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Sample records for assays transcriptional activity

  1. Development of a fluorescent microsphere-based multiplexed high-throughput assay system for profiling of transcription factor activation.

    Science.gov (United States)

    Yaoi, Takuro; Jiang, Xin; Li, Xianqiang

    2006-06-01

    Transcription factors (TFs), which play crucial roles in the regulation of gene expression in the human genome, are highly regulated by a variety of mechanisms. A single extracellular stimulus can trigger multiple signaling pathways, and these in turn can activate multiple TFs to mediate the inducible expression of target genes. Alterations in the activities of TFs are often associated with human diseases, such as altered activating factor 1, estrogen receptor, and p53 function in cancer, nuclear factor kappaB in inflammatory diseases, and peroxisome proliferator-activated receptor gamma in obesity. A systematic assay for profiling the activation of TFs will aid in elucidating the mechanisms of TF activation, reveal altered TFs associated with human diseases, and aid in developing assays for drug discovery. Here, we developed a 24-plex fluorescent microsphere-based TF activation assay system with a 96-well plate format. The assay system enabled high-throughput profiling of the DNA binding activity of TFs in multiple samples with high sensitivity.

  2. High throughput assays for analyzing transcription factors.

    Science.gov (United States)

    Li, Xianqiang; Jiang, Xin; Yaoi, Takuro

    2006-06-01

    Transcription factors are a group of proteins that modulate the expression of genes involved in many biological processes, such as cell growth and differentiation. Alterations in transcription factor function are associated with many human diseases, and therefore these proteins are attractive potential drug targets. A key issue in the development of such therapeutics is the generation of effective tools that can be used for high throughput discovery of the critical transcription factors involved in human diseases, and the measurement of their activities in a variety of disease or compound-treated samples. Here, a number of innovative arrays and 96-well format assays for profiling and measuring the activities of transcription factors will be discussed.

  3. Tartrazine and sunset yellow are xenoestrogens in a new screening assay to identify modulators of human oestrogen receptor transcriptional activity.

    Science.gov (United States)

    Axon, Andrew; May, Felicity E B; Gaughan, Luke E; Williams, Faith M; Blain, Peter G; Wright, Matthew C

    2012-08-16

    Primary biliary cirrhosis (PBC) is a cholestatic liver disease of unknown cause that occurs most frequently in post-menopausal women. Since the female sex hormone oestrogen can be cholestatic, we hypothesised that PBC may be triggered in part by chronic exposure to xenoestrogens (which may be more active on a background of low endogenous oestrogen levels seen in post-menopausal women). A reporter gene construct employing a synthetic oestrogen response element predicted to specifically interact with oestrogen receptors (ER) was constructed. Co-transfection of this reporter into an ER null cell line with a variety of nuclear receptor expression constructs indicated that the reporter gene was trans-activated by ERα and ERβ, but not by the androgen, thyroid, progesterone, glucocorticoid or vitamin D receptors. Chemicals linked to PBC were then screened for xenoestrogen activity in the human ERα-positive MCF-7 breast cancer cell line. Using this assay, the coal-derived food and cosmetic colourings--sunset yellow and tartrazine--were identified as novel human ERα activators, activating the human ER with an EC(50%) concentration of 220 and 160 nM, respectively. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  4. Assessment of androgen receptor agonistic/antagonistic effects on 25 chemicals in household applicants by OECD in vitro stably transfected transcriptional activation assays.

    Science.gov (United States)

    Lee, Hee-Seok; Park, Eun-Jung; Han, Songyi; Oh, Gyeong-Yong; Kang, Hui-Seung; Suh, Jin-Hyang; Shin, Min-Ki; Oh, Hyun-Suk; Hwang, Myung-Sil; Moon, Guiim; Koh, Young-Ho; Park, Yooheon; Hong, Jin-Hwan; Koo, Yong Eui

    2018-01-01

    The aim of this study is to assess the androgen receptor (AR) agonistic/antagonistic effects on various chemicals, which are used in household products including cleaning agents and wetted tissues by in vitro OECD test guideline No. 458 (using AR-EcoScreen™ cell line) and the me-too test method (using 22Rv1cell line), which was adopted as OECD project No. 4.99. All chemicals were not determined as AR agonists. However α-dodecyl-ω-hydroxypoly (oxyethylene) and 3-iodo-2-propynyl butylcarbamate have shown a weak AR antagonistic effects with IC 50 values of 2.18 ± 0.12 and 4.26 ± 0.17 μg/ml via binding affinity to AR in only 22Rv1/mouse mammary tumor virus using AR transcriptional activation assay, because of their different cytotoxicity on each applied cell line. This report firstly provides information about agonistic/antagonistic effects against human AR of various chemicals including surfactants and biocides by OECD in vitro stably transfected transcriptional activation assays. However, further in vivo and human model studies are needed to confirm their adverse effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. In vitro-to-in vivo extrapolation of xenoestrogens using an estrogen responsive in vitro transcriptional activation assay and the in vivo uterotrophic assay

    Science.gov (United States)

    Widespread contamination of waters with both natural and synthetic estrogens is a concern for potential adverse ecological and human health effects. In vitro assays are valuable screening tools for identifying contaminated environmental samples and chemical specific mechanisms o...

  6. In vitro-to-in vivo extrapolation of xenoestrogens using an estrogen responsive in vitro transcriptional activation assay and the in vivo uterotrophic assay##

    Science.gov (United States)

    Widespread contamination of waters with both natural and synthetic estrogens is a concern for potential adverse ecological and human health effects. In vitro assays are valuable screening tools for identifying contaminated environmental samples and chemical specific mechanisms of...

  7. A Robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness.

    Science.gov (United States)

    Gibon, Yves; Blaesing, Oliver E; Hannemann, Jan; Carillo, Petronia; Höhne, Melanie; Hendriks, Janneke H M; Palacios, Natalia; Cross, Joanna; Selbig, Joachim; Stitt, Mark

    2004-12-01

    A platform has been developed to measure the activity of 23 enzymes that are involved in central carbon and nitrogen metabolism in Arabidopsis thaliana. Activities are assayed in optimized stopped assays and the product then determined using a suite of enzyme cycling assays. The platform requires inexpensive equipment, is organized in a modular manner to optimize logistics, calculates results automatically, combines high sensitivity with throughput, can be robotized, and has a throughput of three to four activities in 100 samples per person/day. Several of the assays, including those for sucrose phosphate synthase, ADP glucose pyrophosphorylase (AGPase), ferredoxin-dependent glutamate synthase, glycerokinase, and shikimate dehydrogenase, provide large advantages over previous approaches. This platform was used to analyze the diurnal changes of enzyme activities in wild-type Columbia-0 (Col-0) and the starchless plastid phosphoglucomutase (pgm) mutant, and in Col-0 during a prolongation of the night. The changes of enzyme activities were compared with the changes of transcript levels determined with the Affymetrix ATH1 array. Changes of transcript levels typically led to strongly damped changes of enzyme activity. There was no relation between the amplitudes of the diurnal changes of transcript and enzyme activity. The largest diurnal changes in activity were found for AGPase and nitrate reductase. Examination of the data and comparison with the literature indicated that these are mainly because of posttranslational regulation. The changes of enzyme activity are also strongly delayed, with the delay varying from enzyme to enzyme. It is proposed that enzyme activities provide a quasi-stable integration of regulation at several levels and provide useful data for the characterization and diagnosis of different physiological states. As an illustration, a decision tree constructed using data from Col-0 during diurnal changes and a prolonged dark treatment was used to

  8. Mechanochemical ATPases and transcriptional activation

    National Research Council Canada - National Science Library

    Zhang, X; Chaney, M; Wigneshweraraj, Siva R; Schumacher, J; Bordes, P; Cannon, W; Buck, M

    2002-01-01

    ... transcription from other ATP‐independent activation mechanisms that rely on the recruitment of RNAP by transcription factors. As described below, productive interactions between σ 54 and its a...

  9. Assays for precise quantification of total (including short) and elongated HIV-1 transcripts.

    Science.gov (United States)

    Kaiser, Philipp; Joshi, Sunil K; Kim, Peggy; Li, Peilin; Liu, Hongbing; Rice, Andrew P; Wong, Joseph K; Yukl, Steven A

    2017-04-01

    Despite intensive study, it is unclear which mechanisms are responsible for latent HIV infection in vivo. One potential mechanism is inhibition of HIV transcriptional elongation, which results in short abortive transcripts containing the trans-activation response (TAR) region. Because the relative levels of total (including short) and processive transcripts provide measures of HIV transcriptional initiation and elongation, there is a compelling need for techniques that accurately measure both. Nonetheless, prior assays for total transcripts have been semi-quantitative and have seen limited application to patient samples. This manuscript reports the validation of quantitative reverse transcription (RT) droplet digital PCR assays for measurement of total (TAR) and processive (R-U5/gag) HIV transcripts. Traditional RT priming strategies can efficiently detect the TAR region on long HIV transcripts but detect 10-fold higher than elongated transcripts, implying a substantial block to transcriptional elongation in vivo. This approach may be applied to other difficult-to-prime RNA targets. Published by Elsevier B.V.

  10. Activity Assays for Rhomboid Proteases.

    Science.gov (United States)

    Arutyunova, E; Strisovsky, K; Lemieux, M J

    2017-01-01

    Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of KM and Vmax to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein. © 2017 Elsevier Inc. All rights reserved.

  11. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

    Science.gov (United States)

    Nerys-Junior, Arildo; Costa, Lendel C.; Braga-Dias, Luciene P.; Oliveira, Márcia; Rossi, Átila D.; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S.; Tanuri, Amilcar

    2014-01-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity. PMID:24688299

  12. Isolation of Catharanthus roseus (L.) G. Don Nuclei and Measurement of Rate of Tryptophan decarboxylase Gene Transcription Using Nuclear Run-On Transcription Assay.

    Science.gov (United States)

    Kumar, Santosh; Bhatia, Sabhyata

    2015-01-01

    An accurate assessment of transcription 'rate' is often desired to describe the promoter activity. In plants, isolation of transcriptionally active nuclei and their subsequent use in nuclear run-on assays has been challenging and therefore limit an accurate measurement of gene transcription 'rate'. Catharanthus roseus has emerged as a model medicinal plant as it exhibits an unsurpassed spectrum of chemodiversity, producing over 130 alkaloids through the terpenoid indole alkaloid (TIA) pathway and therefore serves as a 'molecular hub' to understand gene expression profiles. The protocols presented here streamline, adapt and optimize the existing methods of nuclear run-on assay for use in C. roseus. Here, we fully describe all the steps to isolate transcriptionally active nuclei from C. roseus leaves and utilize them to perform nuclear run-on transcription assay. Nuclei isolated by this method transcribed at a level consistent with their response to external stimuli, as transcription rate of TDC gene was found to be higher in response to external stimuli i.e. when seedlings were subjected to UV-B light or to methyl jasmonate (MeJA). However, the relative transcript abundance measured parallel through qRT-PCR was found to be inconsistent with the synthesis rate indicating that some post transcriptional events might have a role in transcript stability in response to stimuli. Our study provides an optimized, efficient and inexpensive method of isolation of intact nuclei and nuclear 'run-on' transcription assay to carry out in-situ measurement of gene transcription rate in Catharanthus roseus. This would be valuable in investigating the transcriptional and post transcriptional response of other TIA pathway genes in C. roseus. Isolated nuclei may also provide a resource that could be used for performing the chip assay as well as serve as the source of nuclear proteins for in-vitro EMSA studies. Moreover, nascent nuclear run-on transcript could be further subjected to RNA

  13. Isolation of Catharanthus roseus (L. G. Don Nuclei and Measurement of Rate of Tryptophan decarboxylase Gene Transcription Using Nuclear Run-On Transcription Assay.

    Directory of Open Access Journals (Sweden)

    Santosh Kumar

    Full Text Available An accurate assessment of transcription 'rate' is often desired to describe the promoter activity. In plants, isolation of transcriptionally active nuclei and their subsequent use in nuclear run-on assays has been challenging and therefore limit an accurate measurement of gene transcription 'rate'. Catharanthus roseus has emerged as a model medicinal plant as it exhibits an unsurpassed spectrum of chemodiversity, producing over 130 alkaloids through the terpenoid indole alkaloid (TIA pathway and therefore serves as a 'molecular hub' to understand gene expression profiles.The protocols presented here streamline, adapt and optimize the existing methods of nuclear run-on assay for use in C. roseus. Here, we fully describe all the steps to isolate transcriptionally active nuclei from C. roseus leaves and utilize them to perform nuclear run-on transcription assay. Nuclei isolated by this method transcribed at a level consistent with their response to external stimuli, as transcription rate of TDC gene was found to be higher in response to external stimuli i.e. when seedlings were subjected to UV-B light or to methyl jasmonate (MeJA. However, the relative transcript abundance measured parallel through qRT-PCR was found to be inconsistent with the synthesis rate indicating that some post transcriptional events might have a role in transcript stability in response to stimuli.Our study provides an optimized, efficient and inexpensive method of isolation of intact nuclei and nuclear 'run-on' transcription assay to carry out in-situ measurement of gene transcription rate in Catharanthus roseus. This would be valuable in investigating the transcriptional and post transcriptional response of other TIA pathway genes in C. roseus. Isolated nuclei may also provide a resource that could be used for performing the chip assay as well as serve as the source of nuclear proteins for in-vitro EMSA studies. Moreover, nascent nuclear run-on transcript could be further

  14. New Applications of the Comet Assay: Comet-FISH and Transcription-Coupled DNA Repair

    OpenAIRE

    Spivak, Graciela; Cox, Rachel A.; Hanawalt, Philip C.

    2008-01-01

    Transcription-coupled repair (TCR) is a pathway dedicated to the removal of damage from the template strands of actively transcribed genes. Although the detailed mechanism of TCR is not yet understood, it is believed to be triggered when a translocating RNA polymerase is arrested at a lesion or unusual structure in the DNA. Conventional assays for TCR require high doses of DNA damage for the statistical analysis of repair in the individual strands of DNA sequences ranging in size from a few h...

  15. Modelling defined mixtures of environmental oestrogens found in domestic animal and sewage treatment effluents using an in vitro oestrogen-mediated transcriptional activation assay (T47D-KBluc).

    Science.gov (United States)

    Bermudez, Dieldrich S; Gray, L Earl; Wilson, Vickie S

    2012-06-01

    There is growing concern of exposure of fish, wildlife and humans to water sources contaminated with oestrogens and the potential impact on reproductive health. Environmental oestrogens can come from various sources including concentrated animal feedlot operations (CAFO), municipal waste, agricultural and industrial effluents. US EPA's drinking water contaminant candidate list 3 (CCL3) includes several oestrogenic compounds. Although these contaminants are currently not subject to any proposed or promulgated national primary drinking water regulations, they are known or anticipated to occur in public water systems and may require future regulation under the Safe Drinking Water Act. Using an in vitro transcriptional activation assay, this study evaluated oestrogens from CCL3 both individually and as a seven oestrogen mixture (fixed ray design) over a broad range of concentrations, including environmentally relevant concentrations. Log EC(50) and Hillslope values for individual oestrogens were as follows: estrone, -11.92, 1.283; estradiol-17α, -9.61, 1.486; estradiol-17β, 11.77, 1.494; estriol, -11.14, 1.074; ethinyl estradiol-17α, -12.63, 1.562; Mestranol, -11.08, 0.809 and Equilin, -11.48, 0.946. In addition, mixtures that mirrored the primary oestrogens found in swine, poultry and dairy CAFO effluent (fixed-ratio ray design), and a ternary mixture (4 × 4 × 4 factorial design) of oestrogens found in hormone replacement therapy and/or oral contraceptives were tested. Mixtures were evaluated for additivity using both the concentration addition (CA) model and oestrogen equivalence (EEQ) model. For each of the mixture studies, a broad range of concentrations were tested, both above and below environmentally relevant concentrations. Results show that the observed data did not vary consistently from either the CA or EEQ predictions for any mixture. Therefore, either the CA or EEQ model should be useful predictors for modelling oestrogen mixtures. © 2012 The Authors

  16. Development of real-time RT-PCR assays for detection of three classes of HHV-6A gene transcripts.

    Science.gov (United States)

    Ihira, Masaru; Urashima, Akiko; Miura, Hiroki; Hattori, Fumihiko; Kawamura, Yoshiki; Sugata, Ken; Yoshikawa, Tetsushi

    2017-10-01

    Human herpesvirus 6 (HHV-6), a member of the betaherpesvirus family, has two distinct species: HHV-6A and HHV-6B. HHV-6B real-time reverse transcription polymerase chain reaction (RT-PCR) has been used to distinguish between active and latent viral infection. In this study, we developed a real-time RT-PCR assay to detect HHV-6A-specific transcripts and evaluated its reliability for analysis of clinical samples. To develop HHV-6A-specific real-time RT-PCR assays, three different classes of gene transcripts (immediate early: U90; early: U12; and late: U100) were selected as targets. Serial d ilutions of plasmid DNAs containing target sequences and RNAs extracted from HHV-6A-infected cells were used to determine assay specificity and sensitivity. Peripheral blood mononuclear cells (PBMCs) collected from patients with either primary or reactivated HHV-6B infection, and one patient with X-linked severe combined immunodeficiency (X-SCID) with HHV-6A reactivation, were used to evaluate assay reliability. The HHV-6A-specific real-time RT-PCR assays amplified plasmids containing the target sequences at concentrations between 10 and 1 × 106 copies per reaction. The intra-assay coefficients of variation were less than 5%. The three classes of HHV-6A gene transcripts were not detected in any HHV-6B sample isolated from the patients. In the X-SCID patient, high copy numbers of HHV-6A U12 and U100 transcripts were detected in PBMC samples during viremia. Thus, we successfully established highly sensitive and reproducible real-time RT-PCR methods targeting three classes of HHV-6A gene transcripts. This method should be useful for discriminating active HHV-6A infection from either latent infection or chromosomally integrated HHV-6A (ciHHV-6A). © 2017 Wiley Periodicals, Inc.

  17. New applications of the Comet assay: Comet-FISH and transcription-coupled DNA repair.

    Science.gov (United States)

    Spivak, Graciela; Cox, Rachel A; Hanawalt, Philip C

    2009-01-01

    Transcription-coupled repair (TCR) is a pathway dedicated to the removal of damage from the template strands of actively transcribed genes. Although the detailed mechanism of TCR is not yet understood, it is believed to be triggered when a translocating RNA polymerase is arrested at a lesion or unusual structure in the DNA. Conventional assays for TCR require high doses of DNA damage for the statistical analysis of repair in the individual strands of DNA sequences ranging in size from a few hundred bases to 30kb. The single cell gel electrophoresis (Comet) assay allows detection of single- or double-strand breaks at a 10-100-fold higher level of resolution. Fluorescence in situ hybridization (FISH) combined with the Comet assay (Comet-FISH) affords a heightened level of sensitivity for the assessment of repair in defined DNA sequences of cells treated with physiologically relevant doses of genotoxins. This approach also reveals localized susceptibility to chromosomal breakage in cells from individuals with hypersensitivity to radiation or chemotherapy. Several groups have reported preferential repair in transcriptionally active genes or chromosomal domains using Comet-FISH. The prevailing interpretation of the behavior of DNA in the Comet assay assumes that the DNA is arranged in loops and matrix-attachment sites; that supercoiled, undamaged loops are contained within the nuclear matrix and appear in Comet "heads", and that Comet "tails" consist of relaxed DNA loops containing one or more breaks. According to this model, localization of FISH probes in Comet heads signifies that loops containing the targeted sequences are free of damage. This implies that preferential repair as detected by Comet-FISH might encompass large chromosomal domains containing both transcribed and non-transcribed sequences. We review the existing evidence and discuss the implications in relation to current models for the molecular mechanism of TCR.

  18. New Applications of the Comet Assay: Comet-FISH and Transcription-Coupled DNA Repair

    Science.gov (United States)

    Cox, Rachel A.; Hanawalt, Philip C.

    2009-01-01

    Transcription-coupled repair (TCR) is a pathway dedicated to the removal of damage from the template strands of actively transcribed genes. Although the detailed mechanism of TCR is not yet understood, it is believed to be triggered when a translocating RNA polymerase is arrested at a lesion or unusual structure in the DNA. Conventional assays for TCR require high doses of DNA damage for the statistical analysis of repair in the individual strands of DNA sequences ranging in size from a few hundred bases to 30 kb. The single cell gel electrophoresis (Comet) assay allows detection of single-or double-strand breaks at a 10 to 100-fold higher level of resolution. Fluorescence in situ hybridization (FISH) combined with the Comet assay (Comet-FISH) affords a heightened level of sensitivity for the assessment of repair in defined DNA sequences of cells treated with physiologically relevant doses of genotoxins. This approach also reveals localized susceptibility to chromosomal breakage in cells from individuals with hypersensitivity to radiation or chemotherapy. Several groups have reported preferential repair in transcriptionally active genes or chromosomal domains using Comet-FISH. The prevailing interpretation of the behavior of DNA in the Comet assay assumes that the DNA is arranged in loops and matrix-attachment sites; that supercoiled, undamaged loops are contained within the nuclear matrix and appear in Comet “heads”, and that Comet “tails” consist of relaxed DNA loops containing one or more breaks. According to this model, localization of FISH probes in Comet heads signifies that loops containing the targeted sequences are free of damage. This implies that preferential repair as detected by Comet-FISH might encompass large chromosomal domains containing both transcribed and non-transcribed sequences. We review the existing evidence and discuss the implications in relation to current models for the molecular mechanism of TCR. PMID:18291710

  19. Cellular Transcription Factor YY1 Mediates the Varicella-Zoster Virus (VZV) IE62 Transcriptional Activation

    Science.gov (United States)

    Khalil, Mohamed I.; Sommer, Marvin; Arvin, Ann; Hay, John; Ruyechan, William T.

    2014-01-01

    Several cellular transcription factors have been shown to be involved in IE62-mediated activation. The YY1 cellular transcription factor has activating and repressive effects on gene transcription. Analysis of the VZV genome revealed 19 postulated YY1 binding sites located within putative promoters of 16 VZV genes. Electrophoretic mobility shift assays (EMSA) confirmed the binding of YY1 to ORF10, ORF28/29 and gI promoters and the mutation of these binding sites inhibited YY1 binding and the promoter activation by IE62 alone or following VZV infection. Mutation of the ORF28/29 YY1 site in the VZV genome displayed insignificant influence on virus growth in melanoma cells; but it inhibited the virus replication significantly at day 5 and 6 post infection in HELF cells. This work suggests a novel role for the cellular factor YY1 in VZV replication through the mediation of IE62 activation of viral gene expression. PMID:24418559

  20. A brain-specific transcription activator.

    Science.gov (United States)

    Korner, M; Rattner, A; Mauxion, F; Sen, R; Citri, Y

    1989-11-01

    We have identified a DNA binding protein, named BETA, that interacts with the same (B) transcriptional regulatory sequence as the known transcription factor NF-kappa B. BETA is found only in gray matter throughout the brain, and not in a variety of other rat tissues. Two binding sites for BETA are present adjacent to the promoter of the rat proenkephalin gene. Transfection of primary brain cultures that express BETA, with a reporter gene driven by the SV40 promoter linked to BETA DNA binding sites, results in transcriptional activation. We infer that BETA is a brain-specific transcription activator.

  1. DNA Methyltransferase Activity Assays: Advances and Challenges.

    Science.gov (United States)

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice.

  2. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

    Science.gov (United States)

    Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

    2015-11-05

    Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Assaying nonspecific phospholipase C activity

    OpenAIRE

    Pejchar, P. (Přemysl); Günther, F.E.S.; Martinec, J. (Jan)

    2013-01-01

    Plant nonspecific phospholipase C (NPC) is a recently described enzyme which plays a role in membrane rearrangement during phosphate starvation. It is also involved in responses of plants to brassinolide, abscisic acid (ABA), elicitors, and salt. The NPC activity is decreased in cells treated with aluminum. In the case of salt stress, the molecular mechanism of NPC action is based on accumulation of diacylglycerol (DAG) by hydrolysis of phospholipids and conversion of DAG, the product of NPC ...

  4. A bioluminescence assay for aldehyde dehydrogenase activity.

    Science.gov (United States)

    Duellman, Sarah J; Valley, Michael P; Kotraiah, Vinayaka; Vidugiriene, Jolanta; Zhou, Wenhui; Bernad, Laurent; Osterman, Jean; Kimball, Joshua J; Meisenheimer, Poncho; Cali, James J

    2013-03-15

    The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin-aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B=64) and Z'=0.75. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Evaluation of Various Real-Time Reverse Transcription Quantitative PCR Assays for Norovirus Detection.

    Science.gov (United States)

    Yoo, Ju Eun; Lee, Cheonghoon; Park, SungJun; Ko, GwangPyo

    2017-04-28

    Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.

  6. An Assay in Microtitre Plates for Absolute Abundance of Chicken Interferon Alpha Transcripts

    Directory of Open Access Journals (Sweden)

    Renata Novak Kujundžić

    2010-01-01

    Full Text Available Immunosuppression of commercial chickens is a serious animal health and economic problem in the poultry industry. The major causes of the immunosuppression are viruses that suppress transcription of interferon genes, especially interferon alpha. There is a need for monitoring immunosuppression in commercially bred chickens. For this purpose, the absolute abundance of interferon alpha transcripts can be measured in blood of chickens by a suitable assay. Such an assay was used to estimate abundance of chicken interferon alpha in a sample of splenic cells induced with polyinosinic polycytidylic acid. The abundance measured was 29 ± 2 attomoles/µg total RNA. This assay can be performed in microtitre plates using samples collected from chickens in poultry houses.

  7. Promoter proximal polyadenylation sites reduce transcription activity

    DEFF Research Database (Denmark)

    Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site......, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites...... RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels....

  8. Endoproteolytic activity assay in malting barley

    Directory of Open Access Journals (Sweden)

    Blanca Gómez Guerrero

    2013-12-01

    Full Text Available Hydrolysis of barley proteins into peptides and amino acids is one of the most important processes during barley germination.The degradation of the endosperm stored proteins facilitates water and enzyme movements, enhances modification, liberates starch granules and increases soluble amino nitrogen. Protease activity is the result of the activities of a mixture of exo- and endo-proteases. The barley proteins are initially solubilized by endo-proteases and the further by exo-proteases. Four classes of endo-proteases have been described: serine-proteases, cysteine-proteases, aspartic-proteases and metallo-proteases. The objective of this work was to develop a rapid and colorimetric enzymatic assay to determine the endo-proteolytic activity of the four endo-protease classes using two different substrates: azo-gelatin and azo-casein. Optimum conditions for the assays such as: pH,reaction time and temperature and absorbance scale were determined. Azo-gelatin presented several difficulties in standardizing an “in solution” assay. On the other hand, azo-casein allowed standardization of the assay for the four enzyme classes to produce consistent results. The endo-proteoteolytic method developed was applied to determine the endo-protease activity in barley, malt and wort.

  9. Methy-sens Comet assay and DNMTs transcriptional analysis as a combined approach in epigenotoxicology.

    Science.gov (United States)

    Perotti, Alessio; Rossi, Valeria; Mutti, Antonio; Buschini, Annamaria

    2015-02-01

    Epigenotoxicology needs simple and fast tools to assess xenobiotic epigenetic load. This work proposes a comet assay modification designed to detect global methylation changes (Methy-sens Comet) through enzymatic digestion with two restriction enzymes (HpaII, MspI). In the methylation-sensitive protocol tested for repeatability on A549 cells, nickel chloride induced hypermethylation and decitabine-induced hypomethylation. A concomitant assessment of DNA methyltransferases (DNMTs) genes transcriptional levels has been performed, to implement a multifunctional approach to epigenotoxicology. Methy-sens Comet showed a general good repeatability and sensitivity to methylation changes while DNMTs transcriptional levels granted additional proof of xenobiotic-induced impairment of methylome maintenance.

  10. Transcriptional peroxisome proliferator-activated receptor γ ...

    African Journals Online (AJOL)

    user

    Peroxisome proliferator-activated receptor γ coactivator (PGC)-1ɑ, a well-known member of PGC-1 transcriptional coactivator's family, plays a key role in various metabolic pathways. Here, we investigated the role of PGC-1ɑ in the transformation of muscle fiber type in Schizothorax prenanti. The expression of PGC-1ɑ was ...

  11. A simple in vitro RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of ndh transcripts.

    Science.gov (United States)

    Sasaki, Tadamasa; Yukawa, Yasushi; Wakasugi, Tatsuya; Yamada, Kyoji; Sugiura, Masahiro

    2006-09-01

    RNA editing is found in various transcripts from land plant chloroplasts. In tobacco chloroplasts, C-to-U conversion occurs at 36 specific sites including two sites identified in this work. Our RNA editing assay system using chloroplast extracts facilitated biochemical analyses of editing reactions but required mRNAs labeled with (32)P at specific sites. Here, we have improved the in vitro system using fluorescence-labeled chain terminators, ddGTP and ddATP, and have measured the editing activity at 19 sites in ndh transcripts. Editing activities varied from site to site. It has been reported that one editing site in ndhA mRNAs is present in spinach but absent in tobacco, but a corresponding editing capacity had been found in vivo in tobacco using biolistic transformation. We confirmed biochemically the existence of this activity in tobacco extracts. Using the non-radioactive assay, we examined sequences essential for editing within a 50-nt mRNA region encompassing an editing site. Editing of the ndhB-2 site requires a short sequence in front of the editing site, while that of the ndhF mRNA requires two separate regions, a sequence surrounding the editing site and a 5' distal sequence. These results suggest that distinct editing mechanisms are present in chloroplasts.

  12. Repressive effects of resveratrol on androgen receptor transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Wen-feng Shi

    Full Text Available BACKGROUND: The chemopreventive effects of resveratrol (RSV on prostate cancer have been well established; the androgen receptor (AR plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity. METHODOLOGY: The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+ cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(- cells serving as controls. AR(+ cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE. RESULTS: AR in the AR (+ stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment. CONCLUSION: We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding.

  13. Carbohydrate microarrays for assaying galactosyltransferase activity.

    Science.gov (United States)

    Park, Sungjin; Shin, Injae

    2007-04-26

    [reaction: see text] Carbohydrate microarrays have been used recently for the rapid analysis of glycan-protein or glycan-cell interactions and for the detection of pathogens. As a demonstration of its significance and versatility, the microarray technology has been applied in this effort to assay glycosyltransferase activities. In addition, carbohydrate microarray based methods have been employed to quantitatively determine binding affinities between lectins and carbohydrates.

  14. Rad51 activates polyomavirus JC early transcription.

    Directory of Open Access Journals (Sweden)

    Martyn K White

    Full Text Available The human neurotropic polyomavirus JC (JCV causes the fatal CNS demyelinating disease progressive multifocal leukoencephalopathy (PML. JCV infection is very common and after primary infection, the virus is able to persist in an asymptomatic state. Rarely, and usually only under conditions of immune impairment, JCV re-emerges to actively replicate in the astrocytes and oligodendrocytes of the brain causing PML. The regulatory events involved in the reactivation of active viral replication in PML are not well understood but previous studies have implicated the transcription factor NF-κB acting at a well-characterized site in the JCV noncoding control region (NCCR. NF-κB in turn is regulated in a number of ways including activation by cytokines such as TNF-α, interactions with other transcription factors and epigenetic events involving protein acetylation--all of which can regulate the transcriptional activity of JCV. Active JCV infection is marked by the occurrence of rapid and extensive DNA damage in the host cell and the induction of the expression of cellular proteins involved in DNA repair including Rad51, a major component of the homologous recombination-directed double-strand break DNA repair machinery. Here we show that increased Rad51 expression activates the JCV early promoter. This activation is co-operative with the stimulation caused by NF-κB p65, abrogated by mutation of the NF-κB binding site or siRNA to NFκB p65 and enhanced by the histone deacetylase inhibitor sodium butyrate. These data indicate that the induction of Rad51 resulting from infection with JCV acts through NF-κB via its binding site to stimulate JCV early transcription. We suggest that this provides a novel positive feedback mechanism to enhance viral gene expression during the early stage of JCV infection.

  15. Development of a NanoString assay to detect leukemogenic fusion transcripts in acute myeloid leukemia.

    Science.gov (United States)

    Hu, D; Zhou, W; Wang, F; Shu, S M; Fan, L L; He, J; Wang, P; He, Y L; Du, W; Zhang, J H; Duan, J X; Sun, L; Zheng, J; Li, X Q; Li, H Y; Feng, X L; Huang, S A

    2016-12-01

    Detection of leukemogenic fusion transcripts in acute myeloid leukemia (AML) is critical for AML diagnosis. NanoString nCounter system is a novel probe-based gene expression platform capable of measuring up to 800 targets with advantages of reproducibility, accuracy, and sample type flexibility. To study the potential application of NanoString in leukemia at clinic, we used this technology to detect AML leukemogenic fusion transcripts and compared the performances with clinical molecular assays. We developed a NanoString assay to detect seven leukemogenic fusion transcripts, namely RUNX1-RUNX1T1 (e5e12), PML-RARA (bcr1, bcr2, and bcr3), and CBFB-MYH11 (e5e12, e5e8, and e5e7). We set up the cut-off value for each fusion transcript and tested 42 de novo AML samples. We compared the results with reverse transcriptase-polymerase chain reaction (RT-PCR) and TaqMan reverse quantitative-polymerase chain reaction (RQ-PCR), the molecular methods standardly used at clinic. We demonstrated that the NanoString and RT-PCR results correlate well (P NanoString compared to RT-PCR and comparable specificity. Furthermore, we showed that NanoString is not as sensitive as TaqMan RQ-PCR in detecting very low level of fusion transcripts. NanoString can serve as a reliable and alternative molecular method to multiplexed RT-PCR for diagnosis of de novo AML with the perspective of screening/quantitation of a large number of leukemogenic fusion transcripts and prognostic genes. However, NanoString may not be an alternative method for monitoring minimal residual disease in AML. © 2016 John Wiley & Sons Ltd.

  16. Repurposing CRISPR System for Transcriptional Activation.

    Science.gov (United States)

    Chen, Meng; Qi, Lei Stanley

    2017-01-01

    In recent years, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most popular one for genome editing. When the nuclease domains of Cas9 protein are mutated into deactivated form (dCas9), CRISPR/dCas9 still retains the ability to bind the targeted DNA sequence, but loses the endonuclease cleavage activity. Taking advantage of the characteristics of this engineered nuclease inactive Cas9, the CRISPR/dCas system has been repurposed into versatile RNA-guided, DNA-targeting platforms, such as genome imaging, gene regulation, and epigenetic modification. Specifically, fusion of dCas9 with activation domains allows specific and efficient transcriptional activation on a genome-wide scale among diverse organisms. The purpose of this chapter is to review most important the recently published literature on CRISPR/dCas9-based transcriptional activation systems. Compared with the conventional approaches for enhancement of the expression of specific genes of interest, CRISPR/Cas9-based system has emerged as a promising technology for genome regulation, allowing specificity, convenience, robustness, and scalability for endogenous gene activation.

  17. Molecular characterization of porcine picobirnaviruses and development of a specific reverse transcription-PCR assay.

    Science.gov (United States)

    Carruyo, Gabriela M; Mateu, Guaniri; Martínez, Laura C; Pujol, Flor H; Nates, Silvia V; Liprandi, Ferdinando; Ludert, Juan E

    2008-07-01

    The molecular characterization of partial- length genomic segment 2 of porcine picobirnavirus (PBV) strains and the development of a specific reverse transcription-PCR (RT-PCR) assay for detection of virus in feces are reported. Phylogenetic analysis indicated that the studied porcine isolates were more closely related (>85% identity) to human PBV belonging to genogroup I than to the other porcine PBV described so far. Analysis by RT-PCR and polyacrylamide gel electrophoresis of fecal samples collected in Venezuela and Argentina showed that PBV circulate at high frequencies in piglets.

  18. WRKY Transcription Factors Involved in Activation of SA Biosynthesis Genes

    Directory of Open Access Journals (Sweden)

    Bol John F

    2011-05-01

    Full Text Available Abstract Background Increased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR. The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA. An important step in SA biosynthesis in Arabidopsis is the conversion of chorismate to isochorismate through the action of isochorismate synthase, encoded by the ICS1 gene. Also AVRPPHB SUSCEPTIBLE 3 (PBS3 plays an important role in SA metabolism, as pbs3 mutants accumulate drastically reduced levels of SA-glucoside, a putative storage form of SA. Bioinformatics analysis previously performed by us identified WRKY28 and WRKY46 as possible regulators of ICS1 and PBS3. Results Expression studies with ICS1 promoter::β-glucuronidase (GUS genes in Arabidopsis thaliana protoplasts cotransfected with 35S::WRKY28 showed that over expression of WRKY28 resulted in a strong increase in GUS expression. Moreover, qRT-PCR analyses indicated that the endogenous ICS1 and PBS3 genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays indentified potential WRKY28 binding sites in the ICS1 promoter, positioned -445 and -460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the ICS1 promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the ICS1 promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA. Conclusions The results obtained here confirm results from our multiple microarray co-expression analyses indicating that WRKY28 and WRKY46 are transcriptional activators of ICS1 and PBS3, respectively, and support this in silico screening as a powerful tool for identifying new components of stress

  19. Clonality assay based on transcriptional analysis of the polymorphic X-chromosome gene encoding the palmitoylated erythrocyte membrane protein, P55

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Y.; Luhovy, M.; Belickova, M. [Univ. of Alabama, Birmingham, AL (United States)] [and others

    1994-09-01

    The concept of clonality has been important for understanding tumor development and hematopoietic stem cell hierarchy. We have developed a highly specific clonality assay utilizing ligase detection reaction (LDR) to distinguish the active X chromosome by a transcriptional polymorphism of a ubiquitous X-chromosome gene that encodes the palmitoylated erythrocyte membrane protein, P55. The GT polymorphism is at position No. 358 in the cDNA sequence (HUMPEMP accession No. M64925). Among 37 random healthy females of Caucasian, African-American and Asian origin examined by this assay, 14 (38%) were heterozygous for the GT polymorphism, 14 were homozygous for the T allele, and 9 were homozygous for the G allele. We also demonstrate that this gene is not transcribed by the inactive X chromosome by determining the expression of a single allele in patients with clonal hematopoiesis (whose clonality was confirmed independently by a G6PD transcriptional analysis assay). Compared to the transcriptional X-chromosome clonality assay we reported previously which used an unlinked exonic polymorphism of G6PD, this new assay renders a larger proportion of females informative for clonality studies than the G6PD assay. The combination of both of these assays can render about 50% of all females informative for the clonality analysis. This new assay also circumvents problems encountered with the other clonality assays based on either X-chromosome coded peptide polymorphisms or DNA methylation differences between the active and inactive X chromosomes, since it is: (1) biologically sound, (2) reproducibly quantitative, (3) applicable to all tissues including non-nucleated cells such as reticulocytes and platelets, and (4) so sensitive that as few as several hundred cells can be analyzed (such as rare cell populations obtained by FACS).

  20. Reverse transcription genome exponential amplification reaction assay for rapid and universal detection of human rhinoviruses.

    Science.gov (United States)

    Guan, Li; Zhao, Lin-Qing; Zhou, Hang-Yu; Nie, Kai; Li, Xin-Na; Zhang, Dan; Song, Juan; Qian, Yuan; Ma, Xue-Jun

    2016-07-01

    Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5' untranslated region (UTR) was developed for pan-HRV detection. The reaction was performed in a single tube in one step at 65 °C for 60 min using a real-time fluorometer (Genie(®)II; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses, including HEV71, CVA16, CVA6, CVA10, CVA24, CVB5, Echo30, and PV1-3 or with other common respiratory viruses including FluA H3, FluB, PIV1-4, ADV3, RSVA, RSVB and HMPV. With in vitro-transcribed RNA containing the amplified regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5, 50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples, and the results were compared with those obtained using a published semi-nested PCR assay followed by sequencing. The tested panel comprised 91 HRV-negative samples and 52 HRV-positive samples (18 HRV-A-positive samples, 3 HRV-B-positive samples and 31 HRV-C-positive samples). The sensitivity and specificity of the pan-HRVs RT-GEAR assay was 98.08 % and 100 %, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie(®)II fluorometer is a sensitive, specific and rapid assay for the universal detection of HRV infection.

  1. A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

    Science.gov (United States)

    Harding, Ethelynda E.; Kimsey, R. Scott

    1998-01-01

    Describes a laboratory protocol optimizing the conditions for the assay of pepsin activity using the Coomasie Blue dye binding assay of protein concentration. The dye bonds through strong, noncovalent interactions to basic and aromatic amino acid residues. (DDR)

  2. SUMO modification regulates the transcriptional activity of FLASH

    Energy Technology Data Exchange (ETDEWEB)

    Alm-Kristiansen, Anne Hege; Norman, Ingrid Louise; Matre, Vilborg [Department of Molecular Biosciences, University of Oslo, N-0316 Oslo (Norway); Gabrielsen, Odd Stokke, E-mail: o.s.gabrielsen@imbv.uio.no [Department of Molecular Biosciences, University of Oslo, N-0316 Oslo (Norway)

    2009-09-25

    FLASH is a huge multifunctional nuclear protein that has been linked to apoptotic signalling, transcriptional control and Cajal body function. To gain further insight into the functions of the FLASH protein, we performed a yeast two-hybrid screening with FLASH as bait and identified the SUMO-conjugating enzyme Ubc9 as an interaction partner. The main interaction surface for Ubc9 was found in the C-terminal part of FLASH, which is also a target for sumoylation. We identified K1813 as the major sumoylation site in FLASH, being enhanced by the SUMO E3 ligases Pc2 and PIASy. Disruption of this SUMO-conjugation site did not change the speckled subnuclear localization of FLASH, but it caused a reduction in FLASH activity as measured in a Gal4-tethering assay. Interestingly, the SUMO-specific protease SENP1 activated FLASH in the same assay. Overall, our results point to a complex involvement of sumoylation in modulating the function of FLASH.

  3. Protein Inhibitors of Activated STAT (Pias1 and Piasy) Differentially Regulate Pituitary Homeobox 2 (PITX2) Transcriptional Activity*

    Science.gov (United States)

    Wang, Jianbo; Sun, Zhao; Zhang, Zichao; Saadi, Irfan; Wang, Jun; Li, Xiao; Gao, Shan; Engle, Jamison J.; Kuburas, Adisa; Fu, Xueyao; Yu, Wenjie; Klein, William H.; Russo, Andrew F.; Amendt, Brad A.

    2013-01-01

    Protein inhibitors of activated STAT (Pias) proteins can act independent of sumoylation to modulate the activity of transcription factors and Pias proteins interacting with transcription factors can either activate or repress their activity. Pias proteins are expressed in many tissues and cells during development and we asked if Pias proteins regulated the pituitary homeobox 2 (PITX2) homeodomain protein, which modulates developmental gene expression. Piasy and Pias1 proteins are expressed during craniofacial/tooth development and directly interact and differentially regulate PITX2 transcriptional activity. Piasy and Pias1 are co-expressed in craniofacial tissues with PITX2. Yeast two-hybrid, co-immunoprecipitation and pulldown experiments demonstrate Piasy and Pias1 interactions with the PITX2 protein. Piasy interacts with the PITX2 C-terminal tail to attenuate its transcriptional activity. In contrast, Pias1 interacts with the PITX2 C-terminal tail to increase PITX2 transcriptional activity. The E3 ligase activity associated with the RING domain in Piasy is not required for the attenuation of PITX2 activity, however, the RING domain of Pias1 is required for enhanced PITX2 transcriptional activity. Bimolecular fluorescence complementation assays reveal PITX2 interactions with Piasy and Pias1 in the nucleus. Piasy represses the synergistic activation of PITX2 with interacting co-factors and Piasy represses Pias1 activation of PITX2 transcriptional activity. In contrast, Pias1 did not affect the synergistic interaction of PITX2 with transcriptional co-factors. Last, we demonstrate that Pias proteins form a complex with PITX2 and Lef-1, and PITX2 and β-catenin. Lef-1, β-catenin, and Pias interactions with PITX2 provide new molecular mechanisms for the regulation of PITX2 transcriptional activity and the activity of Pias proteins. PMID:23515314

  4. Protein inhibitors of activated STAT (Pias1 and Piasy) differentially regulate pituitary homeobox 2 (PITX2) transcriptional activity.

    Science.gov (United States)

    Wang, Jianbo; Sun, Zhao; Zhang, Zichao; Saadi, Irfan; Wang, Jun; Li, Xiao; Gao, Shan; Engle, Jamison J; Kuburas, Adisa; Fu, Xueyao; Yu, Wenjie; Klein, William H; Russo, Andrew F; Amendt, Brad A

    2013-05-03

    Protein inhibitors of activated STAT (Pias) proteins can act independent of sumoylation to modulate the activity of transcription factors and Pias proteins interacting with transcription factors can either activate or repress their activity. Pias proteins are expressed in many tissues and cells during development and we asked if Pias proteins regulated the pituitary homeobox 2 (PITX2) homeodomain protein, which modulates developmental gene expression. Piasy and Pias1 proteins are expressed during craniofacial/tooth development and directly interact and differentially regulate PITX2 transcriptional activity. Piasy and Pias1 are co-expressed in craniofacial tissues with PITX2. Yeast two-hybrid, co-immunoprecipitation and pulldown experiments demonstrate Piasy and Pias1 interactions with the PITX2 protein. Piasy interacts with the PITX2 C-terminal tail to attenuate its transcriptional activity. In contrast, Pias1 interacts with the PITX2 C-terminal tail to increase PITX2 transcriptional activity. The E3 ligase activity associated with the RING domain in Piasy is not required for the attenuation of PITX2 activity, however, the RING domain of Pias1 is required for enhanced PITX2 transcriptional activity. Bimolecular fluorescence complementation assays reveal PITX2 interactions with Piasy and Pias1 in the nucleus. Piasy represses the synergistic activation of PITX2 with interacting co-factors and Piasy represses Pias1 activation of PITX2 transcriptional activity. In contrast, Pias1 did not affect the synergistic interaction of PITX2 with transcriptional co-factors. Last, we demonstrate that Pias proteins form a complex with PITX2 and Lef-1, and PITX2 and β-catenin. Lef-1, β-catenin, and Pias interactions with PITX2 provide new molecular mechanisms for the regulation of PITX2 transcriptional activity and the activity of Pias proteins.

  5. Detection of mycoplasma contamination in cell substrates using reverse transcription-PCR assays.

    Science.gov (United States)

    Peredeltchouk, M; David, S A Wilson; Bhattacharya, B; Volokhov, D V; Chizhikov, V

    2011-01-01

    To assess the limit of detection (LOD) and the feasibility of 16S rRNA-based reverse transcription-PCR (RT-PCR) assays for advanced detection of mycoplasma contamination in cell substrates. The RT-PCR approach is based on detecting the 16S rRNA molecules that, in contrast to genomic bacterial DNA, are represented by multiple copies in mycoplasma cell. The number of 16S rRNA molecules in mycoplasma cells of five species i.e. Mycoplasma arginini, Myc. fermentans, Myc. hyorhinis, Myc. orale and Acholeplasma laidlawii, all known to be frequent cell line contaminants in industrial and research laboratories, was measured using molecular methods. The results of two independently prepared mycoplasma cultures harvested at the stationary phase of their growth showed that the 16S rRNA copy number per cell varied in the range from about 400 to 2000 copies, depending on species, but stayed close between different preparations of one species. The assessment of the LOD of the in-house 16S rRNA-based RT-PCR was performed using samples of MDCK cell culture spiked with different amounts of five aforementioned mycoplasma species. To minimize the bias in methods comparison, the LOD of the RT-PCR assay was expressed in terms of genome equivalents (GEs) and compared with that determined for highly optimized 16S rDNA-based mycoplasma testing methods previously described in scientific literature. The results of the study showed that the in-house 16S rRNA-based RT-PCR assay was able to reliably detect the presence of less than one mycoplasma GE that is at least 10-fold higher of the LOD previously determined for well-optimized 16S rDNA-based assays developed and described by other researchers. The results of the study showed that rapid RT-PCR methods based on the detection of bacterial 16S rRNA are able to expedite mycoplasma testing of cell cultures (1-2 days vs 28 days) and to ensure the limits of detection comparable to that of currently used culture-based mycoplasma testing methods

  6. Engineering Vibrio fischeri transcriptional activator LuxR for diverse transcriptional activities.

    Science.gov (United States)

    Lu, Yang

    2016-09-01

    To alter DNA binding specificity of Vibrio fischeri LuxR and to expand the toolbox for constructing synthetic networks. A mutation library (about 10,000 individuals) of the DNA binding domain of LuxR were generated. A genetic selection was performed to obtain LuxR mutants that recognize three lux box DNA variants that are not recognized by wild-type LuxR. Six LuxR mutants were identified. The evolved LuxR mutants were further characterized by measuring the transcriptional activities of different combinations of LuxR mutants and lux box variants. Varied transcriptional activities were found in these LuxR-lux box pairs. The background expressions of the evolved LuxR-lux box systems are more tightly regulated than the wild-type LuxR-lux box system. The LuxR transcriptional system was evolved to recognize three lux box DNAs which are not recognized by wild-type LuxR.

  7. TaqMan based real time PCR assay targeting EML4-ALK fusion transcripts in NSCLC.

    Science.gov (United States)

    Robesova, Blanka; Bajerova, Monika; Liskova, Kvetoslava; Skrickova, Jana; Tomiskova, Marcela; Pospisilova, Sarka; Mayer, Jiri; Dvorakova, Dana

    2014-07-01

    Lung cancer with the ALK rearrangement constitutes only a small fraction of patients with non-small cell lung cancer (NSCLC). However, in the era of molecular-targeted therapy, efficient patient selection is crucial for successful treatment. In this context, an effective method for EML4-ALK detection is necessary. We developed a new highly sensitive variant specific TaqMan based real time PCR assay applicable to RNA from formalin-fixed paraffin-embedded tissue (FFPE). This assay was used to analyze the EML4-ALK gene in 96 non-selected NSCLC specimens and compared with two other methods (end-point PCR and break-apart FISH). EML4-ALK was detected in 33/96 (34%) specimens using variant specific real time PCR, whereas in only 23/96 (24%) using end-point PCR. All real time PCR positive samples were confirmed with direct sequencing. A total of 46 specimens were subsequently analyzed by all three detection methods. Using variant specific real time PCR we identified EML4-ALK transcript in 17/46 (37%) specimens, using end-point PCR in 13/46 (28%) specimens and positive ALK rearrangement by FISH was detected in 8/46 (17.4%) specimens. Moreover, using variant specific real time PCR, 5 specimens showed more than one EML4-ALK variant simultaneously (in 2 cases the variants 1+3a+3b, in 2 specimens the variants 1+3a and in 1 specimen the variant 1+3b). In one case of 96 EML4-ALK fusion gene and EGFR mutation were detected. All simultaneous genetic variants were confirmed using end-point PCR and direct sequencing. Our variant specific real time PCR assay is highly sensitive, fast, financially acceptable, applicable to FFPE and seems to be a valuable tool for the rapid prescreening of NSCLC patients in clinical practice, so, that most patients able to benefit from targeted therapy could be identified. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  8. Norepinephrine activates NF-κB transcription factor in cultured rat pineal gland.

    Science.gov (United States)

    Villela, Darine; de Sá Lima, Larissa; Peres, Rafael; Peliciari-Garcia, Rodrigo Antonio; do Amaral, Fernanda Gaspar; Cipolla-Neto, José; Scavone, Cristóforo; Afeche, Solange Castro

    2014-01-17

    The circadian rhythm in mammalian pineal melatonin secretion is modulated by norepinephrine (NE) released at night. NE interaction with β1-adrenoceptors activates PKA that phosphorylates the transcription factor CREB, leading to the transcription and translation of the arylalkylamine-N-acetyltransferase (AANAT) enzyme. Several studies have reported the interplay between CREB and the nuclear factor-κB (NF-κB) and a circadian rhythm for this transcription factor was recently described in the rat pineal gland. In this work we studied a direct effect of NE on NF-κB activation and the role played by this factor on melatonin synthesis and Aanat transcription and activity. Cultured rat pineal glands were incubated in the presence of two different NF-κB inhibitors, pyrrolidine-dithiocarbamate or sodium salicylate, and stimulated with NE. Melatonin content was quantified by HPLC with electrochemical detection. AANAT activity was measured by a radiometric assay and the expression of Aanat mRNA was analyzed by real-time PCR. Gel shift assay was performed to study the NF-κB activation in cultured rat pineal glands stimulated by NE. Our results showed that the p50/p50 homodimer of NF-κB is activated by NE and that it has a role in melatonin synthesis, acting on Aanat transcription and activity. Here we present evidence that NF-κB is an important transcription factor that acts, directly or indirectly, on Aanat transcription and activity leading to a modulation of melatonin synthesis. NE plays a role in the translocation of NF-κB p50/p50 homodimer to the nucleus of pinealocytes, thus probably influencing the nocturnal pineal melatonin synthesis. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Bivariate Genomic Footprinting Detects Changes in Transcription Factor Activity

    Directory of Open Access Journals (Sweden)

    Songjoon Baek

    2017-05-01

    Full Text Available In response to activating signals, transcription factors (TFs bind DNA and regulate gene expression. TF binding can be measured by protection of the bound sequence from DNase digestion (i.e., footprint. Here, we report that 80% of TF binding motifs do not show a measurable footprint, partly because of a variable cleavage pattern within the motif sequence. To more faithfully portray the effect of TFs on chromatin, we developed an algorithm that captures two TF-dependent effects on chromatin accessibility: footprinting and motif-flanking accessibility. The algorithm, termed bivariate genomic footprinting (BaGFoot, efficiently detects TF activity. BaGFoot is robust to different accessibility assays (DNase-seq, ATAC-seq, all examined peak-calling programs, and a variety of cut bias correction approaches. BaGFoot reliably predicts TF binding and provides valuable information regarding the TFs affecting chromatin accessibility in various biological systems and following various biological events, including in cases where an absolute footprint cannot be determined.

  10. Bivariate Genomic Footprinting Detects Changes in Transcription Factor Activity.

    Science.gov (United States)

    Baek, Songjoon; Goldstein, Ido; Hager, Gordon L

    2017-05-23

    In response to activating signals, transcription factors (TFs) bind DNA and regulate gene expression. TF binding can be measured by protection of the bound sequence from DNase digestion (i.e., footprint). Here, we report that 80% of TF binding motifs do not show a measurable footprint, partly because of a variable cleavage pattern within the motif sequence. To more faithfully portray the effect of TFs on chromatin, we developed an algorithm that captures two TF-dependent effects on chromatin accessibility: footprinting and motif-flanking accessibility. The algorithm, termed bivariate genomic footprinting (BaGFoot), efficiently detects TF activity. BaGFoot is robust to different accessibility assays (DNase-seq, ATAC-seq), all examined peak-calling programs, and a variety of cut bias correction approaches. BaGFoot reliably predicts TF binding and provides valuable information regarding the TFs affecting chromatin accessibility in various biological systems and following various biological events, including in cases where an absolute footprint cannot be determined. Published by Elsevier Inc.

  11. A multiplex assay to measure RNA transcripts of prostate cancer in urine.

    Directory of Open Access Journals (Sweden)

    Sue-Ing Quek

    Full Text Available The serum prostate-specific antigen (PSA test has a high false positive rate. As a single marker, PSA provides limited diagnostic information. A multi-marker test capable of detecting not only tumors but also the potentially lethal ones provides an unmet clinical need. Using the nanoString nCounter gene expression system, a 20-gene multiplex test was developed based on digital gene counting of RNA transcripts in urine as a means to detect prostate cancer. In this test, voided urine is centrifuged to pellet cells and the purified RNA is amplified for hybridization to preselected probesets. Amplification of test cell line RNA appeared not to introduce significant bias, and the counts matched well with gene abundance levels as measured by DNA microarrays. For data analysis, the individual counts were compared to that of β2 microglobulin, a housekeeping gene. Urine samples of 5 pre-operative cases and 2 non-cancer were analyzed. Pathology information was then retrieved. Signals for a majority of the genes were low for non-cancer and low Gleason scores, and 6/6 known prostate cancer markers were positive in the cases. One case of Gleason 4+5 showed, in contrast, strong signals for all cancer-associated markers, including CD24. One non-cancer also showed signals for all 6 cancer markers, and this man might harbor an undiagnosed cancer. This multiplex test assaying a natural waste product can potentially be used for screening, early cancer detection and patient stratification. Diagnostic information is gained from the RNA signatures that are associated with cell types of prostate tumors.

  12. From DNA binding to transcriptional activation: Is the TALE complete?

    Science.gov (United States)

    Bobola, Nicoletta

    2017-09-04

    How transcription factors (TFs) control enhancer and promoter functions to effect changes in gene expression is an important question. In this issue, Hau et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201701154) show that the TALE TF MEIS recruits the histone modifier PARP1/ARTD1 at promoters to decompact chromatin and activate transcription. © 2017 Bobola.

  13. Human mediator subunit MED15 promotes transcriptional activation.

    Science.gov (United States)

    Nakatsubo, Takuya; Nishitani, Saori; Kikuchi, Yuko; Iida, Satoshi; Yamada, Kana; Tanaka, Aki; Ohkuma, Yoshiaki

    2014-10-01

    In eukaryotes, the Mediator complex is an essential transcriptional cofactor of RNA polymerase II (Pol II). In humans, it contains up to 30 subunits and consists of four modules: head, middle, tail, and CDK/Cyclin. One of the subunits, MED15, is located in the tail module, and was initially identified as Gal11 in budding yeast, where it plays an essential role in the transcriptional regulation of galactose metabolism with the potent transcriptional activator Gal4. For this reason, we investigated the function of the human MED15 subunit (hMED15) in transcriptional activation. First, we measured the effect of hMED15 knockdown on cell growth in HeLa cells. The growth rate was greatly reduced. By immunostaining, we observed the colocalization of hMED15 with the general transcription factors TFIIE and TFIIH in the nucleus. We measured the effects of siRNA-mediated knockdown of hMED15 on transcriptional activation using two different transcriptional activators, VP16 and SREBP1a. Treatment with siRNAs reduced transcriptional activation, and this reduction could be rescued by overexpression of HA/Flag-tagged, wild-type hMED15. To investigate hMED15 localization, we treated human MCF-7 cells with the MDM2 inhibitor Nutlin-3, thus inducing p21 transcription. We found that hMED15 localized to both the p53 binding site and the p21 promoter region, along with TFIIE and TFIIH. These results indicate that hMED15 promotes transcriptional activation.

  14. Development of a sensitive multi-well colorimetric assay for active NFκB

    Science.gov (United States)

    Renard, Patricia; Ernest, Isabelle; Houbion, Andrée; Art, Muriel; Le Calvez, Hervé; Raes, Martine; Remacle, José

    2001-01-01

    The transcription factor nuclear factor κB (NFκB) is a key factor in the immune response triggered by a wide variety of molecules such as inflammatory cytokines, or some bacterial and viral products. This transcription factor represents a new target for the development of anti-inflammatory molecules, but this type of research is currently hampered by the lack of a convenient and rapid screening assay for NFκB activation. Indeed, NFκB DNA-binding capacity is traditionally estimated by radioactive gel shift assay. Here we propose a new DNA-binding assay based on the use of multi-well plates coated with a cold oligonucleotide containing the consensus binding site for NFκB. The presence of the DNA-bound transcription factor is then detected by anti-NFκB antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NFκB, more sensitive than regular radioactive gel shift and very convenient for high throughput screening. PMID:11160941

  15. A Genetic Assay for Transcription Errors Reveals Multilayer Control of RNA Polymerase II Fidelity

    OpenAIRE

    Irvin, Jordan D.; Kireeva, Maria L.; Gotte, Deanna R.; Shafer, Brenda K.; Ingold Huang; Mikhail Kashlev; Strathern, Jeffrey N.

    2014-01-01

    Author Summary Mistakes made during the synthesis of messenger RNAs have been difficult to detect, both because mRNAs can be short lived, and because the translation of mRNAs into proteins has a much higher error rate that masks transcription errors. We present here a highly sensitive genetic screen that detects transcription errors and use it to identify mutations that increase the error rate of RNA polymerase II. The screen incorporates a new principle that allows transient transcription er...

  16. Transcriptional activation of Mina by Sp1/3 factors.

    Science.gov (United States)

    Lian, Shangli; Potula, Hari Hara S K; Pillai, Meenu R; Van Stry, Melanie; Koyanagi, Madoka; Chung, Linda; Watanabe, Makiko; Bix, Mark

    2013-01-01

    Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of Mina gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the Mina 5' region. In order to explore the mechanisms regulating Mina gene expression, we set out to molecularly characterize the Mina promoter in the region encompassing these SNPs. We used three kinds of assays--reporter, gel shift and chromatin immunoprecipitation--to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of Mina promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of Mina gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways.

  17. DNA Methyltransferase Activity Assays: Advances and Challenges

    OpenAIRE

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao,Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modificati...

  18. Peroxisome proliferator-activated receptor gamma recruits the positive transcription elongation factor b complex to activate transcription and promote adipogenesis

    DEFF Research Database (Denmark)

    Iankova, Irena; Petersen, Rasmus K; Annicotte, Jean-Sébastien

    2006-01-01

    Positive transcription elongation factor b (P-TEFb) phosphorylates the C-terminal domain of RNA polymerase II, facilitating transcriptional elongation. In addition to its participation in general transcription, P-TEFb is recruited to specific promoters by some transcription factors such as c...... with and phosphorylation of peroxisome proliferator-activated receptor gamma (PPARgamma), which is the master regulator of this process, on the promoter of PPARgamma target genes. PPARgamma-cdk9 interaction results in increased transcriptional activity of PPARgamma and therefore increased adipogenesis.......-Myc or MyoD. The P-TEFb complex is composed of a cyclin-dependent kinase (cdk9) subunit and a regulatory partner (cyclin T1, cyclin T2, or cyclin K). Because cdk9 has been shown to participate in differentiation processes, such as muscle cell differentiation, we studied a possible role of cdk9...

  19. An Asp7Gly substitution in PPARG is associated with decreased transcriptional activation activity.

    Directory of Open Access Journals (Sweden)

    Liushuai Hua

    Full Text Available As the master regulator of adipogenesis, peroxisome proliferator-activated receptor gamma (PPARG is required for the accumulation of adipose tissue and hence contributes to obesity. A previous study showed that the substitution of +20A>G in PPARG changed the 7(th amino acid from Asp to Gly, creating a mutant referred to as PPARG Asp7Gly. In this study, association analysis indicated that PPARG Asp7Gly was associated with lower body height, body weight and heart girth in cattle (P<0.05. Overexpression of PPARG in NIH3T3-L1 cells showed that the Asp7Gly substitution may cause a decrease in its adipogenic ability and the mRNA levels of CIDEC (cell death-inducing DFFA-like effector c and aP2, which are all transcriptionally activated by PPARG during adipocyte differentiation. A dual-luciferase reporter assay was used to analyze the promoter activity of CIDEC. The results confirmed that the mutant PPARG exhibited weaker transcriptional activation activity than the wild type (P<0.05. These findings likely explain the associations between the Asp7Gly substitution and the body measurements. Additionally, the Asp7Gly mutation may be used in molecular marker assisted selection (MAS of cattle breeding in the future.

  20. Development and validation of quantitative PCR assays to measure cytokine transcript levels in the Florida manatee (Trichechus manatus latirostris)

    Science.gov (United States)

    Ferrante, Jason; Hunter, Margaret; Wellehan, James F.X.

    2018-01-01

    Cytokines have important roles in the mammalian response to viral and bacterial infections, trauma, and wound healing. Because of early cytokine production after physiologic stresses, the regulation of messenger RNA (mRNA) transcripts can be used to assess immunologic responses before changes in protein production. To detect and assess early immune changes in endangered Florida manatees (Trichechus manatus latirostris), we developed and validated a panel of quantitative PCR assays to measure mRNA transcription levels for the cytokines interferon (IFN)-γ; interleukin (IL)-2, -6, and -10; tumor necrosis factor-α, and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (reference genes). Assays were successfully validated using blood samples from free-ranging, apparently healthy manatees from the east and west coasts of central Florida. No cytokine or housekeeping gene transcription levels were significantly different among age classes or sexes. However, the transcription levels for GAPDH, IL-2, IL-6, and IFN-γ were significantly higher (Ptrauma and novel or ongoing environmental stressors.

  1. Dataset of transcriptional landscape of B cell early activation

    Directory of Open Access Journals (Sweden)

    Alexander S. Garruss

    2015-09-01

    Full Text Available Signaling via B cell receptors (BCR and Toll-like receptors (TLRs result in activation of B cells with distinct physiological outcomes, but transcriptional regulatory mechanisms that drive activation and distinguish these pathways remain unknown. At early time points after BCR and TLR ligand exposure, 0.5 and 2 h, RNA-seq was performed allowing observations on rapid transcriptional changes. At 2 h, ChIP-seq was performed to allow observations on important regulatory mechanisms potentially driving transcriptional change. The dataset includes RNA-seq, ChIP-seq of control (Input, RNA Pol II, H3K4me3, H3K27me3, and a separate RNA-seq for miRNA expression, which can be found at Gene Expression Omnibus Dataset GSE61608. Here, we provide details on the experimental and analysis methods used to obtain and analyze this dataset and to examine the transcriptional landscape of B cell early activation.

  2. Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

    Science.gov (United States)

    Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

    2015-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence. Published by Elsevier B.V.

  3. A Simple Assay for Measuring Catalase Activity: A Visual Approach

    Science.gov (United States)

    Iwase, Tadayuki; Tajima, Akiko; Sugimoto, Shinya; Okuda, Ken-ichi; Hironaka, Ippei; Kamata, Yuko; Takada, Koji; Mizunoe, Yoshimitsu

    2013-01-01

    In this study, an assay that combines the ease and simplicity of the qualitative approach for measuring catalase activity was developed. The assay reagents comprised only hydrogen peroxide and Triton X-100. The enzyme-generated oxygen bubbles trapped by Triton X-100 were visualized as foam, whose height was estimated. A calibration plot using the defined unit of catalase activity yielded the best linear fit over a range of 20–300 units (U) (y = 0.3794x − 2.0909, r2 = 0.993). The assay precision and reproducibility at 100 U were 4.6% and 4.8%, respectively. The applicability of the assay for measuring the catalase activity of various samples was assessed using laboratory strains of Escherichia coli, catalase-deficient isogenic mutants, clinically isolated Shiga toxin-producing E. coli, and human cells. The assay generated reproducible results. In conclusion, this new assay can be used to measure the catalase activity of bacterial isolates and human cells. PMID:24170119

  4. In vitro JAK kinase activity and inhibition assays.

    Science.gov (United States)

    Babon, Jeffrey J; Murphy, James M

    2013-01-01

    The discovery that a range of myeloproliferative diseases and leukemias are associated with Janus Kinase (JAK) mutations has highlighted the importance of JAK/STAT signalling in disease and sparked a renewed interest in developing JAK inhibitors. In vitro kinase assays are the most direct and quantitative method to assess mutant forms of JAK for altered enzymatic properties as well as verifying and quantifying the affinity and efficacy of potential inhibitors. Here, we describe protocols for heterologous expression and purification of JAK kinases from insect cells, assays to determine the activity of these purified kinases, and finally inhibition assays to determine the effectiveness of potential inhibitors.

  5. First Exon Length Controls Active Chromatin Signatures and Transcription

    Directory of Open Access Journals (Sweden)

    Nicole I. Bieberstein

    2012-07-01

    Full Text Available Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures.

  6. Pitfalls in the assay of carboxymethylcellulase activity. [Sclerotium rolfsii

    Energy Technology Data Exchange (ETDEWEB)

    Lindner, W.A.; Dennison, C.; Quicke, G.V.

    1983-02-01

    A purified endocellulase from Sclerotium rolfsii and a crude cellulase preparation from Trichoderma reesei are used to illustrate several pitfalls associated with the assay of carboxymethylcellulase activity and the subsequent attainment of linear enzyme dilution curves. It is shown that the nature of both the enzymes and the substrate make the assay unsuitable for use in the calculation of enzyme recovery and purity. (Refs. 16).

  7. A rapid assay for the biological evaluation of helicase activity.

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Dimitrios Vlachakis, Andrea Brancale, Colin Berry & Sophia Kossida ### Abstract A new assay for the measurement of helicase enzyme activity was developed for the evaluation of the potency of potential inhibitors. This assay involves the use of a DNA or RNA duplex substrate and recombinant purified helicase. The DNA duplex consists of a pair of oligonucleotides, one of which is biotinylated and the other is digoxygenin (DIG)-labelled, both at their respective 5’ termini. This ...

  8. Centromeric Transcription Regulates Aurora-B Localization and Activation

    Directory of Open Access Journals (Sweden)

    Michael D. Blower

    2016-05-01

    Full Text Available Centromeric transcription is widely conserved; however, it is not clear what role centromere transcription plays during mitosis. Here, I find that centromeres are transcribed in Xenopus egg extracts into a long noncoding RNA (lncRNA; cen-RNA that localizes to mitotic centromeres, chromatin, and spindles. cen-RNAs bind to the chromosomal passenger complex (CPC in vitro and in vivo. Blocking transcription or antisense inhibition of cen-RNA leads to a reduction of CPC localization to the inner centromere and misregulation of CPC component Aurora-B activation independently of known centromere recruitment pathways. Additionally, transcription is required for normal bipolar attachment of kinetochores to the mitotic spindle, consistent with a role for cen-RNA in CPC regulation. This work demonstrates that cen-RNAs promote normal kinetochore function through regulation of the localization and activation of the CPC and confirm that lncRNAs are components of the centromere.

  9. Resveratrol regulates gene transcription via activation of stimulus-responsive transcription factors.

    Science.gov (United States)

    Thiel, Gerald; Rössler, Oliver G

    2017-03-01

    Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin of grapes and other fruits and plants, is a common constituent of our diet and of dietary supplements. Many health-promoting benefits have been connected with resveratrol in the treatment of cardiovascular diseases, cancer, diabetes, inflammation, neurodegeneration, and diseases connected with aging. To explain the pleiotropic effects of resveratrol, the molecular targets of this compound have to be identified on the cellular level. Resveratrol induces intracellular signal transduction pathways which ultimately lead to changes in the gene expression pattern of the cells. Here, we review the effect of resveratrol on the activation of the stimulus-responsive transcription factors CREB, AP-1, Egr-1, Elk-1, and Nrf2. Following activation, these transcription factors induce transcription of delayed response genes. The gene products of these delayed response genes are ultimately responsible for the changes in the biochemistry and physiology of resveratrol-treated cells. The activation of stimulus-responsive transcription factors may explain many of the intracellular activities of resveratrol. However, results obtained in vitro may not easily be transferred to in vivo systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets.

    Science.gov (United States)

    Babu, Binoy; Washburn, Brian K; Miller, Steven H; Poduch, Kristina; Sarigul, Tulin; Knox, Gary W; Ochoa-Corona, Francisco M; Paret, Mathews L

    2017-02-01

    Rose rosette disease caused by Rose rosette virus (RRV; genus Emaravirus) is the most economically relevant disease of Knock Out® series roses in the U.S. As there are no effective chemical control options for the disease, the most critical disease management strategies include the use of virus free clean plants for propagation and early detection and destruction of infected plants. The current diagnostic techniques for RRV including end-point reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RT-qPCR) are highly sensitive, but limited to diagnostic labs with the equipment and expertise; and is time consuming. To address this limitation, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay based on multiple gene targets for specific detection of RRV was developed. The assay is highly specific and did not cross react with other viruses belonging to the inclusive and exclusive genus. Dilution assays using the in vitro transcripts showed that the primer sets designed (RPA-267, RPA-131, and RPA-321) are highly sensitive, consistently detecting RRV with a detection limit of 1fg/μL. Testing of the infected plants using the primer sets indicated that the virus could be detected from leaves, stems and petals of roses. The primer pair RPA-267 produced 100% positive detection of the virus from infected leaf tissues, while primer set RPA-131 produced 100% detection from stems and petals. The primer set RPA-321 produced 83%, 87.5% and 75% positive detection from leaves, petals and stem tissues, respectively. In addition, the assay has been efficiently used in the detection of RRV infecting Knock Out® roses, collected from different states in the U.S. The assay can be completed in 20min as compared to the end-point RT-PCR assay (3-4h) and RT-qPCR (1.5h). The RT-RPA assay is reliable, rapid, highly sensitive, and can be easily used in diagnostic laboratories for detection of RRV with no need for any special equipment

  11. Comparison of automated von Willebrand factor activity assays

    DEFF Research Database (Denmark)

    Timm, Annette; Hillarp, Andreas; Philips, Malou

    2015-01-01

    INTRODUCTION: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder. Measurement of von Willebrand factor (VWF) activity in plasma is often based on platelet agglutination stimulated by the ristocetin cofactor activity. Novel assays, based on latex beads with recombinant...

  12. Field validation of a transcriptional assay for the prediction of age of uncaged Aedes aegypti mosquitoes in Northern Australia.

    Directory of Open Access Journals (Sweden)

    Leon E Hugo

    Full Text Available BACKGROUND: New strategies to eliminate dengue have been proposed that specifically target older Aedes aegypti mosquitoes, the proportion of the vector population that is potentially capable of transmitting dengue viruses. Evaluation of these strategies will require accurate and high-throughput methods of predicting mosquito age. We previously developed an age prediction assay for individual Ae. aegypti females based on the transcriptional profiles of a selection of age responsive genes. Here we conducted field testing of the method on Ae. aegypti that were entirely uncaged and free to engage in natural behavior. METHODOLOGY/PRINCIPAL FINDINGS: We produced "free-range" test specimens by releasing 8007 adult Ae. aegypti inside and around an isolated homestead in north Queensland, Australia, and recapturing females at two day intervals. We applied a TaqMan probe-based assay design that enabled high-throughput quantitative RT-PCR of four transcripts from three age-responsive genes and a reference gene. An age prediction model was calibrated on mosquitoes maintained in small sentinel cages, in which 68.8% of the variance in gene transcription measures was explained by age. The model was then used to predict the ages of the free-range females. The relationship between the predicted and actual ages achieved an R(2 value of 0.62 for predictions of females up to 29 days old. Transcriptional profiles and age predictions were not affected by physiological variation associated with the blood feeding/egg development cycle and we show that the age grading method could be applied to differentiate between two populations of mosquitoes having a two-fold difference in mean life expectancy. CONCLUSIONS/SIGNIFICANCE: The transcriptional profiles of age responsive genes facilitated age estimates of near-wild Ae. aegypti females. Our age prediction assay for Ae. aegypti provides a useful tool for the evaluation of mosquito control interventions against dengue where

  13. HTLV-1 Tax activates HIV-1 transcription in latency models.

    Science.gov (United States)

    Geddes, Victor Emmanuel Viana; José, Diego Pandeló; Leal, Fabio E; Nixon, Douglas F; Tanuri, Amilcar; Aguiar, Renato Santana

    2017-04-01

    HIV-1 latency is a major obstacle to HIV-1 eradication. Coinfection with HTLV-1 has been associated with faster progression to AIDS. HTLV-1 encodes the transactivator Tax which can activate both HTLV-1 and HIV-1 transcription. Here, we demonstrate that Tax activates HIV transcription in latent CD4(+) T cells. Tax promotes the activation of P-TEFb, releasing CDK9 and Cyclin T1 from inactive forms, promoting transcription elongation and reactivation of latent HIV-1. Tax mutants lacking interaction with the HIV-1-LTR promoter were not able to activate P-TEFb, with no subsequent activation of latent HIV. In HIV-infected primary resting CD4(+) T cells, Tax-1 reactivated HIV-1 transcription up to five fold, confirming these findings in an ex vivo latency model. Finally, our results confirms that HTLV-1/Tax hijacks cellular partners, promoting HIV-1 transcription, and this interaction should be further investigated in HIV-1 latency studies in patients with HIV/HTLV-1 co-infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Radiometric microbiologic assay for the biologically active forms of niacin

    Energy Technology Data Exchange (ETDEWEB)

    Kertcher, J.A.; Guilarte, T.R.; Chen, M.F.; Rider, A.A.; McIntyre, P.A.

    1979-05-01

    A radiometric microbiologic assay has been developed for the determination of niacin in biologic fluids. Lactobacillus plantarum produced /sup 14/CO/sub 2/ from L-(U-/sup 14/C) malic acid in quantities proportional to the amount of niacin present. The assay is specific for the biologically active forms of niacin in humans. Thirty normal hemolysates were analyzed and the values ranged from 13.0 to 17.8 ..mu..g niacin/ml RBC (mean = 15.27 +- 1.33 s.d.). Good recovery and reproducibility studies were obtained with this assay. On thirty blood samples, correlation was excellent between the radiometric and the conventional turbidimetric assays.

  15. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation.

    Science.gov (United States)

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease.

  16. A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections.

    Directory of Open Access Journals (Sweden)

    Sylvie Kemleu

    Full Text Available Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT, and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL. Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar's test: P = 0.0002, and the highest was with RT-PCR (87%, McNemar's test: P = 0.0523. Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay

  17. Chromosome translocation activates heterogeneously initiated, bipolar transcription of a mouse c-myc gene.

    Science.gov (United States)

    Calabi, F; Neuberger, M S

    1985-01-01

    In many mouse plasmacytomas, the active c-myc gene has been truncated by chromosome translocation with the resultant severance of the protein-coding sequence from the normal promoter. Transcripts of such truncated c-myc genes were analyzed by Northern blotting, nuclease S1 mapping, primer extension assays and cDNA cloning. We conclude that transcription originates from multiple initiation sites on both c-myc coding and non-coding strands with the two-sets of transcripts derived from adjacent but essentially non-overlapping regions located greater than 1 kb from the translocation junction. In X63Ag8, where c-myc is translocated to the immunoglobulin C gamma 2b gene, the c-myc non-coding strand transcripts include the translocation junction and then splice directly into the gamma 2b CH1 exon. We propose that chromosome translocation activates a cryptic promoter in the first intron and that the heterogeneously initiated, bipolar transcription reflects the absence of a suitably placed TATA box element. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 7. Fig. 8. PMID:3924591

  18. A naturally occurring truncated form of FosB that inhibits Fos/Jun transcriptional activity.

    Science.gov (United States)

    Nakabeppu, Y; Nathans, D

    1991-02-22

    Fos and Jun transcription factors are induced by a variety of extracellular signaling agents. We describe here an unusual member of the Fos family that is also induced, namely, a truncated form of FosB (delta FosB) missing the C-terminal 101 amino acids of FosB. delta FosB retains the dimerization and DNA-binding activities of FosB but has lost the ability in transfection assays to activate a promoter with an AP-1 site and to repress the c-fos promoter. Rather, delta FosB inhibits gene activation by Jun or Jun + Fos and inhibits repression of the c-fos promoter by FosB or c-Fos, presumably by competing with full-length Fos proteins at the steps of dimerization with Jun and binding to DNA. In stimulated cells delta FosB may act to limit the transcriptional effects of Fos and Jun proteins.

  19. Estrogenic effects of leachates from industrial waste landfills measured by a recombinant yeast assay and transcriptional analysis in Japanese medaka.

    Science.gov (United States)

    Kamata, Ryo; Shiraishi, Fujio; Nakajima, Daisuke; Kageyama, Shiho

    2011-01-25

    In Japan, the leachates from 'stable type' landfills for industrial wastes are not controlled, and this has given rise to concerns about the possible pollution of surrounding environmental waters, especially by endocrine disrupting chemicals leaching from plastic and rubber wastes. To accurately assess the estrogenic potential of the landfill leachates by both in vitro and in vivo approaches, we confirmed gene-transcriptional responses in recombinant yeast cells and in Japanese medaka fish to estrogenic compounds, and applied these transcription assays to leachate samples. The yeast carrying the estrogen receptor (ER) of medaka and an ER-mediated response pathway responded to both the natural estrogen, 17β-estradiol (E2), and an industrial compound, bisphenol A (BPA), and the effective concentration of BPA was about 2.0×10(3) times that of E2. Transcripts of all genes coding for precursors of yolk protein, vitellogenin (vtg1 and vtg2), and precursors of egg envelope subunit proteins, choriogenins (chgh and chgl), increased in a concentration dependent manner in the livers of male medaka exposed to BPA or E2, and, except for chgh, reached peaks at exposure times of 48h. Although many fish in control groups did not have vtg transcripts, the incidence of vtg transcriptions also increased in a concentration dependent manner with exposure. The minimum effective concentrations of BPA at 48h were 0.5mg/L for chgh and vtg2, 2mg/L for vtg1 and 4mg/L for chgl, while those of E2 were 10ng/L for chgh and chgl and 30ng/L for vtg1 and vtg2. All leachates sampled at 3 landfill sites exerted in vitro estrogenic action. The E2 equivalent of the most potent leachate was 375ng/L for the yeast ER assay. This leachate sample significantly increased the transcripts of chgh, vtg1 and vtg2, but not chgl, in the medaka. In addition, chemical analysis showed that bisphenol A, 4-tert-octylphenol and 4-nonylphenol were the main contributors to the estrogenicity of the leachates. This study

  20. CMYB1 Encoding a MYB Transcriptional Activator Is Involved in Abiotic Stress and Circadian Rhythm in Rice

    Directory of Open Access Journals (Sweden)

    Min Duan

    2014-01-01

    Full Text Available Through analysis of cold-induced transcriptome, a novel gene encoding a putative MYB transcription factor was isolated and designated Cold induced MYB 1 (CMYB1. Tissue-specific gene expression analysis revealed that CMYB1 was highly expressed in rice stems and nodes. qRT-PCR assay indicated that CMYB1 was dramatically induced by cold stress (>100-folds and induced by exogenous ABA and osmotic stress. Interestingly, CMYB1 showed rhythmic expression profile in rice leaves at different developmental stages. Subcellular localization assay suggested that CMYB1-GFP (green fluorescent protein fusion protein was localized in the nuclei. Moreover, CMYB1 exhibited the transcriptional activation activity when transiently expressed in rice protoplast cells. Taken together, CMYB1 probably functions as a transcriptional activator in mediating stress and rhythm responsive gene expression in rice.

  1. Internal Ribosome Entry Site-Based Bicistronic In Situ Reporter Assays for Discovery of Transcription-Targeted Lead Compounds.

    Science.gov (United States)

    Lang, Liwei; Ding, Han-Fei; Chen, Xiaoguang; Sun, Shi-Yong; Liu, Gang; Yan, Chunhong

    2015-07-23

    Although transgene-based reporter gene assays have been used to discover small molecules targeting expression of cancer-driving genes, the success is limited due to the fact that reporter gene expression regulated by incomplete cis-acting elements and foreign epigenetic environments does not faithfully reproduce chemical responses of endogenous genes. Here, we present an internal ribosome entry site-based strategy for bicistronically co-expressing reporter genes with an endogenous gene in the native gene locus, yielding an in situ reporter assay closely mimicking endogenous gene expression without disintegrating its function. This strategy combines the CRISPR-Cas9-mediated genome-editing tool with the recombinase-mediated cassette-exchange technology, and allows for rapid development of orthogonal assays for excluding false hits generated from primary screens. We validated this strategy by developing a screening platform for identifying compounds targeting oncogenic eIF4E, and demonstrated that the novel reporter assays are powerful in searching for transcription-targeted lead compounds with high confidence. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Improved Detection of Rhinoviruses in Clinical Samples by Using a Newly Developed Nested Reverse Transcription-PCR Assay

    Science.gov (United States)

    Andeweg, Arno C.; Bestebroer, Theo M.; Huybreghs, Martijn; Kimman, Tjeerd G.; de Jong, Jan C.

    1999-01-01

    This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5′ noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections. PMID:9986806

  3. Model of transcriptional activation by MarA in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Michael E Wall

    2009-12-01

    Full Text Available The AraC family transcription factor MarA activates approximately 40 genes (the marA/soxS/rob regulon of the Escherichia coli chromosome resulting in different levels of resistance to a wide array of antibiotics and to superoxides. Activation of marA/soxS/rob regulon promoters occurs in a well-defined order with respect to the level of MarA; however, the order of activation does not parallel the strength of MarA binding to promoter sequences. To understand this lack of correspondence, we developed a computational model of transcriptional activation in which a transcription factor either increases or decreases RNA polymerase binding, and either accelerates or retards post-binding events associated with transcription initiation. We used the model to analyze data characterizing MarA regulation of promoter activity. The model clearly explains the lack of correspondence between the order of activation and the MarA-DNA affinity and indicates that the order of activation can only be predicted using information about the strength of the full MarA-polymerase-DNA interaction. The analysis further suggests that MarA can activate without increasing polymerase binding and that activation can even involve a decrease in polymerase binding, which is opposite to the textbook model of activation by recruitment. These findings are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. We find that activation involving decreased polymerase binding yields lower latency in gene regulation and therefore might confer a competitive advantage to cells. Our model yields insights into requirements for predicting the order of activation of a regulon and enables us to suggest that activation might involve a decrease in polymerase binding which we expect to be an important theme of gene regulation in E. coli and beyond.

  4. Model of transcriptional activation by MarA in escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Wall, Michael E [Los Alamos National Laboratory; Rosner, Judah L [NATIONAL INSTITUTE OF HEALTH; Martin, Robert G [NATIONAL INSTITUTE OF HEALTH

    2009-01-01

    The AraC family transcription factor MarA activates approximately 40 genes (the marA/soxS/rob regulon) of the Escherichia coli chromosome resulting in different levels of resistance to a wide array of antibiotics and to superoxides. Activation of marA/soxS/rob regulon promoters occurs in a well-defined order with respect to the level of MarA; however, the order of activation does not parallel the strength of MarA binding to promoter sequences. To understand this lack of correspondence, we developed a computational model of transcriptional activation in which a transcription factor either increases or decreases RNA polymerase binding, and either accelerates or retards post-binding events associated with transcription initiation. We used the model to analyze data characterizing MarA regulation of promoter activity. The model clearly explains the lack of correspondence between the order of activation and the MarA-DNA affinity and indicates that the order of activation can only be predicted using information about the strength of the full MarA-polymerase-DNA interaction. The analysis further suggests that MarA can activate without increasing polymerase binding and that activation can even involve a decrease in polymerase binding, which is opposite to the textbook model of activation by recruitment. These findings are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. We find that activation involving decreased polymerase binding yields lower latency in gene regulation and therefore might confer a competitive advantage to cells. Our model yields insights into requirements for predicting the order of activation of a regulon and enables us to suggest that activation might involve a decrease in polymerase binding which we expect to be an important theme of gene regulation in E. coli and beyond.

  5. A promoter polymorphism of the alpha2-HS glycoprotein gene is associated with its transcriptional activity.

    Science.gov (United States)

    Inoue, Mari; Takata, Hiroshi; Ikeda, Yukio; Suehiro, Tadashi; Inada, Shojiro; Osaki, Fumiaki; Arii, Kaoru; Kumon, Yoshitaka; Hashimoto, Kozo

    2008-01-01

    alpha2-Heremans Schmid glycoprotein (AHSG), also designated fetuin-A, is an abundant plasma protein that is expressed in hepatocytes. AHSG/fetuin-A has diverse biological functions including regulation of calcium homeostasis and inhibition of insulin receptor tyrosine kinase activity. The aim of this study was to detect single nucleotide polymorphisms (SNPs) of the AHSG gene that can be involved in regulation of AHSG/fetuin-A expression. By a cycle sequencing method, two common SNPs in the promoter region of AHSG gene, -799A/T (rs2248690, dbSNP ID) and -425G/T (rs2077119), were identified. A reporter gene assay using HepG2 cells showed that the -799A allele had significantly higher promoter activity compared with the -799T allele. The overexpression of c-Fos/c-Jun significantly repressed transcriptional activity and a gel shift assay showed that the -799T DNA fragment had a greater affinity for transcription factor AP-1 than the -799A. In 40 unrelated healthy subjects, serum AHSG/fetuin-A levels increased with the following order of genotypes: -799TTAHSG gene affects the AHSG gene transcription, possibly by producing different association with AP-1.

  6. Exploring cellular memory molecules marking competent and active transcriptions

    Directory of Open Access Journals (Sweden)

    Liu De-Pei

    2007-05-01

    Full Text Available Abstract Background Development in higher eukaryotes involves programmed gene expression. Cell type-specific gene expression is established during this process and is inherited in succeeding cell cycles. Higher eukaryotes have evolved elegant mechanisms by which committed gene-expression states are transmitted through numerous cell divisions. Previous studies have shown that both DNase I-sensitive sites and the basal transcription factor TFIID remain on silenced mitotic chromosomes, suggesting that certain trans-factors might act as bookmarks, maintaining the information and transmitting it to the next generation. Results We used the mouse globin gene clusters as a model system to examine the retention of active information on M-phase chromosomes and its contribution to the persistence of transcriptional competence of these gene clusters in murine erythroleukemia cells. In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome. Moreover, despite mitotic chromatin condensation, the distant regulatory regions and promoters of transcriptionally competent globin gene loci are marked by a preserved histone code consisting in active histone modifications such as H3 acetylation, H3-K4 dimethylation and K79 dimethylation. Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription. Conclusion Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.

  7. Aerobic glycolysis tunes YAP/TAZ transcriptional activity.

    Science.gov (United States)

    Enzo, Elena; Santinon, Giulia; Pocaterra, Arianna; Aragona, Mariaceleste; Bresolin, Silvia; Forcato, Mattia; Grifoni, Daniela; Pession, Annalisa; Zanconato, Francesca; Guzzo, Giulia; Bicciato, Silvio; Dupont, Sirio

    2015-05-12

    Increased glucose metabolism and reprogramming toward aerobic glycolysis are a hallmark of cancer cells, meeting their metabolic needs for sustained cell proliferation. Metabolic reprogramming is usually considered as a downstream consequence of tumor development and oncogene activation; growing evidence indicates, however, that metabolism on its turn can support oncogenic signaling to foster tumor malignancy. Here, we explored how glucose metabolism regulates gene transcription and found an unexpected link with YAP/TAZ, key transcription factors regulating organ growth, tumor cell proliferation and aggressiveness. When cells actively incorporate glucose and route it through glycolysis, YAP/TAZ are fully active; when glucose metabolism is blocked, or glycolysis is reduced, YAP/TAZ transcriptional activity is decreased. Accordingly, glycolysis is required to sustain YAP/TAZ pro-tumorigenic functions, and YAP/TAZ are required for the full deployment of glucose growth-promoting activity. Mechanistically we found that phosphofructokinase (PFK1), the enzyme regulating the first committed step of glycolysis, binds the YAP/TAZ transcriptional cofactors TEADs and promotes their functional and biochemical cooperation with YAP/TAZ. Strikingly, this regulation is conserved in Drosophila, where phosphofructokinase is required for tissue overgrowth promoted by Yki, the fly homologue of YAP. Moreover, gene expression regulated by glucose metabolism in breast cancer cells is strongly associated in a large dataset of primary human mammary tumors with YAP/TAZ activation and with the progression toward more advanced and malignant stages. These findings suggest that aerobic glycolysis endows cancer cells with particular metabolic properties and at the same time sustains transcription factors with potent pro-tumorigenic activities such as YAP/TAZ. © 2015 The Authors.

  8. The transcriptionally active regions in the genome of Bacillus subtilis

    DEFF Research Database (Denmark)

    Rasmussen, Simon; Nielsen, Henrik Bjørn; Jarmer, Hanne Østergaard

    2009-01-01

    The majority of all genes have so far been identified and annotated systematically through in silico gene finding. Here we report the finding of 3662 strand-specific transcriptionally active regions (TARs) in the genome of Bacillus subtilis by the use of tiling arrays. We have measured the genome...

  9. Benzimidazoles diminish ERE transcriptional activity and cell growth in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Payton-Stewart, Florastina [Department of Chemistry, College of Arts and Sciences, Xavier University of Louisiana, New Orleans, LA (United States); Tilghman, Syreeta L. [Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, New Orleans, LA (United States); Williams, LaKeisha G. [Division of Clinical and Administrative Sciences, College of Pharmacy Xavier University of Louisiana, New Orleans, LA (United States); Winfield, Leyte L., E-mail: lwinfield@spelman.edu [Department of Chemistry, Spelman College, Atlanta, GA (United States)

    2014-08-08

    Highlights: • The methyl-substituted benzimidazole was more effective at inhibiting growth in MDA-MB 231 cells. • The naphthyl-substituted benzimidazole was more effective at inhibiting growth in MCF-7 cells than ICI. • The benzimidazole molecules demonstrated a dose-dependent reduction in ERE transcriptional activity. • The benzimidazole molecules had binding mode in ERα and ERβ comparable to that of the co-crystallized ligand. - Abstract: Estrogen receptors (ERα and ERβ) are members of the nuclear receptor superfamily. They regulate the transcription of estrogen-responsive genes and mediate numerous estrogen related diseases (i.e., fertility, osteoporosis, cancer, etc.). As such, ERs are potentially useful targets for developing therapies and diagnostic tools for hormonally responsive human breast cancers. In this work, two benzimidazole-based sulfonamides originally designed to reduce proliferation in prostate cancer, have been evaluated for their ability to modulate growth in estrogen dependent and independent cell lines (MCF-7 and MDA-MB 231) using cell viability assays. The molecules reduced growth in MCF-7 cells, but differed in their impact on the growth of MDA-MB 231 cells. Although both molecules reduced estrogen response element (ERE) transcriptional activity in a dose dependent manner, the contrasting activity in the MDA-MB-231 cells seems to suggest that the molecules may act through alternate ER-mediated pathways. Further, the methyl analog showed modest selectivity for the ERβ receptor in an ER gene expression array panel, while the naphthyl analog did not significantly alter gene expression. The molecules were docked in the ligand binding domains of the ERα-antagonist and ERβ-agonist crystal structures to evaluate the potential of the molecules to interact with the receptors. The computational analysis complimented the results obtained in the assay of transcriptional activity and gene expression suggesting that the molecules

  10. Mining Chemical Activity Status from High-Throughput Screening Assays.

    Science.gov (United States)

    Soufan, Othman; Ba-alawi, Wail; Afeef, Moataz; Essack, Magbubah; Rodionov, Valentin; Kalnis, Panos; Bajic, Vladimir B

    2015-01-01

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare.

  11. Mining Chemical Activity Status from High-Throughput Screening Assays

    KAUST Repository

    Soufan, Othman

    2015-12-14

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare.

  12. [Transcription activator-like effectors(TALEs)based genome engineering].

    Science.gov (United States)

    Zhao, Mei-Wei; Duan, Cheng-Li; Liu, Jiang

    2013-10-01

    Systematic reverse-engineering of functional genome architecture requires precise modifications of gene sequences and transcription levels. The development and application of transcription activator-like effectors(TALEs) has created a wealth of genome engineering possibilities. TALEs are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas species. The DNA-binding domain of each TALE typically consists of tandem 34-amino acid repeat modules rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Such "genome engineering" has now been established in human cells and a number of model organisms, thus opening the door to better understanding gene function in model organisms, improving traits in crop plants and treating human genetic disorders.

  13. Establishment of real-time quantitative reverse transcription polymerase chain reaction assay for transcriptional analysis of duck enteritis virus UL55 gene

    Directory of Open Access Journals (Sweden)

    Zhang Shunchuan

    2011-06-01

    Full Text Available Abstract Background Real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR has become the benchmark for detection and quantification of target gene expression level and been utilized increasingly in detection of viral load and therapy monitoring. The dynamic transcription variation of duck enteritis virus UL55 gene during the life cycle of duck enteritis virus in infected cells has not been reported yet. Results The newly identified duck enteritis virus UL55 gene was amplified and cloned into pMD18-T vector after digestion to generate a recombinant plasmid pMD18-T/UL55 for the establishment of qRT-PCR as standard DNA. The results of agarose gel electrophoresis and melting curve analysis demonstrated the primers we designed for qRT-PCR were specific and available. We used β-actin as a reference gene for normalization and established two standard curves based on pMD18-T/UL55 and pMD18-T/β-actin successfully. Based on that, the transcriptional analysis of DEV UL55 gene was performed, and the result suggested the expression of UL55 mRNA was at a low level from 0 to 8 h post-infection(p.i., then accumulated quickly since 12 h p.i. and peaked at 36 h p.i., it can be detected till 60 h p.i.. Nucleic acid inhibition test was carried out for analyzing a temporal regulation condition of DEV UL55 gene, result revealed that it was sensitive to ganciclovir. Synthesis procedures of DEV UL55 gene can be inhibited by ganciclovir. Conclusions The method we established in this paper can provide quantitative values reflecting the amounts of measured mRNA in samples. It's available for detection and quantification, also can be used in DEV diagnosis. The DEV UL55 gene was produced most abundantly during the late phase of replication in DEV-infected cells and the transcription of it depended on the synthesized DNA. DEV UL55 gene is a γ2 gene which occurs last and have a strict requirement for viral DNA synthesis.

  14. A universal assay for detection of oncogenic fusion transcripts by oligo microarray analysis.

    Science.gov (United States)

    Skotheim, Rolf I; Thomassen, Gard O S; Eken, Marthe; Lind, Guro E; Micci, Francesca; Ribeiro, Franclim R; Cerveira, Nuno; Teixeira, Manuel R; Heim, Sverre; Rognes, Torbjørn; Lothe, Ragnhild A

    2009-01-19

    The ability to detect neoplasia-specific fusion genes is important not only in cancer research, but also increasingly in clinical settings to ensure that correct diagnosis is made and the optimal treatment is chosen. However, the available methodologies to detect such fusions all have their distinct short-comings. We describe a novel oligonucleotide microarray strategy whereby one can screen for all known oncogenic fusion transcripts in a single experiment. To accomplish this, we combine measurements of chimeric transcript junctions with exon-wise measurements of individual fusion partners. To demonstrate the usefulness of the approach, we designed a DNA microarray containing 68,861 oligonucleotide probes that includes oligos covering all combinations of chimeric exon-exon junctions from 275 pairs of fusion genes, as well as sets of oligos internal to all the exons of the fusion partners. Using this array, proof of principle was demonstrated by detection of known fusion genes (such as TCF3:PBX1, ETV6:RUNX1, and TMPRSS2:ERG) from all six positive controls consisting of leukemia cell lines and prostate cancer biopsies. This new method bears promise of an important complement to currently used diagnostic and research tools for the detection of fusion genes in neoplastic diseases.

  15. A universal assay for detection of oncogenic fusion transcripts by oligo microarray analysis

    Directory of Open Access Journals (Sweden)

    Ribeiro Franclim R

    2009-01-01

    Full Text Available Abstract Background The ability to detect neoplasia-specific fusion genes is important not only in cancer research, but also increasingly in clinical settings to ensure that correct diagnosis is made and the optimal treatment is chosen. However, the available methodologies to detect such fusions all have their distinct short-comings. Results We describe a novel oligonucleotide microarray strategy whereby one can screen for all known oncogenic fusion transcripts in a single experiment. To accomplish this, we combine measurements of chimeric transcript junctions with exon-wise measurements of individual fusion partners. To demonstrate the usefulness of the approach, we designed a DNA microarray containing 68,861 oligonucleotide probes that includes oligos covering all combinations of chimeric exon-exon junctions from 275 pairs of fusion genes, as well as sets of oligos internal to all the exons of the fusion partners. Using this array, proof of principle was demonstrated by detection of known fusion genes (such as TCF3:PBX1, ETV6:RUNX1, and TMPRSS2:ERG from all six positive controls consisting of leukemia cell lines and prostate cancer biopsies. Conclusion This new method bears promise of an important complement to currently used diagnostic and research tools for the detection of fusion genes in neoplastic diseases.

  16. Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR.

    Directory of Open Access Journals (Sweden)

    Belete Teferedegne

    Full Text Available A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses. The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours. In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID(50. Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses.

  17. Transcriptional Activity of the FUT1 Gene Promoter Region in Pigs

    Directory of Open Access Journals (Sweden)

    Chen Zi

    2013-12-01

    Full Text Available This study aims to provide a theoretical basis on the regulatory mechanism of the α-l,2-fucosyltransferase (FUT1 gene in pigs by analyzing the transcriptional activity of its promoter region. On the basis of the previously obtained promoter sequence, primers upstream and downstream of the gene were designed using the restriction endonucleases KpnI and HindIII respectively, and the recombinant plasmids of the pGL3-promoter were constructed by inserting promoter sequences with partially missing regions. The resultant mutants were observed by transient transfection assay into HEK293 cells, and the transcriptional activity of the promoter region was determined by luciferase activity. The 5'-flanking region of the FUT1 gene (−1150 to +50 bp exhibited promoter activity. The −1150-bp to −849-bp region showed negative regulation of the gene. The recombinant plasmid pGL3-898 showed the strongest luciferase activity, and the activity showed a decreasing trend when the deleted region was increased. Recombinant plasmids were successfully constructed, verified, and the positive and negative regulation areas and core promoter region were detected, providing a deeper insight into the transcriptional regulatory mechanism of the FUT1 gene.

  18. Real-time reverse transcription-PCR assay for detection of mumps virus RNA in clinical specimens.

    Science.gov (United States)

    Boddicker, Jennifer D; Rota, Paul A; Kreman, Trisha; Wangeman, Andrea; Lowe, Louis; Hummel, Kimberly B; Thompson, Robert; Bellini, William J; Pentella, Michael; Desjardin, Lucy E

    2007-09-01

    The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M-specific antibodies to mumps virus in acute-phase serum samples, the isolation of mumps virus in cell culture, or by detection of the RNA of the mumps virus by reverse transcription (RT)-PCR. We developed and validated a multiplex real-time RT-PCR assay for rapid mumps diagnosis in a clinical setting. This assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene, as well as primers and a probe that targeted the human RNase P gene to assess the presence of PCR inhibitors and as a measure of specimen quality. The test was specific, since it did not amplify a product from near-neighbor viruses, as well as sensitive and accurate. Real-time RT-PCR results showed 100% correlation with results from viral culture, the gold standard for mumps diagnostic testing. Assay efficiency was over 90% and displayed good precision after performing inter- and intraassay replicates. Thus, we have developed and validated a molecular method for rapidly diagnosing mumps infection that may be used to complement existing techniques.

  19. Real-Time Reverse Transcription-PCR Assay for Detection of Mumps Virus RNA in Clinical Specimens▿

    Science.gov (United States)

    Boddicker, Jennifer D.; Rota, Paul A.; Kreman, Trisha; Wangeman, Andrea; Lowe, Louis; Hummel, Kimberly B.; Thompson, Robert; Bellini, William J.; Pentella, Michael; DesJardin, Lucy E.

    2007-01-01

    The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M-specific antibodies to mumps virus in acute-phase serum samples, the isolation of mumps virus in cell culture, or by detection of the RNA of the mumps virus by reverse transcription (RT)-PCR. We developed and validated a multiplex real-time RT-PCR assay for rapid mumps diagnosis in a clinical setting. This assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene, as well as primers and a probe that targeted the human RNase P gene to assess the presence of PCR inhibitors and as a measure of specimen quality. The test was specific, since it did not amplify a product from near-neighbor viruses, as well as sensitive and accurate. Real-time RT-PCR results showed 100% correlation with results from viral culture, the gold standard for mumps diagnostic testing. Assay efficiency was over 90% and displayed good precision after performing inter- and intraassay replicates. Thus, we have developed and validated a molecular method for rapidly diagnosing mumps infection that may be used to complement existing techniques. PMID:17652480

  20. BMP-7 induces TF expression in human monocytes by increasing F3 transcriptional activity.

    Science.gov (United States)

    Sovershaev, T A; Egorina, E M; Unruh, D; Bogdanov, V Y; Hansen, J B; Sovershaev, M A

    2015-02-01

    Bone morphogenetic protein (BMP)-7, a major regulator of bone metabolism, inhibits ectopic calcification in atherosclerotic plaques. We have recently reported that BMP-7 is also a potent inducer of tissue factor (TF) in human mononuclear cells (MNCs). While nuclear factor kappa beta (NF-kB) and activation protein-1 (AP-1) are the transcription factors essential for inducible expression of human TF gene (F3), the mechanisms responsible for TF induction by BMP-7 are not known. To elucidate the molecular mechanisms governing BMP-7-triggered TF expression in human MNCs. Human blood monocytes were stimulated with BMP-7 and western blotting, qRT-PCR, and flow cytometry studies were carried out to assess F3 expression; promoter studies were also performed using a panel of reporter constructs. Procoagulant TF activity was measured using a validated FXa generation assay. The significance of NF-kB transcriptional activity was verified via pharmacological inhibition. BMP-7 increased TF protein levels, procoagulant activity, surface presentation, and TF mRNA expression. This increase was accompanied by activation of NF-kB as evidenced by reduced IkB-α levels and elevated transcriptional activity of an NF-kB-sensitive reporter in transfected MNCs. Although treatment with BMP-7 also led to a strong phosphorylation of c-Jun, activation of AP-1 alone was not sufficient to induce TF expression: JSH-23, a potent and specific NF-kB inhibitor, completely blocked BMP-7-induced TF expression. We report that BMP-7-dependent activation of TF in human MNCs is mediated via increased activity of NF-kB, leading to enhanced F3 transcription in human MNCs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Transcription-dependent association of HDAC2 with active chromatin.

    Science.gov (United States)

    Jahan, Sanzida; Sun, Jian-Min; He, Shihua; Davie, James R

    2018-02-01

    Histone deacetylase 2 (HDAC2) catalyzes deacetylation of histones at the promoter and coding regions of transcribed genes and regulates chromatin structure and transcription. To explore the role of HDAC2 and phosphorylated HDAC2 in gene regulation, we studied the location along transcribed genes, the mode of recruitment and the associated proteins with HDAC2 and HDAC2S394ph in chicken polychromatic erythrocytes. We show that HDAC2 and HDAC2S394ph are associated with transcriptionally active chromatin and located in the interchromatin channels. HDAC2S394ph was present primarly at the upstream promoter region of the transcribed CA2 and GAS41 genes, while total HDAC2 was also found within the coding region of the CA2 gene. Recruitment of HDAC2 to these genes was partially dependent upon on-going transcription. Unmodified HDAC2 was associated with RNA binding proteins and interacted with RNA bound to the initiating and elongating forms of RNA polymerase II. HDAC2S394ph was not associated with RNA polymerase II. These results highlight the differential properties of unmodified and phosphorylated HDAC2 and the organization of acetylated transcriptionally active chromatin in the chicken polychromatic erythrocyte. © 2017 Wiley Periodicals, Inc.

  2. Modeling the Ebolavirus Life Cycle with Transcription and Replication-Competent Viruslike Particle Assays.

    Science.gov (United States)

    Biedenkopf, Nadine; Hoenen, Thomas

    2017-01-01

    Ebolaviruses are the causative agent of a severe hemorrhagic fever with high case fatality rates, for which no approved specific therapy is available. As biosafety level 4 (BSL4) agents, work with live ebolaviruses is restricted to maximum containment laboratories. Transcription and replication-competent viruslike particle (trVLP) systems are reverse genetics-based life cycle modeling systems that allow researchers to model virtually the entire ebolavirus life cycle outside of a maximum containment laboratory. These systems can be used to dissect the virus life cycle, and thus increase our understanding of virus biology, as well as for more applied uses such as the screening and development of novel antivirals, and thus represent powerful tools for work on ebolaviruses.

  3. Mutagenic activity of phthalate esters in bacterial liquid suspension assays.

    OpenAIRE

    Seed, J L

    1982-01-01

    The mutagenic activities of several phthalate esters have been evaluated in an 8-azaguanine resistance assay in Salmonella typhimurium. Three phthalate esters were found to be mutagenic: dimethyl phthalate, diethyl phthalate and di-n-butyl phthalate. A number of other phthalate esters were not found to be mutagenic, including di(2-ethylhexyl) phthalate, di-n-octyl phthalate, diallyl phthalate, diisobutyl phthalate and diisodecyl phthalate. A metabolite of di(2-ethylhexyl) phthalate, 2-ethylhe...

  4. A reporter system for assaying influenza virus RNP functionality based on secreted Gaussia luciferase activity

    Directory of Open Access Journals (Sweden)

    Wu Xiaobing

    2011-01-01

    Full Text Available Abstract Background Influenza A virus can infect a wide variety of animal species including humans, pigs, birds and other species. Viral ribonucleoprotein (vRNP was involved in genome replication, transcription and host adaptation. Currently, firefly luciferase (Fluc reporter system was used in vRNP functional assay. However, its limitation for the testing by virus infection resulted in an increased need for rapid, sensitive, and biosafe techniques. Here, an influenza A virus UTR-driven gene reporter for vRNP assay based on secreted Gaussia luciferase (Gluc activity was evaluated. Results By measuring Gluc levels in supernatants, reporter gene activity could be detected and quantitated after either reconstitution of influenza A virus polymerase complex or viral infection of 293T and A549 cells, respectively. As compared with Fluc reporter, Gluc-based reporter was heat-tolerant (65°C for 30 min and produced 50-fold higher bioluminescent activity at 24 h posttransfection. Signals generated by Gluc reporter gene could be detected as early as 6 h post-infection and accumulated with time. Testing by viral infection, stronger signals were detected by Gluc reporter at a MOI of 0.001 than that of 1 and the effects of PB2-627K/E or amantadine on influenza vRNP activity were elucidated more effectively by the Gluc reporter system. Conclusions This approach provided a rapid, sensitive, and biosafe assay of influenza vRNP function, particularly for the highly pathogenic avian influenza viruses.

  5. Rapid detection of newly isolated Tembusu-related Flavivirus by reverse-transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Wang Youling

    2011-12-01

    Full Text Available Abstract Background From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay for the detection of the new virus related to Tembusu-related Flavivirus. Results The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/μL compared with 190 copies/μL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation. Conclusion The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.

  6. Weak estrogenic transcriptional activities of Bisphenol A and Bisphenol S

    OpenAIRE

    GRIGNARD ELISE; Bremer, Susanne; LAPENNA SILVIA

    2011-01-01

    In 2011, the European Commission has restricted the use of Bisphenol A in plastic infant feeding bottles. In a response to this restriction, Bisphenol S is now often used as a component of plastic substitutes for the production of babybottles. One of the major concerns leading to the restriction of Bisphenol A was its weak estrogenic activity. By using two highly standardised transactivation assays, we could demonstrate that the estrogenic activity of Bisphenol A and Bisphenol S i...

  7. Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise; Snoek, Tim; Kildegaard, Kanchana Rueksomtawin

    2016-01-01

    Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling...... real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily...... of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis...

  8. Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma

    Directory of Open Access Journals (Sweden)

    Dinesh K. Singh

    2017-01-01

    Full Text Available Efforts to identify and target glioblastoma (GBM drivers have primarily focused on receptor tyrosine kinases (RTKs. Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2 transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2 and zinc-finger E-box binding homeobox 1 (ZEB1, which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.

  9. Differential transcriptional activation by v-myb and c-myb in animal cells and Saccharomyces cerevisiae.

    OpenAIRE

    Chen, R H; Lipsick, J S

    1993-01-01

    The v-myb oncogene and its cellular homolog c-myb encode sequence-specific DNA-binding proteins which regulate transcription from promoters containing Myb-binding sites in animal cells. We have developed a Saccharomyces cerevisiae system to assay transcriptional activation by v-Myb and c-Myb. In yeast strains containing integrated reporter genes, activation was strictly dependent upon both the Myb DNA-binding domain and the Myb recognition element. BAS1, an endogenous Myb-related yeast protei...

  10. Transcription Factor YY1 Modulates Lung Cancer Progression by Activating lncRNA-PVT1.

    Science.gov (United States)

    Huang, Tonghai; Wang, Guangsuo; Yang, Lin; Peng, Bin; Wen, Yuxin; Ding, Guanggui; Wang, Zheng

    2017-11-01

    Lung cancer is the leading cause of cancer-related death worldwide. Despite the advancement in surgery and chemotherapy, the prognosis of patients with advanced lung cancer is still poor. Yin Yang-1 (YY1) is a multifunctional transcription factor that exhibits positive and negative control on a large number of cellular and viral genes. In this study, we showed that the expression of YY1 is upregulated in lung cancer tissues as compared to adjacent normal tissues. Patients with higher expression of YY1 had larger tumor size, poor differentiation, higher TNM stage, and lymph node metastasis. Ectopic expression of YY1 in lung cancer cells promoted cell proliferation and invasion. Inversely, siRNA-mediated silencing of YY1 inhibited cell proliferation and induced apoptosis. These results suggested that YY1 may function as an oncogene in lung cancer. Moreover, through luciferase reporter assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay, we showed that YY1 could directly bind to the promoter region of (long noncoding RNA-plasmacytoma variant translocation 1 [lncRNA-PVT1]) and activated its transcription through the consensus YY1 motif. Knockdown of the expression of YY1 reduced cell proliferation in vivo, consistent with the results obtained from silencing the expression of YY1 in lung cancer cells. Collectively, our study showed a critical role of YY1 in the regulation of tumorigenesis, partly through its downstream target PVT1.

  11. Uncoupling evolutionary changes in DNA sequence, transcription factor occupancy and enhancer activity.

    Science.gov (United States)

    Khoueiry, Pierre; Girardot, Charles; Ciglar, Lucia; Peng, Pei-Chen; Gustafson, E Hilary; Sinha, Saurabh; Furlong, Eileen Em

    2017-08-09

    Sequence variation within enhancers plays a major role in both evolution and disease, yet its functional impact on transcription factor (TF) occupancy and enhancer activity remains poorly understood. Here, we assayed the binding of five essential TFs over multiple stages of embryogenesis in two distant Drosophila species (with 1.4 substitutions per neutral site), identifying thousands of orthologous enhancers with conserved or diverged combinatorial occupancy. We used these binding signatures to dissect two properties of developmental enhancers: (1) potential TF cooperativity, using signatures of co-associations and co-divergence in TF occupancy. This revealed conserved combinatorial binding despite sequence divergence, suggesting protein-protein interactions sustain conserved collective occupancy. (2) Enhancer in-vivo activity, revealing orthologous enhancers with conserved activity despite divergence in TF occupancy. Taken together, we identify enhancers with diverged motifs yet conserved occupancy and others with diverged occupancy yet conserved activity, emphasising the need to functionally measure the effect of divergence on enhancer activity.

  12. Fluorescence Assay for Evaluating Microbicidal Activity of Hand Antiseptics

    Science.gov (United States)

    Lopez-Gigosos, Rosa M.; Mariscal-Lopez, Eloisa; Gutierrez-Bedmar, Mario; Fernandez, Joaquin

    2015-01-01

    We developed a fluorescent β-d-glucuronidase activity (BGA)-based assay for detecting and quantifying Escherichia coli in samples to assess the biocide efficacy of hand antiseptics. The fluorescence level is proportional to the number of viable E. coli organisms present. We compared our assay results to those of the E. coli plate count method specified by the European standard for testing hygienic hand rub disinfectant products (EN1500). The plate count method requires excessive handling and materials and is not valid if the number of organisms per plate is too low or high for counting in many of the samples. We optimized the fluorescent assay based on the cleavage of 4-methylumbelliferyl-β-d-glucuronide by adding 4-nitrophenyl-β-d-glucuronide, a nonfluorogenic BGA substrate, to induce glucuronidase activity and reduce assay time. Furthermore, our method can be automated and eliminates the need for multiple dilutions. Fluorescence was temporally monitored, and the time required to reach a specific value of fluorescence was correlated with the initial number of viable E. coli organisms on the samples. There was a positive correlation (P < 0.05) with a high correlation coefficient (R2 = 0.82) between the E. coli counts by plate count and fluorescence methods. Reported effects in fluorescent BGA were compared to the EN1500 plate count method with five hand disinfectants. We found our method more advantageous, because it was as sensitive as the EN1500 method, requires less time to complete, and is less expensive and less laborious than conventional plating techniques. PMID:26276114

  13. Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism

    Science.gov (United States)

    Nader, Nancy; Ng, Sinnie Sin Man; Wang, Yonghong; Abel, Brent S.; Chrousos, George P.; Kino, Tomoshige

    2012-01-01

    GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXRα/RXRα binding to GREs. Endogenous LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of

  14. Post-translational regulation of Oct4 transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Jonathan P Saxe

    Full Text Available Oct4 is a key component of the molecular circuitry which regulates embryonic stem cell proliferation and differentiation. It is essential for maintenance of undifferentiated, pluripotent cell populations, and accomplishes these tasks by binding DNA in multiple heterodimer and homodimer configurations. Very little is known about how formation of these complexes is regulated, or the mechanisms through which Oct4 proteins respond to complex extracellular stimuli which regulate pluripotency. Here, we provide evidence for a phosphorylation-based mechanism which regulates specific Oct4 homodimer conformations. Point mutations of a putative phosphorylation site can specifically abrogate transcriptional activity of a specific homodimer assembly, with little effect on other configurations. Moreover, we performed bioinformatic predictions to identify a subset of Oct4 target genes which may be regulated by this specific assembly, and show that altering Oct4 protein levels affects transcription of Oct4 target genes which are regulated by this assembly but not others. Finally, we identified several signaling pathways which may mediate this phosphorylation and act in combination to regulate Oct4 transcriptional activity and protein stability. These results provide a mechanism for rapid and reversible alteration of Oct4 transactivation potential in response to extracellular signals.

  15. Transcriptionally Active Heterochromatin in Rye B Chromosomes[W

    Science.gov (United States)

    Carchilan, Mariana; Delgado, Margarida; Ribeiro, Teresa; Costa-Nunes, Pedro; Caperta, Ana; Morais-Cecílio, Leonor; Jones, R. Neil; Viegas, Wanda; Houben, Andreas

    2007-01-01

    B chromosomes (Bs) are dispensable components of the genomes of numerous species. Thus far, there is a lack of evidence for any transcripts of Bs in plants, with the exception of some rDNA sequences. Here, we show that the Giemsa banding-positive heterochromatic subterminal domain of rye (Secale cereale) Bs undergoes decondensation during interphase. Contrary to the heterochromatic regions of A chromosomes, this domain is simultaneously marked by trimethylated H3K4 and by trimethylated H3K27, an unusual combination of apparently conflicting histone modifications. Notably, both types of B-specific high copy repeat families (E3900 and D1100) of the subterminal domain are transcriptionally active, although with different tissue type–dependent activity. No small RNAs were detected specifically for the presence of Bs. The lack of any significant open reading frame and the highly heterogeneous size of mainly polyadenylated transcripts indicate that the noncoding RNA may function as structural or catalytic RNA. PMID:17586652

  16. An Ikaros Promoter Element with Dual Epigenetic and Transcriptional Activities.

    Directory of Open Access Journals (Sweden)

    Elizabeth A Perotti

    Full Text Available Ikaros DNA binding factor plays critical roles in lymphocyte development. Changes in Ikaros expression levels during lymphopoiesis are controlled by redundant but also unique regulatory elements of its locus that are critical for this developmental process. We have recently shown that Ikaros binds its own locus in thymocytes in vivo. Here, we evaluated the role of an Ikaros binding site within its major lympho-myeloid promoter. We identified an Ikaros/Ets binding site within a promoter sub-region that was highly conserved in mouse and human. Deletion of this binding site increased the percentage of the reporter-expressing mouse lines, indicating that its loss provided a more permissive chromatin environment. However, once transcription was established, the lack of this site decreased transcriptional activity. These findings implicate a dual role for Ikaros/Ets1 binding on Ikzf1 expression that is exerted at least through its promoter.

  17. Aurora-A interacts with AP-2α and down regulates its transcription activity.

    Directory of Open Access Journals (Sweden)

    Lihui Zou

    Full Text Available Aurora-A is a serine/threonine protein kinase and plays an important role in the control of mitotic progression. Dysregulated expression of Aurora-A impairs centrosome separation and maturation, which lead to disrupted cell cycle progression and tumorigenesis. However, the molecular mechanism by which Aurora-A causes cell malignant transformation remains to be further defined. In this report, using transcription factors array and mRNA expression profiling array, we found that overexpression of Aurora-A suppressed transcription activity of AP-2α, a tumor suppressor that is often downregulated in variety of tumors, and inhibited expression of AP-2α-regulated downstream genes. These array-based observations were further confirmed by microwell colorimetric TF assay and luciferase reporter assay. Downregulated transcription activity of AP-2α by Aurora-A was found to be associated with reduced AP-2α protein stability, which appeared to be mediated by Aurora-A enhanced ubiquitin-dependent proteasomal degradation of AP-2α protein. Interestingly, Aurora-A-mediated AP-2α degradation was likely dependent Aurora-A kinase activity since inhibition of Aurora-A kinase activity was able to rescue Aurora-A-induced degradation of AP-2α. Moreover, we defined a physical interaction between Aurora-A and AP-2α, and such interaction might bridge the suppressive effect of Aurora-A on AP-2α protein stability. These findings provide new insights into molecular mechanism by which Aurora-A acts as an oncogenic molecule in tumor occurrence and malignant development.

  18. Transcriptional Activation of Virulence Genes of Rhizobium etli.

    Science.gov (United States)

    Wang, Luyao; Lacroix, Benoît; Guo, Jianhua; Citovsky, Vitaly

    2017-03-15

    Recently, Rhizobium etli, in addition to Agrobacterium spp., has emerged as a prokaryotic species whose genome encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we show that the signal phenolic molecule acetosyringone (AS) induces R. etli vir gene expression both in an R. etli background and in an Agrobacterium tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter transfer DNA (T-DNA). Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium but not from nonhost plants. The early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a than of T-DNA transfer of pTiC58 of A. tumefaciensIMPORTANCE The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by the genome of a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into the plant cell genome. In this study, we explored the transcriptional

  19. Exosomes from uninfected cells activate transcription of latent HIV-1.

    Science.gov (United States)

    Barclay, Robert A; Schwab, Angela; DeMarino, Catherine; Akpamagbo, Yao; Lepene, Benjamin; Kassaye, Seble; Iordanskiy, Sergey; Kashanchi, Fatah

    2017-07-14

    HIV-1 infection causes AIDS, infecting millions worldwide. The virus can persist in a state of chronic infection due to its ability to become latent. We have previously shown a link between HIV-1 infection and exosome production. Specifically, we have reported that exosomes transport viral proteins and RNA from infected cells to neighboring uninfected cells. These viral products could then elicit an innate immune response, leading to activation of the Toll-like receptor and NF-κB pathways. In this study, we asked whether exosomes from uninfected cells could activate latent HIV-1 in infected cells. We observed that irrespective of combination antiretroviral therapy, both short- and long-length viral transcripts were increased in wild-type HIV-1-infected cells exposed to purified exosomes from uninfected cells. A search for a possible mechanism for this finding revealed that the exosomes increase RNA polymerase II loading onto the HIV-1 promoter in the infected cells. These viral transcripts, which include trans-activation response (TAR) RNA and a novel RNA that we termed TAR-gag, can then be packaged into exosomes and potentially be exported to neighboring uninfected cells, leading to increased cellular activation. To better decipher the exosome release pathways involved, we used siRNA to suppress expression of ESCRT (endosomal sorting complex required for transport) proteins and found that ESCRT II and IV significantly control exosome release. Collectively, these results imply that exosomes from uninfected cells activate latent HIV-1 in infected cells and that true transcriptional latency may not be possible in vivo, especially in the presence of combination antiretroviral therapy.

  20. Automated conductimetric assay of human serum cholinesterase activity.

    Science.gov (United States)

    Duffy, P; Wallach, J M

    1989-01-01

    Serum cholinesterase activity was determined by conductimetry using samples in the microliter range. Butyrylcholine iodide was demonstrated to be a convenient substrate for the conductimetric assay. Validation of the microassay was made by using either purified enzyme or control serum. In the range of 0-60 U/l, a linear relationship was demonstrated. Correlation with a reference spectrophotometric method was obtained with a slope of 1.18. An explanation of this value is proposed, as different hydrolysis rates were obtained with human sera, depending on the substrate used (butyrylthio- or butyryl-choline ester).

  1. A novel and sensitive assay for heme oxygenase activity.

    Science.gov (United States)

    Iwamori, Saki; Sato, Emiko; Saigusa, Daisuke; Yoshinari, Kouichi; Ito, Sadayoshi; Sato, Hiroshi; Takahashi, Nobuyuki

    2015-10-01

    Heme oxygenase (HO) is a renoprotective protein in the microsome that degrades heme and produces biliverdin. Biliverdin is then reduced to a potent antioxidant bilirubin by biliverdin reductase in the cytosol. Because HO activity does not necessarily correlate with HO mRNA or protein levels, a reliable assay is needed to determine HO activity. Spectrophotometric measurement is tedious and requires a relatively large amount of kidney samples. Moreover, bilirubin is unstable and spontaneously oxidized to biliverdin in vitro. We developed a novel and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify biliverdin to measure HO activity in mice. Biliverdin and its internal standard, a deuterated biliverdin-d4, have MS/MS fragments with m/z transitions of 583 to 297 and 587 to 299, respectively. We prepared lysates of mouse kidneys, and added excess hemin, NADPH, and bilirubin oxidase to convert all bilirubin produced to biliverdin. After 30-min incubation at 37 or 4°C, the samples were analyzed by LC-MS/MS. The difference in the amount of biliverdin between the two temperatures is HO activity. Treating mice with cobalt protoporphyrin, which induces the expression of HO, increased HO activity as determined by biliverdin production. Measuring the production of biliverdin using LC-MS/MS is a more sensitive and specific way to determine HO activity than the spectrophotometric method and allows the detection of subtle changes in renal or other HO activity. Copyright © 2015 the American Physiological Society.

  2. Mammalian transcription activation domains of VP16, AP2 and CTF activate transcription in a whole cell extract from Schizosaccharomyces pombe through the SRB/mediator.

    Science.gov (United States)

    Tamayo, Evelyn; Bernal, Giuliano; Maldonado, Edio

    2005-05-01

    The acidic-rich activation domain of VP16 and the proline-rich activation domains of human AP2 and human CTF are able to activate transcription in a whole cell extract from Schizosaccharomyces pombe, whereas the glutamine-rich domains of Sp1 and Oct2 are unable to activate transcription in this system. Immunodepletion experiments of the whole cell extracts using specific antibodies against pombe TAF110, pombe TAF 72, pombe TBP and Srb4 shows that the activation of transcription by VP16, AP2 and CTF is through the mediator, since depletion of Srb4 inhibits activated transcription but does not inhibit basal transcription. Immunodepletion of TBP causes inhibition of both activated and basal transcription. On the other hand, immunodepletion of TAFs does not have an effect on either activated or basal transcription. Purified RNA polymerase holoenzyme is able to rescue the transcriptional activation activity of the anti-Srb4 immunodepleted extract. Moreover, we demonstrate that the mediator is needed for basal transcription of a TATA-less promoter.

  3. Clinical application of transcriptional activators of bile salt transporters☆

    Science.gov (United States)

    Baghdasaryan, Anna; Chiba, Peter; Trauner, Michael

    2014-01-01

    Hepatobiliary bile salt (BS) transporters are critical determinants of BS homeostasis controlling intracellular concentrations of BSs and their enterohepatic circulation. Genetic or acquired dysfunction of specific transport systems causes intrahepatic and systemic retention of potentially cytotoxic BSs, which, in high concentrations, may disturb integrity of cell membranes and subcellular organelles resulting in cell death, inflammation and fibrosis. Transcriptional regulation of canalicular BS efflux through bile salt export pump (BSEP), basolateral elimination through organic solute transporters alpha and beta (OSTα/OSTβ) as well as inhibition of hepatocellular BS uptake through basolateral Na+-taurocholate cotransporting polypeptide (NTCP) represent critical steps in protection from hepatocellular BS overload and can be targeted therapeutically. In this article, we review the potential clinical implications of the major BS transporters BSEP, OSTα/OSTβ and NTCP in the pathogenesis of hereditary and acquired cholestatic syndromes, provide an overview on transcriptional control of these transporters by the key regulatory nuclear receptors and discuss the potential therapeutic role of novel transcriptional activators of BS transporters in cholestasis. PMID:24333169

  4. Activation of p53 transcriptional activity by SMRT: a histone deacetylase 3-independent function of a transcriptional corepressor.

    Science.gov (United States)

    Adikesavan, Anbu Karani; Karmakar, Sudipan; Pardo, Patricia; Wang, Liguo; Liu, Shuang; Li, Wei; Smith, Carolyn L

    2014-04-01

    The silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) is an established histone deacetylase 3 (HDAC3)-dependent transcriptional corepressor. Microarray analyses of MCF-7 cells transfected with control or SMRT small interfering RNA revealed SMRT regulation of genes involved in DNA damage responses, and the levels of the DNA damage marker γH2AX as well as poly(ADP-ribose) polymerase cleavage were elevated in SMRT-depleted cells treated with doxorubicin. A number of these genes are established p53 targets. SMRT knockdown decreased the activity of two p53-dependent reporter genes as well as the expression of p53 target genes, such as CDKN1A (which encodes p21). SMRT bound directly to p53 and was recruited to p53 binding sites within the p21 promoter. Depletion of GPS2 and TBL1, components of the SMRT corepressor complex, but not histone deacetylase 3 (HDAC3) decreased p21-luciferase activity. p53 bound to the SMRT deacetylase activation domain (DAD), which mediates HDAC3 binding and activation, and HDAC3 could attenuate p53 binding to the DAD region of SMRT. Moreover, an HDAC3 binding-deficient SMRT DAD mutant coactivated p53 transcriptional activity. Collectively, these data highlight a biological role for SMRT in mediating DNA damage responses and suggest a model where p53 binding to the DAD limits HDAC3 interaction with this coregulator, thereby facilitating SMRT coactivation of p53-dependent gene expression.

  5. Rapid and sensitive detection of norovirus genomes in oysters by a two-step isothermal amplification assay system combining nucleic acid sequence-based amplification and reverse transcription-loop-mediated isothermal amplification assays.

    Science.gov (United States)

    Fukuda, Shinji; Sasaki, Yukie; Seno, Masato

    2008-06-01

    We developed a two-step isothermal amplification assay system, which achieved the detection of norovirus (NoV) genomes in oysters with a sensitivity similar to that of reverse transcription-seminested PCR. The time taken for the amplification of NoV genomes from RNA extracts was shortened to about 3 h.

  6. Plasma drug activity assay for treatment optimization in tuberculosis patients.

    Science.gov (United States)

    Heysell, Scott K; Mtabho, Charles; Mpagama, Stellah; Mwaigwisya, Solomon; Pholwat, Suporn; Ndusilo, Norah; Gratz, Jean; Aarnoutse, Rob E; Kibiki, Gibson S; Houpt, Eric R

    2011-12-01

    Low antituberculosis (TB) drug levels are common, but their clinical significance remains unclear, and methods of measurement are resource intensive. Subjects initiating treatment for sputum smear-positive pulmonary TB were enrolled from Kibong'oto National TB Hospital, Tanzania, and levels of isoniazid, rifampin, ethambutol, and pyrazinamide were measured at the time of typical peak plasma concentration (C(2 h)). To evaluate the significance of the effect of observed drug levels on Mycobacterium tuberculosis growth, a plasma TB drug activity (TDA) assay was developed using the Bactec MGIT system. Time to detection of plasma-cocultured M. tuberculosis versus time to detection of control growth was defined as a TDA ratio. TDA assays were later performed using the subject's own M. tuberculosis isolate and C(2 h) plasma from the Tanzanian cohort and compared to drug levels and clinical outcomes. Sixteen subjects with a mean age of 37.8 years ± 10.7 were enrolled. Fourteen (88%) had C(2 h) rifampin levels and 11 (69%) had isoniazid levels below 90% of the lower limit of the expected range. Plasma spiked with various concentrations of antituberculosis medications found TDA assay results to be unaffected by ethambutol or pyrazinamide. Yet with a range of isoniazid and rifampin concentrations, TDA exhibited a statistically significant correlation with drug level and drug MIC, and a TDA of ~1.0 indicated the presence of multidrug-resistant TB. In Tanzania, low (≤ 2.0) TDA was significantly associated with both lower isoniazid and rifampin C(2 h) levels, and very low (≤ 1.5) TDA corresponded to a trend toward lack of cure. Study of TDA compared to additional clinical outcomes and as a therapeutic management tool is warranted.

  7. Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses

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    Decai Tuo

    2014-10-01

    Full Text Available Papaya ringspot virus (PRSV, Papaya leaf distortion mosaic virus (PLDMV, and Papaya mosaic virus (PapMV produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay’s specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%, 93/341 (27.3%, and 3/341 (0.9%, for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3% of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.

  8. Modulation of transcriptional mineralocorticoid receptor activity by nitrosative stress.

    Science.gov (United States)

    Ruhs, Stefanie; Strätz, Nicole; Schlör, Kathleen; Meinel, Sandra; Mildenberger, Sigrid; Rabe, Sindy; Gekle, Michael; Grossmann, Claudia

    2012-09-01

    The mineralocorticoid receptor (MR) plays an important role in salt and water homeostasis and pathological tissue modifications, such as cardiovascular and renal fibrosis. Importantly, MR activation by aldosterone per se is not sufficient for the deleterious effects but requires the additional presence of a certain pathological milieu. Phenomenologically, this milieu could be generated by enhanced nitrosative stress. However, little is known regarding the modulation of MR transcriptional activity in a pathological milieu. The glucocorticoid receptor (GR), the closest relative of the MR, binds to the same hormone-response element but elicits protective effects on the cardiovascular system. To investigate the possible modulation of MR and GR by nitrosative stress under controlled conditions we used human embryonic kidney (HEK) cells and measured MR and GR transactivation after stimulation with the nitric oxide (NO)-donor SNAP and the peroxynitrite-donor Sin-1. In the presence of corticosteroids NO led to a general reduced corticosteroid receptor activity by repression of corticosteroid receptor-DNA interaction. The NO-induced diminished transcriptional MR activity was most pronounced during stimulation with physiological aldosterone concentrations, suggesting that NO treatment prevented its pathophysiological overactivation. In contrast, single peroxynitrite administration specifically induced the MR transactivation activity whereas genomic GR activity remained unchanged. Mechanistically, peroxynitrite permitted nuclear MR translocation whereas the cytosolic GR distribution was unaffected. Consequently, peroxynitrite represents a MR-specific aldosterone mimetic. In summary, our data indicate that the genomic function of corticosteroid receptors can be modulated by nitrosative stress which may induce the shift from physiological toward pathophysiological MR effects. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.

    Science.gov (United States)

    O'Brien, Martha; Moehring, Danielle; Muñoz-Planillo, Raúl; Núñez, Gabriel; Callaway, Justin; Ting, Jenny; Scurria, Mike; Ugo, Tim; Bernad, Laurent; Cali, James; Lazar, Dan

    2017-08-01

    Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1-/- mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective

  10. Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

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    Anny Armas Cayarga

    2011-01-01

    Full Text Available Human immunodeficiency virus type-1 (HIV-1 viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (±0.5 log10 unit of the expected results in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation (=0.925 was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux for HIV-1 RNA quantitation with clinical samples (=14. HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load.

  11. Human papillomavirus type 16 E2 protein transcriptionally activates the promoter of a key cellular splicing factor, SF2/ASF.

    Science.gov (United States)

    Mole, Sarah; Milligan, Steven G; Graham, Sheila V

    2009-01-01

    Human papillomavirus (HPV) gene expression is regulated in concert with the epithelial differentiation program. In particular, expression of the virus capsid proteins L1 and L2 is tightly restricted to differentiated epithelial cells. For HPV16, the capsid proteins are encoded by 13 structurally different mRNAs that are produced by extensive alternative splicing. Previously, we demonstrated that upon epithelial differentiation, HPV16 infection upregulates hnRNP A1 and SF2/ASF, both key factors in alternative splicing regulation. Here we cloned a 1-kb region upstream of and including the transcriptional start site of the SF2ASF gene and used it in in vivo transcription assays to demonstrate that the HPV16 E2 transcription factor transactivates the SF2/ASF promoter. The transactivation domain but not the DNA binding domain of the protein is necessary for this. Active E2 association with the promoter was demonstrated using chromatin immunoprecipitation assays. Electrophoretic mobility shift assays indicated that E2 interacted with a region 482 to 684 bp upstream of the transcription initiation site in vitro. This is the first time that HPV16 E2 has been shown to regulate cellular gene expression and the first report of viral regulation of expression of an RNA processing factor. Such E2-mediated control during differentiation of infected epithelial cells may facilitate late capsid protein expression and completion of the virus life cycle.

  12. Quantitation of MYC gene expression in sporadic breast tumors with a real-time reverse transcription-PCR assay.

    Science.gov (United States)

    Bièche, I; Laurendeau, I; Tozlu, S; Olivi, M; Vidaud, D; Lidereau, R; Vidaud, M

    1999-06-15

    MYC gene overexpression was identified recently as a downstream step at the end of the Wnt/APC/beta-catenin pathway dysregulation observed in colorectal cancer (T-C. He et al., Science (Washington DC), 281: 1509-1512, 1998). It thus appears that an excess of c-myc protein is a primary cause of numerous cancers. In breast cancer, MYC has been studied mostly at the DNA level because of the poor quality of available antibodies against the protein product. The renewed interest in MYC calls for a sensitive and accurate method for analyzing MYC overexpression in breast tumors. We have developed a real-time quantitative reverse transcription-PCR assay based on TaqMan fluorescence methodology to quantify the MYC mRNA copy number. We validated the method on a large series of breast tumors. MYC gene overexpression was observed in 29 of 134 (22%) breast tumor RNAs, ranging from 3.2 to 19 times the level in normal breast tissues. These data imply that dysregulated MYC gene expression is potentially involved in the pathogenesis of breast cancer, especially by favoring local cell proliferation. We also found that MYC gene overexpression was rarely due to an increased MYC gene copy number in breast cancer. This new, simple, rapid, and semiautomated method will be useful for screening cancer patients for MYC overexpression and will prove a powerful tool in large, randomized, prospective, cooperative group trials and in the MYC-based therapy project.

  13. Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

    Science.gov (United States)

    Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

    2013-01-01

    WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

  14. Dynamic Mechanism for the Transcription Apparatus Orchestrating Reliable Responses to Activators

    Science.gov (United States)

    Wang, Yaolai; Liu, Feng; Wang, Wei

    2012-05-01

    The transcription apparatus (TA) is a huge molecular machine. It detects the time-varying concentrations of transcriptional activators and initiates mRNA transcripts at appropriate rates. Based on the general structural organizations of the TA, we propose how the TA dynamically orchestrates transcriptional responses. The activators rapidly cycle in and out of a clamp-like space temporarily formed between the enhancer and the Mediator, with the concentration of activators encoded as their temporal occupancy rate (RTOR) within the space. The entry of activators into this space induces allostery in the Mediator, resulting in a facilitated circumstance for transcriptional reinitiation. The reinitiation rate is much larger than the cycling rate of activators, thereby RTOR guiding the amount of transcripts. Based on this mechanism, stochastic simulations can qualitatively reproduce and interpret multiple features of gene expression, e.g., transcriptional bursting is not mere noise as traditionally believed, but rather the basis of reliable transcriptional responses.

  15. Development of reverse transcription loop-mediated isothermal amplification assay as a simple detection method of Chrysanthemum stem necrosis virus in chrysanthemum and tomato.

    Science.gov (United States)

    Suzuki, Ryoji; Fukuta, Shiro; Matsumoto, Yuho; Hasegawa, Toru; Kojima, Hiroko; Hotta, Makiko; Miyake, Noriyuki

    2016-10-01

    For a simple and rapid detection of Chrysanthemum stem necrosis virus (CSNV) from chrysanthemum and tomato, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. A primer set designed to the genome sequences of CSNV worked most efficiently at 63°C and could detect CSNV RNA within 12min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. The result of a specificity test using seven other viruses and one viroid-infectable chrysanthemum or tomato showed that the assay could amplify CSNV specifically, and a sensitivity comparison showed that the RT-LAMP assay was as sensitive as the reverse transcriptase polymerase chain reaction. The RT-LAMP assay using crude RNA, extracted simply, could detect CSNV. Overall, the RT-LAMP assay was found to be a simple, specific, convenient, and time-saving method for CSNV detection. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. NCOA4 transcriptional coactivator inhibits activation of DNA replication origins.

    Science.gov (United States)

    Bellelli, Roberto; Castellone, Maria Domenica; Guida, Teresa; Limongello, Roberto; Dathan, Nina Alayne; Merolla, Francesco; Cirafici, Anna Maria; Affuso, Andrea; Masai, Hisao; Costanzo, Vincenzo; Grieco, Domenico; Fusco, Alfredo; Santoro, Massimo; Carlomagno, Francesca

    2014-07-03

    NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Ludmila R.P. Ferreira

    2008-03-01

    Full Text Available The differentiation of proliferating epimastigote forms of Trypanosoma cruzi , the protozoan parasite that causes Chagas’ disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast - an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.A diferenciação de formas epimastigotas (proliferativas do Trypanosoma cruzi, parasita protozoário causador da doença de Chagas, em formas metacíclicas tripomastigotas (infectivas e não proliferativas, pode ser reproduzida em laborat

  18. Transcription elongation factor GreA has functional chaperone activity.

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    Kun Li

    Full Text Available BACKGROUND: Bacterial GreA is an indispensable factor in the RNA polymerase elongation complex. It plays multiple roles in transcriptional elongation, and may be implicated in resistance to various stresses. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that Escherichia coli GreA inhibits aggregation of several substrate proteins under heat shock condition. GreA can also effectively promote the refolding of denatured proteins. These facts reveal that GreA has chaperone activity. Distinct from many molecular chaperones, GreA does not form stable complexes with unfolded substrates. GreA overexpression confers the host cells with enhanced resistance to heat shock and oxidative stress. Moreover, GreA expression in the greA/greB double mutant could suppress the temperature-sensitive phenotype, and dramatically alleviate the in vivo protein aggregation. The results suggest that bacterial GreA may act as chaperone in vivo. CONCLUSIONS/SIGNIFICANCE: These results suggest that GreA, in addition to its function as a transcription factor, is involved in protection of cellular proteins against aggregation.

  19. The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription

    Energy Technology Data Exchange (ETDEWEB)

    Shlomai, Amir, E-mail: amirsh@tasmc.health.gov.il [Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100 (Israel); Institute for Gastroenterology and Liver disease, Tel-Aviv Sourasky Medical Center, 6 Weizmann street, Tel-Aviv (Israel); Shaul, Yosef [Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100 (Israel)

    2009-04-17

    Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1{alpha} coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1{alpha} coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4{alpha} and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1{alpha} coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1{alpha}, implying that FOXO1 is a target for PGC-1{alpha} coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.

  20. Development of a reverse transcription quantitative polymerase chain reaction-based assay for broad coverage detection of African and Asian Zika virus lineages.

    Science.gov (United States)

    Yang, Yang; Wong, Gary; Ye, Baoguo; Li, Shihua; Li, Shanqin; Zheng, Haixia; Wang, Qiang; Liang, Mifang; Gao, George F; Liu, Lei; Liu, Yingxia; Bi, Yuhai

    2017-06-01

    The Zika virus (ZIKV) is an arbovirus that has spread rapidly worldwide within recent times. There is accumulating evidence that associates ZIKV infections with Guillain-Barré Syndrome (GBS) and microcephaly in humans. The ZIKV is genetically diverse and can be separated into Asian and African lineages. A rapid, sensitive, and specific assay is needed for the detection of ZIKV across various pandemic regions. So far, the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains. To this end, we have developed a one-step quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based on conserved sequences in the ZIKV envelope (E) gene. The detection limit of the assay was determined to be five RNA transcript copies and 2.94 × 10-3 50% tissue culture infectious doses (TCID50) of live ZIKV per reaction. The assay was highly specific and able to detect five different ZIKV strains covering the Asian and African lineages without nonspecific amplification, when tested against other flaviviruses. The assay was also successful in testing for ZIKV in clinical samples. Our assay represents an improvement over the current methods available for the detection ZIKV and would be valuable as a diagnostic tool in various pandemic regions.

  1. An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

    Science.gov (United States)

    Tang, Grace Y.; Pribisko, Melanie A.; Henning, Ryan K.; Lim, Punnajit; Termini, John; Gray, Harry B.; Grubbs, Robert H.

    2015-01-01

    Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy. Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action. Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control. The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide. This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme

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

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    Ichiro Takada

    2013-01-01

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

  3. Interferon Gamma Release Assays in active Tuberculosis: new medical insights

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    Sandro Pierdomenico

    2011-09-01

    Full Text Available Since first presentation, Interferon γ Release Assays (IGRAs have had basic and wide application to LTBI, in accordance with international consensus and CDC recommendations, leaving their use in active TB to the field of study and research.We reviewed the results of 633 patients investigated from 2004 to 2008 targeting active TB, with the objective to highlight immunological data supporting test performances.We evaluated Quantiferon TB Gold (1st generation IGRA kit in association to Culture (MGIT 960 and Lowenstein Jensen and PCR (Probetec-ET having the positivity of culture plus clinical diagnosis as the standard true value to compare. QTB Gold was studied in 69 TB positive patients (42 pulmonary and 27 extra-pulmonary, with Sensitivity, Specificity, PPV and NPV average to 61.8%, 94.5%, 54.3% and 95.9% respectively, after indeterminate results discharging. Significant statistical differences didn’t emerge between pulmonary and extra-pulmonary infections (CI 95%.The overall indeterminate ratio arose up to 20.3% in patients with active TB vs 2.7% of global population (p<0.001. In 22% of patients with active pulmonary disease, IGRA conversed to positivity after 15 days in replicated tests, in spite of current treatment. 4 patients, with pulmonary TB and Quantiferon persistent negativities, underwent 18 months follow-up as not respondent although SIRE phenotypic susceptibilities and enough DOT compliance. Molecular DST documented hetero resistance for rpoB (MUT 1, MUT 3 plus wild lines and katG (MUT 1 plus wild in association to lack of inhA wild lines (Genotype MTBDR plus, Hain Lifescience. These reports suggest a mutational relationship between Rv3874 – 3875 cassette, encoding ESAT-6 / CFP-10, and rpoB, katG, inhA genes plausibly implying weak or absent selective clonal Th 1 activation to IGRA antigens. Our data seem to point out: 1 positive results are able to match true active TB in less than 50% of patients; 2 negative results could leave

  4. Regulation of transcription and activity of Rhizobium etli glutaminase A.

    Science.gov (United States)

    Huerta-Saquero, Alejandro; Calderón-Flores, Arturo; Díaz-Villaseñor, Andrea; Du Pont, Gisela; Durán, Socorro

    2004-08-04

    The present study determines the regulatory mechanisms that operate on Rhizobium etli glutaminase A. glsA gene expression levels were evaluated under several metabolic conditions by fusions of the glsA gene promoter and the transcriptional reporter cassette uidA2-aad. glsA expression was directly correlated to the glutaminase A activity found under the tested growth conditions, reaching its maximum level in the presence of glutamine and during exponential growth phase. Glutamine induces glsA expression. The influence of allosteric metabolites on glutaminase A activity was also determined. The purified enzyme was inhibited by 2-oxoglutarate and pyruvate, whereas oxaloacetate and glyoxylate modulate it positively. Glutaminase A is not inhibited by glutamate and is activated by ammonium. Glutaminase A participates in an ATP-consuming cycle where glutamine is continually degraded and resynthesized by glutamine synthetase (GS). GS and glutaminase A activities appear simultaneously during bacterial growth under different metabolic conditions and their control mechanisms are not reciprocal. Slight overproduction in glutaminase A expression causes a reduction in growth yield and a dramatic decrease in bacterial growth. We propose a model for regulation of glutaminase A, and discuss its contribution to glutamine cycle regulation.

  5. Comprehensive Behavioral Analysis of Activating Transcription Factor 5-Deficient Mice

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    Mariko Umemura

    2017-07-01

    Full Text Available Activating transcription factor 5 (ATF5 is a member of the CREB/ATF family of basic leucine zipper transcription factors. We previously reported that ATF5-deficient (ATF5-/- mice demonstrated abnormal olfactory bulb development due to impaired interneuron supply. Furthermore, ATF5-/- mice were less aggressive than ATF5+/+ mice. Although ATF5 is widely expressed in the brain, and involved in the regulation of proliferation and development of neurons, the physiological role of ATF5 in the higher brain remains unknown. Our objective was to investigate the physiological role of ATF5 in the higher brain. We performed a comprehensive behavioral analysis using ATF5-/- mice and wild type littermates. ATF5-/- mice exhibited abnormal locomotor activity in the open field test. They also exhibited abnormal anxiety-like behavior in the light/dark transition test and open field test. Furthermore, ATF5-/- mice displayed reduced social interaction in the Crawley’s social interaction test and increased pain sensitivity in the hot plate test compared with wild type. Finally, behavioral flexibility was reduced in the T-maze test in ATF5-/- mice compared with wild type. In addition, we demonstrated that ATF5-/- mice display disturbances of monoamine neurotransmitter levels in several brain regions. These results indicate that ATF5 deficiency elicits abnormal behaviors and the disturbance of monoamine neurotransmitter levels in the brain. The behavioral abnormalities of ATF5-/- mice may be due to the disturbance of monoamine levels. Taken together, these findings suggest that ATF5-/- mice may be a unique animal model of some psychiatric disorders.

  6. Butyrate transcriptionally enhances peptide transporter PepT1 expression and activity.

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    Guillaume Dalmasso

    Full Text Available BACKGROUND: PepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte. RESULTS: We found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1 promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by approximately 2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles. CONCLUSIONS: Collectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation.

  7. GATA2 mediates thyrotropin-releasing hormone-induced transcriptional activation of the thyrotropin β gene.

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    Kenji Ohba

    Full Text Available Thyrotropin-releasing hormone (TRH activates not only the secretion of thyrotropin (TSH but also the transcription of TSHβ and α-glycoprotein (αGSU subunit genes. TSHβ expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3. Our prior studies suggest that the main activator of the TSHβ gene is GATA2, not Pit1 or unliganded T3 receptor (TR. In previous studies on the mechanism of TRH-induced activation of the TSHβ gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHβ promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHβ gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHβ promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHβ expression.

  8. GATA2 Mediates Thyrotropin-Releasing Hormone-Induced Transcriptional Activation of the Thyrotropin β Gene

    Science.gov (United States)

    Ohba, Kenji; Sasaki, Shigekazu; Matsushita, Akio; Iwaki, Hiroyuki; Matsunaga, Hideyuki; Suzuki, Shingo; Ishizuka, Keiko; Misawa, Hiroko; Oki, Yutaka; Nakamura, Hirotoshi

    2011-01-01

    Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHβ and α-glycoprotein (αGSU) subunit genes. TSHβ expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3). Our prior studies suggest that the main activator of the TSHβ gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHβ gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHβ promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHβ gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHβ promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHβ expression. PMID:21533184

  9. The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

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    Vidal M.S.

    2002-01-01

    Full Text Available Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22. Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

  10. A Novel Heat Shock Element (HSE) in Entamoeba histolytica that Regulates the Transcriptional Activation of the EhPgp5 Gene in the Presence of Emetine Drug.

    Science.gov (United States)

    Nieto, Alma; Pérez Ishiwara, David G; Orozco, Esther; Sánchez Monroy, Virginia; Gómez García, Consuelo

    2017-01-01

    Transcriptional regulation of the multidrug resistance EhPgp5 gene in Entamoeba histolytica is induced by emetine stress. EhPgp5 overexpression alters the chloride-dependent currents that cause trophozoite swelling, diminishing induced programmed cell death (PCD) susceptibility. In contrast, antisense inhibition of P-glycoprotein (PGP) expression produces synchronous death of trophozoites and the enhancement of the biochemical and morphological characteristics of PCD induced by G418. Transcriptional gene regulation analysis identified a 59 bp region at position -170 to -111 bp promoter as putative emetine response elements (EREs). However, insights into transcription factors controlling EhPgp5 gene transcription are missing; to fill this knowledge gap, we used deletion studies and transient CAT activity assays. Our findings suggested an activating motif (-151 to -136 bp) that corresponds to a heat shock element (HSE). Gel-shift assays, UV-crosslinking, binding protein purification, and western blotting assays revealed proteins of 94, 66, 62, and 51 kDa binding to the EhPgp5 HSE that could be heat shock-like transcription factors that regulate the transcriptional activation of the EhPgp5 gene in the presence of emetine drug.

  11. Comparison of antibacterial activities of root-end filling materials by an agar diffusion assay and Alamar blue assay

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    Tsui-Hsien Huang

    2012-12-01

    Conclusion: We concluded that both the agar diffusion test and Alamar blue assay gave comparable findings of assessing the antimicrobial activity present in root-end filling materials. No antimicrobial activity was detected for mineral trioxide aggregate, calcium silicate cement, or amalgam after coming into contact with S. mutans, S. sanguinis and E. coli. IRM showed high antimicrobial activity against both S. sanguinis and E. coli.

  12. Fast automated online xylanase activity assay using HPAEC-PAD.

    Science.gov (United States)

    Cürten, Christin; Anders, Nico; Juchem, Niels; Ihling, Nina; Volkenborn, Kristina; Knapp, Andreas; Jaeger, Karl-Erich; Büchs, Jochen; Spiess, Antje C

    2018-01-01

    In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35-1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation.

  13. Modulation of transcriptional mineralocorticoid receptor activity by casein kinase 2.

    Science.gov (United States)

    Ruhs, Stefanie; Strätz, Nicole; Quarch, Katja; Masch, Antonia; Schutkowski, Mike; Gekle, Michael; Grossmann, Claudia

    2017-11-10

    The pathogenesis of cardiovascular diseases is a multifunctional process in which the mineralocorticoid receptor (MR), a ligand-dependent transcription factor, is involved as proven by numerous clinical studies. The development of pathophysiological MR actions depends on the existence of additional factors e.g. inflammatory cytokines and seems to involve posttranslational MR modifications e.g. phosphorylation. Casein kinase 2 (CK2) is a ubiquitously expressed multifunctional serine/threonine kinase that can be activated under inflammatory conditions as the MR. Sequence analysis and inhibitor experiments revealed that CK2 acts as a positive modulator of MR activity by facilitating MR-DNA interaction with subsequent rapid MR degradation. Peptide microarrays and site-directed mutagenesis experiments identified the highly conserved S459 as a functionally relevant CK2 phosphorylation site of the MR. Moreover, MR-CK2 protein-protein interaction mediated by HSP90 was shown by co-immunoprecipitation. During inflammation, cytokine stimulation led to a CK2-dependent increased expression of proinflammatory genes. The additional MR activation by aldosterone during cytokine stimulation augmented CK2-dependent NFκB signaling which enhanced the expression of proinflammatory genes further. Overall, in an inflammatory environment the bidirectional CK2-MR interaction aggravate the existing pathophysiological cellular situation.

  14. Activity of polymerase proteins of vaccine and wild-type measles virus strains in a minigenome replication assay.

    Science.gov (United States)

    Bankamp, Bettina; Kearney, Sean P; Liu, Xin; Bellini, William J; Rota, Paul A

    2002-07-01

    The relative activities of five measles virus (MV) polymerase (L) proteins were compared in an intracellular, plasmid-based replication assay. When coexpressed with N and P proteins from an attenuated strain, L proteins from two attenuated viruses directed the production of up to eight times more reporter protein from an MV minigenome than the three wild-type L proteins. Northern blot analysis demonstrated that the differences in reporter protein production correlated with mRNA transcription levels. Increased activity of polymerases from attenuated viruses equally affected mRNA transcription and minigenome replication. The higher level of transcription may be a consequence of increased template availability or may be an independent effect of the elevated activity of the attenuated polymerases. Coexpression of wild-type L proteins with homologous N and P proteins did not affect the activity of the wild-type polymerases, indicating that the differential activity was a function of the L proteins alone. Use of a minigenome that incorporated two nucleotide changes found in the genomic leader of the three wild-type viruses did not raise the activity of the wild-type L proteins. These data demonstrate that increased polymerase activity differentiates attenuated from wild-type viruses and suggest that functions involved in RNA synthesis contribute to the attenuated phenotype of MV vaccine strains.

  15. Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever.

    Science.gov (United States)

    Reddy, Vijayalakshmi; Ravi, Vasanthapuram; Desai, Anita; Parida, Manmohan; Powers, Ann M; Johnson, Barbara W

    2012-11-01

    Chikungunya fever a re-emerging infection with expanding geographical boundaries, can mimic symptoms of other infections like dengue, malaria which makes the definitive diagnosis of the infection important. The present study compares the utility of four laboratory diagnostic methods viz. IgM capture ELISA, an in house reverse transcription PCR for the diagnosis of Chikungunya fever, TaqMan real-time PCR, and a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP). Out of the 70 serum samples tested, 29 (41%) were positive for Chikungunya IgM antibody by ELISA and 50 (71%) samples were positive by one of the three molecular assays. CHIKV specific nucleic acid was detected in 33/70 (47%) by reverse transcription PCR, 46/70 (66%) by TaqMan real-time PCR, and 43/70 (62%) by RT-LAMP assay. A majority of the samples (62/70; 89%) were positive by at least one of the four assays used in the study. The molecular assays were more sensitive for diagnosis in the early stages of illness (2-5 days post onset) when antibodies were not detectable. In the later stages of illness, the IgM ELISA is a more sensitive diagnostic test. In conclusion we recommend that the IgM ELISA be used as an initial screening test followed one of the molecular assays in samples that are collected in the early phase of illness and negative for CHIKV IgM antibodies. Such as approach would enable rapid confirmation of the diagnosis and implementation of public health measures especially during outbreaks. Copyright © 2012 Wiley Periodicals, Inc.

  16. Identification of KX2-391 as an inhibitor of HBV transcription by a recombinant HBV-based screening assay.

    Science.gov (United States)

    Harada, Keisuke; Nishitsuji, Hironori; Ujino, Saneyuki; Shimotohno, Kunitada

    2017-08-01

    Antiviral therapies for chronic hepatitis B virus (HBV) infection that are currently applicable for clinical use are limited to nucleos(t)ide analogs targeting HBV polymerase activity and pegylated interferon alpha (PEG-IFN). Towards establishing an effective therapy for HBV related diseases, it is important to develop a new anti-HBV agent that suppresses and eradicates HBV. This study used recombinant HBV encoding NanoLuc to screen anti-HBV compounds from 1827 US Food and Drug Administration approved compounds and identified several compounds that suppressed HBV infection. Among them, KX2-391, a non-ATP-competitive inhibitor of SRC kinase and tubulin polymerization, was identified as a lead candidate for an anti-HBV drug. Treatment of sodium taurocholate cotransporting polypeptide (NTCP) transduced-HepG2 (HepG2-NTCP) or primary human hepatocytes with KX2-391 suppressed HBV replication in a dose-dependent manner. The anti-HBV activity of KX2-391 appeared not to depend on SRC kinase activity because siRNA for SRC mRNA did not impair the HBV infection/replication. The anti-HBV activity of KX2-391 depended on the inhibitory effect of tubulin polymerization similar to other tubulin polymerization inhibitors, some of which were shown to inhibit HBV replication. KX2-391 inhibited HBV transcription driven by a HBV precore promoter in an HBV X protein-independent manner but did not inhibit the activity of HBV-S1, -S2, -X or cytomegalovirus promoters. Treatment with KX2-391 reduced the expression of several various factors including hepatocyte nuclear factor-4a. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes.

    Science.gov (United States)

    Alpuche Aranda, C M; Swanson, J A; Loomis, W P; Miller, S I

    1992-01-01

    Survival of Salmonella typhimurium within macrophage phagosomes requires the coordinate expression of bacterial gene products. This report examines the contribution of phagosomal pH as a signal for expression of genes positively regulated by the S. typhimurium virulence regulators PhoP and PhoQ. Several hours after bacterial phagocytosis by murine bone marrow-derived macrophages, PhoP-activated gene transcription increased 50- to 77-fold. In contrast, no difference in PhoP-activated gene expression was observed after infection of cultured epithelial cells, suggesting that the membrane sensor PhoQ recognized signals unique to macrophage phagosomes. The increase in PhoP-regulated gene expression was abolished when macrophage culture medium contained NH4Cl or chloroquine, weak bases that raise the pH of acidic compartments. Measurements of pH documented that S. typhimurium delayed and attenuated acidification of its intracellular compartment. Phagosomes containing S. typhimurium required 4-5 hr to reach pH < 5.0. In contrast, within 1 hr vacuoles containing heat-killed bacteria were measured at pH < 4.5. The eventual acidification of phagosomes to pH < 5.0 correlated with the period of maximal PhoP-dependent gene expression. These observations implicate phagosome acidification as an intracellular inducer of PhoP-regulated gene expression and suggest that Salmonella survival is dependent on its ability to attenuate phagosome acidification. Images PMID:1438196

  18. Molecular Dynamics of "Fuzzy" Transcriptional Activator-Coactivator Interactions.

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    Natalie S Scholes

    2016-05-01

    Full Text Available Transcriptional activation domains (ADs are generally thought to be intrinsically unstructured, but capable of adopting limited secondary structure upon interaction with a coactivator surface. The indeterminate nature of this interface made it hitherto difficult to study structure/function relationships of such contacts. Here we used atomistic accelerated molecular dynamics (aMD simulations to study the conformational changes of the GCN4 AD and variants thereof, either free in solution, or bound to the GAL11 coactivator surface. We show that the AD-coactivator interactions are highly dynamic while obeying distinct rules. The data provide insights into the constant and variable aspects of orientation of ADs relative to the coactivator, changes in secondary structure and energetic contributions stabilizing the various conformers at different time points. We also demonstrate that a prediction of α-helical propensity correlates directly with the experimentally measured transactivation potential of a large set of mutagenized ADs. The link between α-helical propensity and the stimulatory activity of ADs has fundamental practical and theoretical implications concerning the recruitment of ADs to coactivators.

  19. Competence of an artificial bent DNA as a transcriptional activator in mouse ES cells.

    Science.gov (United States)

    Tanase, Jun-ichi; Mitani, Tasuku; Udagawa, Koji; Nishikawa, Jun-ichi; Ohyama, Takashi

    2011-01-01

    Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. However, their potency in transgene activation in embryonic stem (ES) cells has not been examined. T20 is an artificial curved DNA of 180 bp that serves as a transcriptional activator. We investigated the effect of T20 on transcription in mouse ES cell lines or hepatocytes differentiated from them. We established 10 sets of cell lines each harboring a single copy of the reporter construct. Each set comprised a T20-harboring cell line and a T20-less control cell line. Analyses showed that in ES cells and in hepatocytes originating from these cells, T20 both activated and repressed transcription in a manner that was dependent on the locus of reporter. The present and previous studies strongly suggest that in cells that have a strict gene regulation system, transcriptional activation by T20 occurs only in a transcriptionally active locus in the genome.

  20. Involvement of the Transcriptional Coactivator ThMBF1 in the Biocontrol Activity of Trichoderma harzianum.

    Science.gov (United States)

    Rubio, M Belén; Pardal, Alonso J; Cardoza, Rosa E; Gutiérrez, Santiago; Monte, Enrique; Hermosa, Rosa

    2017-01-01

    Trichoderma harzianum is a filamentous fungus well adapted to different ecological niches. Owing to its ability to antagonize a wide range of plant pathogens, it is used as a biological control agent in agriculture. Selected strains of T. harzianum are also able to increase the tolerance of plants to biotic and abiotic stresses. However, little is known about the regulatory elements of the T. harzianum transcriptional machinery and their role in the biocontrol by this species. We had previously reported the involvement of the transcription factor THCTF1 in the T. harzianum production of the secondary metabolite 6-pentyl-pyrone, an important volatile compound related to interspecies cross-talk. Here, we performed a subtractive hybridization to explore the genes regulated by THCTF1, allowing us to identify a multiprotein bridging factor 1 (mbf1) homolog. The gene from T. harzianum T34 was isolated and characterized, and the generated Thmbf1 overexpressing transformants were used to investigate the role of this gene in the biocontrol abilities of the fungus against two plant pathogens. The transformants showed a reduced antifungal activity against Fusarium oxysporum f. sp. lycopersici race 2 (FO) and Botrytis cinerea (BC) in confrontation assays on discontinuous medium, indicating that the Thmbf1 gene could affect T. harzianum production of volatile organic compounds (VOC) with antifungal activity. Moreover, cellophane and dialysis membrane assays indicated that Thmbf1 overexpression affected the production of low molecular weight secreted compounds with antifungal activity against FO. Intriguingly, no correlation in the expression profiles, either in rich or minimal medium, was observed between Thmbf1 and the master regulator gene cross-pathway control (cpc1). Greenhouse assays allowed us to evaluate the biocontrol potential of T. harzianum strains against BC and FO on susceptible tomato plants. The wild type strain T34 significantly reduced the necrotic leaf lesions

  1. Involvement of the Transcriptional Coactivator ThMBF1 in the Biocontrol Activity of Trichoderma harzianum

    Directory of Open Access Journals (Sweden)

    M. Belén Rubio

    2017-11-01

    Full Text Available Trichoderma harzianum is a filamentous fungus well adapted to different ecological niches. Owing to its ability to antagonize a wide range of plant pathogens, it is used as a biological control agent in agriculture. Selected strains of T. harzianum are also able to increase the tolerance of plants to biotic and abiotic stresses. However, little is known about the regulatory elements of the T. harzianum transcriptional machinery and their role in the biocontrol by this species. We had previously reported the involvement of the transcription factor THCTF1 in the T. harzianum production of the secondary metabolite 6-pentyl-pyrone, an important volatile compound related to interspecies cross-talk. Here, we performed a subtractive hybridization to explore the genes regulated by THCTF1, allowing us to identify a multiprotein bridging factor 1 (mbf1 homolog. The gene from T. harzianum T34 was isolated and characterized, and the generated Thmbf1 overexpressing transformants were used to investigate the role of this gene in the biocontrol abilities of the fungus against two plant pathogens. The transformants showed a reduced antifungal activity against Fusarium oxysporum f. sp. lycopersici race 2 (FO and Botrytis cinerea (BC in confrontation assays on discontinuous medium, indicating that the Thmbf1 gene could affect T. harzianum production of volatile organic compounds (VOC with antifungal activity. Moreover, cellophane and dialysis membrane assays indicated that Thmbf1 overexpression affected the production of low molecular weight secreted compounds with antifungal activity against FO. Intriguingly, no correlation in the expression profiles, either in rich or minimal medium, was observed between Thmbf1 and the master regulator gene cross-pathway control (cpc1. Greenhouse assays allowed us to evaluate the biocontrol potential of T. harzianum strains against BC and FO on susceptible tomato plants. The wild type strain T34 significantly reduced the

  2. Simultaneously typing nine serotypes of enteroviruses associated with hand, foot, and mouth disease by a GeXP analyzer-based multiplex reverse transcription-PCR assay.

    Science.gov (United States)

    Hu, Xiumei; Zhang, Yong; Zhou, Xiaomian; Xu, Banglao; Yang, Mengjie; Wang, Miao; Zhang, Chen; Li, Jin; Bai, Ruyin; Xu, Wenbo; Ma, Xuejun

    2012-02-01

    Hand, foot, and mouth disease (HFMD) is a contagious enteroviral disease occurring primarily in young children and caused by enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and other serotypes of coxsackievirus and echovirus. In this study, a GeXP analyzer-based multiplex reverse transcription (RT)-PCR assay (GeXP assay) consisting of chimeric primer-based PCR amplification with fluorescent labeling and capillary electrophoresis separation was developed to simultaneously identify nine serotypes of enteroviruses associated with HFMD in China, including EV71, CVA16, CVA4, -5, -9, and -10, and CVB1, -3, and -5. The RNAs extracted from cell cultures of viral isolates and synthetic RNAs via in vitro transcription were used to analyze the specificity and sensitivity of the assay. The GeXP assay detected as little as 0.03 tissue culture infective dose (TCID(50)) of EV71 and CVA16, 10 copies of panenterovirus, EV71, CVA16, CVB1, and CVB5, and 100 copies of 10 (including panenterovirus) premixed RNA templates. A total of 180 stool specimens collected from HFMD patients and persons suspected of having HFMD were used to evaluate the clinical performance of this assay. In comparison with the results of conventional methods, the sensitivities of the GeXP assay for detection of panenterovirus, EV71, and CVA16 were 98.79% (163/165), 91.67% (44/48), and 91.67% (33/36), respectively, and the specificities were 80.00% (12/15), 98.48% (130/132), and 100% (144/144), respectively. The concordance of typing seven other serotypes of enteroviruses with the results of conventional methods was 92.59% (25/27). In conclusion, the GeXP assay is a rapid, cost-effective, and high-throughput method for typing nine serotypes of HFMD-associated enteroviruses.

  3. Building gene expression signatures indicative of transcription factor activation to predict AOP modulation

    Science.gov (United States)

    Building gene expression signatures indicative of transcription factor activation to predict AOP modulation Adverse outcome pathways (AOPs) are a framework for predicting quantitative relationships between molecular initiatin...

  4. A High-Throughput MALDI-TOF Mass Spectrometry-Based Assay of Chitinase Activity

    Science.gov (United States)

    A high-throughput MALDI-TOF mass spectrometric assay is described for assay of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates, and is readily achievable on a microliter scale (2 µL total volume, containing 2 µg of substrate and 1 ng of protein). The speed a...

  5. Evaluation of three influenza A and B real-time reverse transcription-PCR assays and a new 2009 H1N1 assay for detection of influenza viruses.

    Science.gov (United States)

    Selvaraju, Suresh B; Selvarangan, Rangaraj

    2010-11-01

    The performance characteristics of three real-time influenza A/B virus reverse transcription-PCR (RT-PCR) assays and two real-time 2009 H1N1 RT-PCR assays were evaluated using previously characterized clinical specimens. A total of 150 respiratory specimens from children (30 influenza A/H1 virus-, 30 influenza A/H3 virus-, 30 2009 H1N1-, and 30 influenza B virus-positive specimens and 30 influenza virus-negative specimens) were tested with the CDC influenza A/B PCR (CDC), ProFlu(+) multiplex real-time RT-PCR assay (ProFlu(+)), and MGB Alert Influenza A/B & RSV RUO (MGB) assays. A second set of 157 respiratory specimens (100 2009 H1N1-, 22 seasonal influenza A/H1-, and 15 seasonal influenza A/H3-positive specimens and 20 influenza-negative specimens) were tested with a new laboratory-developed 2009 H1N1 RT-PCR and the CDC 2009 H1N1 assay. The overall sensitivities of the CDC, ProFlu(+), and MGB assays for detection of influenza A and B viruses were 100%, 98.3%, and 94%, respectively. The ProFlu(+) assay failed to detect one influenza A/H1 virus-positive specimen and yielded one unresolved result with another influenza A/H1 virus-positive specimen. The MGB assay detected 84/87 (96.5%) of influenza A and B viruses and 26/30 (86.6%) of 2009 H1N1 viruses. The new laboratory-developed 2009 H1N1 RT-PCR assay detected 100/100 (100%) 2009 H1N1 virus-positive specimens, while the CDC SW Inf A and SW H1 PCR assays failed to detect one and three low-positive 2009 H1N1-positive specimens, respectively. The CDC influenza A/B virus assay and the newly developed 2009 H1N1 RT-PCR assay with an internal control can be set up in two separate reactions in the same assay for routine clinical testing to detect influenza A and B viruses and to specifically identify the 2009 H1N1 influenza virus.

  6. Evaluation of Three Influenza A and B Real-Time Reverse Transcription-PCR Assays and a New 2009 H1N1 Assay for Detection of Influenza Viruses ▿

    Science.gov (United States)

    Selvaraju, Suresh B.; Selvarangan, Rangaraj

    2010-01-01

    The performance characteristics of three real-time influenza A/B virus reverse transcription-PCR (RT-PCR) assays and two real-time 2009 H1N1 RT-PCR assays were evaluated using previously characterized clinical specimens. A total of 150 respiratory specimens from children (30 influenza A/H1 virus-, 30 influenza A/H3 virus-, 30 2009 H1N1-, and 30 influenza B virus-positive specimens and 30 influenza virus-negative specimens) were tested with the CDC influenza A/B PCR (CDC), ProFlu+ multiplex real-time RT-PCR assay (ProFlu+), and MGB Alert Influenza A/B & RSV RUO (MGB) assays. A second set of 157 respiratory specimens (100 2009 H1N1-, 22 seasonal influenza A/H1-, and 15 seasonal influenza A/H3-positive specimens and 20 influenza-negative specimens) were tested with a new laboratory-developed 2009 H1N1 RT-PCR and the CDC 2009 H1N1 assay. The overall sensitivities of the CDC, ProFlu+, and MGB assays for detection of influenza A and B viruses were 100%, 98.3%, and 94%, respectively. The ProFlu+ assay failed to detect one influenza A/H1 virus-positive specimen and yielded one unresolved result with another influenza A/H1 virus-positive specimen. The MGB assay detected 84/87 (96.5%) of influenza A and B viruses and 26/30 (86.6%) of 2009 H1N1 viruses. The new laboratory-developed 2009 H1N1 RT-PCR assay detected 100/100 (100%) 2009 H1N1 virus-positive specimens, while the CDC SW Inf A and SW H1 PCR assays failed to detect one and three low-positive 2009 H1N1-positive specimens, respectively. The CDC influenza A/B virus assay and the newly developed 2009 H1N1 RT-PCR assay with an internal control can be set up in two separate reactions in the same assay for routine clinical testing to detect influenza A and B viruses and to specifically identify the 2009 H1N1 influenza virus. PMID:20844230

  7. Cocoa flavanol metabolites activate HNF-3β, Sp1, and NFY-mediated transcription of apolipoprotein AI in human cells.

    Science.gov (United States)

    Oleaga, Carlota; Ciudad, Carlos J; Izquierdo-Pulido, Maria; Noé, Véronique

    2013-06-01

    To identify the mechanisms by which cocoa induces HDL levels and since apolipoprotein AI (ApoAI) is the major protein in HDLs, we analyzed, upon incubation with cocoa metabolites, ApoAI mRNA levels, its transcriptional regulation, and the levels of the transcription factors involved in this process. Epicatechin and cocoa metabolites caused an increase in ApoAI expression in HepG2 cells. Electrophoretic mobility shift assays revealed the involvement of Sites A and B of the ApoAI promoter in the induction of ApoAI mRNA upon incubation with cocoa metabolites. Using supershift assays, we demonstrated the binding of HNF-3β, HNF-4, ER-α, and RXR-α to Site A and the binding of HNF-3β, NFY, and Sp1 to Site B. Luciferase assays performed with a construct containing Site B confirmed its role in the upregulation of ApoAI by cocoa metabolites. Incubation with 3-methyl-epicatechin led to an increase in HNF-3β mRNA, HNF-3β, ER-α, Sp1, and NFY protein levels and the activation of ApoAI transcription mediated by NFY, Sp1, and ER-α. The activation of ApoAI transcription through Site B by cocoa flavanol metabolites is mainly mediated by an increase in HNF-3β, with a significant contribution of Sp1 and NFY, as a mechanism for the protective role of these compounds in cardiovascular diseases. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Transcription factor PIF4 controls the thermosensory activation of flowering

    KAUST Repository

    Kumar, S. Vinod

    2012-03-21

    Plant growth and development are strongly affected by small differences in temperature. Current climate change has already altered global plant phenology and distribution, and projected increases in temperature pose a significant challenge to agriculture. Despite the important role of temperature on plant development, the underlying pathways are unknown. It has previously been shown that thermal acceleration of flowering is dependent on the florigen, FLOWERING LOCUS T (FT). How this occurs is, however, not understood, because the major pathway known to upregulate FT, the photoperiod pathway, is not required for thermal acceleration of flowering. Here we demonstrate a direct mechanism by which increasing temperature causes the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) to activate FT. Our findings provide a new understanding of how plants control their timing of reproduction in response to temperature. Flowering time is an important trait in crops as well as affecting the life cycles of pollinator species. A molecular understanding of how temperature affects flowering will be important for mitigating the effects of climate change. © 2012 Macmillan Publishers Limited. All rights reserved.

  9. Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis.

    Science.gov (United States)

    Sato, Takeo; Maekawa, Shugo; Konishi, Mineko; Yoshioka, Nozomi; Sasaki, Yuki; Maeda, Haruna; Ishida, Tetsuya; Kato, Yuki; Yamaguchi, Junji; Yanagisawa, Shuichi

    2017-01-29

    Nitrate modulates growth and development, functioning as a nutrient signal in plants. Although many changes in physiological processes in response to nitrate have been well characterized as nitrate responses, the molecular mechanisms underlying the nitrate response are not yet fully understood. Here, we show that NLP transcription factors, which are key regulators of the nitrate response, directly activate the nitrate-inducible expression of BT1 and BT2 encoding putative scaffold proteins with a plant-specific domain structure in Arabidopsis. Interestingly, the 35S promoter-driven expression of BT2 partially rescued growth inhibition caused by reductions in NLP activity in Arabidopsis. Furthermore, simultaneous disruption of BT1 and BT2 affected nitrate-dependent lateral root development. These results suggest that direct activation of BT1 and BT2 by NLP transcriptional activators is a key component of the molecular mechanism underlying the nitrate response in Arabidopsis. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Signal transducer and activator of transcription 5B (STAT5B) modulates adipocyte differentiation via MOF.

    Science.gov (United States)

    Gao, Peng; Zhang, Yuchao; Liu, Yuantao; Chen, Jicui; Zong, Chen; Yu, Cong; Cui, Shang; Gao, Weina; Qin, Dandan; Sun, Wenchuan; Li, Xia; Wang, Xiangdong

    2015-12-01

    The role and mechanism of signal transducer and activator of transcription 5B (STAT5B) in adipogenesis remain unclear. In this study, our data showed that Males absent on the first (MOF) protein expression was increased during 3 T3-L1 preadipocytes differentiation accompanied with STAT5B expression increasing. Over-expression STAT5B enhanced MOF promoter trans-activation in HeLa cells. Mutagenesis assay and ChIP analysis exhibited that STAT5B was able to bind MOF promoter. Knocking-down STAT5B in 3 T3-L1 preadipocytes led to decreased expression of MOF, but resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and fatty acid-binding protein 4 (Fabp4), which were important factors or enzymes for adipogenesis. We also found that knocking-down MOF in 3 T3-L1 preadipocytes resulted in increased expression of PPARγ, C/EBPα and Fabp4, which was in the same trend as STAT5B knocking-down. Over-expression MOF resulted in reduced promoter trans-activation activity of C/EBPα. These results suggest that STAT5B and MOF work as negative regulators in adipogenesis, and STAT5B modulates preadipocytes differentiation partially by regulating MOF expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Optimization of a classical aromatase activity assay and application in normal, adenomatous and malignant breast parenchyma

    NARCIS (Netherlands)

    Dikkeschei, LD; Wolthers, BG; BosZuur, [No Value; delaRiviere, GB; Nagel, GT; vanderKolk, DA; Willemse, PHB

    1996-01-01

    The tritium water release assay, originally described for the analysis of aromatase activity in placental tissue, was used to estimate aromatase activity in breast tissue samples. The lower activity in this tissue necessitates longer incubation times and thus optimization of the assay conditions. To

  12. Transcriptional profiling of the dose response: a more powerful approach for characterizing drug activities.

    Directory of Open Access Journals (Sweden)

    Rui-Ru Ji

    2009-09-01

    Full Text Available The dose response curve is the gold standard for measuring the effect of a drug treatment, but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns, but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose, calculate classical pharmacological parameters such as the EC50, and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib, nilotinib, dasatinib and PD0325901 across a six-logarithm dose range, using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets, dose-dependent effects on cellular processes were identified using automated pathway analysis, and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover, these methods are automated, robust to non-optimized assays, and could be applied to other sources of quantitative data.

  13. Diagnosis of enzootic pneumonia in Danish cattle: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle

    DEFF Research Database (Denmark)

    Larsen, Lars Erik; Tjørnehøj, Kirsten; Viuff, B.

    1999-01-01

    A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for detection of bovine respiratory syncytial virus (BRSV) in lung tissue of naturally and experimentally infected cattle. Primers were selected from the gene coding the F fusion protein, which is relatively conserved...... among BRSV isolates. The RT-PCR assay was highly specific, it yielded positive reactions only when performed on BRSV-infected cell cultures or tissues. The detection limit of the RT-PCR assay was assessed as 5 TCID50. BRSV was detected in tissues of the respiratory tract and in the tracheobroncheal...... lymph node of calves euthanized 2-8 days after experimental infection with BRSV, whereas samples of other tissues and samples from mock-infected animals were negative at all time points. Examination of lung samples from 8 different regions of the lungs revealed that although the virus was most often...

  14. Transcriptional activation of the Lats1 tumor suppressor gene in tumors of CUX1 transgenic mice

    Directory of Open Access Journals (Sweden)

    Battat Robert

    2009-08-01

    Full Text Available Abstract Background Lats1 (large tumor suppressor 1 codes for a serine/threonine kinase that plays a role in the progression through mitosis. Genetic studies demonstrated that the loss of LATS1 in mouse, and of its ortholog wts (warts in Drosophila, is associated with increased cancer incidence. There are conflicting reports, however, as to whether overexpression of Lats1 inhibits cell proliferation. CUX1 is a transcription factor that exists in different isoforms as a result of proteolytic processing or alternative transcription initiation. Expression of p110 and p75 CUX1 in transgenic mice increases the susceptibility to cancer in various organs and tissues. In tissue culture, p110 CUX1 was shown to accelerate entry into S phase and stimulate cell proliferation. Results Genome-wide location arrays in cell lines of various cell types revealed that Lats1 was a transcriptional target of CUX1. Scanning ChIP analysis confirmed that CUX1 binds to the immediate promoter of Lats1. Expression of Lats1 was reduced in cux1-/- MEFs, whereas it was increased in cells stably or transiently expressing p110 or p75 CUX1. Reporter assays confirmed that the immediate promoter of Lats1 was sufficient to confer transcriptional activation by CUX1. Lats1 was found to be overexpressed in tumors from the mammary gland, uterus and spleen that arise in p110 or p75 CUX1 transgenic mice. In tissue culture, such elevated LATS1 expression did not hinder cell cycle progression in cells overexpressing p110 CUX1. Conclusion While inactivation of Lats1/wts in mouse and Drosophila can increase cancer incidence, results from the present study demonstrate that Lats1 is a transcriptional target of CUX1 that can be overexpressed in tumors of various tissue-types. Interestingly, two other studies documented the overexpression of LATS1 in human cervical cancers and basal-like breast cancers. We conclude that, similarly to other genes involved in mitotic checkpoint, cancer can be

  15. Hypoxia-Inducible Factor 3 Is an Oxygen-Dependent Transcription Activator and Regulates a Distinct Transcriptional Response to Hypoxia

    Directory of Open Access Journals (Sweden)

    Peng Zhang

    2014-03-01

    Full Text Available Hypoxia-inducible factors (HIFs play key roles in the cellular response to hypoxia. It is widely accepted that whereas HIF-1 and HIF-2 function as transcriptional activators, HIF-3 inhibits HIF-1/2α action. Contrary to this idea, we show that zebrafish Hif-3α has strong transactivation activity. Hif-3α is degraded under normoxia. Mutation of P393, P493, and L503 inhibits this oxygen-dependent degradation. Transcriptomics and chromatin immunoprecipitation analyses identify genes that are regulated by Hif-3α, Hif-1α, or both. Under hypoxia or when overexpressed, Hif-3α binds to its target gene promoters and upregulates their expression. Dominant-negative inhibition and knockdown of Hif-3α abolish hypoxia-induced Hif-3α-promoter binding and gene expression. Hif-3α not only mediates hypoxia-induced growth and developmental retardation but also possesses hypoxia-independent activities. Importantly, transactivation activity is conserved and human HIF-3α upregulates similar genes in human cells. These findings suggest that Hif-3 is an oxygen-dependent transcription factor and activates a distinct transcriptional response to hypoxia.

  16. Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus.

    Science.gov (United States)

    Ambagala, A; Fisher, M; Goolia, M; Nfon, C; Furukawa-Stoffer, T; Ortega Polo, R; Lung, O

    2017-10-01

    Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot-and-mouth disease virus (FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field-deployable assay would support local decision-making during an FMDV outbreak. Here we report validation of a novel reverse transcription-insulated isothermal PCR (RT-iiPCR) assay that can be performed on a commercially available, compact and portable POCKIT™ analyser that automatically analyses data and displays '+' or '-' results. The FMDV RT-iiPCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro-transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross-reactivity with viruses causing similar clinical diseases in cloven-hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory-based real-time RT-PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco™ mini transportable magnetic bead-based, automated extraction system was used. This assay provides a potentially useful field-deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD-free countries or for routine diagnostics in endemic countries with less structured laboratory systems. © 2016 Her Majesty the Queen in Right of Canada.

  17. Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

    Science.gov (United States)

    Maan, S; Maan, N S; Batra, K; Kumar, A; Gupta, A; Rao, Panduranga P; Hemadri, Divakar; Reddy, Yella Narasimha; Guimera, M; Belaganahalli, M N; Mertens, P P C

    2016-08-01

    Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus.

    Science.gov (United States)

    Weyer, Camilla T; Joone, Christopher; Lourens, Carina W; Monyai, Mpho S; Koekemoer, Otto; Grewar, John D; van Schalkwyk, Antoinette; Majiwa, Phelix O A; MacLachlan, N James; Guthrie, Alan J

    2015-10-01

    Blood samples collected as part of routine diagnostic investigations from South African horses with clinical signs suggestive of African horse sickness (AHS) were subjected to analysis with an AHS virus (AHSV) group specific reverse transcription quantitative polymerase chain reaction (AHSV RT-qPCR) assay and virus isolation (VI) with subsequent serotyping by plaque inhibition (PI) assays using AHSV serotype-specific antisera. Blood samples that tested positive by AHSV RT-qPCR were then selected for analysis using AHSV type specific RT-qPCR (AHSV TS RT-qPCR) assays. The TS RT-qPCR assays were evaluated using both historic stocks of the South African reference strains of each of the 9 AHSV serotypes, as well as recently derived stocks of these same viruses. Of the 503 horse blood samples tested, 156 were positive by both AHSV RT-qPCR and VI assays, whereas 135 samples that were VI negative were positive by AHSV RT-qPCR assay. The virus isolates made from the various blood samples included all 9 AHSV serotypes, and there was 100% agreement between the results of conventional serotyping of individual virus isolates by PI assay and AHSV TS RT-qPCR typing results. Results of the current study confirm that the AHSV TS RT-qPCR assays for the identification of individual AHSV serotypes are applicable and practicable and therefore are potentially highly useful and appropriate for virus typing in AHS outbreak situations in endemic or sporadic incursion areas, which can be crucial in determining appropriate and timely vaccination and control strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Global transcriptional regulatory network for Escherichia coli robustly connects gene expression to transcription factor activities

    DEFF Research Database (Denmark)

    Fang, Xin; Sastry, Anand; Mih, Nathan

    2017-01-01

    Transcriptional regulatory networks (TRNs) have been studied intensely for >25 y. Yet, even for the Escherichia coli TRN-probably the best characterized TRN-several questions remain. Here, we address three questions: (i) How complete is our knowledge of the E. coli TRN; (ii) how well can we predict...... were collected from published, validated chromatin immunoprecipitation (ChIP) data and RegulonDB. For 21 different TF knockouts, up to 63% of the differentially expressed genes in the hiTRN were traced to the knocked-out TF through regulatory cascades. Second, we trained supervised machine learning...

  20. A Two-Tube Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Viral and Bacterial Pathogens of Infectious Diarrhea

    Directory of Open Access Journals (Sweden)

    Ji Wang

    2014-01-01

    Full Text Available Diarrhea caused by viral and bacterial infections is a major health problem in developing countries. The purpose of this study is to develop a two-tube multiplex PCR assay using automatic electrophoresis for simultaneous detection of 13 diarrhea-causative viruses or bacteria, with an intended application in provincial Centers for Diseases Control and Prevention, China. The assay was designed to detect rotavirus A, norovirus genogroups GI and GII, human astrovirus, enteric adenoviruses, and human bocavirus (tube 1, and Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia, and Vibrio cholera (tube 2. The analytical specificity was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial tenfold dilutions of in vitro transcribed RNA, recombinant plasmids, or bacterial culture. A total of 122 stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods. The two-tube assay achieved a sensitivity of 20–200 copies for a single virus and 102-103 CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods. In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific, and high throughput method for the simultaneous detection of enteric bacteria and virus.

  1. DAX-1 Inhibits Hepatocellular Carcinoma Proliferation by Inhibiting β-Catenin Transcriptional Activity

    Directory of Open Access Journals (Sweden)

    Hong-Lei Jiang

    2014-08-01

    Full Text Available Background/Aims: Hepatocellular carcinoma (HCC represents the most common type of liver cancer. DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1, an atypical member of the nuclear receptor family due to lack of classical DNA-binding domains, has been known for its fundamental roles in the development, especially in the sex determination and steroidogenesis. Previous studies also showed that DAX-1 played a critical role in endocrine and sex steroid-dependent neoplasms such as adrenocortical, pituitary, endometrial, and ovarian tumors. However, its biological roles in the development of HCC remain largely unexplored. Methods: Real-time PCR and Western blot were used to detect the expression of DAX-1 in HCC tissues and cell lines. Immunoprecipitation (IP assay was used to show the interaction between DAX-1 and β-Catenin. Small interfering RNA (siRNA was used to silence the expression of DAX-1. BrdU incorporation and Cell-cycle assays were used to detect the role of DAX-1 in HCC cells proliferation. Migration and invasion assays were carried out to test the metastasis ability of DAX-1 in HCC cells. Results: In the present study, we found that mRNA and protein levels of DAX-1 were down-regulated in HCC tissues and cell lines. Furthermore, overexpression of DAX-1 could inhibit while its knockdown using small interfering RNA promoted cell proliferation in several HCC cell lines. At the molecular level, we demonstrated that DAX-1 could interact with β-Catenin and attenuate its transcriptional activity. Conclusion: Therefore, our results suggest a previously unknown DAX-1/β-Catenin molecular network controlling HCC development.

  2. The JaCVAM / OECD activities on the comet assay

    Directory of Open Access Journals (Sweden)

    Hajime Kojima

    2015-04-01

    Full Text Available The in vivo alkaline single cell gel electrophoresis assay, also called alkaline comet assay is a method measuring DNA strand breaks in eukaryotic cells. This assay was adopted in the Organisation for Economic Co-operation and Development (OECD Test guideline (TG 489 on September 26, 2014. This TG is part of a series of TGs on genetic toxicology. A formal validation trial of the this assay was performed in 2006-2012, coordinated by the Japanese Center for the Validation of Alternative Methods (JaCVAM, in conjunction with the European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM and the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM . The assay was reviewed by the OECD genotoxicity experts based on the JaCVAM trial (2014 and in Rothfuss et al. (2010. This TG includes the recommended use and limitations of the comet assay, and is based on the final protocol used in the validation trial, and on additional relevant published and unpublished (laboratories proprietary data. The outline of this TG describes below: each treated group is composed of a minimum of 5 animals of one sex (or of each sex as appropriate. A positive and a vehicle control group are also used. Administration of the treatment consists of daily doses over duration of 2 days or more, ensuring the test chemical reaches the target tissue which can be the liver, the kidney or other tissues if justified. Tissues of interest are dissected and single cells/nuclei suspensions are prepared and embedded in agarose on slides. Cells/nuclei are treated with lysis buffer to remove cellular and/or nuclear membranes. The nuclear DNA in the agar is then subjected to electrophoresis at high pH. This results in structures resembling comets which by using suitable fluorescent stain, can be observed by fluorescent microscopy. Based on their size

  3. The adenovirus oncoprotein E1a stimulates binding of transcription factor ETF to transcriptionally activate the p53 gene.

    Science.gov (United States)

    Hale, T K; Braithwaite, A W

    1999-08-20

    Expression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mouse p53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of the p53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53 promoter.

  4. Rapid neurogenesis through transcriptional activation in human stem cells.

    Science.gov (United States)

    Busskamp, Volker; Lewis, Nathan E; Guye, Patrick; Ng, Alex H M; Shipman, Seth L; Byrne, Susan M; Sanjana, Neville E; Murn, Jernej; Li, Yinqing; Li, Shangzhong; Stadler, Michael; Weiss, Ron; Church, George M

    2014-11-17

    Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  5. Role of activating transcription factor-4 in 24-hour rhythm of serotonin transporter expression in the mouse midbrain.

    Science.gov (United States)

    Ushijima, Kentarou; Koyanagi, Satoru; Sato, Yuuki; Ogata, Takamitsu; Matsunaga, Naoya; Fujimura, Akio; Ohdo, Shigehiro

    2012-08-01

    Serotonin (5-HT) transporter (5-HTT) plays a key role in the control of 5-HT neuronal activity by reuptaking extracellular 5-HT from the synapse cleft. We have previously demonstrated that 5-HTT mRNA expression levels and its uptake activity in the mouse midbrain are significantly higher in the dark phase than those in the light phase. However, the molecular mechanisms of time-dependent expression of 5-HTT have not been clarified. In this study, expression of 5-HTT mRNA in the mouse midbrain showed a significant 24-h rhythm and was higher in the dark phase. Although such an oscillation was eliminated by a Clock gene mutation, CLOCK and BMAL1 did not activate 5-HTT transcription in the luciferase reporter assay. Activating transcription factor-4 (ATF4), a member of the ATF/cAMP response element (CRE)-binding protein family, is a component responsible for sustaining circadian oscillations of CRE-mediated gene expression. ATF4 significantly activated 5-HTT transcription in vitro and time dependently bound to the CRE site in the 5-HTT promoter in the mouse midbrain. In addition, mutation of the Clock gene disrupted temporal binding of ATF4 to the CRE site in the 5-HTT promoter. These results indicated that the circuit of circadian-basis molecular regulation between the clockwork system and mouse 5-HTT gene was connected by the ATF4 signaling pathway.

  6. Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay.

    Science.gov (United States)

    Wen, X J; Cheng, A C; Wang, M S; Jia, R Y; Zhu, D K; Chen, S; Liu, M F; Liu, F; Chen, X Y

    2014-09-01

    Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV. © 2014 Poultry Science Association Inc.

  7. Development of a fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification assay for rapid detection of seasonal Japanese B encephalitis outbreaks in pigs.

    Science.gov (United States)

    Tian, C J; Lin, Z X; He, X M; Luo, Q; Luo, C B; Yu, H Q; Chen, R; Wu, X W; Zhu, D Z; Ren, Z J; Bi, Y Z; Ji, J

    2012-08-01

    The standardization and validation of a one-step, single-tube, accelerated fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the NS3 gene of Japanese B encephalitis virus (JEV) is described for rapid, simple, and high-throughput detection of JEV. The amplification can be completed in 35 min under isothermal conditions at 63°C by employing a set of six primers targeting the NS3 gene of JEV. The RT-LAMP assay described demonstrated high sensitivity for detecting JEV, with a detection limit in swine samples of 8.13 PFU/ml. The specificity of the selected primer sets was established by cross-reactivity studies with pathogens that exhibit similar clinical signs and testing of samples from healthy animals. The clinical applicability of the RT-LAMP assay was validated using either spiked samples or samples from seasonal outbreaks. The comparative evaluation of the RT-LAMP assay revealed 79.59 % concordance with conventional RT-PCR targeting the E gene of JEV. The RT-LAMP assay reported here is a valuable tool for rapid real-time and high-throughput seasonal infection surveillance and quarantine after outbreak through blood sampling by using ordinary real-time PCR thermocyclers without purchasing an expensive Loopamp real-time turbidimeter.

  8. Longitudinal noninvasive monitoring of transcription factor activation in cardiovascular regulatory nuclei using bioluminescence imaging.

    Science.gov (United States)

    Peterson, Jeffrey R; Infanger, David W; Braga, Valdir A; Zhang, Yulong; Sharma, Ram V; Engelhardt, John F; Davisson, Robin L

    2008-04-22

    The ability to monitor transcription factor (TF) activation in the central nervous system (CNS) has the potential to provide novel information regarding the molecular mechanisms underlying a wide range of neurobiological processes. However, traditional biochemical assays limit the mapping of TF activity to select time points. In vivo bioluminescence imaging (BLI) has emerged as an attractive technology for visualizing internal molecular events in the same animal over time. Here, we evaluated the utility of BLI, in combination with virally mediated delivery of reporter constructs to cardiovascular nuclei, for monitoring of TF activity in these discrete brain regions. Following viral gene transfer of NF-kappaB-driven luciferase reporter to the subfornical organ (SFO), BLI enabled daily measurements of baseline TF activity in the same animal for 1 mo. Importantly, systemic endotoxin, a stimulator of NF-kappaB activity, induced dramatic and dose-dependent increases in NF-kappaB-dependent bioluminescence in the SFO up to 30 days after gene transfer. Cotreatment with a dominant-negative IkappaBalpha mutant significantly prevented endotoxin-dependent NF-kappaB activation, confirming the specificity of the bioluminescence signal. NF-kappaB-dependent luminescence signals were also stable and inducible 1 mo following delivery of luciferase reporter construct to the paraventricular nucleus or rostral ventrolateral medulla. Lastly, using targeted adenoviral delivery of an AP-1 responsive luciferase reporter, we showed similar baseline and endotoxin-induced AP-1 activity in these same brain regions as with NF-kappaB reporters. These results demonstrate that BLI, in combination with virally mediated gene transfer, is a powerful method for longitudinal monitoring and quantification of TF activity in targeted CNS nuclei in vivo.

  9. SUMOylation of the KRAB zinc-finger transcription factor PARIS/ZNF746 regulates its transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Nishida, Tamotsu, E-mail: nishida@gene.mie-u.ac.jp; Yamada, Yoshiji

    2016-05-13

    Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms. -- Highlights: •PARIS can be SUMOylated in vivo and in vitro. •SUMOylation of PARIS functions in the repression of PGC-1a promoter activity. •PIASy interacts with PARIS and enhances its SUMOylation. •PIASy influences PARIS-mediated repression of PGC-1a promoter activity.

  10. Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis.

    Science.gov (United States)

    Li, S; Zhang, P; Zhang, M; Fu, C; Yu, L

    2013-01-01

    Although the regulation of taxol biosynthesis at the transcriptional level remains unclear, 10-deacetylbaccatin III-10 β-O-acetyl transferase (DBAT) is a critical enzyme in the biosynthesis of taxol. The 1740 bp fragment 5'-flanking sequence of the dbat gene was cloned from Taxus chinensis cells. Important regulatory elements needed for activity of the dbat promoter were located by deletion analyses in T. chinensis cells. A novel WRKY transcription factor, TcWRKY1, was isolated with the yeast one-hybrid system from a T. chinensis cell cDNA library using the important regulatory elements as bait. The gene expression of TcWRKY1 in T. chinensis suspension cells was specifically induced by methyl jasmonate (MeJA). Biochemical analysis indicated that TcWRKY1 protein specifically interacts with the two W-box (TGAC) cis-elements among the important regulatory elements. Overexpression of TcWRKY1 enhanced dbat expression in T. chinensis suspension cells, and RNA interference (RNAi) reduced the level of transcripts of dbat. These results suggest that TcWRKY1 participates in regulation of taxol biosynthesis in T. chinensis cells, and that dbat is a target gene of this transcription factor. This research also provides a potential candidate gene for engineering increased taxol accumulation in Taxus cell cultures. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

  11. STAT3 signaling drives EZH2 transcriptional activation and mediates poor prognosis in gastric cancer.

    Science.gov (United States)

    Pan, Yuan-Ming; Wang, Cheng-Gang; Zhu, Min; Xing, Rui; Cui, Jian-Tao; Li, Wen-Mei; Yu, De-Dong; Wang, Shu-Bin; Zhu, Wei; Ye, Ying-Jiang; Wu, Yun; Wang, Shan; Lu, You-Yong

    2016-12-09

    STAT3 signaling plays the pivotal role in tumorigenesis through EZH2 epigenetic modification, which enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3. Here, another possible feedback mechanism and clinical significance of EZH2 and STAT3 were investigated in gastric cancer (GC). STAT3, p-STAT3 (Tyr 705) and EZH2 expression were examined in 63 GC specimens with matched normal tissues by IHC staining. EZH2 and STAT3 were also identified in five GC cell lines using RT-PCR and western blot analyses. p-STAT3 protein was detected by western blotting. In order to investigate whether EZH2 expression was directly regulated by STAT3, EZH2 expression was further detected using siRNA for STAT3 or IL-6 stimulation, with dual luciferase reporter analyses, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The clinical significance of STAT3, p-STAT3 and EZH2 expression was evaluated by multi-factor COX regression and Kaplan-Meier analyses. Hyper-activation of STAT3, p-STAT3 and EZH2 expression were observed in GC cells and tissues. STAT3 signaling was correlated with EZH2 expression in GC (R = 0.373, P = 0.003), which was consistent with our data showing that STAT3 as the transcriptional factor enhanced EZH2 transcriptional activity by binding the relative promoter region (-214 ~ -206). STAT3 was an independent signature for poor survival (P = 0.002). Patients with STAT3(+)/EZH2(+) or p-STAT3(+)/EZH2(+) had a worse outcome than others (P EZH2 was associated with advanced TNM staging (P = 0.017). Moreover, treatment with a combination of siSTAT3 and EZH2-specific inhibitor, 3-deazaneplanocin A (DZNEP), increased the apoptotic ratio of cells. It is benefit for targeting STAT3-EZH2 interplay in GC treatment. Our results indicate that STAT3 status mediated EZH2 upregulation, associated with advanced TNM stage and poor prognosis, suggesting that combination with knockdown of STAT3 and EZH2 inhibitor

  12. Multiple MAPK cascades regulate the transcription of IME1, the master transcriptional activator of meiosis in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Smadar Kahana-Edwin

    Full Text Available The choice between alternative developmental pathways is primarily controlled at the level of transcription. Induction of meiosis in budding yeasts in response to nutrient levels provides a system to investigate the molecular basis of cellular decision-making. In Saccharomyces cerevisiae, entry into meiosis depends on multiple signals converging upon IME1, the master transcriptional activator of meiosis. Here we studied the regulation of the cis-acting regulatory element Upstream Activation Signal (UASru, which resides within the IME1 promoter. Guided by our previous data acquired using a powerful high-throughput screening system, here we provide evidence that UASru is regulated by multiple stimuli that trigger distinct signal transduction pathways as follows: (i The glucose signal inhibited UASru activity through the cyclic AMP (cAMP/protein kinase A (PKA pathway, targeting the transcription factors (TFs, Com2 and Sko1; (ii high osmolarity activated UASru through the Hog1/mitogen-activated protein kinase (MAPK pathway and its corresponding TF Sko1; (iii elevated temperature increased the activity of UASru through the cell wall integrity pathway and the TFs Swi4/Mpk1 and Swi4/Mlp1; (iv the nitrogen source repressed UASru activity through Sum1; and (v the absence of a nitrogen source was detected and transmitted to UASru by the Kss1 and Fus3 MAPK pathways through their respective downstream TFs, Ste12/Tec1 and Ste12/Ste12 as well as by their regulators Dig1/2. These signaling events were specific to UASru; they did not affect the mating and filamentation response elements that are regulated by MAPK pathways. The complex regulation of UASru through all the known vegetative MAPK pathways is unique to S. cerevisiae and is specific for IME1, likely because it is the master regulator of gametogenesis.

  13. Pokemon decreases the transcriptional activity of RARα in the absence of ligand.

    Science.gov (United States)

    Yang, Yutao; Li, Yueting; Di, Fei; Cui, Jiajun; Wang, Yue; David Xu, Zhi-Qing

    2016-12-20

    Pokemon is a transcriptional repressor that belongs to the POZ and Krüppel (POK) protein family. In this study, we investigated the potential interaction between Pokemon and retinoic acid receptor alpha (RARα) and determined the role of Pokemon in regulation of RARα transcriptional activity in the absence of ligand. We found that Pokemon could directly interact with RARα. Moreover, we demonstrated that Pokemon could decrease the transcriptional activity of RARα in the absence of ligand. Furthermore, we showed that Pokemon could repress the transcriptional activity of RARα by increasing the recruitment of nuclear receptor co-repressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) to the retinoic acid response element (RARE) element. Taken together, these data suggest that Pokemon is a novel partner of RARα that acts as a co-repressor to regulate RARα transcriptional activity in the absence of ligand.

  14. PolyADP-ribose polymerase is a coactivator for AP-2-mediated transcriptional activation.

    OpenAIRE

    Kannan, P.; Yu, Y.; Wankhade, S; Tainsky, M A

    1999-01-01

    Overexpression of transcription factor AP-2 has been implicated in the tumorigenicity of the human teratocarcinoma cell lines PA-1 that contain an activated ras oncogene. Here we show evidence that overexpression of AP-2 sequesters transcriptional coactivators which results in self-inhibition. We identified AP-2-interacting proteins and determined whether these proteins were coactivators for AP-2-mediated transcription. One such interacting protein is polyADP-ribose polymerase (PARP). PARP su...

  15. A transcription activator with restricted tissue distribution regulates cell-specific expression of alpha1(XI) collagen.

    Science.gov (United States)

    Kinoshita, A; Greenwel, P; Tanaka, S; Di Liberto, M; Yoshioka, H; Ramirez, F

    1997-12-12

    Different regulatory programs are likely to control expression of the alpha1(XI) collagen (COL11A1) gene in cartilaginous and non-cartilaginous tissues and in coordination with different collagen genes. Here, we report the identification of a cis-acting element that is required for constitutive and tissue-specific activity of the proximal COL11A1 promoter. The element binds an apparently novel activator whose expression is restricted mostly, but not exclusively, to cells of mesenchymal origin. Transient transfection experiments using wild-type and mutant constructs demonstrated the critical contribution of a 45-base pair upstream element (FP9) to promoter activity. The same functional tests and DNA binding assays narrowed down the critical portion of FP9 to a 20-base pair sequence, which consists of an imperfect palindrome with strong homology to the GATA consensus motif. Despite being able to bind GATA proteins in vitro, FP9 is actually recognized by a distinct approximately 100-kDa polypeptide (FP9C) probably belonging to the zinc-finger family of transcription factors. FP9C binding was mostly identified in nuclei from cells of mesenchymal origin, including those actively engaged in COL11A1 transcription. A positive correlation was also established between the level of FP9C binding and the degree of cell differentiation in vitro. Thus, FP9C represents an unusual example of tissue-specific and differentiation-related transcription factor with overlapping expression in hard and soft connective tissues.

  16. Large-scale transcriptome data reveals transcriptional activity of fission yeast LTR retrotransposons

    Directory of Open Access Journals (Sweden)

    Willerslev Eske

    2010-03-01

    Full Text Available Abstract Background Retrotransposons are transposable elements that proliferate within eukaryotic genomes through a process involving reverse transcription. The numbers of retrotransposons within genomes and differences between closely related species may yield insight into the evolutionary history of the elements. Less is known about the ongoing dynamics of retrotransposons, as analysis of genome sequences will only reveal insertions of retrotransposons that are fixed - or near fixation - in the population or strain from which genetic material has been extracted for sequencing. One pre-requisite for retrotransposition is transcription of the elements. Given their intrinsic sequence redundancy, transcriptome-level analyses of transposable elements are scarce. We have used recently published transcriptome data from the fission yeast Schizosaccharomyces pombe to assess the ability to detect and describe transcriptional activity from Long Terminal Repeat (LTR retrotransposons. LTR retrotransposons are normally flanked by two LTR sequences. However, the majority of LTR sequences in S. pombe exist as solitary LTRs, i.e. as single terminal repeat sequences not flanking a retrotransposon. Transcriptional activity was analysed for both full-length LTR retrotransposons and solitary LTRs. Results Two independent sets of transcriptome data reveal the presence of full-length, polyadenylated transcripts from LTR retrotransposons in S. pombe during growth phase in rich medium. The redundancy of retrotransposon sequences makes it difficult to assess which elements are transcriptionally active, but data strongly indicates that only a subset of the LTR retrotransposons contribute significantly to the detected transcription. A considerable level of reverse strand transcription is also detected. Equal levels of transcriptional activity are observed from both strands of solitary LTR sequences. Transcriptome data collected during meiosis suggests that transcription

  17. DcE2F, a functional plant E2F-like transcriptional activator from Daucus carota.

    Science.gov (United States)

    Albani, D; Mariconti, L; Ricagno, S; Pitto, L; Moroni, C; Helin, K; Cella, R

    2000-06-23

    In animal cells the progression of the cell cycle through G(1)/S transition and S phase is under the control of the pRB/E2F regulatory pathway. The E2F transcription factors are key activators of genes coding for several regulatory proteins and for enzymes involved in nucleotide and DNA synthesis. In this report we have detected the presence of E2F-like DNA binding activities in carrot nuclear extracts, and we have isolated a carrot cDNA (DcE2F) encoding a plant E2F homologue. The DcE2F gene is expressed in proliferating cells and is induced during the G(1)/S transition of the cell cycle. Supershift experiments using anti-DcE2F antiserum have confirmed that the DcE2F protein is a component of the carrot E2F-like nuclear activities. DNA binding assays have demonstrated that the DcE2F protein can recognize a canonical E2F cis-element in association with a mammalian DP protein. Furthermore, transactivation assays have revealed that DcE2F is a functional transcription factor that can transactivate, together with a DP partner, an E2F-responsive reporter gene in both plant and mammalian cells.

  18. Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus.

    Science.gov (United States)

    Warghane, Ashish; Misra, Pragati; Bhose, Sumit; Biswas, Kajal Kumar; Sharma, Ashwani Kumar; Reddy, M Krishna; Ghosh, Dilip Kumar

    2017-12-01

    Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0

  19. Detection of infectious bursal disease virus in various lymphoid tissues of experimentally infected specific pathogen free chickens by different reverse transcription polymerase chain reaction assays

    DEFF Research Database (Denmark)

    Kabell, Susanne; Handberg, Kurt; Kusk, Mette

    2005-01-01

    transcription polymerase chain reaction (RT-PCR) assays, including two recently developed strain-specific assays, were employed for detection of ribonucleic acid (RNA) from three different IBDV strains in bursa tissue samples from experimentally infected specific pathogen free chickens. The virus strains...... included vaccine strain D78, classical strain Faragher 52/70, and the very virulent Danish strain DK01 The presence of the virus infection was confirmed by histopathologic evaluation of bursa lesions. The largest number of positive samples was obtained with a strain-specific two-step multiplex (MPX) RT...... of the IBDV strains used were detected in bursa tissues, whereas only the two virulent strains were detected in bone marrow, spleen, and thymus....

  20. A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Saitou, Yasumasa; Kobayashi, Yuki; Hirano, Shinji; Mochizuki, Nobuyuki; Itou, Takuya; Ito, Fumio H; Sakai, Takeo

    2010-09-01

    At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. Copyright 2010 Elsevier B.V. All rights reserved.

  1. DNA Topoisomerases Maintain Promoters in a State Competent for Transcriptional Activation in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Pedersen, Jakob Madsen; Fredsøe, Jacob Christian; Rødgaard, Morten Terpager

    2012-01-01

    To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down......-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation...

  2. A Novel Single-Cell FISH-Flow Assay Identifies Effector Memory CD4+ T cells as a Major Niche for HIV-1 Transcription in HIV-Infected Patients

    Directory of Open Access Journals (Sweden)

    Judith Grau-Expósito

    2017-07-01

    Full Text Available Cells that actively transcribe HIV-1 have been defined as the “active viral reservoir” in HIV-infected individuals. However, important technical limitations have precluded the characterization of this specific viral reservoir during both treated and untreated HIV-1 infections. Here, we used a novel single-cell RNA fluorescence in situ hybridization-flow cytometry (FISH-flow assay that requires only 15 million unfractionated peripheral blood mononuclear cells (PBMCs to characterize the specific cell subpopulations that transcribe HIV RNA in different subsets of CD4+ T cells. In samples from treated and untreated HIV-infected patients, effector memory CD4+ T cells were the main cell population supporting HIV RNA transcription. The number of cells expressing HIV correlated with the plasma viral load, intracellular HIV RNA, and proviral DNA quantified by conventional methods and inversely correlated with the CD4+ T cell count and the CD4/CD8 ratio. We also found that after ex vivo infection of unstimulated PBMCs, HIV-infected T cells upregulated the expression of CD32. In addition, this new methodology detected increased numbers of primary cells expressing viral transcripts and proteins after ex vivo viral reactivation with latency reversal agents. This RNA FISH-flow technique allows the identification of the specific cell subpopulations that support viral transcription in HIV-1-infected individuals and has the potential to provide important information on the mechanisms of viral pathogenesis, HIV persistence, and viral reactivation.

  3. A Novel Single-Cell FISH-Flow Assay Identifies Effector Memory CD4+ T cells as a Major Niche for HIV-1 Transcription in HIV-Infected Patients

    Science.gov (United States)

    Grau-Expósito, Judith; Serra-Peinado, Carla; Miguel, Lucia; Navarro, Jordi; Curran, Adrià; Burgos, Joaquin; Ocaña, Imma; Ribera, Esteban; Torrella, Ariadna; Planas, Bibiana; Badía, Rosa; Castellví, Josep; Falcó, Vicenç; Crespo, Manuel

    2017-01-01

    ABSTRACT Cells that actively transcribe HIV-1 have been defined as the “active viral reservoir” in HIV-infected individuals. However, important technical limitations have precluded the characterization of this specific viral reservoir during both treated and untreated HIV-1 infections. Here, we used a novel single-cell RNA fluorescence in situ hybridization-flow cytometry (FISH-flow) assay that requires only 15 million unfractionated peripheral blood mononuclear cells (PBMCs) to characterize the specific cell subpopulations that transcribe HIV RNA in different subsets of CD4+ T cells. In samples from treated and untreated HIV-infected patients, effector memory CD4+ T cells were the main cell population supporting HIV RNA transcription. The number of cells expressing HIV correlated with the plasma viral load, intracellular HIV RNA, and proviral DNA quantified by conventional methods and inversely correlated with the CD4+ T cell count and the CD4/CD8 ratio. We also found that after ex vivo infection of unstimulated PBMCs, HIV-infected T cells upregulated the expression of CD32. In addition, this new methodology detected increased numbers of primary cells expressing viral transcripts and proteins after ex vivo viral reactivation with latency reversal agents. This RNA FISH-flow technique allows the identification of the specific cell subpopulations that support viral transcription in HIV-1-infected individuals and has the potential to provide important information on the mechanisms of viral pathogenesis, HIV persistence, and viral reactivation. PMID:28698276

  4. NprR, a moonlighting quorum sensor shifting from a phosphatase activity to a transcriptional activator.

    Science.gov (United States)

    Perchat, Stéphane; Talagas, Antoine; Zouhir, Samira; Poncet, Sandrine; Bouillaut, Laurent; Nessler, Sylvie; Lereclus, Didier

    2016-11-04

    Regulation of biological functions requires factors (proteins, peptides or chemicals) able to sense and translate environmental conditions or any circumstances in order to modulate the transcription of a gene, the stability of a transcript or the activity of a protein. Quorum sensing is a regulation mechanism connecting cell density to the physiological state of a single cell. In bacteria, quorum sensing coordinates virulence, cell fate and commitment to sporulation and other adaptation properties. The critical role of such regulatory systems was demonstrated in pathogenicity and adaptation of bacteria from the Bacillus cereus group (i.e. B. cereus and Bacillus thuringiensis). Furthermore, using insects as a model of infection, it was shown that sequential activation of several quorum sensing systems allowed bacteria to switch from a virulence state to a necrotrophic lifestyle, allowing their survival in the host cadaver, and ultimately to the commitment into sporulation. The chronological development of these physiological states is directed by quorum sensors forming the RNPP family. Among them, NprR combines two distinct functions connecting sporulation to necrotrophism in B. thuringiensis. In the absence of its cognate signaling peptide (NprX), NprR negatively controls sporulation by acting as a phosphatase. In the presence of NprX, it acts as a transcription factor regulating a set of genes involved in the survival of the bacteria in the insect cadaver.

  5. NprR, a moonlighting quorum sensor shifting from a phosphatase activity to a transcriptional activator

    Directory of Open Access Journals (Sweden)

    Stéphane Perchat

    2016-11-01

    Full Text Available Regulation of biological functions requires factors (proteins, peptides or chemicals able to sense and translate environmental conditions or any circumstances in order to modulate the transcription of a gene, the stability of a transcript or the activity of a protein. Quorum sensing is a regulation mechanism connecting cell density to the physiological state of a single cell. In bacteria, quorum sensing coordinates virulence, cell fate and commitment to sporulation and other adaptation properties. The critical role of such regulatory systems was demonstrated in pathogenicity and adaptation of bacteria from the Bacillus cereus group (i.e. B. cereus and Bacillus thuringiensis. Furthermore, using insects as a model of infection, it was shown that sequential activation of several quorum sensing systems allowed bacteria to switch from a virulence state to a necrotrophic lifestyle, allowing their survival in the host cadaver, and ultimately to the commitment into sporulation. The chronological development of these physiological states is directed by quorum sensors forming the RNPP family. Among them, NprR combines two distinct functions connecting sporulation to necrotrophism in B. thuringiensis. In the absence of its cognate signaling peptide (NprX, NprR negatively controls sporulation by acting as a phosphatase. In the presence of NprX, it acts as a transcription factor regulating a set of genes involved in the survival of the bacteria in the insect cadaver.

  6. Functional analysis of the transcriptional activator XlnR from Aspergillus niger

    NARCIS (Netherlands)

    Hasper, A.A.; Trindade, L.M.; Veen, van der D.; Ooyen, van A.J.J.; Graaff, de L.H.

    2004-01-01

    The transcriptional activator XlnR from Aspergillus niger is a zinc binuclear cluster transcription factor that belongs to the GAL4 superfamily. Several putative structural domains in XInR were predicted using database and protein sequence analysis. Thus far, only the functionality of the N-terminal

  7. Extracellular Enzyme Activity assay as indicator of soil microbial functional diversity and activity

    DEFF Research Database (Denmark)

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    Extracellular Enzyme Activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soil enzymes originate from a variety of organisms, notably fungi and bacteria...... and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity reflects the functional diversity and activity of the microorganisms involved in decomposition processes which are essential processes for soil functioning......, experimental conditions of extraction of enzymes from soils, buffer and pH, substrate concentration, temperature and the necessary controls were optimized and standardized. This has resulted in an optimized standard operating procedure of EEA, which are being tested as an indicator of soil functional diversity...

  8. Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity

    DEFF Research Database (Denmark)

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soils provide numerous essential ecosystem services such as carbon cycling......, recycling of nutrients and waste, soil remediation, plant growth support and regulation of above ground biodiversity, resilience, and soil suppressiveness. As such, soil ecosystem services are beneficial and vital for human life and at the same time threatened by anthropogenic activities. Increasing...... of soil microbial functions is still needed. In soil, enzymes originate from a variety of organisms, notably fungi and bacteria and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity will reflect...

  9. Effects of the mycotoxin patulin at the level of nuclear receptor transcriptional activity and steroidogenesis in vitro.

    Science.gov (United States)

    Frizzell, Caroline; Elliott, Christopher T; Connolly, Lisa

    2014-09-02

    Patulin (PAT) is a mycotoxin produced by various species of fungi, with Penicillium expansum being the most commonly occurring. Apples and apple products are the main sources of PAT contamination. This mycotoxin has been shown to induce toxic effects in animals, a few of which include reproductive toxicity and interference with the endocrine system. Here the endocrine disrupting potential of PAT has been investigated in vitro to identify disruption at the level of oestrogen, androgen, progestagen and glucocorticoid nuclear receptor transcriptional activity, and to assess interferences in estradiol, testosterone and progesterone steroid hormone production. At the receptor level, 0.5-5000ng/ml (0.0032-32μM) PAT did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs); however, nuclear transcriptional activity was affected. A >6 fold increase in the glucocorticoid receptor transcriptional activity was observed following treatment with 5000ng/ml PAT in the presence of cortisol. At the hormone production level, despite cytotoxicity being observed after treatment with 5000ng/ml PAT, estradiol levels had increased >2 fold. At 500ng/ml PAT treatment, an increase in progesterone and a decrease in testosterone production were observed. The findings of this study could be considered in assessing the health risks following exposure to PAT. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    Science.gov (United States)

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  11. Differentiation of the virulence potential of Campylobacter jejuni strains by use of gene transcription analysis and a caco-2 assay

    DEFF Research Database (Denmark)

    Poli, Vanessa Fadanelli Schoenardie; Thorsen, Line; Olesen, Inger

    2012-01-01

    Campylobacter jejuni is the leading cause of bacterial diarrheal disease in humans, and contaminated poultry and poultry products are recognized as the main vehicle of infection. Despite the significance of C. jejuni as a foodborne pathogen, little is known about its response to stress, and......, especially, how its virulence is modulated under such conditions. The aim of this study was to assess the effect of temperature shift in a broth model system on virulence expression and cell survival of three different Campylobacter jejuni strains: two clinical (TB1048 and NCTC11168) and one chicken isolate...... properties were evaluated by analyzing transcriptions of the virulence genes cdtB, ciaB, cadF and the stress associated genes clpP, htrB using reverse transcription quantitative PCR (RT-qPCR) and by the ability of the C. jejuni strains to adhere to and invade Caco-2 cells. Similar cell survival and no growth...

  12. Signal Transducer and Activator of Transcription 3 in Liver Diseases: A Novel Therapeutic Target

    OpenAIRE

    Wang, Hua; Lafdil, Fouad; Kong, Xiaoni; Gao, Bin

    2011-01-01

    Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated by many cytokines and growth factors and plays a key role in cell survival, proliferation, and differentiation. STAT3 activation is detected virtually in all rodent models of liver injury and in human liver diseases. In this review, we highlight recent advances of STAT3 signaling in liver injury, steatosis, inflammation, regeneration, fibrosis, and hepatocarcinogenesis. The cytokines and sma...

  13. Specific inhibition of transcriptional activity of the constitutive androstane receptor (CAR) by the splicing factor SF3a3.

    Science.gov (United States)

    Yun, Hye Jin; Kwon, Jungsun; Seol, Wongi

    2008-10-01

    The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily and plays an important role in the degradation of xenobiotics in the liver. Using yeast two-hybrid screening, we identified SF3a3, a 60-kDa subunit of the splicing factor 3a complex, as a specific CAR-interacting protein. We further confirmed their interaction by both co-immunoprecipitation and GST pull-down assay. Functional studies showed that overexpression of SF3a3 inhibited the reporter activity driven by a promoter containing CAR binding sequences by up to 50%, whereas reduced expression of SF3a3 activated the same reporter activity by approximately three-fold. The inhibitory function of SF3a3 is independent of the presence of TCPOBOP, a CAR ligand. These data suggest that SF3a3 functions as a co-repressor of CAR transcriptional activity, in addition to its canonical function.

  14. Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription

    Directory of Open Access Journals (Sweden)

    Luisa S. Hohlefelder

    2013-01-01

    Full Text Available The in vitro proliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D2O. Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, we employ a cell-free expression system for the quantitative analysis of the effect of increasing percentages of D2O on the kinetics of in-vitro GFP expression. Experiments are designed to discriminate the rates of transcription, translation, and protein folding using pDNA and mRNA vectors, respectively. We find that D2O significantly stimulates GFP expression at the transcription level but acts as a suppressor at translation and maturation (folding in a linear dose-dependent manner. At a D2O concentration of 60%, the GFP expression rate was reduced to 40% of an undisturbed sample. We observed a similar inhibition of GFP expression by D2O in a recombinant Escherichia coli strain, although the inhibitory effect is less pronounced. These results demonstrate the suitability of cell-free systems for quantifying the impact of heavy water on gene expression and establish a platform to further assess the potential therapeutic use of heavy water as antiproliferative agent.

  15. Transformation by bovine papillomavirus type 1 E6 is independent of transcriptional activation by E6.

    OpenAIRE

    Ned, R; Allen, S.; Vande Pol, S

    1997-01-01

    We have generated mutants of bovine papillomavirus type 1 E6 (BE6) that are defective for transcriptional activation and have analyzed these mutants for transformation of contact-inhibited cells and association with the mammalian protein E6-AP. These BE6 mutants demonstrate that transformation by BE6 does not require transcriptional activation and that association of BE6 with E6-AP is a function separable from transcriptional activation by BE6. Association of BE6 with E6-AP appears to be nece...

  16. Hepatitis B Virus Activates Signal Transducer and Activator of Transcription 3 Supporting Hepatocyte Survival and Virus ReplicationSummary

    Directory of Open Access Journals (Sweden)

    Marianna Hösel

    2017-11-01

    Full Text Available Background & Aims: The human hepatitis B virus (HBV is a major cause of chronic hepatitis and hepatocellular carcinoma, but molecular mechanisms driving liver disease and carcinogenesis are largely unknown. We therefore studied cellular pathways altered by HBV infection. Methods: We performed gene expression profiling of primary human hepatocytes infected with HBV and proved the results in HBV-replicating cell lines and human liver tissue using real-time polymerase chain reaction and Western blotting. Activation of signal transducer and activator of transcription (STAT3 was examined in HBV-replicating human hepatocytes, HBV-replicating mice, and liver tissue from HBV-infected individuals using Western blotting, STAT3-luciferase reporter assay, and immunohistochemistry. The consequences of STAT3 activation on HBV infection and cell survival were studied by chemical inhibition of STAT3 phosphorylation and small interfering RNA–mediated knockdown of STAT3. Results: Gene expression profiling of HBV-infected primary human hepatocytes detected no interferon response, while genes encoding for acute phase and antiapoptotic proteins were up-regulated. This gene regulation was confirmed in liver tissue samples of patients with chronic HBV infection and in HBV-related hepatocellular carcinoma. Pathway analysis revealed activation of STAT3 to be the major regulator. Interleukin-6–dependent and –independent activation of STAT3 was detected in HBV-replicating hepatocytes in cell culture and in vivo. Prevention of STAT3 activation by inhibition of Janus tyrosine kinases as well as small interfering RNA–mediated knockdown of STAT3-induced apoptosis and reduced HBV replication and gene expression. Conclusions: HBV activates STAT3 signaling in hepatocytes to foster its own replication but also to prevent apoptosis of infected cells. This very likely supports HBV-related carcinogenesis. Keywords: Hepatitis B Virus Infection, STAT3 Signaling

  17. Tissue differences in BER-related incision activity and non-specific nuclease activity as measured by the comet assay.

    Science.gov (United States)

    Gorniak, Joanna P; Cameron, Kerry M; Waldron, Kevin J; von Zglinicki, Thomas; Mathers, John C; Langie, Sabine A S

    2013-11-01

    DNA repair mechanisms are important for genome stability and to prevent accumulation of DNA damage, which contributes to cellular ageing and cancer development. Study of these physiological processes requires robust and practical assays to quantify DNA repair capacity. The in vitro comet-based assay is a simple, yet reliable, assay for measurement of DNA repair and has been modified recently to quantify DNA incision activity in mouse brain and liver. In this study, we applied this assay to assess DNA incision activity in other mouse tissues, i.e. lung and colon, and found that high, non-specific nuclease activity was a problem when measuring DNA incision activity, especially in the colon. We tested the utility of multiple optimisation steps including addition of aphidicolin, ATP and polyAT and used multiple wash steps, which resulted in modest improvements in performance of the assay. Washing the tissues before protein extraction and decreasing the protein concentration in the assay were the most effective steps in reducing non-specific nuclease activity. Using the comet-based assay with these further modifications, we found that base excision repair incision activity changed with age differently in each tissue. This study shows that non-specific nuclease activity in the comet-based assay for DNA repair is more pronounced in some tissues than others so care should be taken to optimise the protocol when applying the assay to a new tissue. Our data suggest the importance of using control cells (noRo cells incubated with extract) in the assay to assess for non-specific nuclease activity. In conclusion, the comet-based DNA repair assay can be easily adapted to study a range of mammalian tissues.

  18. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Rønne, E; Behrendt, N; Ploug, M

    1994-01-01

    Binding of the urokinase plasminogen activator (uPA) to a specific cell surface receptor (uPAR) plays a crucial role in proteolysis during tissue remodelling and cancer invasion. An immunosorbent assay for the quantitation of uPAR has now been developed. This assay is based on two monoclonal anti...

  19. A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

    Science.gov (United States)

    Babu, Binoy; Washburn, Brian K; Ertek, Tülin Sarigül; Miller, Steven H; Riddle, Charles B; Knox, Gary W; Ochoa-Corona, Francisco M; Olson, Jennifer; Katırcıoğlu, Yakup Zekai; Paret, Mathews L

    2017-09-01

    Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (<5min) has been standardized from RRV infected tissue sources, using PBS-T buffer (pH 7.4), which facilitates the virus adsorption onto the PCR tubes at 4°C for 2min, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants using the primer/probe indicated that the virus could be detected from leaves, stems, petals, pollen, primary roots and secondary roots. In addition, the assay was efficiently used in the diagnosis of RRV from different rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes. Copyright © 2017 Elsevier B

  20. Antioxidant activity assays on-line with liquid chromatography

    NARCIS (Netherlands)

    Niederlander, Harm A. G.; van Beek, Teris A.; Bartasiute, Aiste; Koieva, Irina I.

    2008-01-01

    Screening for antioxidants requires simple in vitro model systems to investigate antioxidant activity. High resolution screening (HRS), combining a separation technique like HPLC with fast post-column (bio)chemical detection can rapidly pinpoint active compounds in complex mixtures. In this paper

  1. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and...... process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.......A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co......-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated...

  2. Thyroid hormone receptor inhibits hepatoma cell migration through transcriptional activation of Dickkopf 4

    Energy Technology Data Exchange (ETDEWEB)

    Chi, Hsiang-Cheng; Liao, Chen-Hsin [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China); Huang, Ya-Hui [Medical Research Central, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, ROC (China); Wu, Sheng-Ming; Tsai, Chung-Ying; Liao, Chia-Jung; Tseng, Yi-Hsin; Lin, Yang-Hsiang; Chen, Cheng-Yi; Chung, I-Hsiao; Wu, Tzu-I [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China); Chen, Wei-Jan [First Cardiovascular Division, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, ROC (China); Lin, Kwang-Huei, E-mail: khlin@mail.cgu.edu.tw [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China)

    2013-09-13

    Highlights: •T{sub 3} affects DKK4 mRNA and protein expression in HepG2-TR cells. •Regulation of DKK4 by T{sub 3} is at transcriptional level. •DKK4 overexpression suppresses hepatoma cell metastasis. -- Abstract: Triiodothyronine (T{sub 3}) is a potent form of thyroid hormone mediates several physiological processes including cellular growth, development, and differentiation via binding to the nuclear thyroid hormone receptor (TR). Recent studies have demonstrated critical roles of T{sub 3}/TR in tumor progression. Moreover, long-term hypothyroidism appears to be associated with the incidence of human hepatocellular carcinoma (HCC), independent of other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein that antagonizes the canonical Wnt signaling pathway, is induced by T{sub 3} at both mRNA and protein levels in HCC cell lines. However, the mechanism underlying T{sub 3}-mediated regulation of DKK4 remains unknown. In the present study, the 5′ promoter region of DKK4 was serially deleted, and the reporter assay performed to localize the T{sub 3} response element (TRE). Consequently, we identified an atypical direct repeat TRE between nucleotides −1645 and −1629 conferring T{sub 3} responsiveness to the DKK4 gene. This region was further validated using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Stable DKK4 overexpression in SK-Hep-1 cells suppressed cell invasion and metastatic potential, both in vivo andin vitro, via reduction of matrix metalloproteinase-2 (MMP-2) expression. Our findings collectively suggest that DKK4 upregulated by T{sub 3}/TR antagonizes the Wnt signal pathway to suppress tumor cell progression, thus providing new insights into the molecular mechanism underlying thyroid hormone activity in HCC.

  3. TheRIN-MCFusion of MADS-Box Transcription Factors Has Transcriptional Activity and Modulates Expression of Many Ripening Genes.

    Science.gov (United States)

    Li, Shan; Xu, Huijinlan; Ju, Zheng; Cao, Dongyan; Zhu, Hongliang; Fu, Daqi; Grierson, Donald; Qin, Guozheng; Luo, Yunbo; Zhu, Benzhong

    2018-01-01

    Fruit development and ripening is regulated by genetic and environmental factors and is of critical importance for seed dispersal, reproduction, and fruit quality. Tomato ( Solanum lycopersicum ) ripening inhibitor ( rin ) mutant fruit have a classic ripening-inhibited phenotype, which is attributed to a genomic DNA deletion resulting in the fusion of two truncated transcription factors, RIN and MC In wild-type fruit, RIN, a MADS-box transcription factor, is a key regulator of the ripening gene expression network, with hundreds of gene targets controlling changes in color, flavor, texture, and taste during tomato fruit ripening; MC , on the other hand, has low expression in fruit, and the potential functions of the RIN-MC fusion gene in ripening remain unclear. Here, overexpression of RIN-MC in transgenic wild-type cv Ailsa Craig tomato fruits impaired several ripening processes, and down-regulating RIN-MC expression in the rin mutant was found to stimulate the normal yellow mutant fruit to produce a weak red color, suggesting a distinct negative role for RIN-MC in tomato fruit ripening. By comparative transcriptome analysis of rin and rin 35S :: RIN-MC RNA interference fruits, a total of 1,168 and 1,234 genes were identified as potential targets of RIN-MC activation and inhibition. Furthermore, the RIN-MC fusion gene was shown to be translated into a chimeric transcription factor that was localized to the nucleus and was capable of protein interactions with other MADS-box factors. These results indicated that tomato RIN-MC fusion plays a negative role in ripening and encodes a chimeric transcription factor that modulates the expression of many ripening genes, thereby contributing to the rin mutant phenotype. © 2018 American Society of Plant Biologists. All Rights Reserved.

  4. QUANTITATION OF DNA TOPOISOMERASE-II-ALPHA MESSENGER-RIBONUCLEIC-ACID LEVELS IN A SMALL-CELL LUNG-CANCER CELL-LINE AND 2 DRUG-RESISTANT SUBLINES USING A POLYMERASE CHAIN REACTION-AIDED TRANSCRIPT TITRATION ASSAY

    NARCIS (Netherlands)

    WITHOFF, S; SMIT, EF; MEERSMA, GJ; van den Berg, Anke; TIMMERBOSSCHA, H; KOK, K; POSTMUS, PE; MULDER, NH; DEVRIES, EGE; BUYS, CHCM

    BACKGROUND: We have modified a polymerase chain reaction (PCR)-aided transcript titration assay (1) in order to allow quantitation of low amounts of DNA topoisomerase II alpha mRNA in small RNA samples. EXPERIMENTAL DESIGN: The titration assay was used to quantitate the amount of DNA topoisomerase

  5. A comparative study on basophil activation test, histamine release assay, and passive sensitization histamine release assay in the diagnosis of peanut allergy

    NARCIS (Netherlands)

    Larsen, L. F.; Juel-Berg, N.; Hansen, K. S.; Clare Mills, E. N.; van Ree, R.; Poulsen, L. K.; Jensen, B. M.

    2018-01-01

    BackgroundAllergy can be diagnosed using basophil tests. Several methods measuring basophil activation are available. This study aimed at comparing basophil activation test (BAT), histamine release assay (HR), and passive sensitization histamine release assay (passive HR) in the diagnosis of peanut

  6. The SUMOylation of zinc-fingers and homeoboxes 1 (ZHX1) by Ubc9 regulates its stability and transcriptional repression activity.

    Science.gov (United States)

    Chen, Shuliang; Yu, Xiao; Lei, Quan; Ma, Lixin; Guo, Deyin

    2013-10-01

    Zinc-fingers and homeoboxes protein 1 (ZHX1) belongs to the ZF (zinc-fingers) class of homeodomain transcription factors, and its function remains largely unknown. ZHX1 has been previously found to interact with the activation domain of the nuclear factor Y subunit A (NFYA) and to have a transcriptional repression activity. Here, we report that the SUMO-E2 conjugating enzyme Ubc9 was identified to interact with ZHX1 by an interaction screen using a yeast two-hybrid system. This interaction was confirmed by co-immunoprecipitation and co-localization assays. Further study showed that ZHX1 is SUMOylated by Ubc9 with SUMO1 at the sites K159, K454, and K626. Furthermore, we demonstrated that the SUMOylation of ZHX1 regulated the stability, ubiquitination and transcriptional activity of ZHX1. Copyright © 2013 Wiley Periodicals, Inc.

  7. IRES-based Bicistronic in-situ Reporter Assays for Discovery of Transcription-targeted Lead Compounds

    OpenAIRE

    Lang, Liwei; Ding, Han-Fei; Chen, Xiaoguang; Sun, Shi-Yong; Liu, Gang; Yan, Chunhong

    2015-01-01

    Although transgene-based reporter gene assays have been used to discover small molecules targeting expression of cancer-driving genes, the success is limited due to the fact that reporter gene expression regulated by incomplete cis-acting elements and foreign epigenetic environments does not faithfully reproduce chemical responses of endogenous genes. Here we present an IRES-based strategy for bicistronically co-expressing reporter genes with an endogenous gene in the native gene locus, yield...

  8. Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity.

    Science.gov (United States)

    Menegazzi, Marta; Mariotto, Sofia; Dal Bosco, Martina; Darra, Elena; Vaiana, Nadia; Shoji, Kazuo; Safwat, Abdel-Azeim; Marechal, Jean Didier; Perahia, David; Suzuki, Hisanori; Romeo, Sergio

    2014-02-01

    Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins, characterized by the (-)-gallocatechin-3-gallate stereochemistry, were studied in the human mammary MDA-MB-231 cell line to identify the minimal structural features that preserve the anti-STAT1 activity. We demonstrate that the presence of three hydroxyl groups of B ring and one hydroxyl group in D ring is essential to preserve their inhibitory action. Moreover, a possible molecular target of these compounds in the STAT1 pathway was investigated. Our results demonstrate a direct interaction between STAT1 protein and catechins displaying anti-STAT1 activity. In particular, surface plasmon resonance (SPR) analysis and molecular modeling indicate the presence of two putative binding sites (a and b) with different affinity. Based on docking data, site-directed mutagenesis was performed, and interaction of the most active catechins with STAT1 was studied with SPR to test whether Gln518 on site a and His568 on site b could be important for the catechin-STAT1 interaction. Data indicate that site b has higher affinity for catechins than site a as the highest affinity constant disappears in the H568A-STAT1 mutant. Furthermore, Janus kinase 2 (JAK2) kinase assay data suggest that the contemporary presence in vitro of STAT1 and catechins inhibits JAK2-elicited STAT1 phosphorylation. The very tight catechin-STAT1 interaction prevents STAT1 phosphorylation and represents a novel, specific and efficient molecular mechanism for the inhibition of STAT1 activation. © 2013 FEBS.

  9. Signal Transducer and Activator of Transcription (STAT)-3 Activates Nuclear Factor (NF)-κB in Chronic Lymphocytic Leukemia Cells

    Science.gov (United States)

    Liu, Zhiming; Hazan-Halevy, Inbal; Harris, David M.; Li, Ping; Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J.; Estrov, Zeev

    2014-01-01

    Nuclear factor (NF)-κB plays a major role in the pathogenesis of B-cell neoplasms. A broad array of mostly extracellular stimuli has been reported to activate NF-κB, to various degrees, in chronic lymphocytic leukemia (CLL) cells. Because CLL cells harbor high levels of unphosphorylated (U) signal transducer and activator of transcription (STAT)-3 protein and U-STAT3 was reported to activate NF-κB, we sought to determine whether U-STAT3 activates NF-κB in CLL. Using the electrophoretic mobility shift assay (EMSA) we studied peripheral blood low-density cells from 15 patients with CLL and found that CLL cell nuclear extracts from all the samples bound to an NF-κB DNA probe, suggesting that NF-κB is constitutively activated in CLL. Immunoprecipitation studies showed that STAT3 bound NF-κB p65, and confocal microscopy studies detected U-STAT3/NF-κB complexes in the nuclei of CLL cells, thereby confirming these findings. Furthermore, infection of CLL cells with retroviral STAT3-shRNA attenuated the binding of NF-κB to DNA, as assessed by EMSA, and downregulated mRNA levels of NF-κB-regulated genes, as assessed by quantitative polymerase chain reaction. Taken together, our data suggest that U-STAT3 binds to the NF-κB p50/p65 dimers and that the U-STAT3/NF-κB complexes bind to DNA and activate NF-κB-regulated genes in CLL cells. PMID:21364020

  10. Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity

    KAUST Repository

    Menegazzi, Marta

    2013-12-10

    Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins, characterized by the (-)-gallocatechin-3-gallate stereochemistry, were studied in the human mammary MDA-MB-231 cell line to identify the minimal structural features that preserve the anti-STAT1 activity. We demonstrate that the presence of three hydroxyl groups of B ring and one hydroxyl group in D ring is essential to preserve their inhibitory action. Moreover, a possible molecular target of these compounds in the STAT1 pathway was investigated. Our results demonstrate a direct interaction between STAT1 protein and catechins displaying anti-STAT1 activity. In particular, surface plasmon resonance (SPR) analysis and molecular modeling indicate the presence of two putative binding sites (a and b) with different affinity. Based on docking data, site-directed mutagenesis was performed, and interaction of the most active catechins with STAT1 was studied with SPR to test whether Gln518 on site a and His568 on site b could be important for the catechin-STAT1 interaction. Data indicate that site b has higher affinity for catechins than site a as the highest affinity constant disappears in the H568ASTAT1 mutant. Furthermore, Janus kinase 2 (JAK2) kinase assay data suggest that the contemporary presence in vitro of STAT1 and catechins inhibits JAK2-elicited STAT1 phosphorylation. The very tight catechin-STAT1 interaction prevents STAT1 phosphorylation and represents a novel, specific and efficient molecular mechanism for the inhibition of STAT1 activation. © Copyright 2014 Federation of European Biochemical Societies. All rights reserved.

  11. Sertad1 encodes a novel transcriptional co-activator of SMAD1 in mouse embryonic hearts

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Yin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Zhao, Shaomin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Song, Langying [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Wang, Manyuan [School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Jiao, Kai, E-mail: kjiao@uab.edu [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2013-11-29

    Highlights: •SERTAD1 interacts with SMAD1. •Sertad1 is expressed in mouse embryonic hearts. •SERTAD1 is localized in both cytoplasm and nucleus of cardiomyocytes. •SERTAD1 enhances expression of BMP target cardiogenic genes as a SMAD1 co-activator. -- Abstract: Despite considerable advances in surgical repairing procedures, congenital heart diseases (CHDs) remain the leading noninfectious cause of infant morbidity and mortality. Understanding the molecular/genetic mechanisms underlying normal cardiogenesis will provide essential information for the development of novel diagnostic and therapeutic strategies against CHDs. BMP signaling plays complex roles in multiple cardiogenic processes in mammals. SMAD1 is a canonical nuclear mediator of BMP signaling, the activity of which is critically regulated through its interaction partners. We screened a mouse embryonic heart yeast two-hybrid library using Smad1 as bait and identified SERTAD1 as a novel interaction partner of SMAD1. SERTAD1 contains multiple potential functional domains, including two partially overlapping transactivation domains at the C terminus. The SERTAD1-SMAD1 interaction in vitro and in mammalian cells was further confirmed through biochemical assays. The expression of Sertad1 in developing hearts was demonstrated using RT-PCR, western blotting and in situ hybridization analyses. We also showed that SERTAD1 was localized in both the cytoplasm and nucleus of immortalized cardiomyocytes and primary embryonic cardiomyocyte cultures. The overexpression of SERTAD1 in cardiomyocytes not only enhanced the activity of two BMP reporters in a dose-dependent manner but also increased the expression of several known BMP/SMAD regulatory targets. Therefore, these data suggest that SERTAD1 acts as a SMAD1 transcriptional co-activator to promote the expression of BMP target genes during mouse cardiogenesis.

  12. Role of the σ54 Activator Interacting Domain in Bacterial Transcription Initiation

    Energy Technology Data Exchange (ETDEWEB)

    Siegel, Alexander R. [Univ. of California, Berkeley, CA (United States); Wemmer, David E. [Univ. of California, Berkeley, CA (United States)

    2016-10-11

    Bacterial sigma factors are subunits of RNA polymerase that direct the holoenzyme to specific sets of promoters in the genome and are a central element of regulating transcription. Most polymerase holoenzymes open the promoter and initiate transcription rapidly after binding. However, polymerase containing the members of the σ54 family must be acted on by a transcriptional activator before DNA opening and initiation occur. A key domain in these transcriptional activators forms a hexameric AAA + ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and σ54 are primarily made through an N-terminal σ54 activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the σ54 AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in σ54 alone. In this paper, we identified a minimal construct of the Aquifex aeolicus σ54 AID that consists of two predicted helices and retains native-like binding affinity for the transcriptional activator NtrC1. Using the NtrC1 ATPase domain, bound with the non-hydrolyzable ATP analog ADP-beryllium fluoride, we studied the NtrC1–σ54 AID complex using NMR spectroscopy. We show that the σ54 AID becomes structured after associating with the core loops of the transcriptional activators in their ATP state and that the primary site of the interaction is the first predicted helix. Finally, understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of σ54 bacterial transcription initiation.

  13. Verticillium transcription activator of adhesion Vta2 suppresses microsclerotia formation and is required for systemic infection of plant roots.

    Science.gov (United States)

    Tran, Van-Tuan; Braus-Stromeyer, Susanna A; Kusch, Harald; Reusche, Michael; Kaever, Alexander; Kühn, Anika; Valerius, Oliver; Landesfeind, Manuel; Aßhauer, Kathrin; Tech, Maike; Hoff, Katharina; Pena-Centeno, Tonatiuh; Stanke, Mario; Lipka, Volker; Braus, Gerhard H

    2014-04-01

    Six transcription regulatory genes of the Verticillium plant pathogen, which reprogrammed nonadherent budding yeasts for adhesion, were isolated by a genetic screen to identify control elements for early plant infection. Verticillium transcription activator of adhesion Vta2 is highly conserved in filamentous fungi but not present in yeasts. The Magnaporthe grisea ortholog conidiation regulator Con7 controls the formation of appressoria which are absent in Verticillium species. Vta2 was analyzed by using genetics, cell biology, transcriptomics, secretome proteomics and plant pathogenicity assays. Nuclear Vta2 activates the expression of the adhesin-encoding yeast flocculin genes FLO1 and FLO11. Vta2 is required for fungal growth of Verticillium where it is a positive regulator of conidiation. Vta2 is mandatory for accurate timing and suppression of microsclerotia as resting structures. Vta2 controls expression of 270 transcripts, including 10 putative genes for adhesins and 57 for secreted proteins. Vta2 controls the level of 125 secreted proteins, including putative adhesins or effector molecules and a secreted catalase-peroxidase. Vta2 is a major regulator of fungal pathogenesis, and controls host-plant root infection and H2 O2 detoxification. Verticillium impaired in Vta2 is unable to colonize plants and induce disease symptoms. Vta2 represents an interesting target for controlling the growth and development of these vascular pathogens. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  14. The Runx transcriptional co-activator, CBFβ, is essential for invasion of breast cancer cells

    Directory of Open Access Journals (Sweden)

    Lopez-Camacho Cesar

    2010-06-01

    Full Text Available Abstract Background The transcription factor Runx2 has an established role in cancers that metastasize to bone. In metastatic breast cancer cells Runx2 is overexpressed and contributes to the invasive capacity of the cells by regulating the expression of several invasion genes. CBFβ is a transcriptional co-activator that is recruited to promoters by Runx transcription factors and there is considerable evidence that CBFβ is essential for the function of Runx factors. However, overexpression of Runx1 can partially rescue the lethal phenotype in CBFβ-deficient mice, indicating that increased levels of Runx factors can, in some situations, overcome the requirement for CBFβ. Since Runx2 is overexpressed in metastatic breast cancer cells, and there are no reports of CBFβ expression in breast cells, we sought to determine whether Runx2 function in these cells was dependent on CBFβ. Such an interaction might represent a viable target for therapeutic intervention to inhibit bone metastasis. Results We show that CBFβ is expressed in the metastatic breast cancer cells, MDA-MB-231, and that it associates with Runx2. Matrigel invasion assays and RNA interference were used to demonstrate that CBFβ contributes to the invasive capacity of these cells. Subsequent analysis of Runx2 target genes in MDA-MB-231 cells revealed that CBFβ is essential for the expression of Osteopontin, Matrixmetalloproteinase-13, Matrixmetalloproteinase-9, and Osteocalcin but not for Galectin-3. Chromatin immunoprecipitation analysis showed that CBFβ is recruited to both the Osteopontin and the Galectin-3 promoters. Conclusions CBFβ is expressed in metastatic breast cancer cells and is essential for cell invasion. CBFβ is required for expression of several Runx2-target genes known to be involved in cell invasion. However, whilst CBFβ is essential for invasion, not all Runx2-target genes require CBFβ. We conclude that CBFβ is required for a subset of Runx2-target genes

  15. Global identification and characterization of transcriptionally active regions in the rice genome.

    Directory of Open Access Journals (Sweden)

    Lei Li

    Full Text Available Genome tiling microarray studies have consistently documented rich transcriptional activity beyond the annotated genes. However, systematic characterization and transcriptional profiling of the putative novel transcripts on the genome scale are still lacking. We report here the identification of 25,352 and 27,744 transcriptionally active regions (TARs not encoded by annotated exons in the rice (Oryza. sativa subspecies japonica and indica, respectively. The non-exonic TARs account for approximately two thirds of the total TARs detected by tiling arrays and represent transcripts likely conserved between japonica and indica. Transcription of 21,018 (83% japonica non-exonic TARs was verified through expression profiling in 10 tissue types using a re-array in which annotated genes and TARs were each represented by five independent probes. Subsequent analyses indicate that about 80% of the japonica TARs that were not assigned to annotated exons can be assigned to various putatively functional or structural elements of the rice genome, including splice variants, uncharacterized portions of incompletely annotated genes, antisense transcripts, duplicated gene fragments, and potential non-coding RNAs. These results provide a systematic characterization of non-exonic transcripts in rice and thus expand the current view of the complexity and dynamics of the rice transcriptome.

  16. Optimized UDP-glucuronosyltransferase (UGT) activity assay for trout liver S9 fractions

    Data.gov (United States)

    U.S. Environmental Protection Agency — This publication provides an optimized UGT assay for trout liver S9 fractions which can be used to perform in vitro-in vivo extrapolations of measured UGT activity....

  17. In vitro assay of potential antifungal and antibacterial activities of ...

    African Journals Online (AJOL)

    The anti-inflammatory, antipyretic, and pro-apoptotic properties of extracts of Borassus aethiopum have been reported in the literature. In this study, we investigated the antifungal and antibacterial properties of Borassus aethiopum male inflorescences extracts. The antifungal and antibacterial activity was studied by agar ...

  18. Microtiter plate based colorimetric assay for characterization of dehalogenation activity of GAC/Fe0 composite

    OpenAIRE

    Hwang, Yuhoon; Salatas, Apostolos; Mines, Paul D.; Jakobsen, Mogens Havsteen; Andersen, Henrik Rasmus

    2015-01-01

    Even though nanoscale zero valent iron (nZVI) has been intensively studied for the treatment of a plethora of pollutants through reductive reaction, a quantification of nZVIreactivity has not been standardized. Here, we developed series of colorimetric assays for determining reductive activity of nZVI and its composite with granular activated carbon(GAC). The assay focused on analysis of reaction products rather than its mother compounds, which gives more accurate quantification of reductive ...

  19. Rapid differentiation and identification of potential severe strains of Citrus tristeza virus by real-time reverse transcription-polymerase chain reaction assays.

    Science.gov (United States)

    Yokomi, R K; Saponari, M; Sieburth, P J

    2010-04-01

    A multiplex Taqman-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to identify potential severe strains of Citrus tristeza virus (CTV) and separate genotypes that react with the monoclonal antibody MCA13. Three strain-specific probes were developed using intergene sequences between the major and minor coat protein genes (CPi) in a multiplex reaction. Probe CPi-VT3 was designed for VT and T3 genotypes; probe CPi-T36 for T36 genotypes; and probe CPi-T36-NS to identify isolates in an outgroup clade of T36-like genotypes mild in California. Total nucleic acids extracted by chromatography on silica particles, sodium dodecyl sulfate-potassium acetate, and CTV virion immunocapture all yielded high quality templates for real-time PCR detection of CTV. These assays successfully differentiated CTV isolates from California, Florida, and a large panel of CTV isolates from an international collection maintained in Beltsville, MD. The utility of the assay was validated using field isolates collected in California and Florida.

  20. Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

    Science.gov (United States)

    Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M

    2011-07-01

    Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

  1. Enzyme activity assays within microstructured optical fibers enabled by automated alignment.

    Science.gov (United States)

    Warren-Smith, Stephen C; Nie, Guiying; Schartner, Erik P; Salamonsen, Lois A; Monro, Tanya M

    2012-12-01

    A fluorescence-based enzyme activity assay has been demonstrated within a small-core microstructured optical fiber (MOF) for the first time. To achieve this, a reflection-based automated alignment system has been developed, which uses feedback and piezoelectric actuators to maintain optical alignment. The auto-alignment system provides optical stability for the time required to perform an activity assay. The chosen assay is based on the enzyme proprotein convertase 5/6 (PC6) and has important applications in women's health.

  2. Transcriptional activation in chondrocytes submitted to hydrostatic pressure.

    Science.gov (United States)

    Sironen, R; Elo, M; Kaarniranta, K; Helminen, H J; Lammi, M J

    2000-01-01

    At present, only a little is known about the transcriptional regulation in chondrocytes submitted to various physicomechanical factors known to exist in articular cartilage. Recently, we have investigated the effects of hydrostatic pressure on transcriptional control in chondrocytes using human chondrosarcoma and immortalized chondrocyte cell lines for the experiments. Hydrostatic pressure was applied on the cells in a special computer-controlled, water-filled pressure chamber, where cyclic and static pressures up to 32 MPa can be created. Differential display RT-PCR and probing of cDNA arrays are the methods we have used to study differential gene expression due to hydrostatic pressure. By differential display RT-PCR experiments, we have observed several differentially expressed cDNA bands under continuous 30 MPa hydrostatic pressure, while 30 MPa cyclic pressure at 1 Hz produced much fewer changes. In the first phase of our studies, we have focused on the effects of 30 MPa hydrostatic pressure because it causes a unique hsp70-mediated stress response in immortalized chondrocytes. Differential display RT-PCR screening provided us with several clones that derive from low-abundance mRNAs, such as death-associated protein 3 (DAP3), a nucleotide-binding protein which increases due to interferon-gamma induced cell death; PTZ-17 (or p311), a seizure-related protein; H-NUC, a nuclear DNA binding protein; and one new gene of unknown function. In Northern blots, an induction was confirmed for the new gene, DAP3 and PTZ-17 were down-regulated in some but not in all parallel experiments; however, basal level of H-NUC mRNA was too low to be detected in Northern blots. We then chose to widen our screening to a number of known genes arrayed as cDNA blots. Under 30 MPa continuous hydrostatic pressure, four different time points were chosen (0, 3, 6 and 24 h) for the experiments. The screening of 588 cDNAs showed 15 up-regulated and 6 down-regulated genes. Consistently with our

  3. Proton energy determination using activated yttrium foils and ionization chambers for activity assay

    Science.gov (United States)

    Avila-Rodriguez, M. A.; Rajander, J.; Lill, J.-O.; Gagnon, K.; Schlesinger, J.; Wilson, J. S.; McQuarrie, S. A.; Solin, O.

    2009-05-01

    Excitation functions of the 89Y(p, xn) nuclear reactions were measured up to 18 MeV by the conventional activation method using the stacked-foil technique, and the irradiation of single foils. Activity assays of the irradiated foils were performed via ionization chamber and gamma spectroscopy methods. Activity ratios of the activation products were measured in two different facilities and evaluated for use as a practical and simple method for proton energy determinations. Cross section values measured in this work were compared with published data and with theoretical values as determined by the nuclear reaction model code EMPIRE II. In general, there was a good agreement between the experimental and theoretical values of the cross section data. Activity ratios of the isomeric and ground state of 89Zr measured via ionization chamber were found to be useful for proton energy determinations in the energy range from 7 to 15 MeV. Proton energies above 13 MeV were accurately determined using the 89gZr/ 88Zr and 89gZr/ 88Y activity ratios measured via gamma spectroscopy.

  4. Comparative study of human and mouse pregnane X receptor agonistic activity in 200 pesticides using in vitro reporter gene assays.

    Science.gov (United States)

    Kojima, Hiroyuki; Sata, Fumihiro; Takeuchi, Shinji; Sueyoshi, Tatsuya; Nagai, Tadanori

    2011-02-27

    The nuclear receptor, pregnane X receptor (PXR), is a ligand-dependent transcription factor that regulates genes involved in xenobiotic metabolism. Recent studies have shown that PXR activation may affect energy metabolism as well as the endocrine and immune systems. In this study, we characterized and compared the agonistic activities of a variety of pesticides against human PXR (hPXR) and mouse PXR (mPXR). We tested the hPXR and mPXR agonistic activity of 200 pesticides (29 organochlorines, 11 diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, 12 acid amides, 7 triazines, 7 ureas, and 44 others) by reporter gene assays using COS-7 simian kidney cells. Of the 200 pesticides tested, 106 and 93 activated hPXR and mPXR, respectively, and a total of 111 had hPXR and/or mPXR agonistic activity with greater or lesser inter-species differences. Although all of the pyrethroids and most of the organochlorines and acid amides acted as PXR agonists, a wide range of pesticides with diverse structures also showed hPXR and/or mPXR agonistic activity. Among the 200 pesticides, pyributicarb, pretilachlor, piperophos and butamifos for hPXR, and phosalone, prochloraz, pendimethalin, and butamifos for mPXR, acted as particularly potent activators at low concentrations in the order of 10⁻⁸-10⁻⁷ M. In addition, we found that several organophosphorus oxon- and pyributicarb oxon-metabolites decreased PXR activation potency compared to their parent compounds. These results suggest that a large number of structurally diverse pesticides and their metabolites possess PXR-mediated transcriptional activity, and their ability to do so varies in a species-dependent manner in humans and mice. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  5. The phzA2-G2 transcript exhibits direct RsmA-mediated activation in Pseudomonas aeruginosa M18.

    Directory of Open Access Journals (Sweden)

    Bin Ren

    Full Text Available In bacteria, RNA-binding proteins of the RsmA/CsrA family act as post-transcriptional regulators that modulate translation initiation at target transcripts. The Pseudomonas aeruginosa genome contains two phenazine biosynthetic (phz gene clusters, phzA1-G1 (phz1 and phzA2-G2 (phz2, each of which is responsible for phenazine-1-carboxylic acid (PCA biosynthesis. In the present study, we show that RsmA exhibits differential gene regulation on two phz clusters in P. aeruginosa M18 at the post-transcriptional level. Based on the sequence analysis, four GGA motifs, the potential RsmA binding sites, are found on the 5'-untranslated region (UTR of the phz2 transcript. Studies with a series of lacZ reporter fusions, and gel mobility shift assays suggest that the third GGA motif (S3, located 21 nucleotides upstream of the Shine-Dalgarno (SD sequence, is involved in direct RsmA-mediated activation of phz2 expression. We therefore propose a novel model in which the binding of RsmA to the target S3 results in the destabilization of the stem-loop structure and the enhancement of ribosome access. This model could be fully supported by RNA structure prediction, free energy calculations, and nucleotide replacement studies. In contrast, various RsmA-mediated translation repression mechanisms have been identified in which RsmA binds near the SD sequence of target transcripts, thereby blocking ribosome access. Similarly, RsmA is shown to negatively regulate phz1 expression. Our new findings suggest that the differential regulation exerted by RsmA on the two phz clusters may confer an advantage to P. aeruginosa over other pseudomonads containing only a single phz cluster in their genomes.

  6. Antioxidant Activity of β-Carotene Compounds in Different in Vitro Assays

    Directory of Open Access Journals (Sweden)

    Volker Boehm

    2011-01-01

    Full Text Available β-Carotene (BC is the most abundant carotenoid in human diet, almost solely as (all-E-isomer. Significant amounts of (Z-isomers of BC are present in processed food as well as in mammalian tissues. Differences are described for the activity of various BC isomers in forming retinal and protecting against cancer and cardiovascular diseases. Eccentric cleavage of BC leads to degradation products such as carotenals. A variety of negative consequences were published for the non-vitamin A active BC metabolites, such as inducing the carcinogenesis of benzo[a]pyrene, impairing mitochondrial function, or increasing CYP activity. To increase the knowledge on the antioxidant activity, a variety of BC isomers and metabolites were tested in various in vitro assays.In the present study, no ferric reducing activity (FRAP assay was observed for the BC isomers. Between the major BC isomers (all-E, 9Z, and 13Z no significant differences in bleaching the ABTS●+ (αTEAC assay or in scavenging peroxyl radicals (ROO● generated by thermal degradation of AAPH (using a chemiluminescence assay were detected. However, the (15Z-isomer was less active, maybe due to its low stability. The degradation to β-apo-carotenoids increased FRAP activity and ROO● scavenging activity compared to the parent molecule. Dependence on chain length and character of the terminal function was determined in αTEAC assay with following order of increasing activity: β-apo-8’-carotenal 

  7. Assay of flippase activity in proteoliposomes using fluorescent lipid derivatives

    DEFF Research Database (Denmark)

    Marek, Magdalena; Günther-Pomorski, Thomas

    2016-01-01

    Specific membrane proteins, termed lipid flippases, play a central role in facilitating the movement of lipids across cellular membranes. In this protocol, we describe the reconstitution of ATP-driven lipid flippases in liposomes and the analysis of their in vitro flippase activity based on the use...... of fluorescent lipid derivatives. Working with purified and reconstituted systems provides a well-defined experimental setup and allows to directly characterize these membrane proteins at the molecular level....

  8. The maize Dof protein PBF activates transcription of gamma-zein during maize seed development.

    Science.gov (United States)

    Marzábal, Pau; Gas, Elisabet; Fontanet, Pilar; Vicente-Carbajosa, Jesús; Torrent, Margarita; Ludevid, M Dolores

    2008-07-01

    Maize PBF (prolamin-box binding factor) belongs to the Dof class of plant specific transcription factors containing one highly conserved zinc finger DNA-binding domain, called Dof (DNA binding with one finger) domain. Maize PBF trans-activates the gamma-zein gene (gammaZ) promoter in developing maize seeds as shown by transient expression in maize endosperms. Co-transfection of a gammaZ:GUS construct with 35S:PBF resulted in a sevenfold increase in GUS expression, however, PBF mutation in Cys residues within the Dof domain abolishes both, binding to DNA and the capacity to activate gammaZ promoter. We present two pieces of evidence that PBF transactivates gammaZ promoter by binding to the Pb3 motif (TGTAAAG). First, recombinant Dof domain of PBF (bdPBF) specifically recognized Pb3 site as shown by gel mobility shift assays and second, co-expression of PBF with gammaZ promoter mutated in Pb3 motif suppressed PBF trans-activation capacity. Immunocytochemical analysis on developing endosperm sections shows that PBF is localized in the nuclei of the peripheral layer cells of starchy endosperm, the tissue in which the initial accumulation of gamma-zein protein occurs. By contrast, PBF is detected in the cytosol of the starchy endosperm cells newly differentiated from aleurone daughter cells, where gamma-zein was absent. Taken together these data indicate that maize PBF plays an essential role in the regulation of the temporal and spatial expression of gammaZ gene.

  9. Enhancer of Acetyltransferase Chameau (EAChm Is a Novel Transcriptional Co-Activator.

    Directory of Open Access Journals (Sweden)

    Takeya Nakagawa

    Full Text Available Acetylation of nucleosomal histones by diverse histone acetyltransferases (HAT plays pivotal roles in many cellular events. Discoveries of novel HATs and HAT related factors have provided new insights to understand the roles and mechanisms of histone acetylation. In this study, we identified prominent Histone H3 acetylation activity in vitro and purified its activity, showing that it is composed of the MYST acetyltransferase Chameau and Enhancer of the Acetyltransferase Chameau (EAChm family. EAChm is a negatively charged acidic protein retaining aspartate and glutamate. Furthermore, we identified that Chameau and EAChm stimulate transcription in vitro together with purified general transcription factors. In addition, RNA-seq analysis of Chameu KD and EAChm KD S2 cells suggest that Chameau and EAChm regulate transcription of common genes in vivo. Our results suggest that EAChm regulates gene transcription in Drosophila embryos by enhancing Acetyltransferase Chameau activity.

  10. Enhancer of Acetyltransferase Chameau (EAChm) Is a Novel Transcriptional Co-Activator.

    Science.gov (United States)

    Nakagawa, Takeya; Ikehara, Tsuyoshi; Doiguchi, Masamichi; Imamura, Yuko; Higashi, Miki; Yoneda, Mitsuhiro; Ito, Takashi

    2015-01-01

    Acetylation of nucleosomal histones by diverse histone acetyltransferases (HAT) plays pivotal roles in many cellular events. Discoveries of novel HATs and HAT related factors have provided new insights to understand the roles and mechanisms of histone acetylation. In this study, we identified prominent Histone H3 acetylation activity in vitro and purified its activity, showing that it is composed of the MYST acetyltransferase Chameau and Enhancer of the Acetyltransferase Chameau (EAChm) family. EAChm is a negatively charged acidic protein retaining aspartate and glutamate. Furthermore, we identified that Chameau and EAChm stimulate transcription in vitro together with purified general transcription factors. In addition, RNA-seq analysis of Chameu KD and EAChm KD S2 cells suggest that Chameau and EAChm regulate transcription of common genes in vivo. Our results suggest that EAChm regulates gene transcription in Drosophila embryos by enhancing Acetyltransferase Chameau activity.

  11. Large-scale transcriptome data reveals transcriptional activity of fission yeast LTR retrotransposons

    DEFF Research Database (Denmark)

    Mourier, Tobias; Willerslev, Eske

    2010-01-01

    BACKGROUND: Retrotransposons are transposable elements that proliferate within eukaryotic genomes through a process involving reverse transcription. The numbers of retrotransposons within genomes and differences between closely related species may yield insight into the evolutionary history...... was analysed for both full-length LTR retrotransposons and solitary LTRs. RESULTS: Two independent sets of transcriptome data reveal the presence of full-length, polyadenylated transcripts from LTR retrotransposons in S. pombe during growth phase in rich medium. The redundancy of retrotransposon sequences...... of transcriptional activity are observed from both strands of solitary LTR sequences. Transcriptome data collected during meiosis suggests that transcription of solitary LTRs is correlated with the transcription of nearby protein-coding genes. CONCLUSIONS: Presumably, the host organism negatively regulates...

  12. Cleavage of the JunB Transcription Factor by Caspases Generates a Carboxyl-terminal Fragment That Inhibits Activator Protein-1 Transcriptional Activity*

    Science.gov (United States)

    Lee, Jason K. H.; Pearson, Joel D.; Maser, Brandon E.; Ingham, Robert J.

    2013-01-01

    The activator protein-1 (AP-1) family transcription factor, JunB, is an important regulator of proliferation, apoptosis, differentiation, and the immune response. In this report, we show that JunB is cleaved in a caspase-dependent manner in apoptotic anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma cell lines and that ectopically expressed JunB is cleaved in murine RAW 264.7 macrophage cells treated with the NALP1b inflammasome activator, anthrax lethal toxin. In both cases, we identify aspartic acid 137 as the caspase cleavage site and demonstrate that JunB can be directly cleaved in vitro by multiple caspases at this site. Cleavage of JunB at aspartic acid 137 separates the N-terminal transactivation domain from the C-terminal DNA binding and dimerization domains, and we show that the C-terminal cleavage fragment retains both DNA binding activity and the ability to interact with AP-1 family transcription factors. Furthermore, this fragment interferes with the binding of full-length JunB to AP-1 sites and inhibits AP-1-dependent transcription. In summary, we have identified and characterized a novel mechanism of JunB post-translational modification and demonstrate that the C-terminal JunB caspase cleavage product functions as a potent inhibitor of AP-1-dependent transcription. PMID:23749999

  13. Cleavage of the JunB transcription factor by caspases generates a carboxyl-terminal fragment that inhibits activator protein-1 transcriptional activity.

    Science.gov (United States)

    Lee, Jason K H; Pearson, Joel D; Maser, Brandon E; Ingham, Robert J

    2013-07-26

    The activator protein-1 (AP-1) family transcription factor, JunB, is an important regulator of proliferation, apoptosis, differentiation, and the immune response. In this report, we show that JunB is cleaved in a caspase-dependent manner in apoptotic anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma cell lines and that ectopically expressed JunB is cleaved in murine RAW 264.7 macrophage cells treated with the NALP1b inflammasome activator, anthrax lethal toxin. In both cases, we identify aspartic acid 137 as the caspase cleavage site and demonstrate that JunB can be directly cleaved in vitro by multiple caspases at this site. Cleavage of JunB at aspartic acid 137 separates the N-terminal transactivation domain from the C-terminal DNA binding and dimerization domains, and we show that the C-terminal cleavage fragment retains both DNA binding activity and the ability to interact with AP-1 family transcription factors. Furthermore, this fragment interferes with the binding of full-length JunB to AP-1 sites and inhibits AP-1-dependent transcription. In summary, we have identified and characterized a novel mechanism of JunB post-translational modification and demonstrate that the C-terminal JunB caspase cleavage product functions as a potent inhibitor of AP-1-dependent transcription.

  14. The AIRE -230Y Polymorphism Affects AIRE Transcriptional Activity: Potential Influence on AIRE Function in the Thymus.

    Science.gov (United States)

    Lovewell, Thomas R J; McDonagh, Andrew J; Messenger, Andrew G; Azzouz, Mimoun; Tazi-Ahnini, Rachid

    2015-01-01

    The autoimmune regulator (AIRE) is expressed in the thymus, particularly in thymic medullary epithelial cells (mTECs), and is required for the ectopic expression of a diverse range of peripheral tissue antigens by mTECs, facilitating their ability to perform negative selection of auto-reactive immature T-cells. The expression profile of peripheral tissue antigens is affected not only by AIRE deficiency but also with variation of AIRE activity in the thymus. Therefore we screened 591bp upstream of the AIRE transcription start site including AIRE minimal promoter for single nucleotide polymorphism (SNPs) and identified two SNPs -655R (rs117557896) and -230Y (rs751032) respectively. To study the effect of these variations on AIRE promoter activity we generated a Flp-In host cell line which was stably transfected with a single copy of the reporter vector. Relative promoter activity was estimated by comparing the luciferase specific activity for lysates of the different reporter AIRE promoter-reporter gene constructs including AIRE-655G AIRE-230C, AIRE-655G AIRE-230T and AIRE-655A AIRE-230C. The analysis showed that the commonest haplotype AIRE-655G AIRE-230C has the highest luciferase specific activity (pAIRE-655G AIRE-230T has a luciferase specific activity value that approaches null. Both AIRE promoter polymorphic sites have one allele that forms a CpG methylation site which we determined can be methylated in methylation assays using the M.SssI CpG methyltransferase. AIRE-230Y is in a conserved region of the promoter and is adjacent to a predicted WT1 transcription factor binding site, suggesting that AIRE-230Y affects AIRE expression by influencing the binding of biochemical factors to this region. Our findings show that AIRE-655GAIRE-230T haplotype could dramatically alter AIRE transcription and so have an effect on the process of negative selection and affect susceptibility to autoimmune conditions.

  15. SUMOylation of DRIL1 directs its transcriptional activity towards leukocyte lineage-specific genes.

    Directory of Open Access Journals (Sweden)

    Alexandre Prieur

    Full Text Available DRIL1 is an ARID family transcription factor that can immortalize primary mouse fibroblasts, bypass RAS(V12-induced cellular senescence and collaborate with RAS(V12 or MYC in mediating oncogenic transformation. It also activates immunoglobulin heavy chain transcription and engages in heterodimer formation with E2F to stimulate E2F-dependent transcription. Little, however, is known about the regulation of DRIL1 activity. Recently, DRIL1 was found to interact with the SUMO-conjugating enzyme Ubc9, but the functional relevance of this association has not been assessed. Here, we show that DRIL1 is sumoylated both in vitro and in vivo at lysine 398. Moreover, we provide evidence that PIASy functions as a specific SUMO E3-ligase for DRIL1 and promotes its sumoylation both in vitro and in vivo. Furthermore, consistent with the subnuclear localization of PIASy in the Matrix-Associated Region (MAR, SUMO-modified DRIL1 species are found exclusively in the MAR fraction. This post-translational modification interferes neither with the subcellular localization nor the DNA-binding activity of the protein. In contrast, DRIL1 sumoylation impairs its interaction with E2F1 in vitro and modifies its transcriptional activity in vivo, driving transcription of subset of genes regulating leukocyte fate. Taken together, these results identify sumoylation as a novel post-translational modification of DRIL1 that represents an important mechanism for targeting and modulating DRIL1 transcriptional activity.

  16. A NF-κB-dependent dual promoter-enhancer initiates the lipopolysaccharide-mediated transcriptional activation of the chicken lysozyme in macrophages.

    Directory of Open Access Journals (Sweden)

    James Witham

    Full Text Available The transcriptional activation of the chicken lysozyme gene (cLys by lipopolysaccharide (LPS in macrophages is dependent on transcription of a LPS-Inducible Non-Coding RNA (LINoCR triggering eviction of the CCCTC-binding factor (CTCF from a negative regulatory element upstream of the lysozyme transcription start site. LINoCR is transcribed from a promoter originally characterized as a hormone response enhancer in the oviduct. Herein, we report the characterization of this cis-regulatory element (CRE. In activated macrophages, a 60 bp region bound by NF-κB, AP1 and C/EBPβ controls this CRE, which is strictly dependent on NF-κB binding for its activity in luciferase assays. Moreover, the serine/threonine kinase IKKα, known to be recruited by NF-κB to NF-κB-dependent genes is found at the CRE and within the transcribing regions of both cLys and LINoCR. Such repartition suggests a simultaneous promoter and enhancer activity of this CRE, initiating cLys transcriptional activation and driving CTCF eviction. This recruitment was transient despite persistence of both cLys transcription and NF-κB binding to the CRE. Finally, comparing cLys with other LPS-inducible genes indicates that IKKα detection within transcribing regions can be correlated with the presence of the elongating form of RNA polymerase II or concentrated in the 3' end of the gene.

  17. Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2)

    NARCIS (Netherlands)

    Takano, E.; Gramajo, H.C.; Strauch, E.; White, J.; Bibb, M.J.

    1992-01-01

    Transcription of redD, the activator gene required for production of the red-pigmented antibiotic undecylprodigiosin by Streptomyces coelicolor A3(2), showed a dramatic increase during the transition from exponential to stationary phase. The increase in redD expression was followed by transcription

  18. Active nondestructive assay of nuclear materials: principles and applications

    Energy Technology Data Exchange (ETDEWEB)

    Gozani, Tsahi

    1981-01-01

    The purpose of this book is to present, coherently and comprehensively, the wealth of available but scattered information on the principles and applications of active nondestructive analysis (ANDA). Chapters are devoted to the following: background and overview; interactions of neutrons with matter; interactions of ..gamma..-rays with matter; neutron production and sources; ..gamma..-ray production and sources; effects of neutron and ..gamma..-ray transport in bulk media; signatures of neutron- and photon-induced fissions; neutron and photon detection systems and electronics; representative ANDA systems; and instrument analysis, calibration, and measurement control for ANDA. Each chapter has an introductory section describing the relationship of the topic of that chapter to ANDA. Each chapter ends with a section that summarizes the main results and conclusions of the chapter, and a reference list.

  19. Regulating retrotransposon activity through the use of alternative transcription start sites

    DEFF Research Database (Denmark)

    Persson, Jenna; Steglich, Babett; Smialowska, Agata

    2016-01-01

    Retrotransposons, the ancestors of retroviruses, have the potential for gene disruption and genomic takeover if not kept in check. Paradoxically, although host cells repress these elements by multiple mechanisms, they are transcribed and are even activated under stress conditions. Here, we descri...... retrotransposon transcription from a nonproductive TSS allows for rapid stress-induced activation, while preventing uncontrolled transposon activity in the genome....

  20. In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila.

    Science.gov (United States)

    Lin, Shuailiang; Ewen-Campen, Ben; Ni, Xiaochun; Housden, Benjamin E; Perrimon, Norbert

    2015-10-01

    A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies. Copyright © 2015 by the Genetics Society of America.

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

    DEFF Research Database (Denmark)

    Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper

    2011-01-01

    A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized...

  2. Promoter of CaZF, a chickpea gene that positively regulates growth and stress tolerance, is activated by an AP2-family transcription factor CAP2.

    Directory of Open Access Journals (Sweden)

    Deepti Jain

    Full Text Available Plants respond to different forms of stresses by inducing transcription of a common and distinct set of genes by concerted actions of a cascade of transcription regulators. We previously reported that a gene, CaZF encoding a C2H2-zinc finger family protein from chickpea (Cicer arietinum imparted high salinity tolerance when expressed in tobacco plants. We report here that in addition to promoting tolerance against dehydration, salinity and high temperature, the CaZF overexpressing plants exhibited similar phenotype of growth and development like the plants overexpressing CAP2, encoding an AP2-family transcription factor from chickpea. To investigate any relationship between these two genes, we performed gene expression analysis in the overexpressing plants, promoter-reporter analysis and chromatin immunoprecipitation. A number of transcripts that exhibited enhanced accumulation upon expression of CAP2 or CaZF in tobacco plants were found common. Transient expression of CAP2 in chickpea leaves resulted in increased accumulation of CaZF transcript. Gel mobility shift and transient promoter-reporter assays suggested that CAP2 activates CaZF promoter by interacting with C-repeat elements (CRTs in CaZF promoter. Chromatin immunoprecipitation (ChIP assay demonstrated an in vivo interaction of CAP2 protein with CaZF promoter.

  3. Suppression of estrogen receptor transcriptional activity by connective tissue growth factor.

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    Long Cheng

    Full Text Available Secreted growth factors have been shown to stimulate the transcriptional activity of estrogen receptors (ER that are responsible for many biological processes. However, whether these growth factors physically interact with ER remains unclear. Here, we show for the first time that connective tissue growth factor (CTGF physically and functionally associates with ER. CTGF interacted with ER both in vitro and in vivo. CTGF interacted with ER DNA-binding domain. ER interaction region in CTGF was mapped to the thrombospondin type I repeat, a cell attachment motif. Overexpression of CTGF inhibited ER transcriptional activity as well as the expression of estrogen-responsive genes, including pS2 and cathepsin D. Reduction of endogenous CTGF with CTGF small interfering RNA enhanced ER transcriptional activity. The interaction between CTGF and ER is required for the repression of estrogen-responsive transcription by CTGF. Moreover, CTGF reduced ER protein expression, whereas the CTGF mutant that did not repress ER transcriptional activity also did not alter ER protein levels. The results suggested the transcriptional regulation of estrogen signaling through interaction between CTGF and ER, and thus may provide a novel mechanism by which cross-talk between secreted growth factor and ER signaling pathways occurs.

  4. Assessment of Anaerobic Toluene Biodegradation Activity by bssA Transcript/Gene Ratios

    Science.gov (United States)

    Brow, Christina N.; O'Brien Johnson, Reid; Johnson, Richard L.

    2013-01-01

    Benzylsuccinate synthase (bssA) genes associated with toluene degradation were profiled across a groundwater contaminant plume under nitrate-reducing conditions and were detected in significant numbers throughout the plume. However, differences between groundwater and core sediment samples suggested that microbial transport, rather than local activity, was the underlying cause of the high copy numbers within the downgradient plume. Both gene transcript and reactant concentrations were consistent with this hypothesis. Expression of bssA genes from denitrifying toluene degraders was induced by toluene but only in the presence of nitrate, and transcript abundance dropped rapidly following the removal of either toluene or nitrate. The drop in bssA transcripts following the removal of toluene could be described by an exponential decay function with a half-life on the order of 1 h. Interestingly, bssA transcripts never disappeared completely but were always detected at some level if either inducer was present. Therefore, the detection of transcripts alone may not be sufficient evidence for contaminant degradation. To avoid mistakenly associating basal-level gene expression with actively degrading microbial populations, an integrated approach using the ratio of functional gene transcripts to gene copies is recommended. This approach minimizes the impact of microbial transport on activity assessment and allows reliable assessments of microbial activity to be obtained from water samples. PMID:23811506

  5. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems.

    Directory of Open Access Journals (Sweden)

    Julia K Bialek

    Full Text Available CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs, act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5' long terminal repeat (LTR, for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

  6. DcE2F, a functional plant E2F-like transcriptional activator from Daucus carota

    DEFF Research Database (Denmark)

    Albani, D; Mariconti, L; Ricagno, S

    2000-01-01

    In animal cells the progression of the cell cycle through G(1)/S transition and S phase is under the control of the pRB/E2F regulatory pathway. The E2F transcription factors are key activators of genes coding for several regulatory proteins and for enzymes involved in nucleotide and DNA synthesis...... experiments using anti-DcE2F antiserum have confirmed that the DcE2F protein is a component of the carrot E2F-like nuclear activities. DNA binding assays have demonstrated that the DcE2F protein can recognize a canonical E2F cis-element in association with a mammalian DP protein. Furthermore, transactivation...

  7. PLCz functional haplotypes modulating promoter transcriptional activity are associated with semen quality traits in Chinese Holstein bulls.

    Directory of Open Access Journals (Sweden)

    Qing Pan

    Full Text Available The sperm-specific phospholipase C zeta (PLCz is a candidate sperm-borne oocyte-activating factor that triggers a characteristic series of physiological stimuli via cytoplasmic Ca(2+ oscillations during fertilization. The molecular mechanisms involved in the regulation of PLCz gene expression remain largely unknown. To explore the genetic variations in the 5'-flanking region of the PLCz gene and their common haplotypes in Chinese Holstein bulls, as well as to determine whether these variations affect bovine semen quality traits and transcriptional activity, DNA samples were collected from Chinese Holstein bulls and sequenced for the identification of genetic variants in the 5'-flanking region of PLCz. Two genetic variants were identified, and their haplotypic profiles were constructed. The two novel genetic variations (g. -456 G>A and g. +65 T>C were genotyped in 424 normal Chinese Holstein bulls. Bioinformatics analysis revealed that both loci are in transcription factor binding sites of the core promoter region. The association studies revealed that the two genetic variations and their haplotype combinations significantly affected semen quality traits. Using serially truncated constructs of the bovine PLCz promoters and the luciferase reporter, we found that a 726 bp (-641 nt to +112 nt fragment constitutes the core promoter region. Furthermore, four haplotypes, H1H1 (GTGT, H2H2 (GCGC, H3H3 (ATAT, and H4H4 (ACAC, were significantly associated with semen quality traits and successfully transfected into MLTC-1 cell lines. The luciferase reporter assay showed that the different haplotypes exhibited distinct promoter activities. Maximal promoter activity was demonstrated by the H2H2 haplotypes, as compared with the other haplotypes. To the best of our knowledge, this study is the first report on genetic variants and their respective haplotypes in the 5'-flanking region of PLCz gene that can influence the semen quality of Chinese Holstein bulls as

  8. Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs.

    Directory of Open Access Journals (Sweden)

    Finola E Moore

    Full Text Available Zinc Finger Nucleases (ZFNs made by Context-Dependent Assembly (CoDA and Transcription Activator-Like Effector Nucleases (TALENs provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

  9. New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.

    Directory of Open Access Journals (Sweden)

    Joanna Jońca

    Full Text Available Butyrylcholinesterase (BChE activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman's method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

  10. Reassessment of antioxidant activity of arbutin: multifaceted evaluation using five antioxidant assay systems.

    Science.gov (United States)

    Takebayashi, Jun; Ishii, Rie; Chen, Jianbin; Matsumoto, Teruki; Ishimi, Yoshiko; Tai, Akihiro

    2010-04-01

    Arbutin, a practically used skin-lightening agent, has been reported to possess a weak antioxidant activity compared to that of its precursor, hydroquinone. However, its antioxidant activity has not been systematically evaluated. Hence, this study reassessed its activity using five assay systems. Assays were first performed using model radicals, DPPH radical and ABTS(*+). Arbutin showed weak DPPH radical-scavenging activity compared to that of hydroquinone, but showed strong ABTS(*+)-scavenging activity. Its activity by ORAC assay was then evaluated using a physiologically relevant peroxyl radical. Arbutin exerted weak but long-lasting radical-scavenging activity and showed totally the same antioxidant activity as that of hydroquinone. Finally, it was shown that, in two cell-based antioxidant assays using erythrocytes and skin fibroblasts, arbutin exerted strong antioxidant activity comparable or even superior to that of hydroquinone. These findings indicate that the antioxidant activity of arbutin may have been under-estimated and suggest that it acts as a potent antioxidant in the skin.

  11. Real-time monitoring of PtaHMGB activity in poplar transactivation assays.

    Science.gov (United States)

    Ramos-Sánchez, José M; Triozzi, Paolo M; Moreno-Cortés, Alicia; Conde, Daniel; Perales, Mariano; Allona, Isabel

    2017-01-01

    Precise control of gene expression is essential to synchronize plant development with the environment. In perennial plants, transcriptional regulation remains poorly understood, mainly due to the long time required to perform functional studies. Transcriptional reporters based on luciferase have been useful to study circadian and diurnal regulation of gene expression, both by transcription factors and chromatin remodelers. The high mobility group proteins are considered transcriptional chaperones that also modify the chromatin architecture. They have been found in several species, presenting in some cases a circadian expression of their mRNA or protein. Transactivation experiments have been shown as a powerful and fast method to obtain information about the potential role of transcription factors upon a certain reporter. We designed and validated a luciferase transcriptional reporter using the 5' sequence upstream ATG of Populus tremula × alba LHY2 gene. We showed the robustness of this reporter line under long day and continuous light conditions. Moreover, we confirmed that pPtaLHY2::LUC activity reproduces the accumulation of PtaLHY2 mRNA. We performed transactivation studies by transient expression, using the reporter line as a genetic background, unraveling a new function of a high mobility group protein in poplar, which can activate the PtaLHY2 promoter in a gate-dependent manner. We also showed PtaHMGB2/3 needs darkness to produce that activation and exhibits an active degradation after dawn, mediated by the 26S proteasome. We generated a stable luciferase reporter poplar line based on the circadian clock gene PtaLHY2, which can be used to investigate transcriptional regulation and signal transduction pathway. Using this reporter line as a genetic background, we established a methodology to rapidly assess potential regulators of diurnal and circadian rhythms. This tool allowed us to demonstrate that PtaHMGB2/3 promotes the transcriptional activation of our

  12. A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku

    2013-09-01

    Full Text Available An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR on magnetic beads (MBs and subsequent application of cycling probe technology (CPT is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGGn-3' of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity.

  13. The C-Circle Assay for alternative-lengthening-of-telomeres activity.

    Science.gov (United States)

    Henson, Jeremy D; Lau, Loretta M; Koch, Sylvia; Martin La Rotta, Nancy; Dagg, Rebecca A; Reddel, Roger R

    2017-02-01

    The C-Circle Assay has satisfied the need for a rapid, robust and quantitative ALT assay that responds quickly to changes in ALT activity. The C-Circle Assay involves (i) extraction or simple preparation (Quick C-Circle Preparation) of the cell's DNA, which includes C-Circles (ii) amplification of the self-primed C-Circles with a rolling circle amplification reaction and (iii) sequence specific detection of the amplification products by native telomeric DNA dot blot or telomeric qPCR. Here we detail the protocols and considerations required to perform the C-Circle Assay and its controls, which include exonuclease removal of linear telomeric DNA, production of the synthetic C-Circle C96 and modulation of ALT activity by γ-irradiation. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Antioxidant activity of wines determined by a polarographic assay based on hydrogen peroxide scavenge.

    Science.gov (United States)

    Gorjanović, Stanislava Z; Novaković, Miroslav M; Potkonjak, Nebojsa I; Suznjević, Desanka Z

    2010-04-28

    Antioxidant (AO) activity of various red and white wines of different origin as well as some individual phenolic compounds present in wine has been assessed using a polarographic assay. Direct current polarography has been used to survey hydrogen peroxide scavenge (HPS) upon gradual addition of tested samples. Results expressed as reciprocal value of wine volume required for 50% decrease of anodic limiting current of hydrogen peroxide have been validated through correlation with Folin-Ciocalteau and DPPH assays. All wines exhibit HPS activity analogous with total phenolic content and DPPH scavenge. Reliability and accuracy, low cost, and rapid and direct experimental procedure open a wide area for application of this assay, making it a good alternative to standard, widely accepted AO assays.

  15. LRP16 integrates into NF-κB transcriptional complex and is required for its functional activation.

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    Zhiqiang Wu

    Full Text Available BACKGROUND: Nuclear factor κB (NF-κB-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-κB translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-κB activity inside the nucleus are still poorly understood. Being a special member of macro domain proteins, LRP16 was previously identified as a coactivator of both estrogen receptor and androgen receptor, and as an interactor of NF-κB coactivator UXT. Here, we investigated the regulatory role of LRP16 on NF-κB activation. METHODOLOGY: GST pull-down and coimmunoprecipitation (CoIP assays assessed protein-protein interactions. The functional activity of NF-κB was assessed by luciferase assays, changes in expression of its target genes, and its DNA binding ability. Annexin V staining and flow cytometry analysis were used to evaluate cell apoptosis. Immunohistochemical staining of LRP16 and enzyme-linked immunosorbent assay-based evaluation of active NF-κB were performed on primary human gastric carcinoma samples. RESULTS: We demonstrate that LRP16 integrates into NF-κB transcriptional complex through associating with its p65 component. RNA interference knockdown of the endogenous LRP16 in cells leads to impaired NF-κB activity and significantly attenuated NF-κB-dependent gene expression. Mechanistic analysis revealed that knockdown of LRP16 did not affect tumor necrosis factor α (TNF-α-induced nuclear translocation of NF-κB, but blunted the formation or stabilization of functional NF-κB/p300/CREB-binding protein transcription complex in the nucleus. In addition, knockdown of LRP16 also sensitizes cells to apoptosis induced by TNF-α. Finally, a positive link between LRP16 expression intensity in nuclei of tumor cells and NF-κB activity was preliminarily established in human gastric carcinoma specimens. CONCLUSIONS: Our findings not only

  16. An activating transcription factor of Litopenaeus vannamei involved in WSSV genes Wsv059 and Wsv166 regulation.

    Science.gov (United States)

    Li, Xiao-Yun; Yue, Hai-Tao; Zhang, Ze-Zhi; Bi, Hai-Tao; Chen, Yong-Gui; Weng, Shao-Ping; Chan, Siuming; He, Jian-Guo; Chen, Yi-Hong

    2014-12-01

    Members of activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element binding protein (ATF/CREB) family are induced by various stress signals and function as effector molecules. Consequently, cellular changes occur in response to discrete sets of instructions. In this work, we found an ATF transcription factor in Litopenaeus vannamei designated as LvATFβ. The full-length cDNA of LvATFβ was 1388 bp long with an open reading frame of 939 bp that encoded a putative 313 amino acid protein. The protein contained a basic region-leucine zipper (bZip) domain that was a common feature among ATF/CREB transcription factors. LvATFβ was highly expressed in intestines, gills, and heart. LvATFβ expression was dramatically upregulated by white spot syndrome virus (WSSV) infection. Pull-down assay revealed that LvATFβ had strong affinity to promoters of WSSV genes, namely, wsv059 and wsv166. Dual-luciferase reporter assay showed that LvATFβ could upregulate the expression of wsv059 and wsv166. Knocked down LvATFβ resulted in decreased expression of wsv059 and wsv166 in WSSV-challenged L. vannamei. Knocked down expression of wsv059 and wsv166 by RNA interference inhibited the replication and reduce the mortality of L. vannamei during WSSV challenge inoculation. The copy numbers of WSSV in wsv059 and wsv166 knocked down group were significant lower than in the control. These results suggested that LvATFβ may be involved in WSSV replication by regulating the expression of wsv059 and wsv166. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection

    Directory of Open Access Journals (Sweden)

    Yan B.

    2006-01-01

    Full Text Available We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.

  18. Physiological and Pathological Transcriptional Activation of Endogenous Retroelements Assessed by RNA-Sequencing of B Lymphocytes

    Directory of Open Access Journals (Sweden)

    Jan Attig

    2017-12-01

    Full Text Available In addition to evolutionarily-accrued sequence mutation or deletion, endogenous retroelements (EREs in eukaryotic genomes are subject to epigenetic silencing, preventing or reducing their transcription, particularly in the germplasm. Nevertheless, transcriptional activation of EREs, including endogenous retroviruses (ERVs and long interspersed nuclear elements (LINEs, is observed in somatic cells, variably upon cellular differentiation and frequently upon cellular transformation. ERE transcription is modulated during physiological and pathological immune cell activation, as well as in immune cell cancers. However, our understanding of the potential consequences of such modulation remains incomplete, partly due to the relative scarcity of information regarding genome-wide ERE transcriptional patterns in immune cells. Here, we describe a methodology that allows probing RNA-sequencing (RNA-seq data for genome-wide expression of EREs in murine and human cells. Our analysis of B cells reveals that their transcriptional response during immune activation is dominated by induction of gene transcription, and that EREs respond to a much lesser extent. The transcriptional activity of the majority of EREs is either unaffected or reduced by B cell activation both in mice and humans, albeit LINEs appear considerably more responsive in the latter host. Nevertheless, a small number of highly distinct ERVs are strongly and consistently induced during B cell activation. Importantly, this pattern contrasts starkly with B cell transformation, which exhibits widespread induction of EREs, including ERVs that minimally overlap with those responsive to immune stimulation. The distinctive patterns of ERE induction suggest different underlying mechanisms and will help separate physiological from pathological expression.

  19. Neuraminidase activity provides a practical read-out for a high throughput influenza antiviral screening assay

    Directory of Open Access Journals (Sweden)

    Wu Meng

    2008-09-01

    Full Text Available Abstract Background The emergence of influenza strains that are resistant to commonly used antivirals has highlighted the need to develop new compounds that target viral gene products or host mechanisms that are essential for effective virus replication. Existing assays to identify potential antiviral compounds often use high throughput screening assays that target specific viral replication steps. To broaden the search for antivirals, cell-based replication assays can be performed, but these are often labor intensive and have limited throughput. Results We have adapted a traditional virus neutralization assay to develop a practical, cell-based, high throughput screening assay. This assay uses viral neuraminidase (NA as a read-out to quantify influenza replication, thereby offering an assay that is both rapid and sensitive. In addition to identification of inhibitors that target either viral or host factors, the assay allows simultaneous evaluation of drug toxicity. Antiviral activity was demonstrated for a number of known influenza inhibitors including amantadine that targets the M2 ion channel, zanamivir that targets NA, ribavirin that targets IMP dehydrogenase, and bis-indolyl maleimide that targets protein kinase A/C. Amantadine-resistant strains were identified by comparing IC50 with that of the wild-type virus. Conclusion Antivirals with specificity for a broad range of targets are easily identified in an accelerated viral inhibition assay that uses NA as a read-out of replication. This assay is suitable for high throughput screening to identify potential antivirals or can be used to identify drug-resistant influenza strains.

  20. Variations of isovaline structure related to activity in the formalin foot assay in mice.

    Science.gov (United States)

    Fung, Timothy; Asiri, Yahya I; Wall, Richard; Schwarz, Stephan K W; Puil, Ernest; MacLeod, Bernard A

    2017-07-01

    Current centrally acting analgesics such as opioids are associated with adverse effects that limit their use and threaten patient safety. Isovaline is a novel prototype analgesic that produces peripheral antinociception in several pain models with little or no effect on the central nervous system. The aim of this study was to establish a preliminary structure-activity relationship for isovaline derivatives by assaying efficacy in the formalin foot assay and central adverse effect profile in mice. Selected compounds were tested using the formalin foot assay to determine efficacy in reducing formalin-induced behaviors. Of the compounds tested, R-isovaline, S-isovaline, and 1-amino-1-cyclobutanecarboxylic acid reduced nocifensive behavior in phase II of the assay. These effects occurred without affecting performance on the rotarod, indicating that the reduction in nocifensive behaviors was not due to sedation or motor incoordination. Modifications to isovaline that increased its steric size without a cyclobutane ring formation produced compounds with no activity in the formalin foot assay. These findings indicate that the conformational stability of isovaline or the ability to form a cyclobutane ring is necessary for activity in the formalin foot assay.

  1. The role of proteasome beta subunits in gastrin-mediated transcription of plasminogen activator inhibitor-2 and regenerating protein1.

    Directory of Open Access Journals (Sweden)

    Adrian O'Hara

    Full Text Available The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2 and regenerating protein 1 (Reg1. Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role.

  2. The role of proteasome beta subunits in gastrin-mediated transcription of plasminogen activator inhibitor-2 and regenerating protein1.

    Science.gov (United States)

    O'Hara, Adrian; Howarth, Alice; Varro, Andrea; Dimaline, Rod

    2013-01-01

    The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and regenerating protein 1 (Reg1). Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR) to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP) assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role.

  3. NF-Y recruits both transcription activator and repressor to modulate tissue- and developmental stage-specific expression of human γ-globin gene.

    Directory of Open Access Journals (Sweden)

    Xingguo Zhu

    Full Text Available The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.

  4. Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics

    KAUST Repository

    Roy, S.

    2015-06-27

    Classically or alternatively activated macrophages (M1 and M2, respectively) play distinct and important roles for microbiocidal activity, regulation of inflammation and tissue homeostasis. Despite this, their transcriptional regulatory dynamics are poorly understood. Using promoter-level expression profiling by non-biased deepCAGE we have studied the transcriptional dynamics of classically and alternatively activated macrophages. Transcription factor (TF) binding motif activity analysis revealed four motifs, NFKB1_REL_RELA, IRF1,2, IRF7 and TBP that are commonly activated but have distinct activity dynamics in M1 and M2 activation. We observe matching changes in the expression profiles of the corresponding TFs and show that only a restricted set of TFs change expression. There is an overall drastic and transient up-regulation in M1 and a weaker and more sustainable up-regulation in M2. Novel TFs, such as Thap6, Maff, (M1) and Hivep1, Nfil3, Prdm1, (M2) among others, were suggested to be involved in the activation processes. Additionally, 52 (M1) and 67 (M2) novel differentially expressed genes and, for the first time, several differentially expressed long non-coding RNA (lncRNA) transcriptome markers were identified. In conclusion, the finding of novel motifs, TFs and protein-coding and lncRNA genes is an important step forward to fully understand the transcriptional machinery of macrophage activation.

  5. Comparison of FilmArray Respiratory Panel and laboratory-developed real-time reverse transcription-polymerase chain reaction assays for respiratory virus detection.

    Science.gov (United States)

    Renaud, Christian; Crowley, Janet; Jerome, Keith R; Kuypers, Jane

    2012-12-01

    The FilmArray Respiratory Panel (Idaho Technology) is a highly multiplexed respiratory virus real-time polymerase chain reaction (PCR) assay. Eighty-four respiratory viruses identified by laboratory-developed real-time reverse transcription-PCR assays (LDA) or by viral cultures were mixed and tested by FilmArray to assess its performance. FilmArray identified 72 (90%) of 80 viruses also detected by LDA. Six of the 8 viruses not detected by FilmArray had PCR cycle threshold values >35. Compared to LDA, FilmArray showed comparable sensitivity when used to test serial dilutions of virus mixtures and good agreement with negative samples. With the use of 1 FilmArray instrument, 7 clinical samples could be analyzed and reported in an 8-h shift compared to 20 using LDA and 1 real-time detection instrument. While the FilmArray was rapid and easy to use, its low throughput and qualitative results may be a disadvantage in some clinical settings. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. A novel RT-qPCR assay for quantification of the MLL-MLLT3 fusion transcript in acute myeloid leukaemia

    DEFF Research Database (Denmark)

    Abildgaard, Lotte; Ommen, Hans Beier; Lausen, Birgitte Frederiksen

    2013-01-01

    of the heterogeneity of translocation break points, the MLL-MLLT3 fusion gene is a challenging target. We hypothesised that MRD monitoring using MLL-MLLT3 as a RT-qPCR marker is feasible in the majority of patients with t(9;11)-positive AML. METHODS: Using a locked nucleic acid probe, we developed a sensitive RT......-qPCR assay for quantification of the most common break point region of the MLL-MLLT3 fusion gene. Five paediatric patients with t(9;11)-positive AML were monitored using the MLL-MLLT3 assay. RESULTS: A total of 43 bone marrow (BM) and 52 Peripheral blood (PB) samples were collected from diagnosis until......OBJECTIVES: Patients with acute myeloid leukaemia (AML) of the monocytic lineage often lack molecular markers for minimal residual disease (MRD) monitoring. The MLL-MLLT3 fusion transcript found in patients with AML harbouring t(9;11) is amenable to RT-qPCR quantification but because...

  7. Comparison of the Seeplex reverse transcription PCR assay with the R-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses.

    Science.gov (United States)

    Roh, Kyoung Ho; Kim, Jeeyong; Nam, Myung-Hyun; Yoon, Sooyung; Lee, Chang Kyu; Lee, Kapno; Yoo, Young; Kim, Min Ja; Cho, Yunjung

    2008-01-01

    This study evaluated the clinical usefulness of a newly introduced multiplex reverse transcription PCR assay (Seeplex RV; Seegene, Inc., Seoul, Korea) in patients with respiratory symptoms. Fifty clinical respiratory specimens (45 from children, 5 from adults) were tested for 8 viruses (influenza virus type A and B, parainfluenza virus type 1, 2, 3, respiratory syncytial virus type A and B, and adenovirus) by Seeplex RV (S-RV) and R-mix viral culture with immunofluorescence (VC-IF). Forty (80%) of the 50 samples showed concordant results between S-RV and VC-IF; 24 of these showed the same positive and 16 showed the same negative results. Among the 10 discrepant samples, 9 were S-RV-positive and VC-IF-negative. Six were obtained in patients with lower respiratory tract infection. Only 1 sample was VC-IF-positive and S-RV-negative. This patient had pneumonia. In 3 cases, more than 1 virus was identified by S-RV. The total running time of S-RV was 6 hr, which shortens the detection time for the viral presence by 2 workdays compared to VC-IF. In conclusion, S-RV is reliable, rapid, relatively easy to perform, and able to detect more than 1 virus simultaneously. Therefore, implementation of the S-RV assay in clinical laboratories will aid rapid diagnosis and treatment of major viral infections of the respiratory tract.

  8. Transcriptional Activity of Human Endogenous Retroviruses in Human Peripheral Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Emanuela Balestrieri

    2015-01-01

    Full Text Available Human endogenous retroviruses (HERVs have been implicated in human physiology and in human pathology. A better knowledge of the retroviral transcriptional activity in the general population and during the life span would greatly help the debate on its pathologic potential. The transcriptional activity of four HERV families (H, K, W, and E was assessed, by qualitative and quantitative PCR, in PBMCs from 261 individuals aged from 1 to 80 years. Our results show that HERV-H, HERV-K, and HERV-W, but not HERV-E, are transcriptionally active in the test population already in the early childhood. In addition, the transcriptional levels of HERV-H, HERV-K, and HERV-W change significantly during the life span, albeit with distinct patterns. Our results, reinforce the hypothesis of a physiological correlation between HERVs activity and the different stages of life in humans. Studies aiming at identifying the factors, which are responsible for these changes during the individual’s life, are still needed. Although the observed phenomena are presumably subjected to great variability, the basal transcriptional activity of each individual, also depending on the different ages of life, must be carefully considered in all the studies involving HERVs as causative agents of disease.

  9. Activity-dependent transport of the transcriptional coactivator CRTC1 from synapse to nucleus.

    Science.gov (United States)

    Ch'ng, Toh Hean; Uzgil, Besim; Lin, Peter; Avliyakulov, Nuraly K; O'Dell, Thomas J; Martin, Kelsey C

    2012-07-06

    Long-lasting changes in synaptic efficacy, such as those underlying long-term memory, require transcription. Activity-dependent transport of synaptically localized transcriptional regulators provides a direct means of coupling synaptic stimulation with changes in transcription. The CREB-regulated transcriptional coactivator (CRTC1), which is required for long-term hippocampal plasticity, binds CREB to potently promote transcription. We show that CRTC1 localizes to synapses in silenced hippocampal neurons but translocates to the nucleus in response to localized synaptic stimulation. Regulated nuclear translocation occurs only in excitatory neurons and requires calcium influx and calcineurin activation. CRTC1 is controlled in a dual fashion with activity regulating CRTC1 nuclear translocation and cAMP modulating its persistence in the nucleus. Neuronal activity triggers a complex change in CRTC1 phosphorylation, suggesting that CRTC1 may link specific types of stimuli to specific changes in gene expression. Together, our results indicate that synapse-to-nuclear transport of CRTC1 dynamically informs the nucleus about synaptic activity. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Preliminary assay on the radical scavenging activity of olive wood extracts

    NARCIS (Netherlands)

    Altarejos, J.; Salido, S.; Pérez-Bonilla, M.; Linares-Palomino, P.J.; Beek, van T.A.; Nogueras, M.; Sánchez, A.

    2005-01-01

    The dichloromethane and ethanol extracts of Olea europaea wood (picual olive cultivar) were screened for antioxidant activity, determined by the DPPH free radical scavenging assay. The ethanol extract displayed potent antioxidant activity. (c) 2005 Elsevier B.V. All rights reserved.

  11. In vivo assay to identify bacteria with β-glucosidase activity

    Directory of Open Access Journals (Sweden)

    Erwin Strahsburger

    2017-11-01

    Conclusion: This in vivo β-glucosidase assay can be used as an enzymatic test on living cells without cell disruption. The method is simple, quantitative, and recommended, especially in studies screening for bacteria not only with β-glucosidase activity but also with high β-glucosidase activity.

  12. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    Energy Technology Data Exchange (ETDEWEB)

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G., E-mail: deborah.murdock@vanderbilt.edu

    2013-11-15

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

  13. WRKY transcription factors involved in activation of SA biosynthesis genes

    NARCIS (Netherlands)

    van Verk, Marcel C; Bol, John F; Linthorst, Huub J M

    2011-01-01

    Increased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR). The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA). An important step in SA biosynthesis in Arabidopsis is the

  14. Homeodomain transcription factor Phox2a, via cyclic AMP-mediated activation, induces p27Kip1 transcription, coordinating neural progenitor cell cycle exit and differentiation.

    Science.gov (United States)

    Paris, Maryline; Wang, Wen-Horng; Shin, Min-Hwa; Franklin, David S; Andrisani, Ourania M

    2006-12-01

    Mechanisms coordinating neural progenitor cell cycle exit and differentiation are incompletely understood. The cyclin-dependent kinase inhibitor p27(Kip1) is transcriptionally induced, switching specific neural progenitors from proliferation to differentiation. However, neuronal differentiation-specific transcription factors mediating p27(Kip1) transcription have not been identified. We demonstrate the homeodomain transcription factor Phox2a, required for central nervous system (CNS)- and neural crest (NC)-derived noradrenergic neuron differentiation, coordinates cell cycle exit and differentiation by inducing p27(Kip1) transcription. Phox2a transcription and activation in the CNS-derived CAD cell line and primary NC cells is mediated by combined cyclic AMP (cAMP) and bone morphogenetic protein 2 (BMP2) signaling. In the CAD cellular model, cAMP and BMP2 signaling initially induces proliferation of the undifferentiated precursors, followed by p27(Kip1) transcription, G(1) arrest, and neuronal differentiation. Small interfering RNA silencing of either Phox2a or p27(Kip1) suppresses p27(Kip1) transcription and neuronal differentiation, suggesting a causal link between p27(Kip1) expression and differentiation. Conversely, ectopic Phox2a expression via the Tet-off expression system promotes accelerated CAD cell neuronal differentiation and p27(Kip1) transcription only in the presence of cAMP signaling. Importantly, endogenous or ectopically expressed Phox2a activated by cAMP signaling binds homeodomain cis-acting elements of the p27(Kip1) promoter in vivo and mediates p27(Kip1)-luciferase expression in CAD and NC cells. We conclude that developmental cues of cAMP signaling causally link Phox2a activation with p27(Kip1) transcription, thereby coordinating neural progenitor cell cycle exit and differentiation.

  15. Dynamic mechanism for the transcription apparatus orchestrating reliable responses to activators

    National Research Council Canada - National Science Library

    Wang, Yaolai; Liu, Feng; Wang, Wei

    2012-01-01

    ... between the enhancer and the Mediator, with the concentration of activators encoded as their temporal occupancy rate (RTOR) within the space. The entry of activators into this space induces allostery in theMediator, resulting in a facilitated circumstance for transcriptional reinitiation. The reinitiation rate is much larger than the cycling rate of...

  16. Regulation of the yeast metabolic cycle by transcription factors with periodic activities

    Directory of Open Access Journals (Sweden)

    Pellegrini Matteo

    2011-10-01

    Full Text Available Abstract Background When growing budding yeast under continuous, nutrient-limited conditions, over half of yeast genes exhibit periodic expression patterns. Periodicity can also be observed in respiration, in the timing of cell division, as well as in various metabolite levels. Knowing the transcription factors involved in the yeast metabolic cycle is helpful for determining the cascade of regulatory events that cause these patterns. Results Transcription factor activities were estimated by linear regression using time series and genome-wide transcription factor binding data. Time-translation matrices were estimated using least squares and were used to model the interactions between the most significant transcription factors. The top transcription factors have functions involving respiration, cell cycle events, amino acid metabolism and glycolysis. Key regulators of transitions between phases of the yeast metabolic cycle appear to be Hap1, Hap4, Gcn4, Msn4, Swi6 and Adr1. Conclusions Analysis of the phases at which transcription factor activities peak supports previous findings suggesting that the various cellular functions occur during specific phases of the yeast metabolic cycle.

  17. FBXO3 Protein Promotes Ubiquitylation and Transcriptional Activity of AIRE (Autoimmune Regulator)*

    Science.gov (United States)

    Shao, Wei; Zumer, Kristina; Fujinaga, Koh; Peterlin, B. Matija

    2016-01-01

    The autoimmune regulator (AIRE) is a transcription factor which is expressed in medullary thymic epithelial cells. It directs the expression of otherwise tissue-specific antigens, which leads to the elimination of autoreactive T cells during development. AIRE is modified post-translationally by phosphorylation and ubiquitylation. In this report we connected these modifications. AIRE, which is phosphorylated on two specific residues near its N terminus, then binds to the F-box protein 3 (FBXO3) E3 ubiquitin ligase. In turn, this SCFFBXO3 (SKP1-CUL1-F box) complex ubiquitylates AIRE, increases its binding to the positive transcription elongation factor b (P-TEFb), and potentiates its transcriptional activity. Because P-TEFb is required for the transition from initiation to elongation of transcription, this interaction ensures proper expression of AIRE-responsive tissue-specific antigens in the thymus. PMID:27365398

  18. FBXO3 Protein Promotes Ubiquitylation and Transcriptional Activity of AIRE (Autoimmune Regulator).

    Science.gov (United States)

    Shao, Wei; Zumer, Kristina; Fujinaga, Koh; Peterlin, B Matija

    2016-08-19

    The autoimmune regulator (AIRE) is a transcription factor which is expressed in medullary thymic epithelial cells. It directs the expression of otherwise tissue-specific antigens, which leads to the elimination of autoreactive T cells during development. AIRE is modified post-translationally by phosphorylation and ubiquitylation. In this report we connected these modifications. AIRE, which is phosphorylated on two specific residues near its N terminus, then binds to the F-box protein 3 (FBXO3) E3 ubiquitin ligase. In turn, this SCF(FBXO3) (SKP1-CUL1-F box) complex ubiquitylates AIRE, increases its binding to the positive transcription elongation factor b (P-TEFb), and potentiates its transcriptional activity. Because P-TEFb is required for the transition from initiation to elongation of transcription, this interaction ensures proper expression of AIRE-responsive tissue-specific antigens in the thymus. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. The transcriptional activator LdtR from 'Candidatus Liberibacter asiaticus' mediates osmotic stress tolerance.

    Directory of Open Access Journals (Sweden)

    Fernando A Pagliai

    2014-04-01

    Full Text Available The causal agent of Huanglongbing disease, 'Candidatus Liberibacter asiaticus', is a non-culturable, gram negative, phloem-limited α-proteobacterium. Current methods to control the spread of this disease are still limited to the removal and destruction of infected trees. In this study, we identified and characterized a regulon from 'Ca. L. asiaticus' involved in cell wall remodeling, that contains a member of the MarR family of transcriptional regulators (ldtR, and a predicted L,D-transpeptidase (ldtP. In Sinorhizobium meliloti, mutation of ldtR resulted in morphological changes (shortened rod-type phenotype and reduced tolerance to osmotic stress. A biochemical approach was taken to identify small molecules that modulate LdtR activity. The LdtR ligands identified by thermal shift assays were validated using DNA binding methods. The biological impact of LdtR inactivation by the small molecules was then examined in Sinorhizobium meliloti and Liberibacter crescens, where a shortened-rod phenotype was induced by growth in presence of the ligands. A new method was also developed to examine the effects of small molecules on the viability of 'Ca. Liberibacter asiaticus', using shoots from HLB-infected orange trees. Decreased expression of ldtRLas and ldtPLas was observed in samples taken from HLB-infected shoots after 6 h of incubation with the LdtR ligands. These results provide strong proof of concept for the use of small molecules that target LdtR, as a potential treatment option for Huanglongbing disease.

  20. Anti-oxidative assays as markers for anti-inflammatory activity of flavonoids.

    Science.gov (United States)

    Chanput, Wasaporn; Krueyos, Narumol; Ritthiruangdej, Pitiporn

    2016-11-01

    The complexity of in vitro anti-inflammatory assays, the cost and time consumed, and the necessary skills can be a hurdle to apply to promising compounds in a high throughput setting. In this study, several antioxidative assays i.e. DPPH, ABTS, ORAC and xanthine oxidase (XO) were used to examine the antioxidative activity of three sub groups of flavonoids: (i) flavonol: quercetin, myricetin, (ii) flavanone: eriodictyol, naringenin (iii) flavone: luteolin, apigenin. A range of flavonoid concentrations was tested for their antioxidative activities and were found to be dose-dependent. However, the flavonoid concentrations over 50ppm were found to be toxic to the THP-1 monocytes. Therefore, 10, 20 and 50ppm of flavonoid concentrations were tested for their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated THP-1 monocytes. Expression of inflammatory genes, IL-1β, IL-6, IL-8, IL-10 and TNF-α was found to be sequentially decreased when flavonoid concentration increased. Principle component analysis (PCA) was used to investigate the relationship between the data sets of antioxidative assays and the expression of inflammatory genes. The results showed that DPPH, ABTS and ORAC assays have an opposite correlation with the reduction of inflammatory genes. Pearson correlation exhibited a relationship between the ABTS assay and the expression of three out of five analyzed genes; IL-1β, IL-6 and IL-8. Our findings indicate that ABTS assay can potentially be an assay marker for anti-inflammatory activity of flavonoids. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Transcriptional activation of multiple operons involved in para-nitrophenol degradation by Pseudomonas sp. Strain WBC-3.

    Science.gov (United States)

    Zhang, Wen-Mao; Zhang, Jun-Jie; Jiang, Xuan; Chao, Hongjun; Zhou, Ning-Yi

    2015-01-01

    Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole carbon and energy source. The genes involved in PNP degradation are organized in the following three operons: pnpA, pnpB, and pnpCDEFG. How the expression of the genes is regulated is unknown. In this study, an LysR-type transcriptional regulator (LTTR) is identified to activate the expression of the genes in response to the specific inducer PNP. While the LTTR coding gene pnpR was found to be not physically linked to any of the three catabolic operons, it was shown to be essential for the growth of strain WBC-3 on PNP. Furthermore, PnpR positively regulated its own expression, which is different from the function of classical LTTRs. A regulatory binding site (RBS) with a 17-bp imperfect palindromic sequence (GTT-N11-AAC) was identified in all pnpA, pnpB, pnpC, and pnpR promoters. Through electrophoretic mobility shift assays and mutagenic analyses, this motif was proven to be necessary for PnpR binding. This consensus motif is centered at positions approximately -55 bp relative to the four transcriptional start sites (TSSs). RBS integrity was required for both high-affinity PnpR binding and transcriptional activation of pnpA, pnpB, and pnpR. However, this integrity was essential only for high-affinity PnpR binding to the promoter of pnpCDEFG and not for its activation. Intriguingly, unlike other LTTRs studied, no changes in lengths of the PnpR binding regions of the pnpA and pnpB promoters were observed after the addition of the inducer PNP in DNase I footprinting. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Exercise induces transient transcriptional activation of the PGC-1a gene in human skeletal muscle

    DEFF Research Database (Denmark)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P. Darrell

    2003-01-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1a (PGC-1a) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell...... culture and rodent skeletal muscle. To determine whether PGC-1a transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two......-fold; P trained leg. The present data demonstrate that exercise induces a dramatic transient increase in PGC-1a transcription and mRNA content in human skeletal muscle. Consistent with its role as a transcriptional coactivator...

  3. Yap/Taz transcriptional activity in endothelial cells promotes intramembranous ossification via the BMP pathway

    Science.gov (United States)

    Uemura, Mami; Nagasawa, Ayumi; Terai, Kenta

    2016-01-01

    Osteogenesis is categorized into two groups based on developmental histology, intramembranous and endochondral ossification. The role of blood vessels during endochondral ossification is well known, while their role in intramembranous ossification, especially the intertissue pathway, is poorly understood. Here, we demonstrate endothelial Yap/Taz is a novel regulator of intramembranous ossification in zebrafish. Appropriate blood flow is required for Yap/Taz transcriptional activation in endothelial cells and intramembranous ossification. Additionally, Yap/Taz transcriptional activity in endothelial cells specifically promotes intramembranous ossification. BMP expression by Yap/Taz transactivation in endothelial cells is also identified as a bridging factor between blood vessels and intramembranous ossification. Furthermore, the expression of Runx2 in pre-osteoblast cells is a downstream target of Yap/Taz transcriptional activity in endothelial cells. Our results provide novel insight into the relationship between blood flow and ossification by demonstrating intertissue regulation. PMID:27273480

  4. Expression and Activation of STAT Transcription Factors in Breast Cancer

    Science.gov (United States)

    1998-05-08

    1993). In brea ~t cancer cells, .p-rolactin activates RAS via recruitment qf signaling proteins SHC, GRB2:apd 50S, in a fashion similar to that observed...BRCA-1 genes. In one study of families with evidence of linkage to BReA -l, the lifetime risk of breast cancer was 87% by age 70. The cumulative risk of...in Breast Cancer , 1996). Early pregnancy and oophorectomy lower the incidence of the disease, whereas late menopause and early menarche increase the

  5. Molecular characterization of a transcriptionally active Ty1/copia-like retrotransposon in Gossypium.

    Science.gov (United States)

    Cao, Yuefen; Jiang, Yurong; Ding, Mingquan; He, Shae; Zhang, Hua; Lin, Lifeng; Rong, Junkang

    2015-06-01

    A transcriptionally active Ty1/copia -like retrotransposon was identified in the genome of Gossypium barbadense. The different heat activation of this element was observed in two tetraploid cotton species. Most retrotransposons from plants are transcriptionally silent, or activated under certain conditions. Only a small portion of elements are transcriptionally active under regular condition. A long terminal repeat (LTR) retrotransposon was isolated from the cultivated Sea Island cotton (H7124) genome during the investigation of the function of a homeodomain leucine zipper gene (HD1) in trichome growth. Insertion of this element in HD1 gene of At sub-genome was related to the trichomeless stem in Gossypium barbadense. The element, named as GBRE-1, had all features of a typical Ty1/copia retrotransposon and possessed high similarity to the members of ONSEN retrotransposon family. It was 4997 bp long, comprising a single 4110 bp open reading frame, which encoded 1369 amino acids including the conserved domains of gag and pol. The expression of GBRE-1 was detected under regular condition in G. barbadense and G. hirsutum, and its expression level was increased under heat-stress condition in G. hirsutum. Besides, its expression pattern was similar to that of the ONSEN retrotransposon. Abundant cis-regulatory motifs related to stress-response and transcriptional regulation were found in the LTR sequence. These results suggested that GBRE-1 was a transcriptionally active retrotransposon in Gossypium. To our knowledge, this is the first report of the isolation of a complete Ty1/copia-type retrotransposon with present-day transcriptional activity in cotton.

  6. The GATA transcription factor ELT-2 modulates both the expression and methyltransferase activity of PRMT-1 in Caenorhabditis elegans.

    Science.gov (United States)

    Araoi, Sho; Daitoku, Hiroaki; Yokoyama, Atsuko; Kako, Koichiro; Hirota, Keiko; Fukamizu, Akiyoshi

    2018-01-18

    Protein Arginine Methyltransferase 1 (PRMT1) catalyzes asymmetric arginine dimethylation of cellular proteins and thus modulates various biological processes, including gene regulation, RNA metabolism, cell signaling and DNA repair. Since prmt-1 null mutant completely abolishes asymmetric dimethylarginine in C. elegans, PRMT-1 is thought to play a crucial role in determining levels of asymmetric arginine dimethylation. However, the mechanism underlying the regulation of PRMT-1 activity remains largely unknown. Here we explored for transcription factors that induce the expression of PRMT-1 by an RNAi screen using transgenic C. elegans harboring prmt-1 promoter upstream of gfp. Of 529 clones, we identify a GATA transcription factor elt-2 as a positive regulator of Pprmt-1::gfp expression and show that elt-2 RNAi decreases endogenous PRMT-1 expression at mRNA and protein levels. Nevertheless, surprisingly arginine methylation levels are increased when elt-2 is silenced, implying that ELT-2 may also have ability to inhibit methyltransferase activity of PRMT-1. Supporting this idea, GST pull-down and co-immunoprecipitation assays demonstrate the interaction between ELT-2 and PRMT-1. Furthermore, we find that ELT-2 interferes with PRMT-1-induced arginine methylation in a dose-dependent manner. Collectively, our results illustrate the two modes of PRMT-1 regulation, which could determine the levels of asymmetric arginine dimethylation in C. elegans. © The Authors 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  7. ADAR1 and PACT contribute to efficient translation of transcripts containing HIV-1 trans-activating response (TAR) element.

    Science.gov (United States)

    Chukwurah, Evelyn; Handy, Indhira; Patel, Rekha C

    2017-03-23

    Human immunodeficiency virus type 1 (HIV-1) has evolved various measures to counter the host cell's innate antiviral response during the course of infection. Interferon (IFN)-stimulated gene products are produced following HIV-1 infection to limit viral replication, but viral proteins and RNAs counteract their effect. One such mechanism is specifically directed against the IFN-induced Protein Kinase PKR, which is centrally important to the cellular antiviral response. In the presence of viral RNAs, PKR is activated and phosphorylates the translation initiation factor eIF2α. This shuts down the synthesis of both host and viral proteins, allowing the cell to mount an effective antiviral response. PACT (protein activator of PKR) is a cellular protein activator of PKR, primarily functioning to activate PKR in response to cellular stress. Recent studies have indicated that during HIV-1 infection, PACT's normal cellular function is compromised and that PACT is unable to activate PKR. Using various reporter systems and in vitro kinase assays, we establish in this report that interactions between PACT, ADAR1 and HIV-1-encoded Tat protein diminish the activation of PKR in response to HIV-1 infection. Our results highlight an important pathway by which HIV-1 transcripts subvert the host cell's antiviral activities to enhance their translation. © 2017 The Author(s).

  8. PEA3/ETV4-related transcription factors coupled with active ERK signalling are associated with poor prognosis in gastric adenocarcinoma

    LENUS (Irish Health Repository)

    Keld, R

    2011-06-28

    Background: Transcription factors often play important roles in tumourigenesis. Members of the PEA3 subfamily of ETS-domain transcription factors fulfil such a role and have been associated with tumour metastasis in several different cancers. Moreover, the activity of the PEA3 subfamily transcription factors is potentiated by Ras-ERK pathway signalling, which is itself often deregulated in tumour cells.\\r\

  9. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

    Directory of Open Access Journals (Sweden)

    Wiltrud Haaß

    Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

  10. Spectrophotometric assay for sensitive detection of glycerol dehydratase activity using aldehyde dehydrogenase.

    Science.gov (United States)

    Park, Eul-Soo; Park, Sunghoon; Shin, Jong-Shik

    2017-04-01

    Glycerol dehydratase (GDHt) is a pivotal enzyme for fermentative utilization of glycerol by catalyzing radical-mediated conversion of glycerol into 3-hydroxypropionaldehyde (3-HPA). Precise and sensitive monitoring of cellular GDHt activity during the fermentation process is a prerequisite for reliable metabolic analysis to afford efficient cellular engineering and process optimization. Here we report a new spectrophotometric assay for the sensitive measurement of the GDHt activity with a sub-nanomolar limit of detection (LOD). The assay method employs aldehyde dehydrogenase (ALDH) as a reporter enzyme, so the readout of the GDHt activity is recorded at 340 nm as an increase in UV absorbance which results from NADH generation accompanied by oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP). The GDHt assay was performed under the reaction conditions where the ALDH activity overwhelms the GDHt activity (i.e., 50-fold higher activity of ALDH relative to GDHt activity), affording sensitive detection of GDHt with 360 pM LOD. The ALDH-coupled assay was used to determine kinetic parameters of GDHt for glycerol, leading to KM = 0.73 ± 0.09 mM and kcat = 400 ± 20 s-1 which are in reasonable agreements with the previous reports. Our assay method allowed measurement of even a 104-fold decrease in the cellular GDHt activity during fermentative production of 3-HP, which demonstrates the detection sensitivity much higher than the previous methods. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. Negative Correlation between the Diffusion Coefficient and Transcriptional Activity of the Glucocorticoid Receptor.

    Science.gov (United States)

    Mikuni, Shintaro; Yamamoto, Johtaro; Horio, Takashi; Kinjo, Masataka

    2017-08-25

    The glucocorticoid receptor (GR) is a transcription factor, which interacts with DNA and other cofactors to regulate gene transcription. Binding to other partners in the cell nucleus alters the diffusion properties of GR. Raster image correlation spectroscopy (RICS) was applied to quantitatively characterize the diffusion properties of EGFP labeled human GR (EGFP-hGR) and its mutants in the cell nucleus. RICS is an image correlation technique that evaluates the spatial distribution of the diffusion coefficient as a diffusion map. Interestingly, we observed that the averaged diffusion coefficient of EGFP-hGR strongly and negatively correlated with its transcriptional activities in comparison to that of EGFP-hGR wild type and mutants with various transcriptional activities. This result suggests that the decreasing of the diffusion coefficient of hGR was reflected in the high-affinity binding to DNA. Moreover, the hyper-phosphorylation of hGR can enhance the transcriptional activity by reduction of the interaction between the hGR and the nuclear corepressors.

  12. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L.; Rosen, Barry P.; Tamás, Markus J.

    2015-12-28

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

  13. Gene bookmarking accelerates the kinetics of post-mitotic transcriptional re-activation.

    Science.gov (United States)

    Zhao, Rui; Nakamura, Tetsuya; Fu, Yu; Lazar, Zsolt; Spector, David L

    2011-10-09

    Although transmission of the gene expression program from mother to daughter cells has been suggested to be mediated by gene bookmarking, the precise mechanism by which bookmarking mediates post-mitotic transcriptional re-activation has been unclear. Here, we used a real-time gene expression system to quantitatively demonstrate that transcriptional activation of the same genetic locus occurs with a significantly more rapid kinetics in post-mitotic cells versus interphase cells. RNA polymerase II large subunit (Pol II) and bromodomain protein 4 (BRD4) were recruited to the locus in a different sequential order on interphase initiation versus post-mitotic re-activation resulting from the recognition by BRD4 of increased levels of histone H4 Lys 5 acetylation (H4K5ac) on the previously activated locus. BRD4 accelerated the dynamics of messenger RNA synthesis by de-compacting chromatin and hence facilitating transcriptional re-activation. Using a real-time quantitative approach, we identified differences in the kinetics of transcriptional activation between interphase and post-mitotic cells that are mediated by a chromatin-based epigenetic mechanism.

  14. Development of a novel in vitro assay for the evaluation of integron DNA integrase activity

    Directory of Open Access Journals (Sweden)

    Fatemeh Tohidi

    2016-05-01

    Full Text Available Integrons play an important role in multidrug resistance. The integron platform codes for integrase (intI that is required for gene cassette integration through site-specific recombination. The recombination crossover occurs between the G and TT nucleotides in non-palindromic attI and palindromic attC sites. The aim of this study was to establish an efficient in vitro assay for integrase purification and activity detection. To this end, the intI gene was cloned into the pET-22b plasmid. Then, the resulting recombinant plasmid was transformed into Escherichia coli Origami™ strain. The recombinant protein expression was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE and western blot assays. The recombinant intI protein was purified by nickel–nitrilotriacetic acid (Ni–NTA affinity chromatography, and its activity was measured by a newly introduced assay. Briefly, specific primers for each side of attI and attC were used, thereby, a polymerase chain reaction would be performed, if a fused plasmid containing both attI and attC sites was created upon recombination. SDS-PAGE and western blotting confirmed the presence of a 38-kDa recombinant protein. Optimum conditions were established for the measurement of the integrase activity and a new model assay was conducted to analyse the recombination activity in vitro. Although the electrophoretic mobility shift assay is an efficient and reliable method, the newly introduced assay provided new or enhanced capability to determine the integrase activity, suggesting that there is no need for expensive and advanced equipment.

  15. Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.

    Science.gov (United States)

    Nagashima, Shunta; Maruyama, Junichi; Kawano, Shodai; Iwasa, Hiroaki; Nakagawa, Kentaro; Ishigami-Yuasa, Mari; Kagechika, Hiroyuki; Nishina, Hiroshi; Hata, Yutaka

    2016-06-01

    Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor-suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation. In human cancers, the deregulation of the Hippo pathway and gene amplification enhance TAZ activity. TAZ interacts with TEA domain family members (TEAD), and upregulates genes implicated in epithelial-mesenchymal transition. It also confers stemness to cancer cells. Thus, TAZ activation provides cancer cells with malignant properties and worsens the clinical prognosis. Therefore, TAZ attracts attention as a therapeutic target in cancer therapy. We applied 18 606 small chemical compounds to human osteosarcoma U2OS cells expressing GFP-fused TAZ (GFP-TAZ), monitored the subcellular localization of GFP-TAZ, and selected 33 compounds that shifted GFP-TAZ to the cytoplasm. Unexpectedly, only a limited number of compounds suppressed TAZ-mediated enhancement of TEAD-responsive reporter activity. Moreover, the compounds that weakened TEAD reporter activity did not necessarily decrease the unphosphorylated TAZ. In this study, we focused on three compounds that decreased both TEAD reporter activity and unphosphorylated TAZ, and treated several human cancer cells with these compounds. One compound did not show a remarkable effect, whereas the other two compounds compromised the cell viability in certain cancer cells. In conclusion, the GFP-TAZ-based assay can be used as the first screening for compounds that inhibit TAZ and show anticancer properties. To develop anticancer drugs, we need additional assays to select the compounds. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  16. Krüppel-like factor 4, a novel transcription factor regulates microglial activation and subsequent neuroinflammation

    Directory of Open Access Journals (Sweden)

    Das Sulagna

    2010-10-01

    Full Text Available Abstract Background Activation of microglia, the resident macrophages of the central nervous system (CNS, is the hallmark of neuroinflammation in neurodegenerative diseases and other pathological conditions associated with CNS infection. The activation of microglia is often associated with bystander neuronal death. Nuclear factor-κB (NF-κB is one of the important transcription factors known to be associated with microglial activation which upregulates the expression of inducible nitric oxide synthase (iNOS, cyclooxygenase-2 (Cox-2 and other pro-inflammatory cytokines. Recent studies have focused on the role of Krüppel-like factor 4 (Klf4, one of the zinc-finger transcription factors, in mediating inflammation. However, these studies were limited to peripheral system and its role in CNS is not understood. Our studies focused on the possible role of Klf4 in mediating CNS inflammation. Methods For in vitro studies, mouse microglial BV-2 cell lines were treated with 500 ng/ml Salmonella enterica lipopolysacchride (LPS. Brain tissues were isolated from BALB/c mice administered with 5 mg/kg body weight of LPS. Expressions of Klf4, Cox-2, iNOS and pNF-κB were evaluated using western blotting, quantitative real time PCR, and reverse transcriptase polymerase chain reactions (RT-PCRs. Klf4 knockdown was carried out using SiRNA specific for Klf4 mRNA and luciferase assays and electromobility shift assay (EMSA were performed to study the interaction of Klf4 to iNOS promoter elements in vitro. Co-immunoprecipitation of Klf4 and pNF-κB was done in order to study a possible interaction between the two transcription factors. Results LPS stimulation increased Klf4 expression in microglial cells in a time- and dose-dependent manner. Knockdown of Klf4 resulted in decreased levels of the pro-inflammatory cytokines TNF-α, MCP-1 and IL-6, along with a significant decrease in iNOS and Cox-2 expression. NO production also decreased as a result of Klf4 knockdown

  17. Fluorescence-quenching-based homogeneous caspase-3 activity assay using photon upconversion

    Energy Technology Data Exchange (ETDEWEB)

    Vuojola, Johanna, E-mail: johanna.vuojola@utu.fi [Department of Biotechnology, University of Turku, Tykistoekatu 6A, FI-20520 Turku (Finland); Riuttamaeki, Terhi; Kulta, Essi; Arppe, Riikka; Soukka, Tero [Department of Biotechnology, University of Turku, Tykistoekatu 6A, FI-20520 Turku (Finland)

    2012-05-06

    Highlights: Black-Right-Pointing-Pointer We demonstrate the use of photon upconversion in a caspase-3 activity assay. Black-Right-Pointing-Pointer The separation-free assay uses an internally quenched substrate peptide. Black-Right-Pointing-Pointer UCPs enable simple instrumentation and total elimination of autofluorescence. Black-Right-Pointing-Pointer A sensitive assay with high signal-to-background ratios was achieved. Black-Right-Pointing-Pointer Suitable for high-throughput screening through miniaturization and white plates. - Abstract: Caspase proteases are key mediators in apoptosis and thus of great interest in pharmaceutical industry. Enzyme-activity assays are commonly employed in the screening of protease inhibitors that are potential drug candidates. Conventional homogeneous fluorescence-based assays are susceptible to autofluorescence originating from biological material. This background autofluorescence can be eliminated by using upconverting phosphors (UCPs) that emit visible light upon excitation at near-infrared. In the assay energy was transferred from a UCP-donor to a conventional fluorophore acceptor that resided at one end of a caspase-3-specific substrate peptide. Attached to the other end was a quencher molecule that was used to attenuate the acceptor emission through intramolecular energy transfer in an intact peptide. In non-inhibitory conditions the enzyme reaction separated the fluorophore from the quencher and the emission of the fluorophore was recovered. The method was applied for the detection and characterization of a known caspase-3 inhibitor Z-DEVD-FMK, and the assay gave IC{sub 50} values of approximately 13 nM for this inhibitor. We have demonstrated the applicability of UCPs on a fluorescence-quenching-based homogeneous enzyme-activity assay for the detection of caspase-3 inhibitors. The use of near-infrared excitable UCPs enables inexpensive instrumentation and total elimination of autofluorescence, while the use of an

  18. In Vivo Imaging of Nuclear Receptor Transcriptional Activity.

    Science.gov (United States)

    Dart, D Alwyn; Bevan, Charlotte L

    2016-01-01

    Nuclear receptors drive key processes during development, reproduction, metabolism, and disease. In order to understand and analyze, as well as manipulate, their actions it is imperative that we are able to study them in whole animals and in a spatiotemporal manner. The increasing repertoire of transgenic animals, expressing reporter genes driven by a specific nuclear receptor, enables us to do this. Use of luciferase reporter genes is the method of choice of many researchers as it is well tolerated, relatively easy to use, and robust. Further, luciferase lends itself to the process as it can penetrate tissue and can be manipulated to degrade rapidly thus allowing a dynamic response. However, limited resolution, lack of quantitation, and the largely two-dimensional images acquired make it desirable to support results using ex vivo imaging and enzymatic and/or immunohistochemical analysis of dissected tissue. As well as enabling the visualization of nuclear receptor signaling in wild-type animals, crossing these mouse models with models of disease will provide invaluable information on how such signaling is dysregulated during disease progression, and how we may manipulate nuclear receptor signaling in therapy. The use of in vivo imaging therefore provides the power to determine where and when in development, aging, and disease nuclear receptors are active and how ligands or receptor modulators affect this.

  19. An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria

    DEFF Research Database (Denmark)

    Rasmussen, Randi Engelberth; Erstad, Simon Matthé; Ramos Martinez, Erick Miguel

    2016-01-01

    , lysis of cyanobacterial cells is inefficient and often laborious. In some cases radioisotope-labeled substrates can be fed directly to intact cells; however, label-free assays are often favored due to safety and practical reasons. RESULTS: Here we show an easy and highly efficient method...... and subsequent activity assays were successfully adapted to the 96-well plate system. CONCLUSIONS: An easy, efficient and scalable permeabilization protocol was established for cyanobacteria. The permeabilized cells can be directly applied for measurement of G6PDH and Rubisco activities without using...... radioisotopes and the protocol may be readily adapted to studies of other cyanobacterial species and other intracellular enzymes. The permeabilization and enzyme assays can be performed in 96-well plates in a high-throughput manner....

  20. Community Composition and Transcriptional Activity of Ammonia-Oxidizing Prokaryotes of Seagrass Thalassia hemprichii in Coral Reef Ecosystems

    Directory of Open Access Journals (Sweden)

    Juan Ling

    2018-01-01

    Full Text Available Seagrasses in coral reef ecosystems play important ecological roles by enhancing coral reef resilience under ocean acidification. However, seagrass primary productivity is typically constrained by limited nitrogen availability. Ammonia oxidation is an important process conducted by ammonia-oxidizing archaea (AOA and bacteria (AOB, yet little information is available concerning the community structure and potential activity of seagrass AOA and AOB. Therefore, this study investigated the variations in the abundance, diversity and transcriptional activity of AOA and AOB at the DNA and transcript level from four sample types: the leaf, root, rhizosphere sediment and bulk sediment of seagrass Thalassia hemprichii in three coral reef ecosystems. DNA and complementary DNA (cDNA were used to prepare clone libraries and DNA and cDNA quantitative PCR (qPCR assays, targeting the ammonia monooxygenase-subunit (amoA genes as biomarkers. Our results indicated that the closest relatives of the obtained archaeal and bacterial amoA gene sequences recovered from DNA and cDNA libraries mainly originated from the marine environment. Moreover, all the obtained AOB sequences belong to the Nitrosomonadales cluster. Nearly all the AOA communities exhibited higher diversity than the AOB communities at the DNA level, but the qPCR data demonstrated that the abundances of AOB communities were higher than that of AOA communities based on both DNA and RNA transcripts. Collectively, most of the samples shared greater community composition similarity with samples from the same location rather than sample type. Furthermore, the abundance of archaeal amoA gene in rhizosphere sediments showed significant relationships with the ammonium concentration of sediments and the nitrogen content of plant tissue (leaf and root at the DNA level (P < 0.05. Conversely, no such relationships were found for the AOB communities. This work provides new insight into the nitrogen cycle

  1. Sesamol and sesame (Sesamum indicum) oil enhance macrophage cholesterol efflux via up-regulation of PPARγ1 and LXRα transcriptional activity in a MAPK-dependent manner.

    Science.gov (United States)

    Majdalawieh, Amin F; Ro, Hyo-Sung

    2015-08-01

    Cholesterol clearance by macrophages is a vital process to eliminate excess cholesterol from the body. Internalization of modified cholesterol by macrophages triggers overexpression of peroxisome proliferator-activated receptor γ1 (PPARγ1) and liver X receptor α (LXRα), two transcription factors that are critically involved in macrophage cholesterol efflux. Recent studies demonstrate that oral administration of sesamol derivative (INV-403) and sesame oil leads to a significant attenuation of atherosclerosis in Watanabe heritable hyperlipidemic rabbits and LDLR(-/-) mice, respectively. However, the exact molecular mechanisms underlying such anti-atherogenic effects remain largely unrevealed. Luciferase reporter assays were performed to assess the effects of sesamol and sesame oil on PPARγ1 and LXRα gene expression. The potential of sesamol and sesame oil to modulate cholesterol efflux was evaluated using (3)H-cholesterol efflux assays. Sesamol and sesame oil treatments lead to a significant up-regulation of PPARγ1 and LXRα expression and transcriptional activity in a MAPK-dependent manner. Importantly, primary macrophages display a significantly enhanced cholesterol efflux potential upon treatment with sesamol and sesame oil, and this stimulatory effect is mediated by MAPK signaling. Our findings suggest that the previously reported anti-atherogenic effects of sesamol and sesame oil could be attributed, at least in part, to enhanced PPARγ1 and LXRα expression and transcriptional activity leading to improved macrophage cholesterol efflux. Our study is novel in elucidating the molecular and cellular mechanisms underlying the protective effects of sesamol and sesame oil against atherosclerosis.

  2. Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters.

    Directory of Open Access Journals (Sweden)

    Jessica Marinello

    Full Text Available Topoisomerase I-DNA-cleavage complexes (Top1cc stabilized by camptothecin (CPT have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5'-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning.

  3. DRABAL: novel method to mine large high-throughput screening assays using Bayesian active learning

    KAUST Repository

    Soufan, Othman

    2016-11-10

    Background Mining high-throughput screening (HTS) assays is key for enhancing decisions in the area of drug repositioning and drug discovery. However, many challenges are encountered in the process of developing suitable and accurate methods for extracting useful information from these assays. Virtual screening and a wide variety of databases, methods and solutions proposed to-date, did not completely overcome these challenges. This study is based on a multi-label classification (MLC) technique for modeling correlations between several HTS assays, meaning that a single prediction represents a subset of assigned correlated labels instead of one label. Thus, the devised method provides an increased probability for more accurate predictions of compounds that were not tested in particular assays. Results Here we present DRABAL, a novel MLC solution that incorporates structure learning of a Bayesian network as a step to model dependency between the HTS assays. In this study, DRABAL was used to process more than 1.4 million interactions of over 400,000 compounds and analyze the existing relationships between five large HTS assays from the PubChem BioAssay Database. Compared to different MLC methods, DRABAL significantly improves the F1Score by about 22%, on average. We further illustrated usefulness and utility of DRABAL through screening FDA approved drugs and reported ones that have a high probability to interact with several targets, thus enabling drug-multi-target repositioning. Specifically DRABAL suggests the Thiabendazole drug as a common activator of the NCP1 and Rab-9A proteins, both of which are designed to identify treatment modalities for the Niemann–Pick type C disease. Conclusion We developed a novel MLC solution based on a Bayesian active learning framework to overcome the challenge of lacking fully labeled training data and exploit actual dependencies between the HTS assays. The solution is motivated by the need to model dependencies between existing

  4. DRABAL: novel method to mine large high-throughput screening assays using Bayesian active learning.

    Science.gov (United States)

    Soufan, Othman; Ba-Alawi, Wail; Afeef, Moataz; Essack, Magbubah; Kalnis, Panos; Bajic, Vladimir B

    2016-01-01

    Mining high-throughput screening (HTS) assays is key for enhancing decisions in the area of drug repositioning and drug discovery. However, many challenges are encountered in the process of developing suitable and accurate methods for extracting useful information from these assays. Virtual screening and a wide variety of databases, methods and solutions proposed to-date, did not completely overcome these challenges. This study is based on a multi-label classification (MLC) technique for modeling correlations between several HTS assays, meaning that a single prediction represents a subset of assigned correlated labels instead of one label. Thus, the devised method provides an increased probability for more accurate predictions of compounds that were not tested in particular assays. Here we present DRABAL, a novel MLC solution that incorporates structure learning of a Bayesian network as a step to model dependency between the HTS assays. In this study, DRABAL was used to process more than 1.4 million interactions of over 400,000 compounds and analyze the existing relationships between five large HTS assays from the PubChem BioAssay Database. Compared to different MLC methods, DRABAL significantly improves the F1Score by about 22%, on average. We further illustrated usefulness and utility of DRABAL through screening FDA approved drugs and reported ones that have a high probability to interact with several targets, thus enabling drug-multi-target repositioning. Specifically DRABAL suggests the Thiabendazole drug as a common activator of the NCP1 and Rab-9A proteins, both of which are designed to identify treatment modalities for the Niemann-Pick type C disease. We developed a novel MLC solution based on a Bayesian active learning framework to overcome the challenge of lacking fully labeled training data and exploit actual dependencies between the HTS assays. The solution is motivated by the need to model dependencies between existing experimental confirmatory HTS

  5. The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV).

    Science.gov (United States)

    Burian, Ján; Yim, Grace; Hsing, Michael; Axerio-Cilies, Peter; Cherkasov, Artem; Spiegelman, George B; Thompson, Charles J

    2013-12-01

    Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7's ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the -35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7-SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases.

  6. Comparative investigations of genotoxic activity of five nitriles in the comet assay and the Ames test.

    Science.gov (United States)

    Wu, Jong-C; Hseu, You C; Chen, Chin-H; Wang, Shu-H; Chen, Ssu C

    2009-09-30

    Two short-term assays, the modified Ames test and the comet assay, were carried out to evaluate the genotoxicity of five nitriles (acetonitrile, propionitrile, methacrylonitrile, butyronitrile, and benzonitrile). With the comet assay, all the nitriles studied were found to induce the genotoxicity in human lymphocytes and Hep G2 cells. Except for butyronitrile, the genotoxic potency in lymphocytes was more pronounced than that in Hep G2 cells, and the rank order of genotoxicity induced by these five nitriles in lymphocytes was different from that in Hep G2 cells, indicating that the pathways leading to genotoxicity in both types of cells were different. In the modified Ames test, no tested nitriles showed mutagenic activity on Salmonella typhimurium strain TA 98 and TA 100 with and without metabolic activation. Comparing the results obtained from both tests in this study, the comet assay seems to be more sensitive than the modified Ames test. Thus, the comet assay can be used to detect the genotoxicity of all nitriles.

  7. A novel transcription factor involved in plant defense endowed with protein phosphatase activity

    Science.gov (United States)

    Carrasco, José L.; Ancillo, Gema; Mayda, Esther; Vera, Pablo

    2003-01-01

    In plants, expression of a disease-resistance character following perception of a pathogen involves massive deployment of transcription-dependent defenses. Thus, if rapid and effective defense responses have to be achieved, it is crucial that the pathogenic signal is transduced and amplified through pre-existing signaling pathways. Reversible phosphorylation of specific transcription factors, by a concerted action of protein kinases and phosphatases, may represent a mechanism for rapid and flexible regulation of selective gene expression by environmental stimuli. Here we identified a novel DNA-binding protein from tobacco plants, designated DBP1, with protein phosphatase activity, which binds in a sequence-specific manner to a cis- acting element of a defense-related gene and participates in its transcriptional regulation. This finding helps delineate a terminal event in a signaling pathway for the selective activation of early transcription-dependent defense responses in plants, and suggests that stimulus-dependent reversible phosphorylation of regulatory proteins may occur directly in a transcription protein–DNA complex. PMID:12839999

  8. Correct usage of multiple transcription initiation sites and C/EBP-dependent transcription activation of the rat XDH/XO TATA-less promoter requires downstream elements located in the coding region of the gene.

    Science.gov (United States)

    Clark, M P; Chow, C W; Rinaldo, J E; Chalkley, R

    1998-04-01

    In the present study, we have shown that a downstream element located in the coding region of the TATA-less rat xanthine dehydrogenase/oxidase (XDH/XO) gene (-7 to +42) plays an important role in transcription initiation and C/EBP transcriptional activation. Previous work from our laboratory has shown that the promoter is organized with multiple initiator elements (Inr 1, 2, 3 and 4) which are important for transcription initiation. Additionally, we had identified two C/EBP binding sites upstream of this promoter. Deletional and mutational studies revealed that C/EBP binding was not essential for the basal level of transcriptional initation. However when XO-luciferase constructs include downstream sequence extending to +42 there is development of C/EBP sensitivity as well as a shift in the initiator usage. In the absence of the downstream element, primer extension analyses reveals Inr 3 and 4 to be the major start sites but in the presence of this additional sequence the usage is shifted to Inr 1 and 2. This shift in Inr usage more closely resembles that seen in intact macrophages or liver cells. Gel mobility shift assays indicate the presence of several binding factors located in this downstream region, one of which has been identified as YY-1. We postulate that YY-1 allows DNA bending which permits the upstream C/EBP elements to exhibit a transcriptional activation which is not seen when the downstream element is absent. This study presents a potential model for regulation of the XDH/XO promoter.

  9. Radiochemical assay for determination of dihydropyrimidinase activity using reversed-phase high-performance liquid chromatography

    NARCIS (Netherlands)

    van Kuilenburg, A. B.; van Lenthe, H.; van Gennip, A. H.

    1999-01-01

    A radiochemical assay was developed to measure the activity of dihydropyrimidinase (DHP) in human liver homogenates. The method is based on the separation of radiolabeled dihydrouracil from N-carbamyl-beta-alanine by HPLC with on-line detection of radioactivity combined with detection of 14CO2 by

  10. Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals

    DEFF Research Database (Denmark)

    Körner, Wolfgang; Vinggaard, Anne; Terouanne, B.

    2004-01-01

    ) values ranging from 1.1 x 10(-7) M to 4.7 x 10(-7) M]. In assays 2 and 3, vinclozolin showed partial androgenic activity at the highest concentrations tested. For vinclozolin, calculated antiandrogenic potencies relative to bicalutamide (RAAPs) differed no more than a factor of 10, and IC50 values...

  11. Versatility of cooperative transcriptional activation: a thermodynamical modeling analysis for greater-than-additive and less-than-additive effects.

    Directory of Open Access Journals (Sweden)

    Till D Frank

    Full Text Available We derive a statistical model of transcriptional activation using equilibrium thermodynamics of chemical reactions. We examine to what extent this statistical model predicts synergy effects of cooperative activation of gene expression. We determine parameter domains in which greater-than-additive and less-than-additive effects are predicted for cooperative regulation by two activators. We show that the statistical approach can be used to identify different causes of synergistic greater-than-additive effects: nonlinearities of the thermostatistical transcriptional machinery and three-body interactions between RNA polymerase and two activators. In particular, our model-based analysis suggests that at low transcription factor concentrations cooperative activation cannot yield synergistic greater-than-additive effects, i.e., DNA transcription can only exhibit less-than-additive effects. Accordingly, transcriptional activity turns from synergistic greater-than-additive responses at relatively high transcription factor concentrations into less-than-additive responses at relatively low concentrations. In addition, two types of re-entrant phenomena are predicted. First, our analysis predicts that under particular circumstances transcriptional activity will feature a sequence of less-than-additive, greater-than-additive, and eventually less-than-additive effects when for fixed activator concentrations the regulatory impact of activators on the binding of RNA polymerase to the promoter increases from weak, to moderate, to strong. Second, for appropriate promoter conditions when activator concentrations are increased then the aforementioned re-entrant sequence of less-than-additive, greater-than-additive, and less-than-additive effects is predicted as well. Finally, our model-based analysis suggests that even for weak activators that individually induce only negligible increases in promoter activity, promoter activity can exhibit greater

  12. Targeted deficiency of the transcriptional activator Hnf1alpha alters subnuclear positioning of its genomic targets.

    Directory of Open Access Journals (Sweden)

    Reini F Luco

    2008-05-01

    Full Text Available DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary beta-cells and hepatocytes freshly isolated from mice lacking Hnf1alpha, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3. We show that in Hnf1a-/- cells inactive endogenous Hnf1alpha-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1alpha-targets in Hnf1a-/- cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease.

  13. Cross activity of orthologous WRKY transcription factors in wheat and Arabidopsis

    NARCIS (Netherlands)

    Poietti, S.; Bertini, L.; Ent, S. van der; Leon Reyes, H.A.; Pieterse, C.M.J.; Tucci, M.; Caporale, C.; Caruso, C.

    2011-01-01

    WRKY proteins are transcription factors involved in many plant processes including plant responses to pathogens. Here, the cross activity of TaWRKY78 from the monocot wheat and AtWRKY20 from the dicot Arabidopsis on the cognate promoters of the orthologous PR4-type genes wPR4e and AtHEL of wheat and

  14. Transcription of Mammalian cis-Regulatory Elements Is Restrained by Actively Enforced Early Termination

    NARCIS (Netherlands)

    Austenaa, Liv M I; Barozzi, Iros; Simonatto, Marta; Masella, Silvia; Della Chiara, Giulia; Ghisletti, Serena; Curina, Alessia; de Wit, Elzo; Bouwman, Britta A M|info:eu-repo/dai/nl/41363745X; de Pretis, Stefano; Piccolo, Viviana; Termanini, Alberto; Prosperini, Elena; Pelizzola, Mattia; de Laat, Wouter|info:eu-repo/dai/nl/169934497; Natoli, Gioacchino

    2015-01-01

    Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adaptor protein

  15. dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases.

    Science.gov (United States)

    Duellman, Tyler; Doll, Andrea; Chen, Xi; Wakamiya, Rie; Yang, Jay

    2017-01-01

    Selective gene activation with the dCas9 (deactivated clustered regularly interspaced short palindromic repeats [CRISPR] associated protein 9)/CRISPR targeting of a transcriptional activator effector is now well established. However, the optimal targeting of guide RNA (gRNA) for a given gene is largely a matter of trial and error. We explored the optimal targeting site for tissue inhibitor of metalloproteinases (TIMPs) by first screening multiple gRNA target sites using a luciferase-based promoter-reporter system and next confirmed the effective TIMP induction in the mouse motor neuron-like neuron-enriched spinal cord 34 (NSC34) cells. Screening of many gRNAs targeting the 1-1.9 kB promoter regions of TIMP1-3 identified several hot-spots for optimal gene induction, however, no general pattern defining the optimal target site with respect to the proximity of known transcription factor binding sites or distance from the start ATG was apparent. TIMP2 with a larger basal transcriptional activity showed a greater fold-induction with gRNA compared with TIMP1 or 3 supporting the importance of an open-chromatin for best gRNA-mediated transcriptional induction. The rank order of induction potency for different gRNA identified in the promoter-reporter screening held true for the NSC34 cells. Co-activation with multiple gRNAs greatly increased the gene induction.

  16. Silver nanoclusters-based fluorescence assay of protein kinase activity and inhibition.

    Science.gov (United States)

    Shen, Congcong; Xia, Xiaodong; Hu, Shengqiang; Yang, Minghui; Wang, Jianxiu

    2015-01-06

    A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)-templated silver nanoclusters (Ag NCs). Adenosine-5'-triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates.

  17. Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity

    Directory of Open Access Journals (Sweden)

    Price Brendan D

    2001-07-01

    Full Text Available Abstract Background The p53 protein is activated by genotoxic stress, oncogene expression and during senescence, p53 transcriptionally activates genes involved in growth arrest and apoptosis. p53 activation is regulated by post-translational modification, including phosphorylation of the N-terminal transactivation domain. Here, we have examined how Glycogen Synthase Kinase (GSK3, a protein kinase involved in tumorigenesis, differentiation and apoptosis, phosphorylates and regulates p53. Results The 2 isoforms of GSK3, GSK3α and GSK3β, phosphorylate the sequence Ser-X-X-X-Ser(P when the C-terminal serine residue is already phosphorylated. Several p53 kinases were examined for their ability to create GSK3 phosphorylation sites on the p53 protein. Our results demonstrate that phosphorylation of serine 37 of p53 by DNA-PK creates a site for GSK3β phosphorylation at serine 33 in vitro. GSK3α did not phosphorylate p53 under any condition. GSK3β increased the transcriptional activity of the p53 protein in vivo. Mutation of either serine 33 or serine 37 of p53 to alanine blocked the ability of GSK3β to regulate p53 transcriptional activity. GSK3β is therefore able to regulate p53 function in vivo. p53's transcriptional activity is commonly increased by DNA damage. However, GSK3β kinase activity was inhibited in response to DNA damage, suggesting that GSK3β regulation of p53 is not involved in the p53-DNA damage response. Conclusions GSK3β can regulate p53's transcriptional activity by phosphorylating serine 33. However, GSK3β does not appear to be part of the p53-DNA damage response pathway. Instead, GSK3β may provide the link between p53 and non-DNA damage mechanisms for p53 activation.

  18. Rapid and Sensitive Detection of Norovirus Genomes in Oysters by a Two-Step Isothermal Amplification Assay System Combining Nucleic Acid Sequence-Based Amplification and Reverse Transcription-Loop-Mediated Isothermal Amplification Assays▿

    Science.gov (United States)

    Fukuda, Shinji; Sasaki, Yukie; Seno, Masato

    2008-01-01

    We developed a two-step isothermal amplification assay system, which achieved the detection of norovirus (NoV) genomes in oysters with a sensitivity similar to that of reverse transcription-seminested PCR. The time taken for the amplification of NoV genomes from RNA extracts was shortened to about 3 h. PMID:18456857

  19. Rapid and Sensitive Detection of Norovirus Genomes in Oysters by a Two-Step Isothermal Amplification Assay System Combining Nucleic Acid Sequence-Based Amplification and Reverse Transcription-Loop-Mediated Isothermal Amplification Assays▿

    OpenAIRE

    Fukuda, Shinji; Sasaki, Yukie; Seno, Masato

    2008-01-01

    We developed a two-step isothermal amplification assay system, which achieved the detection of norovirus (NoV) genomes in oysters with a sensitivity similar to that of reverse transcription-seminested PCR. The time taken for the amplification of NoV genomes from RNA extracts was shortened to about 3 h.

  20. RNAscope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma.

    Science.gov (United States)

    Wang, Hongwei; Wang, Mindy Xiao-Ming; Su, Nan; Wang, Li-Chong; Wu, Xingyong; Bui, Son; Nielsen, Allissa; Vo, Hong-Thuy; Nguyen, Nina; Luo, Yuling; Ma, Xiao-Jun

    2014-03-11

    The 'gold standard' for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current 'gold standard' method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

  1. A novel transcriptional activator, tubX, is required for the stability of Bacillus sphaericus mosquitocidal plasmid pBsph.

    Science.gov (United States)

    Ge, Yong; Zhao, Ni; Hu, Xiaomin; Shi, Tingyu; Cai, Quanxin; Yuan, Zhiming

    2014-12-01

    Stable maintenance of the low-copy-number plasmid pBsph in Bacillus sphaericus requires a partitioning (par) system that consists of a filament-forming protein, B. sphaericus TubZ (TubZ-Bs); a centromere-binding protein, TubR-Bs; and a centromere-like DNA site, tubC, composed of three blocks (I, II, and III) of 12-bp degenerate repeats. Previous studies have shown that mini-pBsph replicons encoding the TubZ system are segregationally highly unstable, whereas the native pBsph is stably maintained. However, the mechanism underlying the stability discrepancy between pBsph and its minireplicon is poorly understood. Here orf187 (encoding TubX), a gene downstream of tubZ-Bs, was found to play a role in plasmid stabilization. Null mutation or overexpression of tubX resulted in a defect in pBsph stability and a significant decrease in the level of tubRZ-Bs expression, and the TubX-null phenotype was suppressed by ectopic expression of a wild-type copy of tubX and additional tubRZ-Bs. An electrophoresis mobility shift assay (EMSA) and a DNase I footprinting assay revealed that the TubX protein bound directly to five 8-bp degenerate repeats located in the par promoter region and that TubX competed with TubR-Bs for binding to the par promoter. Further studies demonstrated that TubX significantly stimulated the transcription of the par operon in the absence of tubR-Bs, and a higher level of gene activation was observed when tubR-Bs was present. These results suggested that TubX positively regulates tubRZ-Bs transcription by interfering with TubR-Bs-mediated repression and binding directly to the tubRZ-Bs promoter region. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Detection of anti-tuberculosis activity in some folklore plants by radiometric BACTEC assay.

    Science.gov (United States)

    Gupta, V K; Shukla, C; Bisht, G R S; Saikia, D; Kumar, S; Thakur, R L

    2011-01-01

    The anti-tubercular drugs are less effective because of the emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis, so plants being an alternative source of anti-microbial compounds. The aim of this study was to investigate anti-tuberculosis potential of the plants using Mycobacterium smegmatis as a rapid screening model for detection of anti-mycobacterial activity and further to evaluate the active plants for anti-tuberculosis activity against M. tuberculosis using radiometric BACTEC assay. The 15 plants were screened for anti-mycobacterial activity against M. smegmatis by the disk diffusion assay. The ethanolic extracts of Mallotus philippensis, Vitex negundo, Colebrookea oppositifolia, Rumex hastatus, Mimosa pudica, Kalanchoe integra and Flacourtia ramontchii were active against M. smegmatis in primary screening. The anti-tuberculosis potential was identified in the leaves extracts of Mallotus philippensis by radiometric BACTEC assay. The ethanolic extract of M. philippensis showed anti-tuberculosis activity against virulent and avirulent strains of M. tuberculosis H(37) Rv and M. tuberculosis H(37) Ra with minimum inhibitory concentration 0·25 and 0·125 mg ml(-1), respectively. The inhibition in growth index values of M. tuberculosis was observed in the presence of ethyl acetate fraction at a minimum concentration of 0·05 mg ml(-1). We found that BACTEC radiometric assay is a valuable method for detection of anti-tuberculosis activity of the plant extracts. The results indicate that ethanolic extract and ethyl acetate fraction of M. philippensis exhibited significant anti-mycobacterial activity against M. tuberculosis. These findings provide scientific evidence to support the traditional medicinal uses of M. philippensis and indicate a promising potential of this plant for the development of anti-tuberculosis agent. © 2010 The Authors. Letters in Applied Microbiology © 2010 The Society for Applied

  3. Performance characteristics of the ARCHITECT Active-B12 (Holotranscobalamin) assay.

    Science.gov (United States)

    Merrigan, Stephen D; Owen, William E; Straseski, Joely A

    2015-01-01

    Vitamin B12 (cobalamin) is a necessary cofactor in methionine and succinyl-CoA metabolism. Studies estimate the deficiency prevalence as high as 30% in the elderly population. Ten to thirty percent of circulating cobalamin is bound to transcobalamin (holotranscobalamin, holoTC) which can readily enter cells and is therefore considered the bioactive form. The objective of our study was to evaluate the analytical performance of a high-throughput, automated holoTC assay (ARCHITECT i2000(SR) Active-B12 (Holotranscobalamin)) and compare it to other available methods. Manufacturer-specified limits of blank (LoB), detection (LoD), and quantitation (LoQ), imprecision, interference, and linearity were evaluated for the ARCHITECT HoloTC assay. Residual de-identified serum samples were used to compare the ARCHITECT HoloTC assay with the automated AxSYM Active-B12 (Holotranscobalamin) assay (Abbott Diagnostics) and the manual Active-B12 (Holotranscobalamin) Enzyme Immunoassay (EIA) (Axis-Shield Diagnostics, Dundee, Scotland, UK). Manufacturer's claims of LoB, LoD, LoQ, imprecision, interference, and linearity to the highest point tested (113.4 pmol/L) were verified for the ARCHITECT HoloTC assay. Method comparison of the ARCHITECT HoloTC to the AxSYM HoloTC produced the following Deming regression statistics: (ARCHITECT(HoloTc)) = 0.941 (AxSYM(HoloTC)) + 1.2 pmol/L, S(y/x) = 6.4, r = 0.947 (n = 98). Comparison to the Active-B12 EIA produced: (ARCHITECT(HoloTC)) = 1.105 (EIA(Active-B12)) - 6.8 pmol/L, S(y/x) = 11.0, r = 0.950 (n = 221). This assay performed acceptably for LoB, LoD, LoQ, imprecision, interference, linearity and method comparison to the predicate device (AxSYM). An additional comparison to a manual Active-B12 EIA method performed similarly, with minor exceptions. This study determined that the ARCHITECT HoloTC assay is suitable for routine clinical use, which provides a high-throughput alternative for automated testing of this emerging marker of cobalamin

  4. High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity

    Directory of Open Access Journals (Sweden)

    Rouet Fabien

    2009-10-01

    transcriptional activity, indicating that the controls for transcript generation and transcription are distinct, and require novel tools in order to detect changes in specific transcript quantity. Our results demonstrate that the SpliceArray™ design will provide researchers with a robust platform to detect and quantify specific changes not only in overall gene expression, but also at the individual transcript level.

  5. Oleanane triterpenoids with inhibitory activity against NFAT transcription factor from Liquidambar formosana.

    Science.gov (United States)

    Dat, Nguyen Tien; Lee, Im Seon; Cai, Xing Fu; Shen, Guanghai; Kim, Young Ho

    2004-03-01

    In a search for inhibitory components from natural products against NFAT transcription factor, this study investigated the ethyl acetate extract of the fruits of Liquidambar formosana. Four oleanane triterpenoids were isolated and identified to be liquidambaric acid, oleanolic acid, 3alpha-acetoxy-25-hydroxy-olean-12-en-28-oic acid and lantanolic acid. Of these compounds, 3alpha-acetoxy-25-hydroxy-olean-12-en-28-oic acid (IC50: 4.63 microM) and lantanolic acid (IC50: 12.62 microM) exhibited strong inhibitory activity against the NFAT transcription factor.

  6. Elk3 from hamster--a ternary complex factor with strong transcriptional repressor activity

    DEFF Research Database (Denmark)

    Hjortoe, Gertrud Malene; Weilguny, Dietmar; Willumsen, Berthe Marie

    2005-01-01

    the transcription of genes that are activated during entry into G1. We have isolated the Cricetulus griseus Elk3 gene from the Chinese hamster ovary (CHO) cell line and investigated the transcriptional potential of this factor. Transient transfections revealed that, in addition to its regulation of the c......-fos promoter, Elk3 from CHO cells seems to inhibit other promoters controlling expression of proteins involved in G1/S phase progression; Cyclin D1 and DHFR. As has been described for the Elk3 homologs Net (Mouse) and Sap-2 (Human), the results of the present study further indicate that hamster Elk3...

  7. A Synthetic Transcriptional Activator of Genes Associated with the Retina in Human Dermal Fibroblasts.

    Science.gov (United States)

    Syed, Junetha; Chandran, Anandhakumar; Pandian, Ganesh N; Taniguchi, Junichi; Sato, Shinsuke; Hashiya, Kaori; Kashiwazaki, Gengo; Bando, Toshikazu; Sugiyama, Hiroshi

    2015-07-06

    Small molecules capable of modulating epigenetic signatures can activate the transcription of tissue-restricted genes in a totally unrelated cell type and have potential use in epigenetic therapy. To provide an example for an initial approach, we report here on one synthetic small-molecule compound-termed "SAHA-PIP X"-from our library of conjugates. This compound triggered histone acetylation accompanied by the transcription of retinal-tissue-related genes in human dermal fibroblasts (HDFs). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. In Vitro Enzymatic and Cell Culture-Based Assays for Measuring Activity of HBV RNaseH Inhibitors.

    Science.gov (United States)

    Lomonosova, Elena; Tavis, John E

    2017-01-01

    HBV is a small, enveloped DNA virus that replicates by reverse transcription via an RNA intermediate. Current anti-HBV treatment regiments that include interferon α and nucleos(t)ide analogs have insufficient efficiency, are of long duration and can be accompanied by systemic side effects. Though HBV RNaseH is essential for viral replication, it is unexploited as a drug target against HBV. RNaseH inhibitors that actively block viral replication would represent an important addition to the potential new drugs for treating HBV infection. Here we describe two methods to measure the activity of RNaseH inhibitors. DNA oligonucleotide-directed RNA cleavage assay allows low-throughput screening of compounds for potential anti-HBV RNaseH activity in vitro. Analysis of preferential inhibition of plus-polarity DNA strand synthesis by HBV RNaseH inhibitors in a cell culture model of HBV replication can be used to validate the efficiency of these compounds to block viral replication.

  9. Establishing a Proteomics-Based Monocyte Assay To Assess Differential Innate Immune Activation Responses.

    Science.gov (United States)

    Tarasova, Nataliya K; Ytterberg, A Jimmy; Lundberg, Karin; Zhang, Xing-Mei; Harris, Robert A; Zubarev, Roman A

    2016-07-01

    Innate immune cells are complex systems that can be simultaneously activated in a variety of ways. Common methods currently used to estimate the response of innate immune cells to stimuli are usually biased toward a single mode of activation. The aim of this study was to assess the possibility of designing an assay based on unbiased proteome analysis that would be capable of predicting the complex response of the innate immune system to various challenges. Monocytes were used as representative cells of the innate immune system. The underlying hypothesis was that their proteome response to different activating molecules would reflect the immunogenicity of these molecules. To identify the main modes of response, we treated the human monocytic THP-1 cell line with nine different stimuli. Differentiation and activation were determined to be the two major modes of monocyte response, with PMA causing the strongest differentiation and Pam3CSK4 causing the strongest proinflammatory activation. The established assay was applied to characterize the monocyte response to epidermal growth factor peptide containing isoaspartate, which induced differentiation but not proinflammatory activation. Because of its versatility, robustness, and specificity, this new assay is likely to find a niche among the more established immunological methods.

  10. Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Benoît Couvigny

    Full Text Available The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor, we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health.

  11. Transcriptional and epigenetic signatures of zygotic genome activation during early Drosophila embryogenesis.

    Science.gov (United States)

    Darbo, Elodie; Herrmann, Carl; Lecuit, Thomas; Thieffry, Denis; van Helden, Jacques

    2013-04-05

    In all Metazoa, transcription is inactive during the first mitotic cycles after fertilisation. In Drosophila melanogaster, Zygotic Genome Activation (ZGA) occurs in two waves, starting respectively at mitotic cycles 8 (approximately 60 genes) and 14 (over a thousand genes). The regulatory mechanisms underlying these drastic transcriptional changes remain largely unknown. We developed an original gene clustering method based on discretized transition profiles, and applied it to datasets from three landmark early embryonic transcriptome studies. We identified 417 genes significantly up-regulated during ZGA. De novo motif discovery returned nine motifs over-represented in their non-coding sequences (upstream, introns, UTR), three of which correspond to previously known transcription factors: Zelda, Tramtrack and Trithorax-like (Trl). The nine discovered motifs were combined to scan ZGA-associated regions and predict about 1300 putative cis-regulatory modules. The fact that Trl is known to act as chromatin remodelling factor suggests that epigenetic regulation might play an important role in zygotic genome activation. We thus systematically compared the locations of predicted CRMs with ChIP-seq profiles for various transcription factors, 38 epigenetic marks from ModENCODE, and DNAse1 accessibility profiles. This analysis highlighted a strong and specific enrichment of predicted ZGA-associated CRMs for Zelda, CBP, Trl binding sites, as well as for histone marks associated with active enhancers (H3K4me1) and for open chromatin regions. Based on the results of our computational analyses, we suggest a temporal model explaining the onset of zygotic genome activation by the combined action of transcription factors and epigenetic signals. Although this study is mainly based on the analysis of publicly available transcriptome and ChiP-seq datasets, the resulting model suggests novel mechanisms that underly the coordinated activation of several hundreds genes at a precise

  12. Calmodulin-binding transcription activators and perspectives for applications in biotechnology.

    Science.gov (United States)

    Shen, Chenjia; Yang, Yanjun; Du, Liqun; Wang, Huizhong

    2015-12-01

    In recent years, a novel family of calmodulin-binding transcription activators (CAMTAs) has been reported in various species. The CAMTAs share a conserved domain organization, with a CG-1 DNA-binding domain, a transcription factor immunoglobulin domain, several ankyrin repeats, a calmodulin-binding domain, and a varying number of IQ motifs. CAMTAs participate in transcriptional regulation by recognizing and binding to a specific cis-element: (G/A/C)CGCG(C/G/T). Plants suffer from the environmental challenges, including abiotic and biotic stresses. Investigations in various plant species indicate a broad range of CAMTA functions involved in developmental regulation, environmental stress response, and hormone cross talk. In this review, we focus on the expression patterns and biological functions of CAMTAs to explore their probable applications in biotechnology. Furthermore, the identification and phylogenetic analysis of CAMTAs in crops could open new perspectives for enhancing stress tolerance, which could lead to improved crop production.

  13. The transcriptional regulator Aire binds to and activates super-enhancers.

    Science.gov (United States)

    Bansal, Kushagra; Yoshida, Hideyuki; Benoist, Christophe; Mathis, Diane

    2017-03-01

    Aire is a transcription factor that controls T cell tolerance by inducing the expression of a large repertoire of genes specifically in thymic stromal cells. It interacts with scores of protein partners of diverse functional classes. We found that Aire and some of its partners, notably those implicated in the DNA-damage response, preferentially localized to and activated long chromatin stretches that were overloaded with transcriptional regulators, known as super-enhancers. We also identified topoisomerase 1 as a cardinal Aire partner that colocalized on super-enhancers and was required for the interaction of Aire with all of its other associates. We propose a model that entails looping of super-enhancers to efficiently deliver Aire-containing complexes to local and distal transcriptional start sites.

  14. Tcf7l2/Tcf4 Transcriptional Repressor Function Requires HDAC Activity in the Developing Vertebrate CNS.

    Directory of Open Access Journals (Sweden)

    Hui Wang

    Full Text Available The generation of functionally distinct neuronal subtypes within the vertebrate central nervous system (CNS requires the precise regulation of progenitor gene expression in specific neuronal territories during early embryogenesis. Accumulating evidence has implicated histone deacetylase (HDAC proteins in cell specification, proliferation, and differentiation in diverse embryonic and adult tissues. However, although HDAC proteins have shown to be expressed in the developing vertebrate neural tube, their specific role in CNS neural progenitor fate specification remains unclear. Prior work from our lab showed that the Tcf7l2/Tcf4 transcription factor plays a key role in ventral progenitor lineage segregation by differential repression of two key specification factors, Nkx2.2 and Olig2. In this study, we found that administration of HDAC inhibitors (Valproic Acid (VPA, Trichostatin-A (TSA, or sodium butyrate in chick embryos in ovo disrupted normal progenitor gene segregation in the developing neural tube, indicating that HDAC activity is required for this process. Further, using functional and pharmacological approaches in vivo, we found that HDAC activity is required for the differential repression of Nkx2.2 and Olig2 by Tcf7l2/Tcf4. Finally, using dominant-negative functional assays, we provide evidence that Tcf7l2/Tcf4 repression also requires Gro/TLE/Grg co-repressor factors. Together, our data support a model where the transcriptional repressor activity of Tcf7l2/Tcf4 involves functional interactions with both HDAC and Gro/TLE/Grg co-factors at specific target gene regulatory elements in the developing neural tube, and that this activity is required for the proper segregation of the Nkx2.2 (p3 and Olig2 (pMN expressing cells from a common progenitor pool.

  15. Cardiac preconditioning with sphingosine-1-phosphate requires activation of signal transducer and activator of transcription-3

    Science.gov (United States)

    Kelly-Laubscher, Roisin F; King, Jonathan C; Hacking, Damian; Somers, Sarin; Hastie, Samantha; Stewart, Tessa; Imamdin, Aqeela; Maarman, Gerald; Pedretti, Sarah; Lecour, Sandrine

    2014-01-01

    Summary Aims Sphingosine-1-phosphate (S1P) is a cardioprotective agent. Signal transducer and activator of transcription 3 (STAT-3) is a key mediator of many cardioprotective agents. We aimed to explore whether STAT-3 is a key mediator in S1P-induced preconditioning. Methods Langendorff-perfused hearts from Wistar rats and wild-type or cardiomyocyte-specific STAT-3 knockout mice were pre-treated with S1P (10 nmol/l), with or without the STAT-3 pathway inhibitor AG490, before an ischaemia–reperfusion insult. Triphenyltetrazolium chloride and Evans blue staining were used for the determination of infarct size. Western blot analysis was carried out on the S1P pre-treated hearts for detection of cytosolic, nuclear and mitochondrial phosphorylated and total STAT-3 proteins. Results Pre-treatment with S1P decreased the infarct size in isolated rat (5 ± 3% vs control 26 ± 8%, p < 0.01) and wild-type mouse hearts (13 ± 1% vs control 33 ± 3%, p < 0.05). This protective effect was abolished in the rat hearts pre-treated with AG490 (30 ± 10%, p = ns vs control) and in the hearts from STAT-3 knockout mice (35 ± 4% vs control 30 ± 3%, p = ns). Levels of phosphorylated STAT-3 were significantly increased in both the nuclear (p < 0.05 vs control) and mitochondrial (p < 0.05 vs control) fractions in the S1P pre-treated hearts, but remained unchanged in the cytosolic fraction (p = ns vs control). Conclusion These novel results demonstrate that pharmacological preconditioning with S1P in the isolated heart is mediated by activation of mitochondrial and nuclear STAT-3, therefore suggesting that S1P may be a novel therapeutic target to modulate mitochondrial and nuclear function in cardiovascular disease in order to protect the heart against ischaemia–reperfusion. PMID:25000441

  16. Plant Compounds Enhance the Assay Sensitivity for Detection of Active Bacillus cereus Toxin

    Directory of Open Access Journals (Sweden)

    Reuven Rasooly

    2015-03-01

    Full Text Available Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety.

  17. Plant Compounds Enhance the Assay Sensitivity for Detection of Active Bacillus cereus Toxin

    Science.gov (United States)

    Rasooly, Reuven; Hernlem, Bradley; He, Xiaohua; Friedman, Mendel

    2015-01-01

    Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP) synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety. PMID:25767986

  18. Pyruvate Decarboxylase Activity Assay in situ of Different Industrial Yeast Strains

    Directory of Open Access Journals (Sweden)

    Dorota Kręgiel

    2009-01-01

    Full Text Available Cytoplasmic pyruvate decarboxylase (PDC, EC 4.1.1.1 is one of the key enzymes of yeast fermentative metabolism. PDC is the first enzyme which, under anaerobic conditions, leads to decarboxylation of pyruvate with acetaldehyde as the end product. The aim of this study is to develop a suitable method for PDC activity assay in situ for different industrial yeast strains. Saccharomyces sp. and Debaryomyces sp. yeast strains grew in fermentative medium with 12 % of glucose. Enzymatic assay was conducted in cell suspension treated with digitonin as permeabilisation agent, and with sodium pyruvate as a substrate, at temperature of 30 °C. Metabolites of PDC pathway were detected using gas chromatographic (GC technique. Various parameters like type and molar concentration of the substrate, minimal effective mass fraction of digitonin, cell concentration, reaction time and effect of pyrazole (alcohol dehydrogenase inhibitor were monitored to optimize PDC enzymatic assay in situ. In the concentration range of yeast cells from 1⋅10^7 to 1⋅10^8 per mL, linear correlation between the produced acetaldehyde and cell density was noticed. Only pyruvate was the specific substrate for pyruvate decarboxylase. In the presence of 0.05 M sodium pyruvate and 0.05 % digitonin, the enzymatic reaction was linear up to 20 min of the assay. During incubation, there was no formation of ethanol and, therefore, pyrazole was not necessary for the assay.

  19. RIT1 GTPase Regulates Sox2 Transcriptional Activity and Hippocampal Neurogenesis.

    Science.gov (United States)

    Mir, Sajad; Cai, Weikang; Andres, Douglas A

    2017-02-10

    Adult neurogenesis, the process of generating mature neurons from neuronal progenitor cells, makes critical contributions to neural circuitry and brain function in both healthy and disease states. Neurogenesis is a highly regulated process in which diverse environmental and physiological stimuli are relayed to resident neural stem cell populations to control the transcription of genes involved in self-renewal and differentiation. Understanding the molecular mechanisms governing neurogenesis is necessary for the development of translational strategies to harness this process for neuronal repair. Here we report that the Ras-related GTPase RIT1 serves to control the sequential proliferation and differentiation of adult hippocampal neural progenitor cells, with in vivo expression of active RIT1 driving robust adult neurogenesis. Gene expression profiling analysis demonstrates increased expression of a specific set of transcription factors known to govern adult neurogenesis in response to active RIT1 expression in the hippocampus, including sex-determining region Y-related HMG box 2 (Sox2), a well established regulator of stem cell self-renewal and neurogenesis. In adult hippocampal neuronal precursor cells, RIT1 controls an Akt-dependent signaling cascade, resulting in the stabilization and transcriptional activation of phosphorylated Sox2. This study supports a role for RIT1 in relaying niche-derived signals to neural/stem progenitor cells to control transcription of genes involved in self-renewal and differentiation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Identification of the GTPase-activating protein DEP domain containing 1B (DEPDC1B) as a transcriptional target of Pitx2.

    Science.gov (United States)

    Wu, Di; Zhu, Xiaoxi; Jimenez-Cowell, Kevin; Mold, Alexander J; Sollecito, Christopher C; Lombana, Nicholas; Jiao, Meng; Wei, Qize

    2015-04-10

    Pitx2 is a bicoid-related homeobox transcription factor implicated in regulating left-right patterning and organogenesis. However, only a limited number of Pitx2 downstream target genes have been identified and characterized. Here we demonstrate that Pitx2 is a transcriptional repressor of DEP domain containing 1B (DEPDC1B). The first intron of the human and mouse DEP domain containing 1B genes contains multiple consensus DNA-binding sites for Pitx2. Chromatin immunoprecipitation assays revealed that Pitx2, along with histone deacetylase 1, was recruited to the first intron of Depdc1b. In contrast, RNAi-mediated depletion of Pitx2 not only enhanced the acetylation of histone H4 in the first intron of Depdc1b, but also increased the protein level of Depdc1b. Luciferase reporter assays also showed that Pitx2 could repress the transcriptional activity mediated by the first intron of human DEPDC1B. The GAP domain of DEPDC1B interacted with nucleotide-bound forms of RAC1 in vitro. In addition, exogenous expression of DEPDC1B suppressed RAC1 activation and interfered with actin polymerization induced by the guanine nucleotide exchange factor TRIO. Moreover, DEPDC1B interacted with various signaling molecules such as U2af2, Erh, and Salm. We propose that Pitx2-mediated repression of Depdc1b expression contributes to the regulation of multiple molecular pathways, such as Rho GTPase signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. A comparative evaluation of a new automated assay for von Willebrand factor activity.

    Science.gov (United States)

    Lawrie, A S; Stufano, F; Canciani, M T; Mackie, I J; Machin, S J; Peyvandi, F

    2013-03-01

    The ristocetin cofactor assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity in the diagnosis of von Willebrand's Disease (VWD). However, the assay suffers from poor reproducibility and sensitivity at low levels of VWF and is labour intensive. We have undertaken an evaluation of a new immunoturbidimetric VWF activity (VWF:Ac) assay (INNOVANCE(®) VWF Ac. Siemens Healthcare Diagnostics, Marburg, Germany) relative to an established platelet-based VWF:RCo method. Samples from 50 healthy normal subjects, 80 patients with VWD and 50 samples that exhibited 'HIL' (i.e. Haemolysis, Icterus or Lipaemia) were studied. VWF:Ac, VWF:RCo and VWF:Ag were performed on a CS-analyser (Sysmex UK Ltd, Milton Keynes, UK), all reagents were from Siemens Healthcare Diagnostics. The VWF:Ac assay, gave low intra- and inter-assay imprecision (over a 31-day period, n = 200 replicate readings) using commercial normal (Mean 96.2 IU dL(-1), CV < 3.0%) and pathological (Mean 36.1 IU dL(-1), CV < 3.5%) control plasmas. The normal and clinical samples exhibited good correlation between VWF:RCo (range 3-753 IU dL(-1)) and VWF:Ac (rs = 0.97, P < 0.0001), with a mean bias of 5.6 IU dL(-1). Ratios of VWF:Ac and VWF:RCo to VWF:Ag in the VWD samples were comparable, although VWF:Ac had a superior lower level of detection to that of VWF:RCo (3% and 5% respectively). A subset (n = 97) of VWD and HIL samples were analysed for VWF:Ac at two different dilutions to assess the effect on relative potency, no significant difference was observed (P = 0.111). The INNOVANCE(®) VWF Ac assay was shown to be reliable and precise. © 2012 Blackwell Publishing Ltd.

  2. Hemolytic plate assay for quantification of active human complement component C3 using methylamine-treated plasma as complement source

    DEFF Research Database (Denmark)

    Ploug, M; Jessen, T E; Welinder, K. G.

    1985-01-01

    A hemolytic plate assay specific for active human complement component C3 is described. The method is well suited for tracing active C3 during preparative purification or for screening of plasma samples. The assay is based on activation of the alternative pathway of complement by unmodified rabbi...

  3. Mouse Dfa Is a Repressor of TATA-box Promoters and Interacts with the Abt1 Activator of Basal Transcription*

    Science.gov (United States)

    Brower, Christopher S.; Veiga, Lucia; Jones, Richard H.; Varshavsky, Alexander

    2010-01-01

    Our study of the mouse Ate1 arginyltransferase, a component of the N-end rule pathway, has shown that Ate1 pre-mRNA is produced from a bidirectional promoter that also expresses, in the opposite direction, a previously uncharacterized gene (Hu, R. G., Brower, C. S., Wang, H., Davydov, I. V., Sheng, J., Zhou, J., Kwon, Y. T., and Varshavsky, A. (2006) J. Biol. Chem. 281, 32559–32573). In this work, we began analyzing this gene, termed Dfa (divergent from Ate1). Mouse Dfa was found to be transcribed from both the bidirectional PAte1/Dfa promoter and other nearby promoters. The resulting transcripts are alternatively spliced, yielding a complex set of Dfa mRNAs that are present largely, although not exclusively, in the testis. A specific Dfa mRNA encodes, via its 3′-terminal exon, a 217-residue protein termed DfaA. Other Dfa mRNAs also contain this exon. DfaA is sequelogous (similar in sequence) to a region of the human/mouse HTEX4 protein, whose physiological function is unknown. We produced an affinity-purified antibody to recombinant mouse DfaA that detected a 35-kDa protein in the mouse testis and in several cell lines. Experiments in which RNA interference was used to down-regulate Dfa indicated that the 35-kDa protein was indeed DfaA. Furthermore, DfaA was present in the interchromatin granule clusters and was also found to bind to the Ggnbp1 gametogenetin-binding protein-1 and to the Abt1 activator of basal transcription that interacts with the TATA-binding protein. Given these results, RNA interference was used to probe the influence of Dfa levels in luciferase reporter assays. We found that DfaA acts as a repressor of TATA-box transcriptional promoters. PMID:20356838

  4. Mouse Dfa is a repressor of TATA-box promoters and interacts with the Abt1 activator of basal transcription.

    Science.gov (United States)

    Brower, Christopher S; Veiga, Lucia; Jones, Richard H; Varshavsky, Alexander

    2010-05-28

    Our study of the mouse Ate1 arginyltransferase, a component of the N-end rule pathway, has shown that Ate1 pre-mRNA is produced from a bidirectional promoter that also expresses, in the opposite direction, a previously uncharacterized gene (Hu, R. G., Brower, C. S., Wang, H., Davydov, I. V., Sheng, J., Zhou, J., Kwon, Y. T., and Varshavsky, A. (2006) J. Biol. Chem. 281, 32559-32573). In this work, we began analyzing this gene, termed Dfa (divergent from Ate1). Mouse Dfa was found to be transcribed from both the bidirectional P(Ate1/Dfa) promoter and other nearby promoters. The resulting transcripts are alternatively spliced, yielding a complex set of Dfa mRNAs that are present largely, although not exclusively, in the testis. A specific Dfa mRNA encodes, via its 3'-terminal exon, a 217-residue protein termed Dfa(A). Other Dfa mRNAs also contain this exon. Dfa(A) is sequelogous (similar in sequence) to a region of the human/mouse HTEX4 protein, whose physiological function is unknown. We produced an affinity-purified antibody to recombinant mouse Dfa(A) that detected a 35-kDa protein in the mouse testis and in several cell lines. Experiments in which RNA interference was used to down-regulate Dfa indicated that the 35-kDa protein was indeed Dfa(A). Furthermore, Dfa(A) was present in the interchromatin granule clusters and was also found to bind to the Ggnbp1 gametogenetin-binding protein-1 and to the Abt1 activator of basal transcription that interacts with the TATA-binding protein. Given these results, RNA interference was used to probe the influence of Dfa levels in luciferase reporter assays. We found that Dfa(A) acts as a repressor of TATA-box transcriptional promoters.

  5. Validation of a ligand-binding assay for active protein drug quantification following the 'free analyte QC concept'.

    Science.gov (United States)

    Schick, Eginhard; Staack, Roland F; Haak, Markus; Jordan, Gregor; Dahl, Uwe; Heinrich, Julia; Birnboeck, Herbert; Papadimitriou, Apollon

    2016-12-01

    Active drug assays are becoming increasingly important in protein drug development. We describe the validation of a ligand-binding assay for active protein drug quantification and address practical challenges as well as regulatory implications. A bioanalytical method for active protein drug quantification was successfully validated. Validation data prove that this method can be routinely used applying the commonly accepted acceptance criteria for ligand-binding assays. Active drug assays are a powerful tool to elucidate the pharmacokinetic/pharmacodynamic relationship as they take into consideration the influence of various matrix components, such as soluble ligand and anti-drug antibodies. However, not all aspects of the validation concept described in the guidelines for pharmacokinetic assays can be applied to active drug assays and thus regulatory guidelines should be adapted in consequence.

  6. [Resonance scattering spectral assay of papain enzymatic activity with sodium dodecyl benzene sulfonate].

    Science.gov (United States)

    Huang, Guo-xia; Jiang, Zhi-liang; Liang, Ai-hui; Deng, Ye-cheng

    2007-05-01

    In pH 6.5 phosphate buffer solutions, dodecyl benzene sulfonate (SDBS) was combined with casein to form association particles, which exhibited five Rayleigh scattering peaks at 470, 360, 400, 420 and 520 nm, respectively. Under suitable conditions, papain has catalytic effect on the hydrolysis of casein, and SDBS can stop the catalytic reaction and be combined with the excess casein to form association particles. The scattering peak at 470 nm decreased with the activity of papain. The delta I470 value was linear with the papain activity in the range of 0.048-4.8 USP x mL(-1). Its regress equation is delta I(SDBS) = 1.972c + 2.31, with a related coefficient of 0.9999 and detection limit of 0.020 USP x mL(-1). This new assay has been applied to the assay of the papain activity in food additive with satisfactory results.

  7. Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sang-pil [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Chung, Sung Woon [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Hong, Ki Whan; Kim, Chi Dae [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Bae, Sun Sik, E-mail: sunsik@pusan.ac.kr [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of)

    2010-08-13

    Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.

  8. Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes

    Directory of Open Access Journals (Sweden)

    Groscurth Peter

    2007-06-01

    Full Text Available Abstract Background Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522 were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. Results In this study we investigated generation of granulysin in lymphokine activated killer (LAK cells and antigen (Listeria specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation. Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. Conclusion This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing.

  9. Rapid Cell-Based Assay for Detection and Quantification of Active Staphylococcal Enterotoxin Type D.

    Science.gov (United States)

    Rasooly, Reuven; Do, Paula M; Hernlem, Bradley J

    2017-03-01

    Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences, including a recent outbreak in Guam affecting 300 children. Current immunology methods for SED detection cannot distinguish between the biologically active form of the toxin, which poses a threat, from the inactive form, which poses no threat. In vivo bioassays that measure emetic activity in kitten and monkeys have been used, but these methods rely upon expensive procedures using live animals and raising ethical concerns. A rapid (5 h) quantitative bioluminescence assay, using a genetically engineered T-cell Jurkat cell line expressing luciferase under regulation of nuclear factor of activated T cells response elements, in combination with the lymphoblastoid B-cell line Raji for antigen presentation, was developed. In this assay, the detection limit of biologically active SED is 100 ng/mL, which is 10 times more sensitive than the splenocyte proliferation assay, and 105 times more sensitive than monkey or kitten bioassay. Pasteurization or repeated freeze-thaw cycles had no effect on SED activity, but reduction in SED activity was shown with heat treatment at 100°C for 5 min. It was also shown that milk exhibits a protective effect on SED. This bioluminescence assay may also be used to rapidly evaluate antibodies to SED for potential therapeutic application as a measurement of neutralizing biological effects of SED. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  10. Phorate-induced oxidative stress, DNA damage and transcriptional activation of p53 and caspase genes in male Wistar rats

    Energy Technology Data Exchange (ETDEWEB)

    Saquib, Quaiser [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Attia, Sabry M. [Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh (Saudi Arabia); Siddiqui, Maqsood A. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Aboul-Soud, Mourad A.M. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Biochemistry Department, Faculty of Agriculture, Cairo University, 12613 Giza (Egypt); Al-Khedhairy, Abdulaziz A. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Giesy, John P. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Department of Biomedical and Veterinary Biosciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Canada S7N 5B3 (Canada); Zoology Department and Center for Integrative Toxicology, Michigan State University, East Lansing 48824 (United States); Musarrat, Javed, E-mail: musarratj1@yahoo.com [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Department of Microbiology, Faculty of Agricultural Sciences, AMU, Aligarh (India)

    2012-02-15

    Male Wistar rats exposed to a systemic organophosphorus insecticide, phorate [O,O-diethyl S-[(ethylthio) methyl] phosphorothioate] at varying oral doses of 0.046, 0.092 or 0.184 mg phorate/kg bw for 14 days, exhibited substantial oxidative stress, cellular DNA damage and activation of apoptosis-related p53, caspase 3 and 9 genes. The histopathological changes including the pyknotic nuclei, inflammatory leukocyte infiltrations, renal necrosis, and cardiac myofiber degeneration were observed in the liver, kidney and heart tissues. Biochemical analysis of catalase and glutathione revealed significantly lesser activities of antioxidative enzymes and lipid peroxidation in tissues of phorate exposed rats. Furthermore, generation of intracellular reactive oxygen species and reduced mitochondrial membrane potential in bone marrow cells confirmed phorate-induced oxidative stress. Significant DNA damage was measured through comet assay in terms of the Olive tail moment in bone marrow cells of treated animals as compared to control. Cell cycle analysis also demonstrated the G{sub 2}/M arrest and appearance of a distinctive SubG{sub 1} peak, which signified induction of apoptosis. Up-regulation of tumor suppressor p53 and caspase 3 and 9 genes, determined by quantitative real-time PCR and enzyme-linked immunosorbent assay, elucidated the activation of intrinsic apoptotic pathways in response to cellular stress. Overall, the results suggest that phorate induces genetic alterations and cellular toxicity, which can adversely affect the normal cellular functioning in rats. -- Highlights: ► This is the first report on molecular toxicity of phorate in an in vivo test system. ► Phorate induces biochemical and histological changes in liver, kidney and heart. ► Rats treated with phorate exhibited DNA damage in bone marrow cells. ► Phorate induces apoptosis, oxidative stress and alters mitochondrial fluorescence. ► Phorate induces transcriptional changes and enhanced

  11. Functional assay and structure-activity relationships of new third-generation P-glycoprotein inhibitors.

    Science.gov (United States)

    Müller, Henrik; Pajeva, Ilza K; Globisch, Christoph; Wiese, Michael

    2008-03-01

    Twenty-eight compounds, including 24 structurally related derivatives of tariquidar synthesized in our laboratory, and four XR compounds, reported by Xenova group Ltd, were investigated by the Hoechst 33342 and Calcein AM functional assays for estimation of their inhibitory effects on the transport activity of P-glycoprotein (P-gp). A high correlation between the effects obtained in both assays was observed at the substrate concentrations used. The analyses of kinetics data from experiments at different substrate concentrations revealed non-competitive inhibition in the Calcein AM assay and competitive inhibition in the Hoechst 33342 assay. The 3D structures of the compounds were further aligned on Hoechst 33342 using flexible and pharmacophore alignments. The results suggested that inhibitors could interact with the H-binding site of P-gp and this could potentially be achieved by different ways of binding. The best 3D-QSAR models, generated by CoMFA and CoMSIA, yielded an internal predictive squared correlation coefficient higher than 0.8 and included electrostatic, steric, hydrogen bond acceptor, and hydrophobic fields. Validation of the models on an external test set of 30 XR compounds gave predictive squared correlation coefficients of up to 0.66. An excellent correspondence between the experimental and modeled activities of the test compounds was observed. The models can be used for prediction and rational design of new P-gp inhibitors.

  12. Evaluation of antioxidant activity of crocin, podophyllotoxin and kaempferol by chemical, biochemical and electrochemical assays

    Directory of Open Access Journals (Sweden)

    Riyaz A. Dar

    2017-02-01

    Full Text Available The present study was designed to evaluate the antioxidant potential of three natural origin drugs, namely crocin, kaempferol and podophyllotoxin by chemical, biochemical and electrochemical assays. The chemical assay was carried out by DPPH and reducing power assays while the biochemical assay evaluated the lipid peroxidation inhibition capacity, using brain cells as models; the electrochemical characterization was performed by cyclic voltammetry and differential pulse voltammetry using multi-walled carbon nanotube paste electrode (MWCNTPE in 0.02 M acetate buffer (pH 4.5. The superoxide radical scavenging activity was performed at dropping mercury electrode (DME in 0.1 M KCl. All the species proved to have antioxidant activity, and particularly, by the electrochemical techniques, it has been shown that these drugs showed scavenging ability on superoxide anion produced by electrochemical reduction of oxygen. The highest scavenging property of crocin may be due to the hydroxyl and glucose moieties that could provide the necessary component as a radical scavenger.

  13. Resveratrol induces growth arrest and apoptosis through activation of FOXO transcription factors in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Qinghe Chen

    2010-12-01

    Full Text Available Resveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol.Resveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27/KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins (FOXO1-TM, FOXO3A-TM and FOXO4-TM induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and apoptosis, and inhibition of cyclin D1 by

  14. Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin.

    Science.gov (United States)

    Escobar-Ramirez, Adelma; Vercoutter-Edouart, Anne-Sophie; Mortuaire, Marlène; Huvent, Isabelle; Hardivillé, Stephan; Hoedt, Esthelle; Lefebvre, Tony; Pierce, Annick

    2015-01-01

    Delta-lactoferrin is a transcription factor, the expression of which is downregulated or silenced in case of breast cancer. It possesses antitumoral activities and when it is re-introduced in mammary epithelial cancer cell lines, provokes antiproliferative effects. It is posttranslationally modified and our earlier investigations showed that the O-GlcNAcylation/phosphorylation interplay plays a major role in the regulation of both its stability and transcriptional activity. Here, we report the covalent modification of delta-lactoferrin with the small ubiquitin-like modifier SUMO-1. Mutational and reporter gene analyses identified five different lysine residues at K13, K308, K361, K379 and K391 as SUMO acceptor sites. The SUMOylation deficient M5S mutant displayed enhanced transactivation capacity on a delta-lactoferrin responsive promoter, suggesting that SUMO-1 negatively regulates the transactivation function of delta-lactoferrin. K13, K308 and K379 are the main SUMO sites and among them, K308, which is located in a SUMOylation consensus motif of the NDSM-like type, is a key SUMO site involved in repression of delta-lactoferrin transcriptional activity. K13 and K379 are both targeted by other posttranslational modifications. We demonstrated that K13 is the main acetylation site and that favoring acetylation at K13 reduced SUMOylation and increased delta-lactoferrin transcriptional activity. K379, which is either ubiquitinated or SUMOylated, is a pivotal site for the control of delta-lactoferrin stability. We showed that SUMOylation competes with ubiquitination and protects delta-lactoferrin from degradation by positively regulating its stability. Collectively, our results indicate that multi-SUMOylation occurs on delta-lactoferrin to repress its transcriptional activity. Reciprocal occupancy of K13 by either SUMO-1 or an acetyl group may contribute to the establishment of finely regulated mechanisms to control delta-lactoferrin transcriptional activity. Moreover

  15. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

    Energy Technology Data Exchange (ETDEWEB)

    Iordanskiy, Sergey [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Van Duyne, Rachel [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Romerio, Fabio [Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Kashanchi, Fatah, E-mail: fkashanc@gmu.edu [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States)

    2015-11-15

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4{sup +} T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4{sup +} T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4{sup +} T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. - Highlights: • X-ray irradiation

  16. The silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor is required for full estrogen receptor alpha transcriptional activity.

    Science.gov (United States)

    Peterson, Theresa J; Karmakar, Sudipan; Pace, Margaret C; Gao, Tong; Smith, Carolyn L

    2007-09-01

    Multiple factors influence estrogen receptor alpha (ERalpha) transcriptional activity. Current models suggest that the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor functions within a histone deactylase-containing protein complex that binds to antiestrogen-bound ERalpha and contributes to negative regulation of gene expression. In this report, we demonstrate that SMRT is required for full agonist-dependent ERalpha activation. Chromatin immunoprecipitation assays demonstrate that SMRT, like ERalpha and the SRC-3 coactivator, is recruited to an estrogen-responsive promoter in estrogen-treated MCF-7 cells. Depletion of SMRT, but not histone deacetylases 1 or 3, negatively impacts estradiol-stimulated ERalpha transcriptional activity, while exogenous expression of SMRT's receptor interaction domains blocks ERalpha activity, indicating a functional interaction between this corepressor and agonist-bound ERalpha. Stimulation of estradiol-induced ERalpha activity by SMRT overexpression occurred in HeLa and MCF-7 cells, but not HepG2 cells, indicating that these positive effects are cell type specific. Similarly, the ability of SMRT depletion to promote the agonist activity of tamoxifen was observed for HeLa but not MCF-7 cells. Furthermore, impairment of agonist-stimulated activity by SMRT depletion is specific to ERalpha and not observed for receptors for vitamin D, androgen, or thyroid hormone. Nuclear receptor corepressor (N-CoR) depletion increased the transcriptional activity of all four tested receptors. SMRT is required for full expression of the ERalpha target genes cyclin D1, BCL-2, and progesterone receptor but not pS2, and its depletion significantly attenuated estrogen-dependent proliferation of MCF-7 cells. Taken together, these data indicate that SMRT, in conjunction with gene-specific and cell-dependent factors, is required for positively regulating agonist-dependent ERalpha transcriptional activity.

  17. RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Jong-Jin Park

    Full Text Available Clustered regularly interspaced short palindromic repeats (CRISPR and the CRISPR associated protein 9 (Cas9 system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9 offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF activation domain to dCas9 bound with the VP64 (tetramer of VP16 activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of production of anthocyanin pigment 1 (PAP1 and Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1. The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels.

  18. RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis.

    Science.gov (United States)

    Park, Jong-Jin; Dempewolf, Emma; Zhang, Wenzheng; Wang, Zeng-Yu

    2017-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9) offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF) activation domain to dCas9 bound with the VP64 (tetramer of VP16) activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of production of anthocyanin pigment 1 (PAP1) and Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1). The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels.

  19. Technical advance: transcriptional activator TGV mediates dexamethasone-inducible and tetracycline-inactivatable gene expression

    Science.gov (United States)

    Bohner; Lenk; Rieping; Herold; Gatz

    1999-07-01

    A chemically regulated gene expression system that can be switched on with dexamethasone and switched off with tetracycline was constructed. It is based on a transcriptional activator (TGV) that consists of the Tn10 encoded Tet repressor, the rat glucocorticoid receptor hormone binding domain and the transcriptional activation domain of Herpes simplex virion protein VP16. When stably expressed in transgenic tobacco plants, it mediates dexamethasone-inducible transcription from a synthetic promoter (PTop10) consisting of seven tet operators upstream of a TATA-box. Tetracycline interferes with induction by negatively regulating the DNA-binding activity of the TetR moiety of TGV. The boundaries of the expression window of the TGV-driven PTop10 reach from undetectable levels of the reporter enzyme beta-glucuronidase in the absence of dexa- methasone to induced levels reaching 15-20% of the Cauliflower Mosaic Virus 35S promoter (PCaMV35S). By modifying the sequence of PTop10, we generated a new target promoter (PTax) that is stably expressed over several generations and that can be activated to levels comparable to PCaMV35S, while yielding only slightly elevated background activities.

  20. Rat tenascin-R gene: structure, chromosome location and transcriptional activity of promoter and exon 1.

    Science.gov (United States)

    Leprini, A; Gherzi, R; Vecchi, E; Borsi, L; Zardi, L; Siri, A

    1998-01-01

    Tenascin-R is an extracellular matrix protein expressed exclusively in the central nervous system where it is thought to play a relevant role in regulating neurite outgrowth. We have i) cloned the cDNA of the rat tenascin-R 5' region; ii) defined its genomic organization, obtaining the sequence of two novel untranslated exons; iii) mapped the gene to rat chromosome 13q23 and suggested a previously unreported synteny between rat chromosome 13q23, human chromosome 1q24, and mouse chromosome 4E; and iv) sequenced and characterized the elements responsible for its neural cell-restricted transcription. We found that two discrete regions of the rat gene (the first in the proximal promoter, the second in the first exon) are independently able to activate to a high degree the transcription of a reporter gene in either human or rat neuroblastoma cell lines but not in other cell lines. Based on this observation, we re-evaluated the arrangement of transcriptionally active regions in the human tenascin-R gene we recently cloned and found that the human gene also contains an exon sequence able to initiate and sustain transcription independently of promoter sequences.

  1. The Calmodulin-Binding Transcription Activator CAMTA1 Is Required for Long-Term Memory Formation in Mice

    Science.gov (United States)

    Bas-Orth, Carlos; Tan, Yan-Wei; Oliveira, Ana M. M.; Bengtson, C. Peter; Bading, Hilmar

    2016-01-01

    The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by…

  2. Antioxidant activity by DPPH assay of potential solutions to be applied on bleached teeth.

    Science.gov (United States)

    Garcia, Eugenio José; Oldoni, Tatiane Luiza Cadorin; Alencar, Severino Matias de; Reis, Alessandra; Loguercio, Alessandro D; Grande, Rosa Helena Miranda

    2012-01-01

    The aim of this study was to assess, using the DPPH assay, the antioxidant activity of several substances that could be proposed to immediately revert the problems caused by bleaching procedures. The percentage of antioxidant activity (AA%) of 10% ascorbic acid solution (AAcidS), 10% ascorbic acid gel (AAcidG), 10% sodium ascorbate solution (SodAsS), 10% sodium ascorbate gel (SodAsG), 10% sodium bicarbonate (Bicarb), Neutralize(®) (NE), Desensibilize(®) (DES), catalase C-40 at 10 mg/mL (CAT), 10% alcohol solution of alpha-tocopherol (VitE), Listerine(®) (LIS), 0.12% chlorhexidine (CHX), Croton Lechleri (CL), 10 % aqueous solution of Uncaria Tomentosa (UT), artificial saliva (ArtS) and 0.05% sodium fluoride (NaF) was assessed in triplicate by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical assay. All substances exhibited antioxidant activity, except for CL. AAcidS, AAcidG and VitE exhibited the highest AA% (p<0.05). On the contrary, CHX, NE, LIS and NaF showed the lowest AA% (p<0.05). In conclusion, AAcidS, AAcidG, SodAsS, SodAsG and VitE presented the highest antioxidant activity among substances tested in this study. The DPPH assay provides an easy and rapid way to evaluate potential antioxidants.

  3. Serum based fluorescent assay for evaluating dipeptidyl peptidase I activity in collagen induced arthritis rat model.

    Science.gov (United States)

    Liu, Xiaoqian; Wang, Jingjing; Chu, Yi; Zhou, Xiaoying

    2017-04-01

    Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease and derived from immune granule cells. It has been suggested playing an important role in the development of rheumatoid arthritis. In this study, a coumarin based fluorescent probe (GF-AFC) was designed and synthesized to evaluate DPPI activity in serum or tissue homogenates of collagen-induced arthritis (CIA) rats, an inflammatory arthropathy model. It was revealed that the fluorescent intensity was significantly increased in a very short time after specific substrate GF-AFC reacted with the DPPI. The fluorophore (AFC) was released to shine after the cleavage reaction which was examined by 19F NMR spectroscopy. It has been shown that DPPI hydrolyzed the GF-AFC in a robust, linear, and time dependent manner at a significant high rate. A serum-based DPPI activity assay was validated by spiking and gradient dilution methods, there were no interferences or auto-fluorescence observed. The Coefficient of Variance calculated for serum-based DPPI activity assays indicates the good reproducibility. The good correlation has been seen between serum DPPI levels and the severity of arthritis during RA development in CIA rats. Our study has demonstrated a new serum based diagnostic assay for detecting DPPI activity using coumarin conjugated fluorescent (GF-AFC) as a substrate. The successful implementation of the case would provide beneficial experience in rheumatoid arthritis research. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Determining antioxidant activities of lactobacilli cell-free supernatants by cellular antioxidant assay: a comparison with traditional methods.

    Science.gov (United States)

    Xing, Jiali; Wang, Gang; Zhang, Qiuxiang; Liu, Xiaoming; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Wei

    2015-01-01

    Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA) assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs) of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS), reducing power (RP), and inhibition of linoleic acid peroxidation (ILAP). Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs.

  5. Determining antioxidant activities of lactobacilli cell-free supernatants by cellular antioxidant assay: a comparison with traditional methods.

    Directory of Open Access Journals (Sweden)

    Jiali Xing

    Full Text Available Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS, reducing power (RP, and inhibition of linoleic acid peroxidation (ILAP. Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs.

  6. A purified truncated form of yeast Gal4 expressed in Escherichia coli and used to functionalize poly(lactic acid) nanoparticle surface is transcriptionally active in cellulo.

    Science.gov (United States)

    Legaz, Sophie; Exposito, Jean-Yves; Borel, Agnès; Candusso, Marie-Pierre; Megy, Simon; Montserret, Roland; Lahaye, Vincent; Terzian, Christophe; Verrier, Bernard

    2015-09-01

    Gal4/UAS system is a powerful tool for the analysis of numerous biological processes. Gal4 is a large yeast transcription factor that activates genes including UAS sequences in their promoter. Here, we have synthesized a minimal form of Gal4 DNA sequence coding for the binding and dimerization regions, but also part of the transcriptional activation domain. This truncated Gal4 protein was expressed as inclusion bodies in Escherichia coli. A structured and active form of this recombinant protein was purified and used to cover poly(lactic acid) (PLA) nanoparticles. In cellulo, these Gal4-vehicles were able to activate the expression of a Green Fluorescent Protein (GFP) gene under the control of UAS sequences, demonstrating that the decorated Gal4 variant can be delivery into cells where it still retains its transcription factor capacities. Thus, we have produced in E. coli and purified a short active form of Gal4 that retains its functions at the surface of PLA-nanoparticles in cellular assay. These decorated Gal4-nanoparticles will be useful to decipher their tissue distribution and their potential after ingestion or injection in UAS-GFP recombinant animal models. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. EVI-1 zinc finger protein works as a transcriptional activator via binding to a consensus sequence of GACAAGATAAGATAAN1-28 CTCATCTTC.

    Science.gov (United States)

    Morishita, K; Suzukawa, K; Taki, T; Ihle, J N; Yokota, J

    1995-05-18

    Previously, the DNA-binding consensus sequences for domains 1 (GACAAGATAAGATAA) and 2 (GAAGATGAG) of the EVI-1 protein were identified using GST fusion proteins of each domain in binding and amplification reactions. We have utilized full-length EVI-1 protein to confirm these consensus sequences and determine the spacial and orientation requirements for binding. Our data demonstrate that full-length EVI-1 can independently bind the consensus sequences in gel mobility shift assays. In binding and amplification reactions only the domain 1 consensus sequence (D1-CONS) was obtained with full-length EVI-1 protein. However, by using constructs in which D1-CONS was anchored, products were obtained in which the domain 2 consensus sequence (D2-CONS) was observed with the spacing and orientation of GACAAGATAATATAAN1-28 CTCATCTTC. Using this consensus sequence we show that EVI-1 can activate transcription from reporter constructs. No transcriptional activation was seen with the reporter construct containing D1-CONS alone while activation was seen with the construct-containing D2-CONS alone. These results indicate that the EVI-1 protein works as a transcriptional activator and the binding of the domain 2 with D2-CONS is essential for its activation.

  8. The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, F.; Petrovicova, I.

    2008-01-01

    was lacking and clustering of nucleolar proteins was hampered. In conclusion, rDNA transcription is not required for targeting of rRNA processing proteins, rRNA is maternally inherited and target to rDNA independent of transcription, and de novo transcription is required for proper nucleologenesis in cattle.......The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition......, Ad 0.2 µg/ml; total transcriptional inhibition, AD 2.0 µg/ml). Late four-cell embryos were cultured to late eight-cell stage in 0.2 µg/ml AD (MTA prevention, ADLT (long-term total transcriptional inhibition group). Embryos were processed for autoradiography, transmission electron microscopy...

  9. Activation of the Escherichia coli marA/soxS/rob regulon in response to transcriptional activator concentration.

    Science.gov (United States)

    Martin, Robert G; Bartlett, Emily S; Rosner, Judah L; Wall, Michael E

    2008-07-04

    The paralogous transcriptional activators MarA, SoxS, and Rob activate a common set of promoters, the marA/soxS/rob regulon of Escherichia coli, by binding a cognate site (marbox) upstream of each promoter. The extent of activation varies from one promoter to another and is only poorly correlated with the in vitro affinity of the activator for the specific marbox. Here, we examine the dependence of promoter activation on the level of activator in vivo by manipulating the steady-state concentrations of MarA and SoxS in Lon protease mutants and by measuring promoter activation using lacZ transcriptional fusions. We found that: (i) the MarA concentrations needed for half-maximal stimulation varied by at least 19-fold among the 10 promoters tested; (ii) most marboxes were not saturated when there were 24,000 molecules of MarA per cell; (iii) the correlation between the MarA concentration needed for half-maximal promoter activity in vivo and marbox binding affinity in vitro was poor; and (iv) the two activators differed in their promoter activation profiles. The marRAB and sodA promoters could both be saturated by MarA and SoxS in vivo. However, saturation by MarA resulted in greater marRAB and lesser sodA transcription than did saturation by SoxS, implying that the two activators interact with RNA polymerase in different ways at the different promoters. Thus, the concentration and nature of activator determine which regulon promoters are activated, as well as the extent of their activation.

  10. Estrogenic and mutagenic activities of Crotalaria pallida measured by recombinant yeast assay and Ames test.

    Science.gov (United States)

    Boldrin, Paula; Resende, Flávia; Höhne, Ana; de Camargo, Mariana; Espanha, Lívia; Nogueira, Catarine; Melo, Maria; Vilegas, Wagner; Varanda, Eliana

    2013-09-04

    Crotalaria pallida Ailton is a plant belonging to the Fabaceae family, popularly known as "rattle or rattlesnake" and used in traditional medicine to treat swelling of the joints and as a vermifuge. Previous pharmacological studies have also reported anti-inflammatory, antimicrobial and antifungal activities. Nevertheless, scientific information regarding this species is scarce, and there are no reports related to its possible estrogenic and mutagenic effects. Thus, the purpose of the present study was to investigate the estrogenic potential of C. pallida leaves by means of the Recombinant Yeast Assay (RYA), seeking an alternative for estrogen replacement therapy during menopause; and to reflect on the safe use of natural products to assess the mutagenic activity of the crude extract from C. pallida leaves, the dichloromethane fraction and stigmasterol by means of the Ames test. The recombinant yeast assay with the strain BY4741 of Saccharomyces cerevisiae, was performed with the ethanolic extract, dichloromethane fraction and stigmasterol isolated from the leaves of C. pallida. Mutagenic activity was evaluated by the Salmonella/microsome assay (Ames test), using the Salmonella typhimurium tester strains TA100, TA98, TA97 and TA102, with (+S9) and without (-S9) metabolization, by the preincubation method. All samples showed estrogenic activity, mainly stigmasterol. The ethanolic extract from C. pallida leaves showed mutagenic activity in the TA98 strain (-S9), whereas dichloromethane fraction and stigmasterol were found devoid of activity. Considering the excellent estrogenic activity performed by stigmasterol in the RYA associated with the absence of mutagenic activity when evaluated by the Ames test, stigmasterol becomes a strong candidate to be used in hormone replacement therapy during menopause.

  11. Structural and functional analysis of myostatin-2 promoter alleles from the marine fish Sparus aurata: evidence for strong muscle-specific promoter activity and post-transcriptional regulation.

    Science.gov (United States)

    Nadjar-Boger, Elisabeth; Hinits, Yaniv; Funkenstein, Bruria

    2012-09-25

    Myostatin (MSTN) is a negative regulator of skeletal muscle growth. In contrast to mammals, fish possess at least two paralogs of MSTN: MSTN-1 and MSTN-2. In this study, we analyzed the structural-functional features of the four variants of Sparus aurata MSTN-2 5'-flanking region: saMSTN-2a, saMSTN-2as, saMSTN-2b and saMSTN-2c. In silico analysis revealed numerous putative cis regulatory elements including several E-boxes known as binding sites to myogenic transcription factors. Transient transfection experiments using non-muscle and muscle cell lines showed surprisingly high transcriptional activity in muscle cells, suggesting the presence of regulatory elements unique to differentiated myotubes. These observations were confirmed by in situ intramuscular injections of promoter DNA followed by reporter gene assays. Moreover, high promoter activity was found in differentiated neural cell, in agreement with MSTN-2 expression in brain. Progressive 5'-deletion analysis, using reporter gene assays, showed that the core promoter is located within the first -127 bp upstream of the ATG, and suggested the presence of regulatory elements that either repress or induce transcriptional activity. Transient transgenic zebrafish provided evidence for saMSTN-2 promoter ability to direct GFP expression to myofibers. Finally, our data shows that although no mature saMSTN-2 mRNA is observed in muscle; unspliced forms accumulate, confirming high level of transcription. In conclusion, our study shows for the first time that MSTN-2 promoter is a very robust promoter, especially in muscle cells. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  12. Modulatory role of phospholipase D in the activation of signal transducer and activator of transcription (STAT-3 by thyroid oncogenic kinase RET/PTC

    Directory of Open Access Journals (Sweden)

    Kim Dong Wook

    2008-05-01

    Full Text Available Abstract Background RET/PTC (rearranged in transformation/papillary thyroid carcinomas gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although it has been established that RET/PTC kinase plays a crucial role in intracellular signaling pathways that regulate cellular transformation, growth, and proliferation in thyroid epithelial cells, the upstream signaling that leads to the activation of RET/PTC is largely unknown. Based on the observation of high levels of PLD expression in human papillary thyroid cancer tissues, we investigated whether PLD plays a role in the regulating the RET/PTC-induced STAT3 activation. Methods Cancer tissue samples were obtained from papillary thyroid cancer patients (n = 6. The expression level of PLD was examined using immunohistochemistry and western blotting. Direct interaction between RET/PTC and PLD was analyzed by co-immunoprecipitation assay. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol, the product of PLD-mediated transphosphatidylation, in the presence of n-butanol. The transcriptional activity of STAT3 was assessed by m67 luciferase reporter assay. Results In human papillary thyroid cancer, the expression levels of PLD2 protein were higher than those in the corresponding paired normal tissues. PLD and RET/PTC could be co-immunoprecipitated from cells where each protein was over-expressed. In addition, the activation of PLD by pervanadate triggered phosphorylation of tyrosine 705 residue on STAT-3, and its phosphorylation was dramatically higher in TPC-1 cells (from papillary carcinoma that have an endogenous RET/PTC1 than in ARO cells (from anaplastic carcinoma without alteration of total STAT-3 expression. Moreover, the RET/PTC-mediated transcriptional activation of STAT-3 was synergistically increased by over-expression of PLD, whereas the PLD activity as a lipid hydrolyzing enzyme was not affected by RET

  13. O-GlcNAc modification of PPAR{gamma} reduces its transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Suena; Park, Sang Yoon [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Roth, Juergen [Department of Integrated OMICS for Biomedical Science, Graduate School, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Hoe Suk, E-mail: hoeskim@snu.ac.kr [Institute of Radiation Medicine, Medical Research Center, Seoul National University, Seoul 110-744 (Korea, Republic of); Cho, Jin Won, E-mail: chojw311@yonsei.ac.kr [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Department of Integrated OMICS for Biomedical Science, Graduate School, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer We found that PPAR{gamma} is modified by O-GlcNAc in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer The Thr54 of PPAR{gamma}1 is the major O-GlcNAc site. Black-Right-Pointing-Pointer Transcriptional activity of PPAR{gamma}1 was decreased on treatment with the OGA inhibitor. -- Abstract: The peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a member of the nuclear receptor superfamily, is a key regulator of adipogenesis and is important for the homeostasis of the adipose tissue. The {beta}-O-linked N-acetylglucosamine (O-GlcNAc) modification, a posttranslational modification on various nuclear and cytoplasmic proteins, is involved in the regulation of protein function. Here, we report that PPAR{gamma} is modified by O-GlcNAc in 3T3-L1 adipocytes. Mass spectrometric analysis and mutant studies revealed that the threonine 54 of the N-terminal AF-1 domain of PPAR{gamma} is the major O-GlcNAc site. Transcriptional activity of wild type PPAR{gamma} was decreased 30% by treatment with the specific O-GlcNAcase (OGA) inhibitor, but the T54A mutant of PPAR{gamma} did not respond to inhibitor treatment. In 3T3-L1 cells, an increase in O-GlcNAc modification by OGA inhibitor reduced PPAR{gamma} transcriptional activity and terminal adipocyte differentiation. Our results suggest that the O-GlcNAc state of PPAR{gamma} influences its transcriptional activity and is involved in adipocyte differentiation.

  14. Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity.

    Science.gov (United States)

    Andrysik, Zdenek; Galbraith, Matthew D; Guarnieri, Anna L; Zaccara, Sara; Sullivan, Kelly D; Pandey, Ahwan; MacBeth, Morgan; Inga, Alberto; Espinosa, Joaquín M

    2017-10-01

    The tumor suppressor TP53 is the most frequently mutated gene product in human cancer. Close to half of all solid tumors carry inactivating mutations in the TP53 gene, while in the remaining cases, TP53 activity is abrogated by other oncogenic events, such as hyperactivation of its endogenous repressors MDM2 or MDM4. Despite identification of hundreds of genes regulated by this transcription factor, it remains unclear which direct target genes and downstream pathways are essential for the tumor suppressive function of TP53. We set out to address this problem by generating multiple genomic data sets for three different cancer cell lines, allowing the identification of distinct sets of TP53-regulated genes, from early transcriptional targets through to late targets controlled at the translational level. We found that although TP53 elicits vastly divergent signaling cascades across cell lines, it directly activates a core transcriptional program of ∼100 genes with diverse biological functions, regardless of cell type or cellular response to TP53 activation. This core program is associated with high-occupancy TP53 enhancers, high levels of paused RNA polymerases, and accessible chromatin. Interestingly, two different shRNA screens failed to identify a single TP53 target gene required for the anti-proliferative effects of TP53 during pharmacological activation in vitro. Furthermore, bioinformatics analysis of thousands of cancer genomes revealed that none of these core target genes are frequently inactivated in tumors expressing wild-type TP53. These results support the hypothesis that TP53 activates a genetically robust transcriptional program with highly distributed tumor suppressive functions acting in diverse cellular contexts. © 2017 Andrysik et al.; Published by Cold Spring Harbor Laboratory Press.

  15. SUMOylation regulates the transcriptional repression activity of FOG-2 and its association with GATA-4.

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    José Perdomo

    Full Text Available Friend of GATA 2 (FOG-2, a co-factor of several GATA transcription factors (GATA-4, -5 and 6, is a critical regulator of coronary vessel formation and heart morphogenesis. Here we demonstrate that FOG-2 is SUMOylated and that this modification modulates its transcriptional activity. FOG-2 SUMOylation occurs at four lysine residues (K324, 471, 915, 955 [corrected]. Three of these residues are part of the characteristic SUMO consensus site (ψKXE, while K955 is found in the less frequent TKXE motif. Absence of SUMOylation did not affect FOG-2's nuclear localization. However, mutation of the FOG-2 SUMOylation sites, or de-SUMOylation, with SENP-1 or SENP-8 resulted in stronger transcriptional repression activity in both heterologous cells and cardiomyocytes. Conversely, increased FOG-2 SUMOylation by overexpression of SUMO-1 or expression of a SUMO-1-FOG-2 fusion protein rendered FOG-2 incapable of repressing GATA-4-mediated activation of the B-type natriuretic peptide (BNP promoter. Moreover, we demonstrate both increased interaction between a FOG-2 SUMO mutant and GATA-4 and enhanced SUMOylation of wild-type FOG-2 by co-expression of GATA-4. These data suggest a new dynamics in which GATA-4 may alter the activity of FOG-2 by influencing its SUMOylation status.

  16. DNA recognition by a σ(54) transcriptional activator from Aquifex aeolicus.

    Science.gov (United States)

    Vidangos, Natasha K; Heideker, Johanna; Lyubimov, Artem; Lamers, Meindert; Huo, Yixin; Pelton, Jeffrey G; Ton, Jimmy; Gralla, Jay; Berger, James; Wemmer, David E

    2014-10-23

    Transcription initiation by bacterial σ(54)-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain (DBD). The structurally characterized DBDs from activators all belong to the Fis (factor for inversion stimulation) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DBD of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ(54) activators. Two NtrC4-binding sites were identified upstream (-145 and -85bp) from the start of the lpxC gene, which is responsible for the first committed step in lipid A biosynthesis. This is the first experimental evidence for σ(54) regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145-binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homolog, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base-specific contacts contributing to affinity than for Fis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. An improved 96-well turbidity assay for T4 lysozyme activity

    Science.gov (United States)

    2015-05-13

    room temperature, according to the manufacturer’s instructions. 4. Wash resin twice with 10 bed volumes of wash buffer (30mM potassium phosphate pH...measurements for T4L. First, the 30minute time allowance between cell suspension and the start of the assay was optimal for cells to rehydrate and settle...Determination of lysozyme activities in a microplate format, Anal. Biochem. 310 (2002) 223–224. [3] D. Christybapita, M. Divyagnaneswari, R.D. Michael, Oral

  18. A high-throughput, modified ALS activity assay for Cyperus difformis and Schoenoplectus mucronatus seedlings.

    Science.gov (United States)

    Pedroso, Rafael M; Al-Khatib, Kassim; Hanson, Bradley D; Fischer, Albert J

    2017-01-01

    Cyperus difformis L. (CYPDI) and Schoenoplectus mucronatus (L.) Palla (SCHMU) are major weeds of California (CA) rice, where resistance to acetolactate synthase (ALS)-inhibitors was identified in several CYPDI and SCHMU populations that have also evolved resistance to photosystem II (PSII)-inhibiting herbicides. The mechanism of ALS resistance in these populations remains to be clarified but this information is crucial in a weed management program, especially in a scenario where resistance to multiple herbicides has been identified. ALS activity assays are commonly used to diagnose resistance to ALS-inhibitors, but protocols currently available are burdensome for the study of CYPDI and SCHMU, as they require large amounts of plant material from young seedlings and have low yields. Our objective was to investigate the ALS resistance mechanism in suspected ALS-resistant (R) CYPDI and SCHMU biotypes using a modified ALS activity assay that requires less plant material. ALS enzymes from suspected R biotypes were at least 10,000-fold less sensitive to bensulfuron-methyl than susceptible (S) cohorts, indicating ALS resistance that is likely due to an altered target-site. Protein concentration (mgg(-1) tissue) did not differ between R and S biotypes within each species, suggesting that R biotypes do not over produce ALS enzymes. CYPDI biotypes had up to 4-fold more protein per mg of tissue than SCHMU biotypes, but up to 7-fold more acetoin per mg(-1) protein was quantified in SCHMU, suggesting greater ALS catalytic ability in SCHMU biotypes, regardless of their herbicide resistance status. Our optimized protocol to measure ALS activity allowed for up to a 3-fold increase in the number of assays performed per g of leaf tissue. The modified assay may be useful for measuring ALS activity in other weed species that also produce small amount of foliage in early growth stages when protein in tissue is most abundant. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Activation of the Nrf2 Pathway by Inorganic Arsenic in Human Hepatocytes and the Role of Transcriptional Repressor Bach1

    Directory of Open Access Journals (Sweden)

    Dan Liu

    2013-01-01

    Full Text Available Previous studies have proved that the environmental toxicant, inorganic arsenic, activates nuclear factor erythroid 2-related factor 2 (Nrf2 pathway in many different cell types. This study tried to explore the hepatic Nrf2 pathway upon arsenic treatment comprehensively, since liver is one of the major target organs of arsenical toxicity. Our results showed that inorganic arsenic significantly induced Nrf2 protein and mRNA expression in Chang human hepatocytes. We also observed a dose-dependent increase of antioxidant response element- (ARE- luciferase activity. Both the mRNA and protein levels of NAD(PH:quinone oxidoreductase 1 (NQO1 and heme oxygenase-1 (HO-1 were all upregulated dramatically. On the other hand, entry and accumulation of Nrf2 protein in the nucleus, while exportting the transcriptional repressor BTB and CNC homology 1 (Bach1 from nucleus to cytoplasm, were also confirmed by western blot and immunofluorescence assay. Our results therefore confirmed the arsenic-induced Nrf2 pathway activation in hepatocytes and also suggested that the translocation of Bach1 was associated with the regulation of Nrf2 pathway by arsenic. Hepatic Nrf2 pathway plays indispensable roles for cellular defenses against arsenic hepatotoxicity, and the interplay of Bach1 and Nrf2 may be helpful to understand the self-defensive responses and the diverse biological effects of arsenicals.

  20. Activation of the Nrf2 Pathway by Inorganic Arsenic in Human Hepatocytes and the Role of Transcriptional Repressor Bach1

    Science.gov (United States)

    Liu, Dan; Duan, Xiaoxu; Dong, Dandan; Bai, Caijun; Li, Xin; Sun, Guifan; Li, Bing

    2013-01-01

    Previous studies have proved that the environmental toxicant, inorganic arsenic, activates nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in many different cell types. This study tried to explore the hepatic Nrf2 pathway upon arsenic treatment comprehensively, since liver is one of the major target organs of arsenical toxicity. Our results showed that inorganic arsenic significantly induced Nrf2 protein and mRNA expression in Chang human hepatocytes. We also observed a dose-dependent increase of antioxidant response element- (ARE-) luciferase activity. Both the mRNA and protein levels of NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) were all upregulated dramatically. On the other hand, entry and accumulation of Nrf2 protein in the nucleus, while exportting the transcriptional repressor BTB and CNC homology 1 (Bach1) from nucleus to cytoplasm, were also confirmed by western blot and immunofluorescence assay. Our results therefore confirmed the arsenic-induced Nrf2 pathway activation in hepatocytes and also suggested that the translocation of Bach1 was associated with the regulation of Nrf2 pathway by arsenic. Hepatic Nrf2 pathway plays indispensable roles for cellular defenses against arsenic hepatotoxicity, and the interplay of Bach1 and Nrf2 may be helpful to understand the self-defensive responses and the diverse biological effects of arsenicals. PMID:23738048

  1. Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases.

    Directory of Open Access Journals (Sweden)

    Hanae Takatsuki

    Full Text Available The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt-Jakob disease patients demonstrated that 50% seeding dose (SD50 is reached approximately 10(10/g brain (values varies 10(8.79-10.63/g. A genetic case (GSS-P102L yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6-5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06-0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.

  2. Heme polymerization inhibition activity (HPIA) assay of synthesized xanthone derivative as antimalarial compound

    Science.gov (United States)

    Fitriastuti, Dhina; Jumina, Priatmoko

    2017-03-01

    Xanthone is a phenolic secondary metabolite of Garcinia and Calophyllum herbs which has been clinically proven to display anti malaria activity. In the present paper, 2,3,4-trihydroxy-5-methyl xanthone which has been synthesized from gallic acid and o-cresol in Eaton's reagent was tested for its activity as antimalarial. Thus, HPIA assay of the synthesized xanthones was successfully conducted. The HPIA assay was carried out towards the xanthone, chloroquine diphosphate as positive control and distilled water as negative control in various concentration. The samples were reacted with hematin (ferriprotoporphyrin IX hydroxide) and the absorbance of the precipitate was observed by using Elisa reader. The results of HPIA assay showed that 2,3,4-trihydroxy-5-methyl xanthone and chloroquine have IC50 values of 0.755 and 1.462 mg/mL or 2.92 and 4.57 mM, respectively. 2,3,4-Trihydroxy-5-methyl xanthone displayed better antimalarial activity than chloroquine.

  3. A nanostructure-initiator mass spectrometry-based enzyme activity assay

    Energy Technology Data Exchange (ETDEWEB)

    Siuzdak, Gary; Northen, Trent R.; Lee, Jinq-Chyi; Hoang, Linh; Raymond, Jason; Hwang, Der-Ren; Yannone, Steven M.; Wong, Chi-Huey; Siuzdak, Gary

    2008-03-10

    We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorous-phase interactions. This 'soft' immobilization allows efficient desorption/ionization while also enabling the use of surface-washing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of the Nimzyme assay to analyze complex mixtures is illustrated by identifying and directly characterizing {beta}-1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65 C and 5.5, respectively, and the activity was inhibited by both phenylethyl-{beta}-d-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced {gamma}-proteobacterium. In general, this assay provides an efficient method for detection and characterization of enzymatic activities in complex biological mixtures prior to sequencing or cloning efforts. More generally, this approach may have important applications for screening both enzymatic and inhibitor libraries, constructing and screening glycan microarrays, and complementing fluorous-phase organic synthesis. The interest in leveraging mass spectrometry for studying enzyme activities in complex biological samples derives from its high sensitivity and specificity; however, signal suppression and significant sample preparation requirements limit its overall utility (1). Here we describe a Nanostructure-Initiator Mass Spectrometry (NIMS

  4. Generation of knockout rabbits using transcription activator-like effector nucleases

    Directory of Open Access Journals (Sweden)

    Yu Wang

    2014-01-01

    Full Text Available Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

  5. Effects of a methanolic fraction of soybean seeds on the transcriptional activity of peroxisome proliferator-activated receptors (PPAR

    Directory of Open Access Journals (Sweden)

    V.S. Carrara

    2009-06-01

    Full Text Available Since the anti-inflammatory, antidiabetic and hypolipidemic effects of soy isoflavones may be mediated by activation of peroxisome proliferator-activated receptors (PPAR, the present study investigated whether the methanolic fractions obtained from soybean seeds (E1 and soybean seed coats with hypocotyls (E2 could influence PPARα, PPARγ and PPARβ/δ transcriptional activity. The isoflavones from E1 and E2 were quantified by HPLC analysis. E1 and E2 were rich in isoflavones (daidzin, glycitin, genistin, malonyldaidzin, malonylglycitin, malonylgenistin, daidzein, glycitein, and genistein. Moreover, E1 and E2 showed no evidence of genetically modified material containing the gene CP4 EPSPS. To investigate PPAR transcriptional activity, human promonocytic U-937 cells were treated with E1 and E2 (200, 400, 800, and 1600 µg/mL, positive controls or vehicle. Data are reported as fold-activation of the luciferase reporter driven by the PPAR-responsive element. Dose-response analysis revealed that E1 and E2 induced the transcriptional activity of PPARα (P < 0.001, with activation comparable to that obtained with 0.1 mM bezafibrate (positive control at 1600 µg/mL (4-fold and 800 µg/mL (9-fold, respectively. In addition, dose-response analysis revealed that E1 and E2 activated PPARβ/δ (P < 0.05, and the activation at 800 µg/mL (4- and 9-fold, respectively was comparable to that of 0.1 mM bezafibrate (positive control. However, no effect on PPARγ was observed. Activation of PPARα is consistent with the lipid-lowering activity of soy isoflavones in vivo, but further studies are needed to determine the physiological significance of PPARβ/δ activation.

  6. Wavelet-based detection of transcriptional activity on a novel Staphylococcus aureus tiling microarray

    Directory of Open Access Journals (Sweden)

    Segura Víctor

    2012-09-01

    Full Text Available Abstract Background High-density oligonucleotide microarray is an appropriate technology for genomic analysis, and is particulary useful in the generation of transcriptional maps, ChIP-on-chip studies and re-sequencing of the genome.Transcriptome analysis of tiling microarray data facilitates the discovery of novel transcripts and the assessment of differential expression in diverse experimental conditions. Although new technologies such as next-generation sequencing have appeared, microarrays might still be useful for the study of small genomes or for the analysis of genomic regions with custom microarrays due to their lower price and good accuracy in expression quantification. Results Here, we propose a novel wavelet-based method, named ZCL (zero-crossing lines, for the combined denoising and segmentation of tiling signals. The denoising is performed with the classical SUREshrink method and the detection of transcriptionally active regions is based on the computation of the Continuous Wavelet Transform (CWT. In particular, the detection of the transitions is implemented as the thresholding of the zero-crossing lines. The algorithm described has been applied to the public Saccharomyces cerevisiae dataset and it has been compared with two well-known algorithms: pseudo-median sliding window (PMSW and the structural change model (SCM. As a proof-of-principle, we applied the ZCL algorithm to the analysis of the custom tiling microarray hybridization results of a S. aureus mutant deficient in the sigma B transcription factor. The challenge was to identify those transcripts whose expression decreases in the absence of sigma B. Conclusions The proposed method archives the best performance in terms of positive predictive value (PPV while its sensitivity is similar to the other algorithms used for the comparison. The computation time needed to process the transcriptional signals is low as compared with model-based methods and in the same range to those

  7. High hydrostatic pressure activates transcription factors involved in Saccharomyces cerevisiae stress tolerance.

    Science.gov (United States)

    Bravim, Fernanda; da Silva, Lucas F; Souza, Diego T; Lippman, Soyeon I; Broach, James R; Fernandes, A Alberto R; Fernandes, Patricia M B

    2012-12-01

    A number of transcriptional control elements are activated when Saccharomyces cerevisiae cells are submitted to various stress conditions, including high hydrostatic pressure (HHP). Exposure of Saccharomyces cerevisiae cells to HHP results in global transcriptional reprogramming, similar to that observed under other industrial stresses, such as temperature, ethanol and oxidative stresses. Moreover, treatment with a mild hydrostatic pressure renders yeast cells multistress tolerant. In order to identify transcriptional factors involved in coordinating response to high hydrostatic pressure, we performed a time series microarray expression analysis on a wild S. cerevisiae strain exposed to 50 MPa for 30 min followed by recovery at atmospheric pressure (0.1 MPa) for 5, 10 and 15 min. We identified transcription factors and corresponding DNA and RNA motifs targeted in response to hydrostatic pressure. Moreover, we observed that different motif elements are present in the promoters of induced or repressed genes during HHP treatment. Overall, as we have already published, mild HHP treatment to wild yeast cells provides multiple protection mechanisms, and this study suggests that the TFs and motifs identified as responding to HHP may be informative for a wide range of other biotechnological and industrial applications, such as fermentation, that may utilize HHP treatment.

  8. FATS is a transcriptional target of p53 and associated with antitumor activity

    Directory of Open Access Journals (Sweden)

    Zhang Xifeng

    2010-09-01

    Full Text Available Abstract Frequent mutations of p53 in human cancers exemplify its crucial role as a tumor suppressor transcription factor, and p21, a transcriptional target of p53, plays a central role in surveillance of cell-cycle checkpoints. Our previous study has shown that FATS stabilize p21 to preserve genome integrity. In this study we identified a novel transcript variant of FATS (GenBank: GQ499374 through screening a cDNA library from mouse testis, which uncovered the promoter region of mouse FATS. Mouse FATS was highly expressed in testis. The p53-responsive elements existed in proximal region of both mouse and human FATS promoters. Functional study indicated that the transcription of FATS gene was activated by p53, whereas such effect was abolished by site-directed mutagenesis in the p53-RE of FATS promoter. Furthermore, the expression of FATS increased upon DNA damage in a p53-dependent manner. FATS expression was silent or downregulated in human cancers, and overexpression of FATS suppressed tumorigenicity in vivo independently of p53. Our results reveal FATS as a p53-regulated gene to monitor genomic stability.

  9. A promoter polymorphism rs2075824 within IMPA2 gene affecting the transcription activity: possible relationship with schizophrenia.

    Science.gov (United States)

    Li, Jia; Huang, Sheng; Dai, Hui-Rong; Wang, Juan; Lin, Li-Hui; Xiao, Hui; Peng, Xia; Li, Fei; Wang, Yu-Ping; Yuan, Jian-Min; Li, Li

    2017-04-01

    Previous studies with biological and genetic evidence indicate that the myo-inositol monophosphatase 2 (IMPA2) gene may influence schizophrenia. We performed a genetic association study in Han Chinese cohorts. Five single nucleotide polymorphisms within IMPA2 promoter region (rs971363, rs971362, rs2075824, rs111410794 and rs111610121), as well as one (rs45442994, in intron 1) that was positively associated in another study, were selected for genotyping in 1397 patients with schizophrenia and 1285 mentally healthy controls. Genotype and allele frequencies were assessed by gender stratification. Interestingly, rs2075824 showed a strong association with schizophrenia (P = 4.1 × 10 -4 ), and the T allele was more frequent in cases than controls [P = 5.6 × 10 -5 , OR (95% CI) = 1.26 (1.13-1.41)]. In vitro promoter assay showed that the transcription activity of the T allele promoter was higher than that of the C allele promoter and the T allele of rs2075824 contributed to risk for schizophrenia. By stratifying males and females, we found a gender-specific association for IMPA2 and schizophrenia: the T allele of rs2075824 was more frequent in male cases compared with male controls [P = 1.4 × 10 -4 , OR (95% CI) = 1.33 (1.15-1.55)]. Our data suggest that a promoter polymorphism of IMPA2 possibly contributed to risk for schizophrenia by elevating transcription activity in Han Chinese individuals. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  10. Automated locomotor activity monitoring as a quality control assay for mass-reared tephritid flies.

    Science.gov (United States)

    Dominiak, Bernard C; Fanson, Benjamin G; Collins, Samuel R; Taylor, Phillip W

    2014-02-01

    The Sterile Insect Technique (SIT) requires vast numbers of consistently high quality insects to be produced over long periods. Quality control (QC) procedures are critical to effective SIT, both providing quality assurance and warning of operational deficiencies. We here present a potential new QC assay for mass rearing of Queensland fruit flies (Bactrocera tryoni Froggatt) for SIT; locomotor activity monitoring. We investigated whether automated locomotor activity monitors (LAMs) that simply detect how often a fly passes an infrared sensor in a glass tube might provide similar insights but with much greater economy. Activity levels were generally lower for females than for males, and declined over five days in the monitor for both sexes. Female activity levels were not affected by irradiation, but males irradiated at 60 or 70 Gy had reduced activity levels compared with unirradiated controls. We also found some evidence that mild heat shock of pupae results in adults with reduced activity. LAM offers a convenient, effective and economical assay to probe such changes. © 2013 Society of Chemical Industry.

  11. New antibacterial hydrophobic assay reveals Abies balsamea oleoresin activity against Staphylococcus aureus and MRSA.

    Science.gov (United States)

    Coté, Héloïse; Boucher, Marie-Anne; Pichette, André; Roger, Benoit; Legault, Jean

    2016-12-24

    Oleoresin of Abies balsamea (L.) Mill. was used by Native Americans of the boreal forest of Canada and French Canadians to treat various infections, suggesting that oleoresin has antibacterial properties. In this study, the antibacterial activity of whole oleoresin from A. balsamea was investigated against E. coli, S. aureus and two methicillin-resistant S. aureus (MRSA) strains using a new sensitive assay developed to evaluate hydrophobic matrix and compounds. Antibacterial activity of oleoresin was first investigated using dilution and disk diffusion methods against E. coli and S. aureus, and compared to a new sensitive assay for hydrophobic matrix. Moreover, whole oleoresin was analyzed by GC-MS to characterize the composition and to identify the compounds responsible of the antibacterial activity. The results showed that whole oleoresin was inactive against Gram-negative E. coli (MIC90 >90µg/ml) but active against Gram-positive S. aureus and MRSA with MIC90 ranging from 18.2 to 30µg/ml. The oleoresin is mainly composed of monoterpene (28%), sesquiterpenes (2%), and diterpenes (45%). Resin acids were found, in part, responsible for the antibacterial activity of whole oleoresin. Isopimaric acid and levopimaric acid are the most active with a MIC90 of respectively 9.7µg/ml and 10µg/ml. This study supports the use of oleoresin of A. balsamea by the Native Americans and French Canadians to treat bacterial infections due to S. aureus. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Yeast Estrogen Screen Assay as a Tool for Detecting Estrogenic Activity in Water Bodies

    Directory of Open Access Journals (Sweden)

    Mirjana Bistan

    2012-01-01

    Full Text Available The presence of endocrine-disrupting compounds in wastewater, surface water, groundwater and even drinking water has become a major concern worldwide, since they negatively affect wildlife and humans. Therefore, these substances should be effectively removed from effluents before they are discharged into surface water to prevent pollution of groundwater, which can be a source of drinking water. Furthermore, an efficient control of endocrine-disrupting compounds in wastewater based on biological and analytical techniques is required. In this study, a yeast estrogen screen (YES bioassay has been introduced and optimized with the aim to assess potential estrogenic activity of waters. First, assay duration, concentration of added substrate to the assay medium and wavelength used to measure the absorbance of the substrate were estimated. Several compounds, such as 17-β-estradiol, 17-α-ethinylestradiol, bisphenol A, nonylphenol, genisteine, hydrocortisone, dieldrin, atrazine, methoxychlor, testosterone and progesterone were used to verify its specificity and sensitivity. The optimized YES assay was sensitive and responded specifically to the selected estrogenic and nonestrogenic compounds in aqueous samples. Potential estrogenicity of influent and effluent samples of two wastewater treatment plants was assessed after the samples had been concentrated by solid-phase extraction (SPE procedure using Oasis® HLB cartridges and methanol as eluting solvent. Up to 90 % of relative estrogenic activity was detected in concentrated samples of influents to wastewater treatment plants and estrogenic activity was still present in the concentrated effluent samples. We found that the introduced YES assay is a suitable screening tool for monitoring the potential estrogenicity of effluents that are discharged into surface water.

  13. "Singing in the Tube"--audiovisual assay of plant oil repellent activity against mosquitoes (Culex pipiens).

    Science.gov (United States)

    Adams, Temitope F; Wongchai, Chatchawal; Chaidee, Anchalee; Pfeiffer, Wolfgang

    2016-01-01

    Plant essential oils have been suggested as a promising alternative to the established mosquito repellent DEET (N,N-diethyl-meta-toluamide). Searching for an assay with generally available equipment, we designed a new audiovisual assay of repellent activity against mosquitoes "Singing in the Tube," testing single mosquitoes in Drosophila cultivation tubes. Statistics with regression analysis should compensate for limitations of simple hardware. The assay was established with female Culex pipiens mosquitoes in 60 experiments, 120-h audio recording, and 2580 estimations of the distance between mosquito sitting position and the chemical. Correlations between parameters of sitting position, flight activity pattern, and flight tone spectrum were analyzed. Regression analysis of psycho-acoustic data of audio files (dB[A]) used a squared and modified sinus function determining wing beat frequency WBF ± SD (357 ± 47 Hz). Application of logistic regression defined the repelling velocity constant. The repelling velocity constant showed a decreasing order of efficiency of plant essential oils: rosemary (Rosmarinus officinalis), eucalyptus (Eucalyptus globulus), lavender (Lavandula angustifolia), citronella (Cymbopogon nardus), tea tree (Melaleuca alternifolia), clove (Syzygium aromaticum), lemon (Citrus limon), patchouli (Pogostemon cablin), DEET, cedar wood (Cedrus atlantica). In conclusion, we suggest (1) disease vector control (e.g., impregnation of bed nets) by eight plant essential oils with repelling velocity superior to DEET, (2) simple mosquito repellency testing in Drosophila cultivation tubes, (3) automated approaches and room surveillance by generally available audio equipment (dB[A]: ISO standard 226), and (4) quantification of repellent activity by parameters of the audiovisual assay defined by correlation and regression analyses.

  14. Identification of the sequences recognized by phage phi 29 transcriptional activator: possible interaction between the activator and the RNA polymerase.

    Science.gov (United States)

    Nuez, B; Rojo, F; Barthelemy, I; Salas, M

    1991-05-11

    Expression of Bacillus subtilis phage phi 29 late genes requires the transcriptional activator protein p4. This activator binds to a region of the late A3 promoter spanning nucleotides -56 to -102 relative to the transcription start site, generating a strong bending Tin the DNA. In this work the target sequences recognized by protein p4 in the phage phi 29 late A3 promoter have been characterized. The binding of protein p4 to derivatives of the late A3 promoter harbouring deletions in the protein p4 binding site has been studied. When protein p4 recognition sequences were altered, the activator could only bind to the promoter in the presence of RNA polymerase. This strong cooperativity in the binding of protein p4 and RNA polymerase to the promoter suggests the presence of direct protein-protein contacts between them.

  15. Development and validation of a 3-Plex RT-qPCR assay for the simultaneous detection and quantitation of the three PML-RARa fusion transcripts in acute promyelocytic leukemia.

    Directory of Open Access Journals (Sweden)

    Zhanguo Chen

    Full Text Available Rapid diagnosis of acute promyelocytic leukemia (APL with promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa contributes to a highly effective therapy with all-trans retinoic acid (ATRA. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR is a valuable tool to diagnose APL with PML-RARa. However, a single RT-qPCR analysis, which is laborious and costly, has to be performed in three reactions to determine whether one of the three PML-RARa transcripts is present and to quantify the involved transcript. This paper describes a novel TaqMan MGB probe-based 3-plex RT-qPCR assay in a single reaction to detect simultaneously the three PML-RARa transcripts. Specific primers and probe were designed, and the results were further normalized to the Abelson gene. The detection results for the serially diluted plasmid indicate that the analytical sensitivity was 10 copies per reaction for PML-RARa bcr1, bcr2, and bcr3. A relatively high sensitivity of 10-4 was achieved with this assay when analyzing the bcr1 transcripts obtained from the NB4 cell line. The reproducibility was satisfactory because the coefficients of variation of cycle threshold values were less than 3% for both inter- and intra-assays. After testing 319 newly diagnosed patients with leukemia (including 61 APL cases, the results of the 3-plex RT-qPCR assay completely agreed with the traditional methods used for the detection of PML-RARa. The quantitative results of the 3-plex RT-qPCR were highly correlated with the single RT-qPCR and showed similar assay sensitivity for 60 PML-RARa positive APL samples at diagnosis and 199 samples from 57 patients during follow-up. Interestingly, one PML-RARa bcr2 case at diagnosis with breakpoint at 1579, which was not detected by the single RT-q-PCR, was detected by the 3-plex RT-qPCR assay. The 3-plex RT-qPCR assay is a specific, sensitive, stable, and cost-effective method that can be used for the rapid diagnosis and

  16. A critique on nuclear factor-kappa B and signal transducer and activator of transcription 3: The key transcription factors in periodontal pathogenesis

    Directory of Open Access Journals (Sweden)

    Ranjith Ambili

    2017-01-01

    Full Text Available Periodontal disease is initiated by microorganisms in dental plaque, and host immunoinflammatory response to the microbial challenge helps in disease progression. Conventional periodontal therapy was mainly targeted on the elimination of microbial component. However, a better understanding of molecular aspects in host response will enable the clinicians to formulate effective host modulation therapy (HMT for the periodontal management. Inflammatory mediators were the main targets for HMT in the past. Transcription factors can regulate the production of multiple mediators simultaneously, and inhibition of these factors will be more beneficial than blocking individual molecule. Two important transcription factors implicated in chronic inflammatory diseases are nuclear factor kappa B (NF-κB and signal transducers and activators of transcription 3. The role of these factors in periodontal disease is a less explored area. This comprehensive review is aimed at unveiling the critical role of NF-κB and signal transducers and activators of transcription 3 in periodontal pathogenesis. An online search was performed using MEDLINE/PubMed database. All publications till 2016 related to NF-κB, signal transducer and activator of transcription 3 (STAT3, and inflammation were included in writing this review. A total of 27,390 references were published based on the search terms used. Out of these, 507 were related to the periodontal research published in English till 2016. Relevant papers were chosen after carefully reading the abstract. This review has attempted to comprehend the existing knowledge regarding the role of transcription factors NF-κB and STAT3 in periodontal disease. Moreover, it also provides a connecting molecular link for the periodontal medicine concept.

  17. Low prevalence of transcriptionally active human papilloma virus in Indian patients with HNSCC and leukoplakia.

    Science.gov (United States)

    Bhosale, Priyanka G; Pandey, Manishkumar; Desai, Rajiv S; Patil, Asawari; Kane, Shubhada; Prabhash, Kumar; Mahimkar, Manoj B

    2016-11-01

    In the present study, we comprehensively analyzed the prevalence of transcriptionally active human papilloma virus (HPV) in tissue samples of Indian patients with leukoplakia, predominantly hyperplastic lesions and head and neck squamous cell carcinoma (HNSCC). In addition, saliva samples from patients with HNSCC were screened for HPV detection. P16 overexpression was analyzed by immunohistochemistry. Tissue samples of leukoplakia (n = 121) and HNSCC (n = 427) and saliva from patients with HNSCC (n = 215) were tested for HPV using nested polymerase chain reaction. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in situ hybridization. P16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, in saliva or tissue. E6/E7 mRNA positivity was observed in 8 HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx, with very little concordance between p16 overexpression and HPV positivity. All patients with HPV-positive saliva samples had transcriptionally active HPV present in their tumors. The presence of HPV DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV-associated tumors. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Presence and transcriptional activity of anaerobic fungi in agricultural biogas plants.

    Science.gov (United States)

    Dollhofer, Veronika; Callaghan, Tony M; Griffith, Gareth W; Lebuhn, Michael; Bauer, Johann

    2017-07-01

    Bioaugmentation with anaerobic fungi (AF) is promising for improved biogas generation from lignocelluloses-rich substrates. However, before implementing AF into biogas processes it is necessary to investigate their natural occurrence, community structure and transcriptional activity in agricultural biogas plants. Thus, AF were detected with three specific PCR based methods: (i) Copies of their 18S genes were found in 7 of 10 biogas plants. (ii) Transcripts of a GH5 endoglucanase gene were present at low level in two digesters, indicating transcriptional cellulolytic activity of AF. (iii) Phylogeny of the AF-community was inferred with the 28S gene. A new Piromyces species was isolated from a PCR-positive digester. Evidence for AF was only found in biogas plants operated with high proportions of animal feces. Thus, AF were most likely transferred into digesters with animal derived substrates. Additionally, high process temperatures in combination with long retention times seemed to impede AF survival and activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Domain-specific c-Myc ubiquitylation controls c-Myc transcriptional and apoptotic activity

    Science.gov (United States)

    Zhang, Qin; Spears, Erick; Boone, David N.; Li, Zhaoliang; Gregory, Mark A.; Hann, Stephen R.

    2013-01-01

    The oncogenic transcription factor c-Myc causes transformation and tumorigenesis, but it can also induce apoptotic cell death. Although tumor suppressors are necessary for c-Myc to induce apoptosis, the pathways and mechanisms are unclear. To further understand how c-Myc switches from an oncogenic protein to an apoptotic protein, we examined the mechanism of p53-independent c-Myc–induced apoptosis. We show that the tumor suppressor protein ARF mediates this switch by inhibiting ubiquitylation of the c-Myc transcriptional domain (TD). Whereas TD ubiquitylation is critical for c-Myc canonical transcriptional activity and transformation, inhibition of ubiquitylation leads to the induction of the noncanonical c-Myc target gene, Egr1, which is essential for efficient c-Myc–induced p53-independent apoptosis. ARF inhibits the interaction of c-Myc with the E3 ubiquitin ligase Skp2. Overexpression of Skp2, which occurs in many human tumors, inhibits the recruitment of ARF to the Egr1 promoter, leading to inhibition of c-Myc–induced apoptosis. Therapeutic strategies could be developed to activate this intrinsic apoptotic activity of c-Myc to inhibit tumorigenesis. PMID:23277542

  20. Antioxidant activity by DPPH assay of potential solutions to be applied on bleached teeth

    OpenAIRE

    GARCIA, Eugenio Jose; Oldoni, Tatiane Luiza Cadorin; Alencar, Severino Matias de; Reis, Alessandra; Loguercio, Alessandro D.; GRANDE, Rosa Helena Miranda

    2012-01-01

    The aim of this study was to assess, using the DPPH assay, the antioxidant activity of several substances that could be proposed to immediately revert the problems caused by bleaching procedures. The percentage of antioxidant activity (AA%) of 10% ascorbic acid solution (AAcidS), 10% ascorbic acid gel (AAcidG), 10% sodium ascorbate solution (SodAsS), 10% sodium ascorbate gel (SodAsG), 10% sodium bicarbonate (Bicarb), Neutralize® (NE), Desensibilize® (DES), catalase C-40 at 10 mg/mL (CAT), 1...

  1. Activities of various cobalamins for Euglena gracilis with reference to vitamin B12 assay with Euglena

    Science.gov (United States)

    Adams, J. F.; McEwan, Fiona

    1971-01-01

    Coenzyme B12 and methylcobalamin in water are less active in promoting growth of Euglena gracilis Z strain than the same concentrations of cyanocobalamin and hydroxocobalamin which are equally active. When bound to human serum or human liver homogenate, however, the activities of these four cobalamins do not differ significantly with one exception. The results suggest that the Euglena assay using cyanocobalamin standards is not satisfactory for quantitation of coenzyme B12 and methylcobalamin in water but acceptable when coenzyme B12 and methylcobalamin are bound to serum or liver. Sulphitocobalamin in water is as active as cyanocobalamin and hydroxocobalamin but nitritocobalamin is less active. Factor B, the monocarboxylic acids of cyanocobalamin and hydroxocobalamin, and the dicarboxylic acid of cyanocobalamin in water were inactive. PMID:5572999

  2. The phosphatase calcineurin PP2BAβ mediates part of mineralocorticoid receptor transcriptional activity.

    Science.gov (United States)

    Seiferth, Anja; Ruhs, Stefanie; Mildenberger, Sigrid; Gekle, Michael; Grossmann, Claudia

    2012-06-01

    Recently it was shown that the mineralocorticoid receptor (MR) may exert part of its transcriptional activity by mediation of calcineurin (PP2B). Here we investigated the mechanism of interaction of MR with calcineurin and provide a new MR signaling pathway with potential physiological and pathophysiological relevance. MR → calcineurin crosstalk was assessed in a heterologous expression system (human embryonic kidney cells), which provides the opportunity for detailed mechanistic investigation. SiRNA knockdown experiments show that activated MR, but not GR, reduces CREB- and enhances NFaT-mediated transcriptional activation via the catalytic calcineurin subunit PP2BAβ but not via PP2BAα. Altered PP2BAβ expression, elevated cytosolic Ca(2+), activation of mitogen-activated kinase [p38, extracellular signal-regulated kinase (ERK) 1/2], or protein kinase C do not seem to be involved, whereas inhibition of the chaperone heat-shock protein 90 (HSP90) abrogated the effect of MR. Coimmunoprecipitation indicates the existence of protein complexes harboring MR and PP2BAβ independent of MR activation but dependent on HSP90. Activated MR alters the subcellular distribution of PP2BAβ, enhancing its nuclear fraction, and reduces mRNA expression of the endogenous inhibitor CAIN (calcineurin inhibitor) but not of RCAN1 (regulator of calcineurin). Overall, transcriptional relevant MR → calcineurin crosstalk occurs via the catalytic subunit PP2BAβ, enables glucocorticoid response element-independent genomic signaling of MR, and is of potential pathophysiological relevance. Mechanistically, the crosstalk results from HSP90-mediated cytosolic protein complex formation, altered subcellular distribution, and altered endogenous inhibitor expression.

  3. Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells.

    Science.gov (United States)

    Kojima, Hiroyuki; Katsura, Eiji; Takeuchi, Shinji; Niiyama, Kazuhito; Kobayashi, Kunihiko

    2004-01-01

    We tested 200 pesticides, including some of their isomers and metabolites, for agonism and antagonism to two human estrogen receptor (hER) subtypes, hERalpha and hERbeta, and a human androgen receptor (hAR) by highly sensitive transactivation assays using Chinese hamster ovary cells. The test compounds were classified into nine groups: organochlorines, diphenyl ethers, organophosphorus pesticides, pyrethroids, carbamates, acid amides, triazines, ureas, and others. These pesticides were tested at concentrations methiocarb were predominantly hERbeta rather than hERalpha agonistic. Weak antagonistic effects toward hERalpha and hERbeta were shown in five and two pesticides, respectively. On the other hand, none of tested pesticides showed hAR-mediated androgenic activity, but 66 of 200 pesticides exhibited inhibitory activity against the transcriptional activity induced by 5alpha-dihydrotestosterone. In particular, the antiandrogenic activities of two diphenyl ether herbicides, chlornitrofen and chlomethoxyfen, were higher than those of vinclozolin and p,p -dichlorodiphenyl dichloroethylene, known AR antagonists. The results of our ER and AR assays show that 34 pesticides possessed both estrogenic and antiandrogenic activities, indicating pleiotropic effects on hER and hAR. We also discussed chemical structures related to these activities. Taken together, our findings suggest that a variety of pesticides have estrogenic and/or antiandrogenic potential via ER and/or AR, and that numerous other manmade chemicals may also possess such estrogenic and antiandrogenic activities. PMID:15064155

  4. Enhanced NFκB and AP-1 transcriptional activity associated with antiestrogen resistant breast cancer

    Directory of Open Access Journals (Sweden)

    Moore Dan H

    2007-04-01

    Full Text Available Abstract Background Signaling pathways that converge on two different transcription factor complexes, NFκB and AP-1, have been identified in estrogen receptor (ER-positive breast cancers resistant to the antiestrogen, tamoxifen. Methods Two cell line models of tamoxifen-resistant ER-positive breast cancer, MCF7/HER2 and BT474, showing increased AP-1 and NFκB DNA-binding and transcriptional activities, were studied to compare tamoxifen effects on NFκB and AP-1 regulated reporter genes relative to tamoxifen-sensitive MCF7 cells. The model cell lines were treated with the IKK inhibitor parthenolide (PA or the proteasome inhibitor bortezomib (PS341, alone and in combination with tamoxifen. Expression microarray data available from 54 UCSF node-negative ER-positive breast cancer cases with known clinical outcome were used to search for potential genes signifying upregulated NFκB and AP-1 transcriptional activity in association with tamoxifen resistance. The association of these genes with patient outcome was further evaluated using node-negative ER-positive breast cancer cases identified from three other published data sets (Rotterdam, n = 209; Amsterdam, n = 68; Basel, n = 108, each having different patient age and adjuvant tamoxifen treatment characteristics. Results Doses of parthenolide and bortezomib capable of sensitizing the two endocrine resistant breast cancer models to tamoxifen were capable of suppressing NFκB and AP-1 regulated gene expression in combination with tamoxifen and also increased ER recruitment of the transcriptional co-repressor, NCoR. Transcript profiles from the UCSF breast cancer cases revealed three NFκB and AP-1 upregulated genes – cyclin D1, uPA and VEGF – capable of dichotomizing node-negative ER-positive cases into early and late relapsing subsets despite adjuvant tamoxfien therapy and most prognostic for younger age cases. Across the four independent sets of node-negative ER-positive breast cancer cases

  5. Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications

    Science.gov (United States)

    Nader, Nancy; Chrousos, George P.; Kino, Tomoshige

    2009-01-01

    Glucocorticoids, end products of the hypothalamic-pituitary-adrenal axis, influence functions of virtually all organs and tissues through the glucocorticoid receptor (GR). Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion and regulate the transcriptional activity of GR in target tissues. The basic helix-loop-helix protein CLOCK, a histone acetyltransferase (HAT), and its heterodimer partner BMAL1 are self-oscillating transcription factors that generate circadian rhythms in both the central nervous system and periphery. We found that CLOCK/BMAL1 repressed GR-induced transcriptional activity in a HAT-activity- dependent fashion. In serum-shock-synchronized cells, transactivational activity of GR, accessed by mRNA expression of an endogenous-responsive gene, fluctuated spontaneously in a circadian fashion in reverse phase with CLOCK/BMAL1 mRNA expression. CLOCK and GR interacted with each other physically, and CLOCK suppressed binding of GR to its DNA recognition sequences by acetylating multiple lysine residues located in its hinge region. These findings indicate that CLOCK/BMAL1 functions as a reverse-phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing biological actions of diurnally fluctuating circulating glucocorticoids. Further, these results suggest that a peripheral target tissue circadian rhythm indirectly influences the functions of every organ and tissue inside the body through modulation of the ubiquitous and diverse actions of glucocorticoids.—Nader, N., Chrousos, G. P., Kino, T. Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications. PMID:19141540

  6. Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

    Science.gov (United States)

    Koh, Hye Ran; Wang, Xinlei; Myong, Sua

    2016-08-01

    TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

    Science.gov (United States)

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan

    2015-05-15

    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Development of a keratinase activity assay using recombinant chicken feather keratin substrates.

    Directory of Open Access Journals (Sweden)

    Hyeon-Su Jin

    Full Text Available Poultry feathers consist mainly of the protein keratin, which is rich in β-pleated sheets and consequently resistant to proteolysis. Although many keratinases have been identified, the reasons for their substrate specificity towards β-keratin remain unclear due to difficulties in preparing a soluble feather keratin substrate for use in activity assays. In the present study, we overexpressed Gallus gallus chromosomes 2 and 27 β-keratin-encoding genes in Escherichia coli, purified denatured recombinant proteins by Ni2+ affinity chromatography, and refolded by stepwise dialysis to yield soluble keratins. To assess the keratinolytic activity, we compared the proteolytic activity of crude extracts from the feather- degrading bacterium Fervidobacterium islandicum AW-1 with proteinase K, trypsin, and papain using purified recombinant keratin and casein as substrates. All tested proteases showed strong proteolytic activities for casein, whereas only F. islandicum AW-1 crude extracts and proteinase K exhibited pronounced keratinolytic activity for the recombinant keratin. Moreover, LC-MS/MS analysis of keratin hydrolysates allowed us to predict the P1 sites of keratinolytic enzymes in the F. islandicum AW-1 extracts, thereby qualifying and quantifying the extent of keratinolysis. The soluble keratin-based assay has clear therapeutic and industrial potential for the development of a high-throughput screening system for proteases hydrolyzing disease-related protein aggregates, as well as mechanically resilient keratin-based polymers.

  9. MYRF is a membrane-associated transcription factor that autoproteolytically cleaves to directly activate myelin genes.

    Directory of Open Access Journals (Sweden)

    Helena Bujalka

    Full Text Available The myelination of axons is a crucial step during vertebrate central nervous system (CNS development, allowing for rapid and energy efficient saltatory conduction of nerve impulses. Accordingly, the differentiation of oligodendrocytes, the myelinating cells of the CNS, and their expression of myelin genes are under tight transcriptional control. We previously identified a putative transcription factor, Myelin Regulatory Factor (Myrf, as being vital for CNS myelination. Myrf is required for the generation of CNS myelination during development and also for its maintenance in the adult. It has been controversial, however, whether Myrf directly regulates transcription, with reports of a transmembrane domain and lack of nuclear localization. Here we show that Myrf is a membrane-associated transcription factor that undergoes an activating proteolytic cleavage to separate its transmembrane domain-containing C-terminal region from a nuclear-targeted N-terminal region. Unexpectedly, this cleavage event occurs via a protein domain related to the autoproteolytic intramolecular chaperone domain of the bacteriophage tail spike proteins, the first time this domain has been found to play a role in eukaryotic proteins. Using ChIP-Seq we show that the N-terminal cleavage product directly binds the enhancer regions of oligodendrocyte-specific and myelin genes. This binding occurs via a defined DNA-binding consensus sequence and strongly promotes the expression of target genes. These findings identify Myrf as a novel example of a membrane-associated transcription factor and provide a direct molecular mechanism for its regulation of oligodendrocyte differentiation and CNS myelination.

  10. Microtiter plate based colorimetric assay for characterization of dehalogenation activity of GAC/Fe0 composite

    DEFF Research Database (Denmark)

    Hwang, Yuhoon; Salatas, Apostolos; Mines, Paul D.

    2015-01-01

    Even though nanoscale zero valent iron (nZVI) has been intensively studied for the treatment of a plethora of pollutants through reductive reaction, a quantification of nZVI reactivity has not been standardized. Here, we developed series of colorimetric assays for determining reductive activity......, phenol, and aniline, generated as results of reduction of nitrate, halophenols, and nitrobenzene, respectively. The color reactions are simple and versatile since same types of reagents are able to be applied for all reactions. The colorimetric assays were further miniaturized and optimized into 96-well...... microplate having 230 μL of sample volume and 2 h of reaction time. The three groups of compounds, nitrate, nitrobenzene, and para-positioned halogenated phenols, showed graduated reactivity and were possible to distinguish a reaction mechanism between normal reduction and catalytic behaviour of second metal...

  11. Reciprocal activation of transcription factors underlies the dichotomy between proliferation and invasion of glioma cells.

    Directory of Open Access Journals (Sweden)

    Harshil D Dhruv

    Full Text Available Histology of malignant glioma depicts dense proliferative areas rich in angiogenesis as well as dissemination of neoplastic cells into adjacent brain tissue. Although the mechanisms that trigger transition from proliferative to invasive phenotypes are complex, the dichotomy of cell proliferation and migration, the "Go or Grow" hypothesis, argues for specific and coordinated regulation of these phenotypes. We investigated transcriptional elements that accompany the phenotypes of migration and proliferation, and consider the therapeutic significance of the "Go or Grow" hypothesis. Interrogation of matched core and rim regions from human glioblastoma biopsy specimens in situ (n = 44 revealed higher proliferation (Ki67 labeling index in cells residing at the core compared to the rim. Profiling activated transcription factors in a panel of migration-activated versus migration-restricted GBM cells portrayed strong NF-κB activity in the migratory cell population. In contrast, increased c-Myc activity was found in migration-restricted proliferative cells. Validation of transcriptional activity by NF-κB- or c-Myc-driven GFP or RFP, respectively, showed an increased NF-κB activity in the active migrating cells, whereas the proliferative, migration restricted cells displayed increased c-Myc activity. Immunohistochemistry on clinical specimens validated a robust phosphorylated c-Myc staining in tumor cells at the core, whereas increased phosphorylated NF-κB staining was detected in the invasive tumor cells at the rim. Functional genomics revealed that depletion of c-Myc expression by siRNA oligonucleotides reduced cell proliferation in vitro, but surprisingly, cell migration was enhanced significantly. Conversely, inhibition of NF-κB by pharmacological inhibitors, SN50 or BAY-11, decreased both cell migration in vitro and invasion ex vivo. Notably, inhibition of NF-κB was found to have no effect on the proliferation rate of glioma cells. These

  12. Cardiac hypertrophy induced by active Raf depends on Yorkie-mediated transcription.

    Science.gov (United States)

    Yu, Lin; Daniels, Joseph P; Wu, Huihui; Wolf, Matthew J

    2015-02-03

    Organ hypertrophy can result from enlargement of individual cells or from cell proliferation or both. Activating mutations in the serine-threonine kinase Raf cause cardiac hypertrophy and contribute to Noonan syndrome in humans. Cardiac-specific expression of activated Raf also causes hypertrophy in Drosophila melanogaster. We found that Yorkie (Yki), a transcriptional coactivator in the Hippo pathway that regulates organ size, is required for Raf-induced cardiac hypertrophy in flies. Although aberrant activation of Yki orthologs stimulates cardiac hyperplasia in mice, cardiac-specific expression of an activated mutant form of Yki in fruit flies caused cardiac hypertrophy without hyperplasia. Knockdown of Yki caused cardiac dilation without loss of cardiomyocytes and prevented Raf-induced cardiac hypertrophy. In flies, Yki-induced cardiac hypertrophy required the TEA domain-containing transcription factor Scalloped, and, in mammalian cells, expression of mouse Raf(L613V), an activated form of Raf with a Noonan syndrome mutation, increased Yki-induced Scalloped activity. Furthermore, overexpression of Tgi (a Tondu domain-containing Scalloped-binding corepressor) in the fly heart abrogated Yki- or Raf-induced cardiac hypertrophy. Thus, crosstalk between Raf and Yki occurs in the heart and can influence Raf-mediated cardiac hypertrophy. Copyright © 2015, American Association for the Advancement of Science.

  13. Preferential Arc transcription at rest in the active ensemble during associative learning.

    Science.gov (United States)

    Hashikawa, Koichi; Matsuki, Norio; Nomura, Hiroshi

    2011-05-01

    Information processing in the central nervous system (CNS) during periods of rest is crucial for lasting memories but the precise off-line neuronal population activity that contributes to long-term memory formation remains unclear. This pattern of neuronal activity during rest triggers transcription of immediate early genes such as activity regulated cytoskeletal gene (Arc). We compared the active neuronal population in the lateral amygdala of C57BL/6J mice during fear conditioning and rest periods using a large scale imaging technique, Arc cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH). We found that the neuronal population transcribing Arc during fear conditioning was more similar to that the population transcribing Arc after fear conditioning than before fear conditioning. The overlapping population was larger in conditioned mice that acquired associative memory than in unshocked mice and in latent inhibited mice that received shocks but did not form associative memory. Moreover, these results were confirmed using Arc/Homer 1a catFISH. Our findings indicate that Arc is preferentially transcribed in neurons that are active during fear conditioning after associative learning. This preferential transcription may contribute to the formation of long-lasting memory. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells

    Science.gov (United States)

    Berezhnaya, Elena; Neginskaya, Marya; Kovaleva, Vera; Sharifulina, Svetlana; Ischenko, Irina; Komandirov, Maxim; Rudkovskii, Mikhail; Uzdensky, Anatoly B.

    2015-07-01

    Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT.

  15. Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta-cells

    DEFF Research Database (Denmark)

    Friedrichsen, Birgitte N; Richter, Henrijette E; Hansen, Johnny A

    2003-01-01

    Signal transducer and activator of transcription 5 (STAT5) activation plays a central role in GH- and prolactin-mediated signal transduction in the pancreatic beta-cells. In previous experiments we demonstrated that STAT5 activation is necessary for human (h)GH-stimulated proliferation of INS-1...... cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and beta-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced...... in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aDelta749, partially inhibited cyclin D2 protein levels. INS-1...

  16. A new chronometric assay to determine plasma antifactor Xa activity which is insensitive to the antithrombin activity of low molecular weight heparins.

    Science.gov (United States)

    Dignac, M; Gabaig, A M; Cambus, J P; Boneu, B

    1994-01-01

    Some commercially available chronometric assays are influenced by the residual antithrombin activity of low molecular weight heparins (LMWH) and they underestimate the ex vivo anti Xa activity. We have evaluated a new kit (Staclot-Heparin) highly specific for the anti Xa activity of LMWH. A comparison with the results given by a reference chromogenic method (Stachrom-Heparin) indicates a very good correlation between the 2 assays (r = 0.95, n = 59). This new assay is not influenced by vitamin K antagonist treatments. Clinical biologists now have the possibility of determining the anti Xa activity generated by LMWH easily and accurately, using a chronometric assay.

  17. Activation of the transcription factor NF-kappaB by Campylobacter jejuni.

    Science.gov (United States)

    Mellits, Kenneth H; Mullen, Joseph; Wand, Matthew; Armbruster, Gisèle; Patel, Amit; Connerton, Phillippa L; Skelly, Maeve; Connerton, Ian F

    2002-09-01

    Campylobacter jejuni is a food-borne pathogen responsible for infectious enterocolitis. The early-response transcription factor NF-kappa B triggers the expression of genes associated with cellular immune and inflammatory responses. Co-incubation of HeLa cells with viable C. jejuni leads to the activation of the transcription factor NF-kappa B as determined by specific induction of a cellular luciferase-based reporter. Boiled cell-free extracts of C. jejuni are also potent dose-dependent stimulators of NF-kappa B-dependent transcription, the levels of which can reach up to 1000-fold as compared with independent controls. Using both cultured HeLa cells and human colonic epithelial (HCA-7) cells, the activation of NF-kappa B by C. jejuni boiled extract has been monitored through the degradation of IKB alpha and DNA binding of the nuclear translocated p50/p65 heterodimer of NF-kappa B. These events are co-ordinated with elaboration of the pro-inflammatory cytokine interleukin-8. Fractionation of the boiled C. jejuni extract suggests that the majority of the bioactive component has a molecular mass of 3 kDa or less, which is insensitive to proteinase K treatment.

  18. Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Courtney M. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Hu, Jianxin [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Thomas, Reuben [Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Gainous, T. Blair [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Celona, Barbara [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Sinha, Tanvi [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Dickel, Diane E. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Genomics Division; Heidt, Analeah B. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Xu, Shan-Mei [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Bruneau, Benoit G. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Pollard, Katherine S. [Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Pennacchio, Len A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Genomics Division; Black, Brian L. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Univ. of California, San Francisco, CA (United States). Dept. of

    2017-03-28

    Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here in this paper, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by the SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo. Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.

  19. Endocrine Activity of Bisphenol S (BPS) Using In Vitro Estrogenic/Anti-Androgenic Transcriptional Activation Assays and the In Vivo Uterotrophic Assay

    Science.gov (United States)

    Bisphenol A (BPA) is gradually being phased out of many consumer products and processes leading to potential increases in human and environmental exposures to relatively understudied replacement compounds, including Bisphenol S (BPS). Research from our lab has shown that BPA and...

  20. WRKY6 Transcription Factor Restricts Arsenate Uptake and Transposon Activation in Arabidopsis[W

    Science.gov (United States)

    Castrillo, Gabriel; Sánchez-Bermejo, Eduardo; de Lorenzo, Laura; Crevillén, Pedro; Fraile-Escanciano, Ana; TC, Mohan; Mouriz, Alfonso; Catarecha, Pablo; Sobrino-Plata, Juan; Olsson, Sanna; Leo del Puerto, Yolanda; Mateos, Isabel; Rojo, Enrique; Hernández, Luis E.; Jarillo, Jose A.; Piñeiro, Manuel; Paz-Ares, Javier; Leyva, Antonio

    2013-01-01

    Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing. PMID:23922208

  1. WRKY6 transcription factor restricts arsenate uptake and transposon activation in Arabidopsis.

    Science.gov (United States)

    Castrillo, Gabriel; Sánchez-Bermejo, Eduardo; de Lorenzo, Laura; Crevillén, Pedro; Fraile-Escanciano, Ana; Tc, Mohan; Mouriz, Alfonso; Catarecha, Pablo; Sobrino-Plata, Juan; Olsson, Sanna; Leo Del Puerto, Yolanda; Mateos, Isabel; Rojo, Enrique; Hernández, Luis E; Jarillo, Jose A; Piñeiro, Manuel; Paz-Ares, Javier; Leyva, Antonio

    2013-08-01

    Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing.

  2. Fucose-Mediated Transcriptional Activation of the fcs Operon by FcsR in Streptococcus pneumoniae.

    Science.gov (United States)

    Manzoor, Irfan; Shafeeq, Sulman; Afzal, Muhammad; Kuipers, Oscar P

    2015-01-01

    In this study, we explore the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5'-ATTTGAACATTATTCAAGT-3') in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon. © 2015 S. Karger AG, Basel.

  3. Molecular genetic analysis of activation-tagged transcription factors thought to be involved in photomorphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Neff, Michael M.

    2011-06-23

    This is a final report for Department of Energy Grant No. DE-FG02-08ER15927 entitled “Molecular Genetic Analysis of Activation-Tagged Transcription Factors Thought to be Involved in Photomorphogenesis”. Based on our preliminary photobiological and genetic analysis of the sob1-D mutant, we hypothesized that OBP3 is a transcription factor involved in both phytochrome and cryptochrome-mediated signal transduction. In addition, we hypothesized that OBP3 is involved in auxin signaling and root development. Based on our preliminary photobiological and genetic analysis of the sob2-D mutant, we also hypothesized that a related gene, LEP, is involved in hormone signaling and seedling development.

  4. Ketamine inhibits transcription factors activator protein 1 and nuclear factor-kappaB, interleukin-8 production, as well as CD11b and CD16 expression: studies in human leukocytes and leukocytic cell lines.

    Science.gov (United States)

    Welters, Ingeborg D; Hafer, Georg; Menzebach, Axel; Mühling, Jörg; Neuhäuser, Christoph; Browning, Paul; Goumon, Yannick

    2010-03-01

    Recent data indicate that ketamine exerts antiinflammatory actions. However, little is known about the signaling mechanisms involved in ketamine-induced immune modulation. In this study, we investigated the effects of ketamine on lipopolysaccharide-induced activation of transcription factors activator protein 1 (AP-1) and nuclear factor-kappaB (NF-kappaB) in human leukocyte-like cell lines and in human blood neutrophils. Electric mobility shift assays were used to investigate ketamine's effects on nuclear binding activity of both transcription factors in U937 cells, and a whole blood flow cytometric technique was used for AP-1 and NF-kappaB determination in leukocytes. Cell lines with different expression patterns of opioid and N-methyl-D-aspartate receptors were used for reverse transcription-polymerase chain reaction to investigate receptors involved in ketamine signaling. Ketamine's effect on interleukin-8 production was assessed in a whole blood assay. Ketamine inhibited both transcription factors in a concentration-dependent manner. These effects did not depend on opiate or N-methyl-D-aspartate receptors. Ketamine also reduced interleukin-8 production in whole blood and expression of CD11b and CD16 on neutrophils. The immunoinhibitory effects of ketamine are at least in part caused by inhibition of transcription factors NF-kappaB and AP-1, which regulate production of proinflammatory mediators. However, signaling mechanisms different from those present in the central nervous system are responsible for ketamine-mediated immunomodulation.

  5. The Saccharomyces cerevisiae Srb8-Srb11 Complex Functions with the SAGA Complex during Gal4-Activated Transcription

    OpenAIRE

    Larschan, Erica; Winston, Fred

    2005-01-01

    The Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase) complex functions as a coactivator during Gal4-activated transcription. A functional interaction between the SAGA component Spt3 and TATA-binding protein (TBP) is important for TBP binding at Gal4-activated promoters. To better understand the role of SAGA and other factors in Gal4-activated transcription, we selected for suppressors that bypass the requirement for SAGA. We obtained eight complementation groups and identified t...

  6. The Transcriptional Signature of Active Tuberculosis Reflects Symptom Status in Extra-Pulmonary and Pulmonary Tuberculosis.

    Directory of Open Access Journals (Sweden)

    Simon Blankley

    Full Text Available Mycobacterium tuberculosis infection is a leading cause of infectious death worldwide. Gene-expression microarray studies profiling the blood transcriptional response of tuberculosis (TB patients have been undertaken in order to better understand the host immune response as well as to identify potential biomarkers of disease. To date most of these studies have focused on pulmonary TB patients with gene-expression profiles of extra-pulmonary TB patients yet to be compared to those of patients with pulmonary TB or sarcoidosis.A novel cohort of patients with extra-pulmonary TB and sarcoidosis was recruited and the transcriptional response of these patients compared to those with pulmonary TB using a variety of transcriptomic approaches including testing a previously defined 380 gene meta-signature of active TB.The 380 meta-signature broadly differentiated active TB from healthy controls in this new dataset consisting of pulmonary and extra-pulmonary TB. The top 15 genes from this meta-signature had a lower sensitivity for differentiating extra-pulmonary TB from healthy controls as compared to pulmonary TB. We found the blood transcriptional responses in pulmonary and extra-pulmonary TB to be heterogeneous and to reflect the extent of symptoms of disease.The transcriptional signature in extra-pulmonary TB demonstrated heterogeneity of gene expression reflective of symptom status, while the signature of pulmonary TB was distinct, based on a higher proportion of symptomatic individuals. These findings are of importance for the rational design and implementation of mRNA based TB diagnostics.

  7. Activity-Based Anorexia Alters the Expression of BDNF Transcripts in the Mesocorticolimbic Reward Circuit.

    Directory of Open Access Journals (Sweden)

    Emily V Ho

    Full Text Available Anorexia nervosa (AN is a complex eating disorder with severe dysregulation of appetitive behavior. The activity-based anorexia (ABA paradigm is an animal model in which rodents exposed to both running wheels and scheduled feeding develop aspects of AN including paradoxical hypophagia, dramatic weight loss, and hyperactivity, while animals exposed to only one condition maintain normal body weight. Brain-derived neurotrophic factor (BDNF, an activity-dependent modulator of neuronal plasticity, is reduced in the serum of AN patients, and is a known regulator of feeding and weight maintenance. We assessed the effects of scheduled feeding, running wheel access, or both on the expression of BDNF transcripts within the mesocorticolimbic pathway. We also assessed the expression of neuronal cell adhesion molecule 1 (NCAM1 to explore the specificity of effects on BDNF within the mesocorticolimbic pathway. Scheduled feeding increased the levels of both transcripts in the hippocampus (HPC, increased NCAM1 mRNA expression in the ventral tegmental area (VTA, and decreased BDNF mRNA levels in the medial prefrontal cortex (mPFC. In addition, wheel running increased BDNF mRNA expression in the VTA. No changes in either transcript were observed in the nucleus accumbens (NAc. Furthermore, no changes in either transcript were induced by the combined scheduled feeding and wheel access condition. These data indicate that scheduled feeding or wheel running alter BDNF and NCAM1 expression levels in specific regions of the mesocorticolimbic pathway. These findings contribute to our current knowledge of the molecular alterations induced by ABA and may help elucidate possible mechanisms of AN pathology.

  8. ZmMYB14 is an important transcription factor involved in the regulation of the activity of the ZmBT1 promoter in starch biosynthesis in maize.

    Science.gov (United States)

    Xiao, Qianlin; Wang, Yayun; Du, Jia; Li, Hui; Wei, Bin; Wang, Yongbin; Li, Yangping;