alpha (HIF-1α) to the OSTα-OSTβ gene promoters was studied in electrophoretic mobility shift assays (EMSA). Results Expression of OSTα and OSTβ increased in PHH under conditions of hypoxia. Exposure of Huh7 cells or PHH to CDCA (50 μM) enhanced the effect of hypoxia on OSTα mRNA levels. In luciferase assays and EMSA, the inducing effect of low oxygen could be assigned to HIF-1α, which binds to hypoxia responsive elements (HRE) in the OSTα and OSTβ gene promoters. Site-directed mutagenesis of either the predicted HRE or the bile acid responsive FXR binding site abolished inducibility of the OSTα promoter, indicating that both elements need to be intact for induction by hypoxia and CDCA. In a rat model of chronic renal failure, the known increase in hepatic OSTα expression was associated with an increase in HIF-1α protein levels. Conclusion OSTα-OSTβ expression is induced by hypoxia. FXR and HIF-1α bind in close proximity to the OSTα gene promoter and produce synergistic effects on OSTα expression. PMID:24703425

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

PubMed

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

2017-01-01

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

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

PubMed Central

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

2017-01-01

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

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.

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

PubMed

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohtera, Anna; Miyamae, Yusaku; Nakai, Naomi

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

High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

PubMed Central

Han, Xiao; Guo, Jinhai; Deng, Weiwei; Zhang, Chenying; Du, Peige; Shi, Taiping; Ma, Dalong

2008-01-01

Background Estrogen receptor α (ERα) is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE) with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. Results From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131) as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE). In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene. Conclusion We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells. PMID:18847501

Bioluminescent system for dynamic imaging of cell and animal behavior

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hara-Miyauchi, Chikako; Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, Saitama 351-0198; Department of Biophysics and Biochemistry, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Tokyo 113-8510

2012-03-09

Highlights: Black-Right-Pointing-Pointer We combined a yellow variant of GFP and firefly luciferase to make ffLuc-cp156. Black-Right-Pointing-Pointer ffLuc-cp156 showed improved photon yield in cultured cells and transgenic mice. Black-Right-Pointing-Pointer ffLuc-cp156 enabled video-rate bioluminescence imaging of freely-moving animals. Black-Right-Pointing-Pointer ffLuc-cp156 mice enabled tracking real-time drug delivery in conscious animals. -- Abstract: The current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, ffLuc-cp156, which consists of a yellow variant of Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over threemore » orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies.« less

Trehalose radial networks protect Renilla luciferase helical layers against thermal inactivation.

PubMed

Liyaghatdar, Zahra; Emamzadeh, Rahman; Rasa, Sayed Mohammad Mahdi; Nazari, Mahboobeh

2017-12-01

Renilla luciferase (Rluc) from Renilla reniformis is an appropriate protein reporter for the detection of specific molecular targets due to its bioluminescent feature, although its relatively low stability limits the application. To investigate the effects of trehalose and sucrose as chemical chaperones on the kinetic stability of Rluc, we assayed the activity of the enzyme in the presence of these additives at high temperatures and to comprehend the mechanism of stability, molecular dynamic (MD) simulation was carried out. In the presence of trehalose a thermostabilizing effect which was considerable in comparison with other systems was observed. It is proposed that a wide radial like network of trehalose molecules supports α-helix structures that are located in the N-terminus and C-terminus of the protein. However, in the water simulation box, these helices alter to instable structures at high temperatures. Reduction of the fluctuation of these helices in the presence of trehalose molecules, may prevent the protein from unfolding and increase its shelf life. Copyright © 2017 Elsevier B.V. All rights reserved.

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. Copyright © 2016 John Wiley & Sons, Inc.

Ethanolic extract of dandelion (Taraxacum mongolicum) induces estrogenic activity in MCF-7 cells and immature rats.

PubMed

Oh, Seung Min; Kim, Ha Ryong; Park, Yong Joo; Lee, Yong Hwa; Chung, Kyu Hyuck

2015-11-01

Plants of the genus Taraxacum, commonly known as dandelions, are used to treat breast cancer in traditional folk medicine. However, their use has mainly been based on empirical findings without sufficient scientific evidence. Therefore, we hypothesized that dandelions would behave as a Selective estrogen receptor modulator (SERM) and be effective as hormone replacement therapy (HRT) in the postmenopausal women. In the present study, in vitro assay systems, including cell proliferation assay, reporter gene assay, and RT-PCR to evaluate the mRNA expression of estrogen-related genes (pS2 and progesterone receptor, PR), were performed in human breast cancer cells. Dandelion ethanol extract (DEE) significantly increased cell proliferation and estrogen response element (ERE)-driven luciferase activity. DEE significantly induced the expression of estrogen related genes such as pS2 and PR, which was inhibited by tamoxifen at 1 μmol·L(-1). These results indicated that DEE could induce estrogenic activities mediated by a classical estrogen receptor pathway. In addition, immature rat uterotrophic assay was carried out to identify estrogenic activity of DEE in vivo. The lowest concentration of DEE slightly increased the uterine wet weight, but there was no significant effect with the highest concentration of DEE. The results demonstrate the potential estrogenic activities of DEE, providing scientific evidence supporting their use in traditional medicine. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Real-time monitoring of artemin in vivo chaperone activity using luciferase as an intracellular reporter.

PubMed

Takalloo, Zeinab; Sajedi, Reza H; Hosseinkhani, Saman; Asghari, S Mohsen

2016-11-15

Artemin is an abundant thermostable protein in Artemia encysted embryos and considered as a stress protein, as its highly regulated expression is associated with stress resistance. Artemin cDNA was previously isolated and cloned from Artemia urmiana and artemin was found as an efficient molecular chaperone in vitro. Here, co-transformation of E. coli was performed with two expression vectors containing artemin and firefly luciferase for in vivo studies. The time-course of luciferase inactivation at low and elevated temperatures showed that luciferase was rapidly inactivated in control cells, but it was found that luciferase was protected significantly in artemin expressing cells. More interestingly, luciferase activity was completely regained in heat treated artemin expressing cells at room temperature. In addition, in both stress conditions, similar to residual activity of luciferase, cell viability in induced cultures over-expressing artemin was significantly higher than non-expressed artemin cells. It can be suggested that artemin confers impressive resistance in stressful conditions when introduced into E. coli cells, which is due to that it protects proteins against aggregation. Such luciferase co-expression system can be used as a real-time reporter to investigate the activity of chaperone proteins in vivo and provide a rapid and simple test for molecular chaperones. Copyright © 2016 Elsevier Inc. All rights reserved.

Hypoxia promotes apoptosis of neuronal cells through hypoxia-inducible factor-1α-microRNA-204-B-cell lymphoma-2 pathway

PubMed Central

Wang, Xiuwen; Li, Ji; Wu, Dongjin; Bu, Xiangpeng

2015-01-01

Neuronal cells are highly sensitive to hypoxia and may be subjected to apoptosis when exposed to hypoxia. Several apoptosis-related genes and miRNAs involve in hypoxia-induced apoptosis. This study aimed to examine the role of HIF1α-miR-204-BCL-2 pathway in hypoxia-induced apoptosis in neuronal cells. Annexin V/propidium iodide assay was performed to analyze cell apoptosis in AGE1.HN and PC12 cells under hypoxic or normoxic conditions. The expression of BCL-2 and miR-204 were determined by Western blot and qRT-PCR. The effects of miR-204 overexpression or knockdown on the expression of BCL-2 were evaluated by luciferase assay and Western blot under hypoxic or normoxic conditions. HIF-1α inhibitor YC-1 and siHIF-1α were employed to determine the effect of HIF-1α on the up-regulation of miR-204 and down-regulation of BCL-2 induced by hypoxia. Apoptosis assay showed the presence of apoptosis induced by hypoxia in neuronal cells. Moreover, we found that hypoxia significantly down-regulated the expression of BCL-2, and increased the mRNA level of miR-204 in neuronal cells than that in control. Bioinformatic analysis and luciferase reporter assay demonstrated that miR-204 directly targeted and regulated the expression of BCL-2. Specifically, the expression of BCL-2 was inhibited by miR-204 mimic and enhanced by miR-204 inhibitor. Furthermore, we detected that hypoxia induced cell apoptosis via HIF-1α/miR-204/BCL-2 in neuronal cells. This study demonstrated that HIF-1α-miR-204-BCL-2 pathway contributed to apoptosis of neuronal cells induced by hypoxia, which could potentially be exploited to prevent spinal cord ischemia–reperfusion injury. PMID:26350953

Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension

PubMed Central

Sonawane, Parshuram J.; Sahu, Bhavani S.; Sasi, Binu K.; Geedi, Parimala; Lenka, Govinda; Mahapatra, Nitish R.

2011-01-01

3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5′-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure. PMID:21304971

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

PubMed

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

2004-12-15

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

Nonaminoglycoside compounds induce readthrough of nonsense mutations

PubMed Central

Damoiseaux, Robert; Nahas, Shareef; Gao, Kun; Hu, Hailiang; Pollard, Julianne M.; Goldstine, Jimena; Jung, Michael E.; Henning, Susanne M.; Bertoni, Carmen

2009-01-01

Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) might be exploited as a potential treatment strategy. We have successfully developed a sensitive and quantitative high-throughput screening (HTS) assay, protein transcription/translation (PTT)–enzyme-linked immunosorbent assay (ELISA), for identifying novel PTC-readthrough compounds using ataxia-telangiectasia (A-T) as a genetic disease model. This HTS PTT-ELISA assay is based on a coupled PTT that uses plasmid templates containing prototypic A-T mutated (ATM) mutations for HTS. The assay is luciferase independent. We screened ∼34,000 compounds and identified 12 low-molecular-mass nonaminoglycosides with potential PTC-readthrough activity. From these, two leading compounds consistently induced functional ATM protein in ATM-deficient cells containing disease-causing nonsense mutations, as demonstrated by direct measurement of ATM protein, restored ATM kinase activity, and colony survival assays for cellular radiosensitivity. The two compounds also demonstrated readthrough activity in mdx mouse myotube cells carrying a nonsense mutation and induced significant amounts of dystrophin protein. PMID:19770270

Effects of COL8A1 on the proliferation of muscle-derived satellite cells.

PubMed

Li, Xiaofan; Wang, Zhao; Tong, Huili; Yan, Yunqin; Li, Shufeng

2018-04-25

Collagen type VIII alpha 1 chain (COL8A1) is a component of the extracellular matrix. Our previous studies suggested that COL8A1 is associated with the proliferation of muscle-derived satellite cells (MDSCs). Additionally, it has been demonstrated that COL8A1 promotes the proliferation of smooth muscle cells and liver cancer cells. Therefore, we predicted that COL8A1 is associated with the proliferation of bovine MDSCs, which have potential applications in research. In this study, we constructed vectors to activate and repress COL8A1 in bovine MDSCs using the CRISPR/Cas9 technique and determined the effects of COL8A1 modulation by EdU labeling, western blotting, and dual-luciferase reporter assays. The results showed that activation of COL8A1 increased the number of EdU-positive cells and expression of the proliferation markers cyclin B1 (CCNB1) and P-AKT. The expression of P-Akt was unchanged after addition of LY294002 (a protein kinase inhibitor capable of blocking the signal transduction pathway of the phosphoinositide 3-kinase). In contrast, repression of COL8A1 reduced the number of EdU-positive cells and expression of CCNB1 and P-AKT. We also observed upregulation and downregulation of COL8A1 following the overexpression and repression of EGR1, respectively. The dual-luciferase reporter assay revealed that EGR1 regulates the promoter activity of COL8A1. To our knowledge, this is the first study demonstrating that EGR1 positively regulates the expression of COL8A1, which in turn promotes the proliferation of bovine MDSCs via the PI3K/AKT signaling pathway. This article is protected by copyright. All rights reserved.

MicroRNA-34b/c inhibits aldosterone-induced vascular smooth muscle cell calcification via a SATB2/Runx2 pathway.

PubMed

Hao, Jianbing; Zhang, Lei; Cong, Guangting; Ren, Liansheng; Hao, Lirong

2016-12-01

Increasing evidence shows that aldosterone and specific microRNAs (miRs) contribute to vascular smooth muscle cell (VSMC) calcification. In this study, we aim to explore the mechanistic links between miR-34b/c and aldosterone in VSMC calcification. VSMC calcification models were established both in vitro and in vivo. First, the levels of aldosterone, miR-34b/c and special AT-rich sequence-binding protein 2 (SATB2) were measured. Then, miR-34b/c mimics or inhibitors were transfected into VSMCs to evaluate the function of miR-34b/c. Luciferase reporter assays were used to demonstrate whether SATB2 was a direct target of miR-34b/c. Aldosterone and SATB2 were found to be markedly upregulated during VSMC calcification, whereas miR-34b/c expression was downregulated. Treatment with the mineralocorticoid receptor (MR) antagonist eplerenone inhibited VSMC calcification. In aldosterone-induced VSMC calcification, miR-34b/c levels were downregulated and SATB2 protein was upregulated. Furthermore, miR-34b/c overexpression alleviated aldosterone-induced VSMC calcification as well as inhibited the expression of SATB2 protein, whereas miR-34b/c inhibition markedly enhanced VSMC calcification and upregulated SATB2 protein. In addition, luciferase reporter assays showed that SATB2 is a direct target of miR-34b/c in VSMCs. Overexpression of SATB2 induced Runx2 overproduction and VSMC calcification. Therefore, miR-34b/c participates in aldosterone-induced VSMC calcification via a SATB2/Runx2 pathway. As miR-34b/c appears to be a negative regulator, it has potential as a therapeutic target of VSMC calcification.

Bakuchiol Is a Phenolic Isoprenoid with Novel Enantiomer-selective Anti-influenza A Virus Activity Involving Nrf2 Activation*

PubMed Central

Shoji, Masaki; Arakaki, Yumie; Esumi, Tomoyuki; Kohnomi, Shuntaro; Yamamoto, Chihiro; Suzuki, Yutaka; Takahashi, Etsuhisa; Konishi, Shiro; Kido, Hiroshi; Kuzuhara, Takashi

2015-01-01

Influenza represents a substantial threat to human health and requires novel therapeutic approaches. Bakuchiol is a phenolic isoprenoid compound present in Babchi (Psoralea corylifolia L.) seeds. We examined the anti-influenza viral activity of synthetic bakuchiol using Madin-Darby canine kidney cells. We found that the naturally occurring form, (+)-(S)-bakuchiol, and its enantiomer, (−)-(R)-bakuchiol, inhibited influenza A viral infection and growth and reduced the expression of viral mRNAs and proteins in these cells. Furthermore, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-β and myxovirus-resistant protein 1. Interestingly, (+)-(S)-bakuchiol had greater efficacy than (−)-(R)-bakuchiol, indicating that chirality influenced anti-influenza virus activity. In vitro studies indicated that bakuchiol did not strongly inhibit the activities of influenza surface proteins or the M2 ion channel, expressed in Chinese hamster ovary cells. Analysis of luciferase reporter assay data unexpectedly indicated that bakuchiol may induce some host cell factor(s) that inhibited firefly and Renilla luciferases. Next generation sequencing and KeyMolnet analysis of influenza A virus-infected and non-infected cells exposed to bakuchiol revealed activation of transcriptional regulation by nuclear factor erythroid 2-related factor (Nrf), and an Nrf2 reporter assay showed that (+)-(S)-bakuchiol activated Nrf2. Additionally, (+)-(S)-bakuchiol up-regulated the mRNA levels of two Nrf2-induced genes, NAD(P)H quinone oxidoreductase 1 and glutathione S-transferase A3. These findings demonstrated that bakuchiol had enantiomer-selective anti-influenza viral activity involving a novel effect on the host cell oxidative stress response. PMID:26446794

Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus

PubMed Central

Elek, Zsuzsanna; Németh, Nóra; Nagy, Géza; Németh, Helga; Somogyi, Anikó; Hosszufalusi, Nóra; Sasvári-Székely, Mária; Rónai, Zsolt

2015-01-01

The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ 2-test in combination with correction for multiple testing. For functional analysis, the entire 3’ UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3’ UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function. PMID:26426397

The role of miR-370 in fibrosis after myocardial infarction

PubMed Central

Yuan, Hui; Gao, Jie

2017-01-01

In the present study, we investigated the expression of miR-370 in the border area of infarction after myocardial infarction and its role in the process of post-infarction fibrosis. A myocardial infarction model in Sprague-Dawley rats was established. After two weeks, the mRNA levels of transforming growth factor-β1 (TGFβ1), TGFβRII, ColIa1, ColIIIa1 and miR-370 and the expression of TGFβ1, TGFβRII and α-smooth muscle actin (α-SMA) proteins in the border area of infarction were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and western blot analysis. Cardiac fibroblasts in neonatal rat were isolated and cultured, and the changes in the above indicators were detected after AngII and miR-370 intervention. Luciferase reporter gene assay was conducted to verify whether TGFβRII was a target gene of miR-370. In the border area after myocardial infarction, the expression of miR-370 decreased, while mRNA levels of TGFβ1, TGFβRII, ColIa1 and ColIIIa1 and levels of TGFβ1, TGFβRII and α-SMA proteins were all increased. Luciferase reporter gene assay confirmed that TGFβRII was the target gene of miR-370. miR-370 reduced the expression of TGFβRII and inhibited the increased expression of TGFβRII and collagen protein caused by AngII. As well, its inhibited the differentiation effect of muscle fibroblasts while it did not inhibit the expression of TGFβ1. miR-370 inhibited the expression of TGFβRII protein by combining with TGFβRII mRNA. miR-370 also partially blocked TGFβ1-TGFβRII and induced the downstream signal transduction pathways, thus exerting anti-fibrotic effects. PMID:28350072

Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus.

PubMed

Elek, Zsuzsanna; Németh, Nóra; Nagy, Géza; Németh, Helga; Somogyi, Anikó; Hosszufalusi, Nóra; Sasvári-Székely, Mária; Rónai, Zsolt

2015-01-01

The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ2-test in combination with correction for multiple testing. For functional analysis, the entire 3' UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3' UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function.

E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

PubMed

Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

2017-01-01

Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

Berberine Alleviates Oxidative Stress in Islets of Diabetic Mice by Inhibiting miR-106b Expression and Up-Regulating SIRT1.

PubMed

Chen, Dong-Liang; Yang, Ke-Ya

2017-12-01

Mounting studies have indicated the role of berberine, SIRT1, and oxidative stress in diabetes, respectively. However, few studies have demonstrated their correlation and regulation function in diabetes. Therefore, the protective effect of berberine in diabetic and the underlying core mechanism were investigated in the current study. Diabetic mice model in vivo were established. Mouse pancreatic beta-cell line NIT-1 cells were treated with 30 mM high glucose to induce diabetic condition in vitro. Serum biochemical parameters (glucose, total cholesterol, and triglycerides) were detected. Oxidative stress indicators (MDA, SOD1), along with miR-106b and SIRT1 expression in islets and cells were also assessed. Direct targeting relationship between miR-106b and SIRT1 was discussed by dual luciferase reporter gene assay. Diabetic model in vivo and in vitro were both established successfully. The expression of serum biochemical parameters was increased, and oxidative stress parameters, and miR-106b, SIRT1 were abnormally expressed in diabetic mice and NIT-1 cells. Meanwhile, berberine could alleviate oxidative stress injury in diabetic progression. Through dual luciferase reporter gene assay, we found that SIRT1 was a target gene of miR-106b. In addition, miR-106b over-expression could reverse the protection of berberine in NIT-1 cells against from oxidative stress induced by high glucose. Berberine could attenuate oxidative stress of diabetic mice at least partly through miR-106b/SIRT1 pathway and affecting the function of islets, which might be beneficial in reducing the cardiovascular risk factors in diabetes. J. Cell. Biochem. 118: 4349-4357, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Downregulation of microRNA‑574 in cancer stem cells causes recurrence of prostate cancer via targeting REL.

PubMed

Lai, Xiaodong; Guo, Yanchun; Guo, Zhitao; Liu, Ruibao; Wang, Xunguo; Wang, Fang

2016-12-01

miR‑574‑5p has been reported involved in the pathogenesis of numerous human malignancies such as colorectal and lung cancer. In this study, we aimed to explore the roles of REL and miR‑574 in the recurrence of prostate cancer (PCa) and to identify the underlying molecular mechanisms. Our literature search found that miR‑574 is regulated in cancer stem cells (CSCs), and next we used the microRNA (miRNA) database (www.mirdb.org) to find REL as a target of miR‑574. Luciferase assay was performed to verify the miRNA/target relationship. Oligo-transfection, real‑time PCR and western blot analysis were used to support the conclusions. We validated REL to be the direct gene via luciferase reporter assay system, and real‑time PCR and western blot analysis were also conducted to study the mRNA and protein expression level of REL between different groups (recurrence and non‑recurrence) or cells treated with scramble control, miR‑574 mimics, REL siRNA and miR‑574 inhibitors, indicating the negative regulatory relationship between miR‑574 and REL. We also investigated the relative viability of prostate CSCs when transfected with scramble control, miR‑574 mimics, REL siRNA and miR‑574 inhibitors to validate miR‑574 to be positively interfering with the viability of prostate CSCs. We then investigated the relative apoptosis of prostate CSCs when transfected with scramble control, miR‑574 mimics, REL siRNA and miR‑574 inhibitors. The results showed miR‑574 inhibited apoptosis. In conclusion, miR‑574 might be a novel prognostic and therapeutic target in the management of PCa recurrence.

A Pro-Inflammatory Role for Nuclear Factor Kappa B in Childhood Obstructive Sleep Apnea Syndrome

PubMed Central

Israel, Lee P.; Benharoch, Daniel; Gopas, Jacob; Goldbart, Aviv D.

2013-01-01

Study Objectives: Childhood obstructive sleep apnea syndrome (OSAS) is associated with an elevation of inflammatory markers such as C-reactive protein (CRP) that correlates with specific morbidities and subsides following intervention. In adults, OSAS is associated with activation of the transcription factor nuclear factor kappa B (NF-kB). We explored the mechanisms underlying NF-kB activation, based on the hypothesis that specific NF-kB signaling is activated in children with OSAS. Design: Adenoid and tonsillar tissues from children with OSAS and matched controls were immunostained against NF-kB classical (p65 and p50) and alternative (RelB and p52) pathway subunits, and NF-kB-dependent cytokines: interleukin (IL)- 1α, IL-1β, tumor necrosis factor-α, and IL-8). Serum CRP levels were measured in all subjects. NF-kB induction was evaluated by a luciferase-NF-kB reporter assay in L428 cells constitutively expressing NF-kB and in Jurkat cells with inducible NF-kB expression. p65 translocation to the nucleus, reflecting NF-kB activation, was measured in cells expressing fluorescent NF-kB-p65-GFP (green fluorescent protein). Setting: Sleep research laboratory. Patients or Participants: Twenty-five children with OSAS and 24 without OSAS. Interventions: N/A. Measurements and Results: Higher expression of IL-1α and classical NF-kB subunits p65 and p50 was observed in adenoids and tonsils of children with OSAS. Patient serum induced NF-kB activity, as measured by a luciferase-NF-kB reporter assay and by induction of p65 nuclear translocation in cells permanently transfected with GFP-p65 plasmid. IL-1β showed increased epithelial expression in OSAS tissues. Conclusions: Nuclear factor kappa B is locally and systemically activated in children with obstructive sleep apnea syndrome. This observation may motivate the search for new anti-inflammatory strategies for controlling nuclear factor kappa B activation in obstructive sleep apnea syndrome. Citation: Israel LP; Benharoch D; Gopas J; Goldbart AD. A pro-inflammatory role for nuclear factor kappa B in childhood obstructive sleep apnea syndrome. SLEEP 2013;36(12):1947-1955. PMID:24293770

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. Copyright © 2015 Elsevier B.V. All rights reserved.

Tiered High-Throughput Screening Approach to Identify ...

EPA Pesticide Factsheets

High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the US EPA ToxCast screening assay portfolio. To fill one critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast Phase I and II chemical libraries, comprised of 1,074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single concentration screen were retested in concentration-response. Due to high false positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed two additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using

Modulation of the response of prostate cancer cell lines to cisplatin treatment using small interfering RNA.

PubMed

Parra, Eduardo; Ferreira, Jorge

2013-10-01

Cisplatin is one of the most effective and widely used chemotherapeutic agents against several types of human cancers. However, the underlying mechanisms of action are not fully understood. We aimed to investigate the possible molecular mechanism(s) of acquired chemoresistance observed in prostate cancer cells treated with cisplatin. Human LNCaP cells (bearing wild-type p53) and PC-3 cells (lacking p53) were used. The expression levels of protein were determined by western blotting, and the mRNA levels were determined by reverse transcription-polymerase chain reaction (RT-PCR). Cell viability was measured by MTT assay, and the transcriptional effect of small interfering RNA (siRNA) was measured by luciferase reporter gene. We showed that cisplatin treatment increased JNK-1 and JNK-2 activity and expression in both LNCaP and PC-3 cells. In addition, the knockdown of JNK-1 expression by siRNA-JNK-1 or siRNA-JNK-2 significantly impaired the upregulation of AP-1 luciferase reporter gene, but failed to decrease the levels of AP-1 reporter gene expression induced by TPA treatment. Our observations indicate that JNK-1 and JNK-2 may be involved in the chemoresistance observed in prostate cancer cells treated with cisplatin and that blocking the stimulation of Jun kinase (JNK) signaling may be important for regulating the susceptibility to cisplatin of prostate cancer.

Mechanistic insights into the link between visfatin gene C-1535T polymorphism and coronary artery disease: an in vitro study.

PubMed

Wang, Yong-Sheng; Gao, Wei; Li, Hong-Fen; Wang, Ze-Mu; Zhu, Jun; Zhao, Huan; Yan, Jian-Jun; Jia, En-Zhi; Yang, Zhi-Jian; Wang, Lian-Sheng

2012-04-01

Visfatin, a pro-inflammatory cytokine predominantly released from leucocytes, is correlated with coronary artery disease (CAD). We have previously reported that the -1535C>T polymorphism (rs1330082), which located on the promoter region of visfatin, was associated with decreased risk of CAD. Here, we investigated the underlying mechanism by which this polymorphism affects the genetic susceptibility to CAD. The difference of the promoter activities between -1535T variant and -1535C allele was tested by luciferase reporter gene assay. The difference of transcription factor binding activities between T and C allele was evaluated by electrophoretic mobility shift assay. In reporter gene assay, we showed that the T variant had a significantly reduced transcriptional activity compared with the C allele. The T-variant significantly attenuated the promoter binding affinity to nuclear transcription factors and this effect became much obvious after treatment with TNF-α. Moreover, competition experiment revealed that the retarded complex formed by T-1535- or C-1535-probe binding to nuclear extracts was nearly completely inhibited by unlabeled activator protein-1 (AP-1) specific probe, indicating that AP-1 might be the target nuclear effector. Taken together, our data provided potential mechanistic link between the visfatin -1535C>T polymorphism and reduced CAD risk.

AR-NCoR Interaction as a Therapeutic Target for Prostate Cancer Prevention and Treatment

DTIC Science & Technology

2007-10-01

reporter and a control CMV regulated Renilla luciferase plasmid (pRL-CMV). Cells were incubated for 24 hrs in steroid-hormone depleted medium (DMEM...5% CDS-FBS) treated with A, 10 nM DHT; B, 1 µM androstenedione; C, 1 µM progesterone; or D, 10 µM cyproterone acetate. Luciferase versus Renilla ...and luciferase versus Renilla luciferase activities were determined from triplicate samples. The data are expressed as relative light units (RLU

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

Sensitive Dual Color In Vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase Laura...20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that...spectral emissions using a suitable spectral unmixing algorithm . This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future

“Glowing Head” Mice: A Genetic Tool Enabling Reliable Preclinical Image-Based Evaluation of Cancers in Immunocompetent Allografts

PubMed Central

Day, Chi-Ping; Carter, John; Ohler, Zoe Weaver; Bonomi, Carrie; El Meskini, Rajaa; Martin, Philip; Graff-Cherry, Cari; Feigenbaum, Lionel; Tüting, Thomas; Van Dyke, Terry; Hollingshead, Melinda; Merlino, Glenn

2014-01-01

Preclinical therapeutic assessment currently relies on the growth response of established human cell lines xenografted into immunocompromised mice, a strategy that is generally not predictive of clinical outcomes. Immunocompetent genetically engineered mouse (GEM)-derived tumor allograft models offer highly tractable preclinical alternatives and facilitate analysis of clinically promising immunomodulatory agents. Imageable reporters are essential for accurately tracking tumor growth and response, particularly for metastases. Unfortunately, reporters such as luciferase and GFP are foreign antigens in immunocompetent mice, potentially hindering tumor growth and confounding therapeutic responses. Here we assessed the value of reporter-tolerized GEMs as allograft recipients by targeting minimal expression of a luciferase-GFP fusion reporter to the anterior pituitary gland (dubbed the “Glowing Head” or GH mouse). The luciferase-GFP reporter expressed in tumor cells induced adverse immune responses in wildtype mouse, but not in GH mouse, as transplantation hosts. The antigenicity of optical reporters resulted in a decrease in both the growth and metastatic potential of the labeled tumor in wildtype mice as compared to the GH mice. Moreover, reporter expression can also alter the tumor response to chemotherapy or targeted therapy in a context-dependent manner. Thus the GH mice and experimental approaches vetted herein provide concept validation and a strategy for effective, reproducible preclinical evaluation of growth and response kinetics for traceable tumors. PMID:25369133

Transcriptional regulation of podoplanin expression by Prox1 in lymphatic endothelial cells.

PubMed

Pan, Yanfang; Wang, Wen-di; Yago, Tadayuki

2014-07-01

Transcription factor prospero homeobox 1 (Prox-1) and podoplanin (PDPN), mucin-type transmembane protein, are both constantly expressed in lymphatic endothelial cells (LECs) and appear to function in an LEC-autonomous manner. Mice globally lacking PDPN (Pdpn(-/-)) develop abnormal and blood-filled lymphatic vessels that highly resemble those in inducible mice lacking Prox-1 (Prox1(-/-)). Prox1 has also been reported to induce PDPN expression in cultured ECs. Thus, we hypothesize that PDPN functions downstream of Prox1 and that its expression is regulated by Prox1 in LECs at the transcriptional level. We first identified four putative binding elements for Prox1 in the 5' upstream regulatory region of Pdpn gene and found that Prox1 directly binds to the 5' regulatory sequence of Pdpn gene in LECs by chromatin immunoprecipitation assay. DNA pull down assay confirmed that Prox1 binds to the putative binding element. In addition, luciferase reporter assay indicated that Prox1 binding to the 5' regulatory sequence of Pdpn regulates Pdpn gene expression. We are therefore the first to experimentally demonstrate that Prox1 regulates PDPN expression at the transcriptional level in the lymphatic vascular system. Copyright © 2014 Elsevier Inc. All rights reserved.

[Regulation of microRNA-199a on adhesion, migration and invasion ability of human endometrial stromal cells].

PubMed

Dai, Lan; Gu, Li-ying; Zhu, Jie; Shi, Jun; Wang, Yao; Ji, Fang; Di, Wen

2011-11-01

To study the regulation of microRNA 199a (miR-199a) on adhesion, migration and invasion ability of human eutopic endometrial stromal cells (ESC) from patients with endometriosis. ESC were transfected with miR-199a mimics or negative control (NC) RNA by lipofectamine 2000. The adhesion, migration and invasion ability of ESC were detected by cell adhesion assay, scratch assay, cell migration assay and matrigel invasion assay, respectively. Luciferase reporter assay was used to evaluate whether IKKβ was the target gene of miR-199a. The expression of ikappa B kinase beta (IKKβ), inhibitory kappa B alpha (IκB-α), phospho-IκB-α(p-IκB-α) and nuclear factor-kappa B (NF-κB) protein were measured by western blot. (1) Adhesion potential: the adhesion inhibitory rates were (14 ± 4)% in miR-199a group and 0 in control group, which showed significant difference (P < 0.01). (2) Migration and invasion: in the scratch assay, ESC transfected with miR-199a exhibited a lower scratch closure rate than that of controls. In migration and invasion assays, the migration and invasion ability of miR-199a group were significantly decreased compared with those of NC group [130 ± 31 vs. 247 ± 36 (P < 0.01); 63 ± 15 vs. 133 ± 17 (P < 0.01), respectively]. (3) The luciferase activity of miR-199a group was significantly lowered than that of control group [0.160 ± 0.006 vs. 0.383 ± 0.083 (P < 0.01)]. The protein levels of IKKβ, p-IκB-α, IκB-α and NF-κB of 0.350 ± 0.195, 0.443 ± 0.076, 1.970 ± 0.486 and 0.454 ± 0.147 in miR-199a group were significantly different compared with the NC group in which the protein levels were set at 1.000 (P < 0.01). miR-199a can inhibit the adhesion, migration and invasion of the ESC. IKKβ is the target gene of miR-199a in ESC. One of the mechanisms of the inhibition effect is probably that miR-199a inhibits the activation of NF-κB signaling pathway by targeting IKKβ gene.

Baicalin Protects Mice from Aristolochic Acid I-Induced Kidney Injury by Induction of CYP1A through the Aromatic Hydrocarbon Receptor

PubMed Central

Wang, Ke; Feng, Chenchen; Li, Chenggang; Yao, Jun; Xie, Xiaofeng; Gong, Likun; Luan, Yang; Xing, Guozhen; Zhu, Xue; Qi, Xinming; Ren, Jin

2015-01-01

Exposure to aristolochic acid I (AAI) can lead to aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and urothelial cancer. The induction of hepatic CYP1A, especially CYP1A2, was considered to detoxify AAI so as to reduce its nephrotoxicity. We previously found that baicalin had the strong ability to induce CYP1A2 expression; therefore in this study, we examined the effects of baicalin on AAI toxicity, metabolism and disposition, as well as investigated the underlying mechanisms. Our toxicological studies showed that baicalin reduced the levels of blood urea nitrogen (BUN) and creatinine (CRE) in AAI-treated mice and attenuated renal injury induced by AAI. Pharmacokinetic analysis demonstrated that baicalin markedly decreased AUC of AAI in plasma and the content of AAI in liver and kidney. CYP1A induction assays showed that baicalin exposure significantly increased the hepatic expression of CYP1A1/2, which was completely abolished by inhibitors of the Aromatic hydrocarbon receptor (AhR), 3ʹ,4ʹ-dimethoxyflavone and resveratrol, in vitro and in vivo, respectively. Moreover, the luciferase assays revealed that baicalin significantly increased the luciferase activity of the reporter gene incorporated with the Xenobiotic response elements recognized by AhR. In summary, baicalin significantly reduced the disposition of AAI and ameliorated AAI-induced kidney toxicity through AhR-dependent CYP1A1/2 induction in the liver. PMID:26204831

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. Published by Elsevier B.V.

Development of a rapid immunoassay system: Luminescent detection of antigen-associated antibody-luciferase in the presence of a dye that absorbs light from free antibody-luciferase.

PubMed

Mori, Akihiro; Ojima-Kato, Teruyo; Fuchi, Shingo; Kaiya, Shinichi; Kojima, Takaaki; Nakano, Hideo

2017-12-01

In this report, we developed a rapid immunoassay system, designated the bioluminescent interference gathering optical (BINGO) assay, which required no time-consuming washing steps for removal of unbound antibodies. This system employed a luciferase (Luc)-conjugated antibody (LucAb) and a dye that absorbed light from the LucAb. The antigen-associated LucAb was localized by transfer of an antigen to the detector-side of a chamber where a detector photomultiplier tube (PMT) was installed. In contrast, the free LucAb was distributed throughout the solution, and the light emitted by the free LucAb was absorbed by the dye. Therefore, only light from LucAb associated with antigen could be detected by the PMT. The new system could be used to rapidly detect the amount of antigen-antibody-Luc complex by collecting steps, such as centrifugation or magnetic collection of antibody-coated magnetic beads. Proof-of-principle experiments were performed using a model system with streptavidin beads and biotinylated Luc. The feasibility of the system was demonstrated using magnetic beads coated with anti-Escherichia coli O157 antibody, enabling detection of 4 × 10 3  cells in only 15 min. Thus, this system may have applications in a variety of biomedical fields. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Opposing effects of bile acids deoxycholic acid and ursodeoxycholic acid on signal transduction pathways in oesophageal cancer cells.

PubMed

Abdel-Latif, Mohamed M; Inoue, Hiroyasu; Reynolds, John V

2016-09-01

Ursodeoxycholic acid (UDCA) was reported to reduce bile acid toxicity, but the mechanisms underlying its cytoprotective effects are not fully understood. The aim of the present study was to examine the effects of UDCA on the modulation of deoxycholic acid (DCA)-induced signal transduction in oesophageal cancer cells. Nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activity was assessed using a gel shift assay. NF-κB activation and translocation was performed using an ELISA-based assay and immunofluorescence analysis. COX-2 expression was analysed by western blotting and COX-2 promoter activity was assessed by luciferase assay. DCA induced NF-κB and AP-1 DNA-binding activities in SKGT-4 and OE33 cells. UDCA pretreatment inhibited DCA-induced NF-κB and AP-1 activation and NF-κB translocation. This inhibitory effect was coupled with a blockade of IκB-α degradation and inhibition of phosphorylation of IKK-α/β and ERK1/2. Moreover, UDCA pretreatment inhibited COX-2 upregulation. Using transient transfection of the COX-2 promoter, UDCA pretreatment abrogated DCA-induced COX-2 promoter activation. In addition, UDCA protected oesophageal cells from the apoptotic effects of deoxycholate. Our findings indicate that UDCA inhibits DCA-induced signalling pathways in oesophageal cancer cells. These data indicate a possible mechanistic role for the chemopreventive actions of UDCA in oesophageal carcinogenesis.

Crystal Structures of the Luciferase and Green Fluorescent Protein from Renilla reniformis

PubMed Central

Loening, Andreas Markus; Fenn, Timothy David; Gambhir, Sanjiv Sam

2009-01-01

Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in both cell culture experiments and small animal imaging. To accomplish this bioluminesce, the 37 KDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8), and present the first structures for any coelenterazine-using luciferase. These structures are based on high resolution data measured to 1.4 Å and demonstrate a classic α/β-hydrolase fold. We also present data of a coe-lenteramide bound-luciferase, and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase’s accessory green fluorescent protein (RrGFP) was determined as well and shown to be highly similar to that of Aequorea GFP. PMID:17980388

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 Central

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

2015-01-01

Summary 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 M2 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, are necessary to induce the transcription of sulfate assimilation genes during early cadmium stress. PMID:22283708

High-throughput screening and mechanism-based evaluation of estrogenic botanical extracts

PubMed Central

Overk, Cassia R.; Yao, Ping; Chen, Shaonong; Deng, Shixing; Imai, Ayano; Main, Matthew; Schinkovitz, Andreas; Farnsworth, Norman R.; Pauli, Guido F.; Bolton, Judy L.

2009-01-01

Symptoms associated with menopause can greatly affect the quality of life for women. Botanical dietary supplements have been viewed by the public as safe and effective despite a lack of evidence indicating a urgent necessity to standardize these supplements chemically and biologically. Seventeen plants were evaluated for estrogenic biological activity using standard assays: competitive estrogen receptor (ER) binding assay for both alpha and beta subtypes, transient transfection of the estrogen response element luciferase plasmid into MCF-7 cells expressing either ER alpha or ER beta, and the Ishikawa alkaline phosphatase induction assay for both estrogenic and antiestrogenic activities. Based on the combination of data pooled from these assays, the following was determined: a) a high rate of false positive activity for the competitive binding assays, b) some extracts had estrogenic activity despite a lack of ability to bind the ER, c) one extract exhibited selective estrogen receptor modulator (SERM) activity, and d) several extracts show additive/synergistic activity. Taken together, these data indicate a need to reprioritize the order in which the bioassays are performed for maximal efficiency of programs involving bioassay-guided fractionation. In addition, possible explanations for the conflicts in the literature over the estrogenicity of Cimicifuga racemosa (black cohosh) are suggested. PMID:18473738

Prader-Willi region non-protein coding RNA 1 suppressed gastric cancer growth as a competing endogenous RNA of miR-425-5p.

PubMed

Chen, Zihao; Ju, Hongping; Yu, Shan; Zhao, Ting; Jing, Xiaojie; Li, Ping; Jia, Jing; Li, Nan; Tan, Bibo; Li, Yong

2018-05-23

Gastric cancer (GC) is one of the major global health problems, especially in Asia. Nowadays, long non-coding RNA (lncRNA) has gained significant attention in the current research climate such as carcinogenesis. This research desires to explore the mechanism of Prader-Willi region non-protein coding RNA 1 (PWRN1) on regulating GC process. Differentially expressed lncRNAs in GC tissues were screened out through microarray analysis. The RNA and protein expression level were detected by quantitative real-time PCR (qRT-PCR) and Western blot. Cell proliferation, apoptosis rate, metastasis abilities were respectively determined by cell counting kit 8 (CCK8), flow cytometry, wound healing, and transwell assay. The luciferase reporter system was used to verify the targetting relationships between PWRN1, miR-425-5p , and phosphatase and tensin homolog ( PTEN ). RNA-binding protein immunoprecipitation (RIP) assay was performed to prove whether PWRN1 acted as a competitive endogenous RNA (ceRNA) of miR-425-5p Tumor xenograft model and immunohistochemistry (IHC) were developed to study the influence of PWRN1 on tumor growth in vivo Microarray analysis determined that PWRN1 was differently expressed between GC tissues and adjacent tissues. qRT-PCR revealed PWRN1 low expression in GC tissues and cells. Up-regulated PWRN1 could reduce proliferation and metastasis and increase apoptosis in GC cells, while miR-425-5p had reverse effects. The RIP assay indicated that PWRN1 may target an oncogene, miR-425-5p The tumor xenograft assay found that up-regulated PWRN1 suppressed the tumor growth. The bioinformatics analysis, luciferase assay, and Western blot indicated that PWRN1 affected PTEN / Akt / MDM2 / p53 axis via suppressing miR-425-5p Our findings suggested that PWRN1 functioned as a ceRNA targetting miR-425-5p and suppressed GC development via p53 signaling pathway. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.